Toxoplasma gondii, in mice, but the mechanism of resistance is poorly understood. We show that the p47 GTPases, including IIGP1, accumulate at vacuoles containing T. gondii. The accumulation is GTP-dependent and requires live parasites. Vacuolar IIGP1 accumulations undergo a maturation-like process accompanied by vesiculation of the parasitophorous vacuole membrane. This culminates in disruption of the parasitophorous vacuole and finally of the parasite itself. Over-expression of IIGP1 leads to accelerated vacuolar disruption whereas a dominant negative form of IIGP1 interferes with interferon-γ-mediated killing of intracellular parasites. Targeted deletion of the IIGP1 gene results in partial loss of the IFN-γ-mediated T. gondii growth restriction in mouse astrocytes.The p47 GTPases are essential for interferon-γ-induced cell-autonomous immunity against the protozoan parasite,  Toxoplasma gondii is a small unicellular parasite infecting virtually every warm-blooded animal including humans. After infection, T. gondii does not stay in extracellular fluids such as the blood, but actively invades body cells. The parasite has developed elaborate mechanisms enabling it to form a so-called parasitophorous vacuole (PV) within the cell it invades. Within this vacuole the parasite multiplies until the host cell ruptures and the progeny are released into the extracellular space to infect further cells. Host cells have developed several special mechanisms to combat the parasite. In mice, these mechanisms include a protein family, the p47 GTPases, which are induced by immune-alert factors called interferons. This study begins to address how the mouse p47 GTPases function. The study shows that the p47 GTPases assemble on the PV very shortly after infection, apparently to form a “membrane attack complex.” Within an hour the PV membrane shows signs of damage, bulging into small out-foldings that separate from the membrane in small vesicles. Shortly afterward the PV membrane ruptures and the parasite deteriorates. The p47 GTPase have several properties in common with the dynamin GTPases, which deform cellular membranes, suggesting that the p47 GTPases function in a mechanistically similar manner. Toxoplasma gondii and Plasmodium which enter host cells by active invasion , th, thT. goi growth E. IIGP1  vacuole . The inc vacuole , act witT. gondii from interferon-stimulated astrocytes. An LC3 construct labeled N-terminally with GFP was transfected into interferon-stimulated cells that were subsequently infected with T. gondii. At 2 hour after infection smooth vacuoles with strong IIGP1 accumulation showed no accumulation of LC3 , which are primarily located at the ER in interferon-treated cells in the absence of infection -uracil, astrocytes were inoculated with T. gondii at a multiplicity of infection (MOI) of 1  for an additional 20 h (Murine astrocytes were stimulated with IFNOI) of 1 A or MOI OI) of 1 E. After OI) of 1 A or 24 hnal 20 h A or 24 hnal 20 h E. The amT. gondii at a MOI of 5 or 10 for a maximum of 2 h. At that time point, extracellular parasites were removed by extensive washing with PBS. Cells were then either fixed or incubated further (up to 24 h post-infection) in fresh medium in the absence or presence of IFN-γ before fixation.For immunostaining, astrocytes were inoculated with T. gondii dense granule protein GRA7, a 29 kDa-predicted transmembrane protein that is released into the vacuole by intracellular parasites shortly after invasion and associates with the intravacuolar network and the PV membrane [Vacuoles containing intracellular parasites were identified by immunostaining for the membrane ,42. IntrThe coding regions of IIGP1, TGTP, and LC3 were amplified by PCR from full-length cDNAs described in  from IFN-T. gondii rabbit antiserum , anti-LAMP1 1D4B rat mAb , anti-GRA7 5–241–178 mouse mAb  [Serological reagents used were: Anti-IIGP1 165 rabbit antiserum , anti-II States) , anti-ctCells were washed with PBS and fixed in 3% paraformaldehyde for 20 min at room temperature. Cells were permeabilized with 0.1% saponin and blocked with 3% BSA (Roth). The cells were analyzed using an Axioplan II fluorescence microscope  equipped with a cooled CCD camera . Image processing and 2D deconvolution was done with the Metamorph software . For 3D deconvolution the Auto Deblur software  was used.For cryo-electron microscopy astrocytes were treated as indicated and fixed at 6 h post infection with 4% paraformaldehyde in 0.2M HEPES (pH 7.4). Cells were released from the plastic with a Teflon edge, pelleted in 1% gelatin in PBS and washed several times with 100 mM glycine in PBS. Pellets were incubated overnight in 2.3 M sucrose in PBS and shock frozen in liquid nitrogen. Ultra-thin sections were cut at −120 C and placed on formvar coated grids. Immunogold labeling was performed as follows: Sections were blocked in PBG  followed by incubation with the primary antibody diluted in PBG. After seven washes with PBS, grids were incubated with protein A coupled to 10 nm gold particles diluted in PBG. Grids were then washed ten times with PBS and ten times with water, and finally contrasted with methylcellulose/uranacetate (8.5 parts/1.5 parts).Astrocytes were treated as indicated and lysed in 1% T×100/PBS containing “complete mini” protease inhibitor cocktail . Lysates were spun at 23,000 × g for 15 min at 4C and supernatants were subjected to SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and probed for IIGP1 by incubation with the anti-IIGP1 165 antiserum. Bound primary antiserum was detected using goat anti-rabbit-Alexa-680 antiserum and blots were scanned using the Odyssey system  (IIGP1) or by conventional chemiluminescence.Figure S1, or left untreated and infected with T. gondii 24 h later for 2 h. Cells were fixed and stained for the indicated p47 GTPase. With the exception of the A19 anti-LRG-47 antiserum (E) no or a very low signal was detected in uninduced cells. The arrowheads point to representative vacuoles in the uninduced cells. The images for the −FN-γ controls were taken with the same exposure time as the +IFN-γ images.Astrocytes were induced with IFN-γ(1.0 MB JPG)Click here for additional data file.Figure S2Click here for additional data file.GTPI Localizes to the Golgi Apparatus, fixed 24 h later, and stained for GTPI and Gm130. The GTPI signal accurately, though not perfectly, overlaps with Gm130 localizing to the cis-Golgi. An additional vesicular signal throughout the cytoplasm and a weak signal at plasma membrane ruffles are also apparent. The nucleus was stained with DAPI.(A) Astrocytes were induced with IFN-γ(B) Astrocytes were induced with IFN-γ and simultaneously transfected with an IIGP1ctag1-expression construct. Cells were fixed 24 h later and stained for ctag1 using the anti-ctag1 2600 antiserum.(200 KB JPG)Figure S3(A) Lysates of astrocytes induced with the indicated concentrations of IFN-γ for 24 h were probed for the indicated proteins by Western blotting.T. gondii 6 h post-infection. Cells were labeled with the 165 anti-IIGP1 antiserum and protein A coupled to 10 nm gold particles  Bars: 0.5μm and 0.25μm (inset).(B) Transmission electron micrograph of ultra-thin cryosectioned uninduced astrocytes infected with (2.5 MB JPG)Click here for additional data file.Figure S4T. gondii stained for IIGP1 (green) and ROP2/3/4 (red). The arrowheads point to IIGP1-positive, but ROP2/3/4-low vacuoles.(A) IFN-γ- induced astrocytes 2 h post-infection with T. gondii. Cells were stained for IIGP1 with 10E7 (green) and an anti-T. gondii antiserum (red). The white arrowhead points to a disrupted parasite. Nuclei were stained with DAPI.(B) IFN-γ- induced astrocytes 6 h post-infection with (628 KB JPG)Click here for additional data file.Figure S5T. gondii and fixed 2 h (A) or 6 h (B) post-infection. Cells were stained with 10D7 for IIGP1 (green) and A20 for TGTP1 (red). (C) Astrocytes were transfected with IIGP1ctag1 and stimulated with IFN-γ. Cells were infected 24 h later with T. gondii, fixed 2 h post-infection, and stained for ctag1 (green) and GRA7 (red). (D) Astrocytes were transfected with IIGP1K82Actag1 and stimulated with IFN-γ. Cells were infected 24 h later with T. gondii, fixed 2 h post-infection, and stained for ctag1 (green) and TGTP1 (red). (E) Astrocytes were transfected with the indicated expression constructs and stimulated with IFN-γ. Cells were infected 24 h later with T. gondii at a MOI of 5, fixed at the indicated time points, stained for TGTP1 and ctag1. The number of TGTP1-positive vacuoles in transfected cells displaying a smooth, rough, or disrupted morphology was counted.  Nuclei were stained with DAPI. (B) Shows a maximum projection of a 3D deconvoluted Z-series.IFN-γ induced astrocytes were infected with (2.8 MB JPG)Click here for additional data file.Table S1(28 KB DOC)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for genes and gene products discussed in this paper are GTPI , IGTP , IIGP1 , IRG-47 , LRG-47 , and TGTP .The Mouse Genome Initiative Database (


SGNE1 is located. The aim of this study is to analyze associations of SGNE1 genetic variation with obesity and metabolism related quantitative traits.7B2 is a regulator/activator of the prohormone convertase 2 which is involved in the processing of numerous neuropeptides, including insulin, glucagon and pro-opiomelanocortin. We have previously described a suggestive genetic linkage peak with childhood obesity on chr15q12-q14, where the 7B2 encoding gene, SGNE1 for genetic variants in obese children and genotyped 12 frequent single nucleotide polymorphisms (SNPs). Case control analyses were performed in 1,229 obese (534 children and 695 adults), 1,535 individuals with type 2 diabetes and 1,363 controls, all French Caucasians. We also studied 4,922 participants from the D.E.S.I.R prospective population-based cohort.We screened SGNE1 SNPs and childhood or adult obesity. However, the 5' region SNP -1,701A>G associated with higher area under glucose curve after oral glucose tolerance test (p = 0.0005), higher HOMA-IR (p = 0.005) and lower insulinogenic index (p = 0.0003) in obese children. Similar trends were found in obese adults. SNP -1,701A>G did not associate with risk of T2D but tends to associate with incidence of type 2 diabetes  in the prospective cohort.We did not find any association between SGNE1 genetic variation does not contribute to obesity and common forms of T2D but may worsen glucose intolerance and insulin resistance, especially in the background of severe and early onset obesity. Further molecular studies are required to understand the molecular bases involved in this process. We directly sequenced coding regions including exons, exons/introns boundaries, 3 kb upstream ATG codon (including non coding exon1 and intron1) and 1 Kb downstream the stop codon. We used DNA from 48 unrelated obese children and 24 unrelated control adults. PCR amplifications were purified with Montage PCR384 Multiscreen®S384PCR (Millipore). Sequencing was performed using the automated ABI Prism 3730xl DNA sequencer in combination with the Big Dye Terminator cycle (Applied Biosystems) and purification sequencing reaction with MultiScreen®SEQ384 filter plates (Millipore). Data from HapMap phase I was used to select TagSNPs (MAF≥0.05 in CEU population) to improve the genetic coverage of SGNE1.SNPs identified by direct sequencing were genotyped using either the LightCycler™ or LightTyper™ technologies (Roche). TagSNPs selected from HAPMAP I, and SNP -1,071A>G in the cohort were genotyped by the Applied Biosystems SNPlex™ technology based on the Oligonucleotide Ligation Assay (OLA) combined with multiplex PCR target amplification. Allelic discrimination was performed through capillary electrophoresis analysis using an Applied Biosystems 3730xl DNA Analyzer and GeneMapper3.7 software. All SNPs were re-genotyped in a subset of 379 individuals by direct sequencing to check for genotyping discrepancies. Genotyping error rates were ≤ 0.01 for all SNPs. Hardy-Weinberg equilibrium (HWE) was tested in controls and was verified for all SNPs (0.11 ≤ p ≤ 0.68).Tests for deviation from HWE and for association were performed by the De Finetti program . Case coSGNE1, we selected TagSNPs from the HapMap phase I. SNPs with a low genotype call rate (<70%) were not analyzed. Thus, eight TagSNPs all located in introns were added and 12 SNPs  and eight rare SNPs by direct sequencing. Frequent SNPs  were all located in the 5' region. In order to improve the genetic coverage of SGNE1 SNPs or haplotype combination (data not shown) studied showed a significant association with severe forms of obesity in our populations. Thus, as expected, the contribution of these SNPs to linkage with childhood obesity observed on chromosome 15q12-q14 was not significant (data not shown). We also analyzed association of SGNE1 SNPs with moderate obesity (30 ≤ BMI ≤ 40). Case control analyses in additional 620 subjects did not show significant association with this less severe form of obesity .Clinical characteristics of the populations studied are presented in Table SGNE1 SNPs and obesity related quantitative traits in obese subjects. SNP-1,701A>G, located in intron 1 which is part of the 5' region of SGNE1  and tended to be associated with higher levels at 90 min (p = 0.06) and 120 min (p = 0.085) Figure . Accordi8 and tenSGNE1 SNPs, including -1,701A>G in an independent population of 1,535 type 2 diabetics. SGNE1 SNPs or halpotypes (data not shown) did not associate with T2D  but not HbA1C levels .We then investigated the role of SNP -1,701A>G in the risk of T2D. We genotyped We then aimed to assess if the SNP -1,701A>G modulates traits related to obesity and glucose intolerance in a population-based cohort. Thus, SNP-1,701A>G was also genotyped in 4,922 participants from the D.E.S.I.R prospective study. Cross sectional analyses at baseline did not show an association of SNP -1,701A>G with fasting glucose, fasting insulin, HbA1c levels, HOMA-IR or BMI (data not shown). Only fasting glucose and fasting insulin levels were available for the cohort participants. After 9 years follow-up, allele -1,701G carriers tend to be at lower risk of incidence of overweight , obesity  and T2D  , where SNPs were associated with higher glucose after OGTT, although allelic frequencies were similar between T2D cases and controls [Our results support that SNP -1,071A>G, although not associated with obesity, may contribute to higher glucose levels after OGTT. Similar findings were previously reported for controls . SNP -1,controls . Howevercontrols .SGNE1. It has been shown that intron 1 sequences are essential for transcriptional activity of SGNE1 in human cell lines [SGNE1 is hypermethylated and epigenetically silenced in medulloblastomas [SGNE1 and a potential functional role of SNP -1,701A>G in the regulation of SGNE1. According to in silico analyses [TCF/LEF1 (Transcription Factor T-Cell specific/Lymphoid Enhancer binding Factor1). To our knowledge, TCF/LEF1 role in metabolism has not been described. We note that TCF/LEF1 belongs to the Wnt signaling pathway that includes TCL7L2, which harbours highly associated SNPs with T2D [SGNE1 and ultimately impaired metabolism. An increase of 7B2 has been reported in pancreas from ob/ob mice compared to wild type mice [SGNE1 functionality and consequently, glucose tolerance and insulin resistance.SNP-1,701A>G is located in intron 1 and is part of the 5' region of ll lines . A recenlastomas . This daanalyses , SNP -1,with T2D . Furtherype mice  suggestiype mice , analyseAs a protein helper and activator of PC2, 7B2 is involved in the maturation of a large spectrum of molecules playing central roles in weight and glucose homeostasis . 7B2 andSGNE1 genetic variation in glucose intolerance and insulin resistance in the background of young onset obesity. Further analyses are required to replicate our findings and to understand the molecular bases of SGNE1 role in the genetic susceptibility to the risk of glucose intolerance in response to glucose load.In summary, we report genetic evidence for the role of AUC Area under curveBMI Body mass indexCART Cocaine amphetamine regulated transcriptCCK CholecystokininHOMA-IR Homeostasis model assessment – insulin resistanceIFG Intolerant fasting glucoseNGT Normal glucose tolerantOGTT Oral glucose tolerance testPC2 Proprotein convertase 2PCR Pomymerase chain reactionPOMC Pro-opiomelanocortinSGNE1 Secretory granule neuroendocrine protein 1SNP Single nucleotide polymorphismT2D Type 2 diabetesTCF/LEF1 Transcription factor t-cell specific/lymphoid enhancer binding factor 1TCF7L2 Transcription factor t-cell specific 7-like 2USF1 Upstream Stimulatory Factor 1The author(s) declare that they have no competing interests.NBN and VV performed the genotyping. NBN, CL, JV, CP and DM performed the statistical analyses. DNA was provided by GC, BH, JT, MM and BB. NBN drafted the manuscript under PF and DM supervision. DM and PF supervised the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Dicer is a ribonuclease that mediates RNA interference both at the transcriptional and the post-transcriptional levels. Human dicer gene expression is regulated in different tissues. Dicer is responsible for the synthesis of microRNAs and short temporal (st)RNAs that regulate the expression of many genes. Thus, understanding the control of the expression of the dicer gene is essential for the appreciation of double-stranded (ds)RNA-mediated pathways of gene expression. Human dicer mRNA has many upstream open reading frames (uORFs) at the 5'-leader sequences (the nucleotide sequence between the 5'-end and the start codon of the major ORF), and we studied whether these elements at the 5'-leader sequences regulate the expression of the dicer gene.in vitro and in vivo.We determined the 5'-leader sequences of the dicer mRNAs in human breast cells by 5'-RACE and S1-nuclease protection analysis. We have analyzed the functions of the 5'-leader variants by reporter gene expression We found that the dicer transcripts in human breast cells vary in the sequence of their 5'-leader sequences, and that alternative promoter selection along with alternative splicing of the 5'-terminal exons apparently generate these variations. The breast cell has at least two predominant forms of dicer mRNAs, one of which has an additional 110 nucleotides at the 5'-end. Sequence comparison revealed that the first 80 nucleotides of these mRNA isoforms are encoded by a new exon located approximately 16 kb upstream of the reported start site. There are 30 extra nucleotides added to the previously reported exon 1. The human breast cells studied predominantly express two 5'-leader variants of dicer mRNAs, one with the exons 2 and 3 (long form) and the other without them (short form). By reporter gene expression analysis we found that the exon 2 and 3 sequences at the 5'-leader sequences are greatly inhibitory for the translation of the mRNA into protein.Dicer gene expression in human breast cells is regulated by alternative promoter selection to alter the length and composition of the 5'-leader sequence of its mRNA. Furthermore, alternative splicing of its exon 2 and 3 sequences of their pre-mRNA creates a more translationally competent mRNA in these cells. RNA interference (RNAi), a process of silencing gene expression, involves the generation of short, double-stranded RNA (dsRNA) molecules by an enzyme called dicer, which cleaves RNA duplexes into 21–23 base-pair oligomers . These oDicer gene expression is regulated in different tissues in humans . BecauseWe used a series of commercially available human lines of breast cells, including human mammary epithelial (HME) cells , MDA-MB-231, MCF-7, MDA-MB-468, MCF-10A, and BT549. We also used non-breast cells such as HeLa and HepG2. All cells, other than the HME cells, were purchased from American Type Culture Collection . HMEC cells were grown in medium purchased from Clonetics under their recommended conditions. Human breast carcinoma MDA-MD-231 and MDA-MD-468 cells were maintained in Leibovitz's L-15 medium supplemented with 1% antibiotic/antimycotic and 10% fetal bovine serum. MCF-10A cells were maintained in a 1:1 mixture of Ham's F12 medium, Dulbecco's modified Eagle's medium supplemented with 1% antibiotic/antimycotic, 0.098 mg/ml cholera toxin, 0.02 μg/μl epidermal growth factor, 0.5 μg/ml hydrocortisone, and 10% horse donor heard serum. BT-549 cells were maintained following standard ATCC recommended media. All cells were maintained in a humidified CO2 (5%) incubator at 37°C. Other cells were maintained and grown in ATCC recommended media and conditions ,18.Complementary DNAs (cDNA) were made from total RNA (5 μg) using random primers following standard protocols ,19. The Oligonucleotides that were biotinylated at the 5'-end  spanning the exon 2 and exon 3 boundary of the long 5'-variant and the exon 1 and exon 4 boundary of the short 5'-variant of the dicer mRNA, respectively, were custom synthesized and gel-purified (Invitrogen). The standard S1-nuclease protection protocol was followed  with theRenilla luciferase that was amplified with forward primer 5'-GCTGTGCTAGAACAAAAATGCAATGACTTCGAAAGTTTATGATCC-3' and reverse primer 5'-CTCGAAGCGGCCGCTCTAG-3' [Renilla luciferase ORF alone was amplified using forward primer 5'-ATGACTTCGAAAGTTTATGATCC-3' and reverse primer 5'-CTCGAAGCGGCCGCTCTAG-3'. SOEing products were then ligated into the pCRII-Topo vector (Invitrogen), digested with EcoRI (Promega) and cloned at the EcoRI site of pcDNA3.1(+) (Invitrogen) and sequenced with T7 primer to determine the orientation of the cloned insert. It is anticipated from the vector information (Invitrogen) that the Renilla luciferase transcript from these constructs will have a 133 nucleotide vector derived sequence before the 5'-leader sequences from the dicer gene at their 5'-end. Thus, the nucleotide sequence that is common to the control and the experimental transcripts is 5'-TAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTT-3'. This sequence does not have any ATG codon.The long and short form dicer 5'-leader sequences were amplified using the forward primer 5'-GCGGAAGTGGGTGTTTGTTATTTCC-3' and the reverse primer 5'-GGATCATAAACTTTCGAAGTCATTGCATTTTTGTTCTAGCACAGC-3'. Gene splicing by overlapping extension (SOE) was used to fuse dicer variants to TCTAG-3' . The RenRenilla luciferase activities using Dual Luciferase Assay Reagents (Promega) following suggested protocols [Renilla luciferase activity was normalized with respect to firefly luciferase activity and presented as a ratio (relative light units). Protein contents of the extract, when needed, were determined using RC-DC reagents and protocol from BioRad Laboratories Hercules, CA, USA [Cells were seeded at 80% confluency in a 24-well plate for 24 h in their growth media before co-transfection with one of the pcDNA3.1(+) test plasmid constructs and pGL3-control plasmid. The latter was used as a transfection normalization control. Plasmids were mixed at 0.5 μg per well and transfection was done using the Lipofectamine Plus transfection reagents (Invitrogen) using the protocol suggested by the supplier. After 20 h of incubation in complete medium at 37°C, the cells were lysed in 100 μl passive lysis buffer (Promega) and 5–20 μl of the lysate was assayed  for firerotocols . Renilla CA, USA .EcoRI site in proper orientation on the translatability of the mRNA, we used the T7 RNA polymerase/rabbit reticulocyte lysate in vitro transcription/translation system . Each plasmid DNA (1 μg) was added to 40 μl of TNT Quick Master Mix containing 1 mM of methionine (Promega). The reactions were incubated at 30°C for 75 min then cooled to 4°C. The TNT reactions were diluted 1:2 with 1x passive lysis buffer (Promega) and incubated at room temperature for 15 min. An aliquot (5–20 μl) was assayed in 100 μl of Renilla Luciferase Assay Substrate (Promega).The pcDNA3.1(+) constructs containing the cloned inserts also have a T7 RNA polymerase promoter (Invitrogen). To evaluate whether there is any effect of the dicer 5'-leader sequence insert at the P value [P value was less than 0.05, the difference was considered to be significant [Each data set is presented as mean ± SEM (N = 12). Statistical significance of a difference between two series of data was tested by determining the P value . If the nificant .The expression of the dicer gene is differentially regulated in many organisms that have the RNAi mechanism -26. In hThe long form of the 5'-leader sequences contains nine upstream AUG codons Fig. . The shoTo ensure that the existence of the different 5'-leader sequences in dicer mRNA as revealed by the 5'-RACE analysis was not a PCR artifact, and also to determine relative quantities of each of the mRNA isoforms, we verified the levels of each of the isoforms by S1-nuclease protection assays Fig. . Data frRenilla luciferase mRNAs with the general structure shown in Fig. Renilla luciferase activity in the cell lysate. The plasmid that constitutively expresses firefly luciferase from SV40 late promoter was used as transfection control and the Renilla luciferase data were normalized with respect to the firefly luciferase activity in the extract. Tthe construct with the long form of the 5'-leader sequence expressed no significant Renilla luciferase activity compared to the controls  declare that they have no competing interests.CVIW executed the experiments described in the manuscript and wrote the initial drafts of the manuscript. GC conceived and coordinated the study. All authors read and approved the final manuscript.


Uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP) act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5'-triphosphate (ATP) acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems.2+-dependent, Cl- current  activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA) and large (LPA) intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student's t test or one way ANOVA.The perforated-patch clamp technique was used to record the Ca-4M) evoked oscillating, inward currents (peak = 13–727 pA) in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P < 0.05). Subsequent currents tended to decrease in amplitude, with a variable time-course, to a level that was significantly smaller for ATP (P < 0.05), UTP (P < 0.001) and UDP (P < 0.05) in the SPA. The frequency of oscillations was similar for each agonist (mean≈6–11.min-1) and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10-4M) abolished currents evoked by ATP in SPA (n = 4) and LPA (n = 4), but pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (10-4M), also a non-selective P2 antagonist, had no effect . Currents elicited by UTP (n = 37) or UDP (n = 14) were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4) and abolished by suramin (n = 5). Both antagonists abolished the contractions in LPA.ATP, UTP and UDP (10Cl,Ca in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y11 receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction.At least two P2Y subtypes couple to I After incubation in DM containing 0.6 – 0.8 mg.ml-1 papain, 0.04% BSA and 0.4 mM dithiothreitol at 37°C , collagenase  was added and the tissues incubated for a further 10 (LPA) or 5 (SPA) min. Cells were then dispersed by mild trituration in enzyme-free solution and used within 7 hours.Male Sprague-Dawley rats (150 – 250 g) were killed by the approved Schedule 1 method of cervical dislocation and exsanguination. After thoracotomy, the heart and lungs were removed 2PO4 0.5; NaH2PO4 0.5; MgCl2 1; glucose 11; CaCl2 1.8; titrated to pH 7.3 with NaOH. Electrophysiological responses of isolated smooth muscle cells were studied in the whole-cell, perforated-patch mode with amphotericin B (150 μg.ml-1) added to a pipette solution of the following composition (mM): KCl 125; MgCl2 4; HEPES 10; ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) 0.02, titrated to pH 7.3 with KOH. Pipette resistance was 4–8 MΩ. The cells were voltage-clamped at -50 mV using an Axopatch 200A amplifier (Axon Instruments). Data were recorded and analysed with a personal computer interfaced with a Digidata 1200 A/D converter (Axon Instruments) using Axotape and pClamp (V5) software (Axon Instruments). Current responses to -10 mV hyperpolarizing steps were used to measure cell capacitance.Cells were placed in a 50 μl chamber and superfused at room temperature with physiological salt solution (PSS) composed of (mM): NaCl 122; KCl 5; HEPES 10; KH-4 M ATP, UTP and UDP each evoked pronounced contractions of rat isolated SPA and LPA and that 10-4 M suramin and PPADS produced maximum inhibition of these responses   . Thus, t11 receptor. The site of action of UTP and UDP is less clear. Its pharmacology is inconsistent with our present understanding of P2Y2,4 & 6 receptors, so a novel receptor or receptor complex may be involved. These different P2Y receptors are likely to play a significant role in nucleotide-induced pulmonary vasoconstriction as ATP, UTP and UDP each induce contractions of the rat pulmonary artery with matching pharmacological profiles.The results of the present study indicate the presence of at least two different subtypes of P2Y receptors mediating oscillating inward currents in rat SPA and LPA smooth muscle cells. ATP acts via a suramin-sensitive, PPADS-insensitive site, which most resembles the P2YThe author(s) declare that they have no competing interests.KC was involved in the planning of the experiments described and carried them out. She also analysed the data, drafted the manuscript and was involved in its revision. AMG was involved in the planning of the experiments and revision of the manuscript. CK was involved in the planning of the experiments, the analysis of the data and revision of the manuscript. All authors read and approved the final manuscript.


We established a vindesine-resistant (x 11.6) human small-cell lung cancer cell line (H69/VDS) by stepwise exposure of parent line H69 to vindesine. H69/VDS showed cross-resistance to taxol (x 10.1), vincristine (x 6.9) and colchicine (x 3.4) but not to doxorubicin, cisplatin or etoposide. There was no significant difference in intracellular [3H]-vincristine and doxorubicin accumulation between H69 and H69/VDS cells. The human mdr1 mRNA was not detected in either of the cell lines. These results indicated that H69/VDS did not express a typical multidrug resistant phenotype. Addition of 20 microM verapamil enhanced the growth inhibitory effect of vindesine on both H69/VDS (x 12.0) and H69 cells (x 3.8). The amount of total tubulin in H69/VDS cells was lower than that in the H69 parental cells. No significant increase was observed in the amount of total and polymerised tubulins of H69 cells. In H69/VDS cells, however, verapamil increased the amount of total tubulin to the level of parental cells, but decreased the amount of polymerised tubulin. Modulation of tubulin may play a role in the resistance to vindesine.


CUL5 gene (encoding Cullin 5 protein) on AIDS disease progression in five HIV-1 longitudinal cohorts. A total of 12 single nucleotide polymorphisms (SNPs) spanning 93 kb in the CUL5 locus were genotyped and their haplotypes inferred. A phylogenetic network analysis revealed that CUL5 haplotypes were grouped into two clusters of evolutionarily related haplotypes. Cox survival analysis and mixed effects models were used to assess time to AIDS outcomes and CD4+ T cell trajectories, respectively. Relative to cluster I haplotypes, the collective cluster II haplotypes were associated with more rapid CD4+ T cell loss (relative hazards [RH] = 1.47 and p = 0.009), in a dose-dependent fashion. This effect was mainly attributable to a single cluster II haplotype (Hap10) (RH = 2.49 and p = 0.00001), possibly due to differential nuclear protein–binding efficiencies of a Hap10-specifying SNP as indicated by a gel shift assay. Consistent effects were observed for CD4+ T cell counts and HIV-1 viral load trajectories over time. The findings of both functional and genetic epidemiologic consequences of CUL5 polymorphism on CD4+ T cell and HIV-1 levels point to a role for Cullin 5 in HIV-1 pathogenesis and suggest interference with the Vif-Cullin 5 pathway as a possible anti-HIV-1 therapeutic strategy.Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (Apobec3) antiretroviral factors cause hypermutation of proviral DNA leading to degradation or replication-incompetent HIV-1. However, HIV-1 viral infectivity factor (Vif) suppresses Apobec3 activity through the Cullin 5-Elongin B-Elongin C E3 ubiquitin ligase complex. We examined the effect of genetic polymorphisms in the  CUL5 could tip the balance between HIV-1 and Apobec3G and modify the course of HIV-1 infection. They showed that genetic variation in the CUL5 gene encoding Cullin 5 affected the rate of CD4+ T cell loss in patients infected with HIV-1. CUL5 haplotypes formed two clusters of evolutionarily related haplotypes with opposing effects—cluster I delayed and the cluster II accelerated CD4+ T cell loss. The effect was mainly attributable to a single haplotype or its tagging-SNP, which demonstrated differential binding of transcription factors. This finding highlights the epidemiologic importance of the HIV-1 and Cullin 5 interaction and suggests that the factors in the HIV-1 Vif-Apobec3G degradation pathway may be targets for antiviral drugs.Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 G (Apobec3G) is an innate antiviral protein that inhibits HIV type 1 (HIV-1) replication by causing deleterious mutations in the HIV-1 genome. Unfortunately, HIV-1 has a strategy to defeat the antiviral activity of Apobec3G. The HIV-1 viral infectivity factor (Vif) binds to Apobec3G leading to the degradation of Apobec3G through a complex containing Cullin 5 and the proteins Elongin B and Elongin C. Since Cullin 5 directly interacts with Vif and is critical to the Apobec3G degradation pathway, the authors asked if genetic variation of  Members of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (Apobec3) family of cytidine deaminases are innate cellular anti-HIV-1 factors . In the APOBEC3G gene may be associated with altered AIDS progression adj = 1.47 and p = 0.009). No significant associations were found for AIDS-1987   A. These luster 1 A.f ≈ 5%) was a strong risk factor for CD4+ T cell depletion  , and all A/G heterozgotes developed CD4+ < 200 within nine years of seroconversion  SNP6 G allele carried only on Hap10 ( e model) . In a Kailcoxon) B.adj = 1.25 and p = 0.170), suggesting that most of the association of ctSNP5 G allele and other cluster II haplotypes with CD4+ T cell loss was due to htSNP6. When considering ctSNP5 as a confounding covariate, the association remained robust for htSNP6 (RHadj = 2.16 and p = 0.0009). However, not all of the ctSNP5 association with CD4+ T cell decline could be attributed to htSNP6. This can be readily observed in p = 0.0002, log-rank). This suggests that one or more ctSNP5 G-bearing haplotypes and the htSNP6 G-bearing Hap10 may be tracking the same functional allele elsewhere in the CUL5 gene.Because Hap10 carries both the htSNP6 G allele and the ctSNP5 G allele, we tested ctSNP5 adjusting for the effects of htSNP6 within a Cox model. The ctSNP5 effect became nonsignificant   . None ofctively) .+ < 200, the Akaike Information Criteria (AIC) were used to select the best Cox proportional hazards model. The covariates HLA homozygosity and HLA B57 were used in the base mode (model 1). The smallest AIC was achieved for model 5 where all three haplotypes were added in model 5 , and the cluster I Hap7 had a slight accelerating effect  (p = 0.03), SNP7 , and SNP12   . Small e = 0.02) .+ T cell counts and HIV-1 RNA levels were measured multiple times during the follow-up period for the AIDS Link to the Intravenous Experience (ALIVE) SC participants. An average of 7.8 and 6.8 measurements of CD4+ T cell counts and viral load, respectively, per patient for up to nine years from the seroconversion to 1997-censoring date were available for analysis. We evaluated the effects of ctSNP5  and htSNP6 (Hap10) on the longitudinal slope of CD4+ T cell count and HIV-1 RNA level over the clinical course using the linear mixed random effects model.CD4+ T cell slopes stratified by genotype (p= 0.05) or −1.23 (p = 0.01) lower mean CD4+ T cell trajectory, over the observation periods from seroconversion to July 31, 1997  or to July 31, 2004, respectively  or −2.63 (p = 0.01) lower mean CD4+ T cell trajectory, for the observation periods from seroconversion to July 31, 1997 or to July 31, 2004, respectively . No effect was detected for SNP6, possibly due to the low frequency of the minor allele and smaller number of HIV-1 RNA measurements available , in the data censored in July 31, 1997. A stronger association  was obtained using the extended followed-up time to July 31, 2004 . The FDR was calculated incorporating all p-values from 122 tests performed for SNPs and haplotypes in the Cox model and mixed effects model. The q-value was obtained for each and all of the p-values, and the q-values for those with p ≤ 0.05 were presented. The associations for ctSNP5, htSNP6 with CD4+ < 200, and CD4+ T cell slope in AA as well as Hap1, Hap7 in AA, and Hap3 in EA had q-values below the stringent cutoff of 0.05 indicating a FDR of 5% for a given p-value -4 in an electrophoretic mobility-shift assay (EMSA). A 1.85-fold or 1.75-fold increase in the band density was observed for the SNP6 G allele in comparison with the common A allele from two independent experiments, although no pattern changes were observed . A similCUL5 gene product Cullin 5, leading to the polyubiquitination and degradation of Apobec3G and Apobec3F through the Cullin 5-Elongin B-Elongin C E3 ubiquitin ligase pathway [+ T cell decline as indicated by the Cox model analysis of survival to the endpoint of CD4+ < 200 and reaffirmed by the mixed effects model assessing CD4+ T cell trajectory over time. Individuals who were carriers of any two cluster II haplotypes progressed to CD4+ < 200 about 5.7 years faster than those who carried any two cluster I haplotypes. The effect of cluster II was largely attributable to Hap10, carrying both ctSNP5 G and htSNP6 G alleles. On the other hand, cluster I contains the two protective haplotypes, Hap1 and Hap6, which delayed CD4+ T cell loss. We also observed an additive interaction between CUL5 cluster II haplotypes and APOBEC3G-H186R on CD4+ T cell slope, suggesting that Apobec3G and Cullin 5 likely confer independent effects on CD4+ T cell loss [The ability of Apobec3G to restrict HIV-1 replication through hypermutation is suppressed by HIV-1 Vif binding to  pathway ,14,21. TCUL5 likely modifies the rate of disease progression of HIV-1 with the effects being stronger and more consistent among AA than EA. The differences observed between the two racial groups suggest that there are additional functional alleles in LD with the SNP markers assessed in this study.The genetic effects derived from multiple SNPs and haplotypes, and from the haplotype clusters, provide evidence that genetic variation in p < 0.05) SNP and haplotype associations in both AA and EA was 5% or less. However, the gold standard for validation of genetic associations remains confirmation in other adequately powered studies.To assess the possibility that the observed associations were due to the multiple tests, we estimated the FDR for all the tests performed. Although this test is quite conservative as there is considerable correlation among SNPs, haplotypes, and disease endpoints, the FDR for significant  was detected between genotypes (unpublished data), though a more subtle difference could not be excluded. Little is known about CUL5 regulation in different cell types or within CD4+ T cells infected by HIV-1. It is possible that SNPs in CUL5 influence HIV-1 disease by alternative splicing, by affecting mRNA stability, or by unknown protein structural change, in addition to altered gene expression. The underlying mechanisms of CUL5 genetic variation on HIV-1 warrant further investigation.In an in vitro electromobility gel shift assay, the DNA fragment carrying the G allele of SNP6, compared to the more common A allele, was found to show an increased binding affinity to nuclear proteins from human T lymphocytes. This suggests that SNP6 is a functional SNP that may affect the gene regulation or interaction. A higher level of Cullin 5 due to a potential up-regulation of CUL5 gene variation is associated with modified rates of CD4+ T cell loss. If confirmed, these findings demonstrate the epidemiologic importance of the interaction between HIV-1 Vif and human Cullin 5. The antiviral activity of both Apobec3G and Apobec3F have been shown to have antiviral activity that is fully suppressed by HIV-1 Vif through the Cullin 5 containing E3 ubiquitin ligase [CUL5 genetic variants may affect the efficiency of Vif-mediated degradation of Apobec3F and Apobec3G. These in vitro studies, together with our findings that CUL5 genetic factors modify the rate of CD4+ T cell loss, provide support for the development of inhibitors that block HIV-1 Vif and Cullin 5-Elongin B-Elongin C E3 ligase complex binding, aiming to limit or prevent degradation of these potent antiviral host factors.This study provides evidence that n ligase ,14. DeplStudy participants were enrolled in five United States-based natural history HIV/AIDS cohorts. ALIVE is a community-based cohort of intravenous injection drug users in Baltimore enrolled in 1988–1989 , consistCUL5 genetic studies in this population. This group was not used in the association analyses.The date of seroconversion after study enrollment was estimated as the midpoint between the last seronegative and first seropositive HIV-1 antibody test; only individuals with less than two years' elapsed time between the two tests were included in the seroconverter progression analysis. The censoring date was the earliest of the date of the last recorded visit, or December 31, 1995 for the MACS, MHCS, HGDS, and SFCC, or July 31, 1997 for the ALIVE cohort, to avoid potential confounding by HAART. The censoring date was extended in the ALIVE cohort because of delayed administration of HAART to this group ,29. The The study was approved by the Institutional Review Boards of participating institutes, and informed consent was obtained from the participants.CUL5 polymorphisms. PCR primers were designed based on GenBank DNA sequence AP003307 and mRNA sequence NM_003478 to cover the putative promoter region, 5′ UTR, exons 15–19 encoding the C terminus that was shown to confer major function [CUL5 gene. SNPs were obtained from dbSNP, HapMap (http://www.hapmap.org), and TaqMan SNP Genotyping Assay databases (http://myscience.appliedbiosystems.com). A total of 12 SNPs were selected for genotyping by considering SNP location, spacing, and at least 5% allele frequency . TaqMan primer and probes were designed by using the Primer Express software or by the Assay-by-Demand service of Applied Biosystems . Conformity to the genotype frequencies expected under Hardy-Weinberg equilibrium was examined for each SNP. The genetic effects of SNPs on HIV-1 infection susceptibility were assessed by comparing allelic frequencies between HIV-1 HREU and HIV-1 SC participants using the Fisher's exact test. Kaplan-Meier survival statistics and the Cox proportional hazards model were used to assess the effects of SNPs and haplotypes on the rate of progression to AIDS. We considered three separate endpoints reflecting advancing AIDS pathogenesis for SC: (1) HIV-1 infection plus a decline of CD4+ T cell counts < 200 cells/mm3 (CD4+ < 200); (2) the 1993 Centers for Disease Control and Prevention (CDC) definition of AIDS (AIDS-93): HIV-1 infection plus a CD4+ T cell count of <200/mm3 or AIDS-defining conditions [p-values were 2-tailed. Genetic factors previously shown to affect progression to AIDS in EA and AA groups were included as confounding covariates in the adjusted Cox model analysis: CCR5 Δ32 [CCR2-64I [CCR5-P1 [HLA-B*27 [HLA-B*57 [HLA-B*35Px group  [HLA class I homozygosity [HLA-B*57 and HLA class I homozygosity for AA. CCR2-64I, HLA-B*27, and HLA-B*35Px were not considered as covariates in AA due to absent or weak effects in our AA participants, and CCR5 Δ32 was not considered due to its rarity in AA. Participants were stratified by sex and by age at seroconversion: 0–20, >20–40, and >40 years [Analyses were conducted using the statistical packages SAS version 9.0  thenditions . The sigCCR2-64I ; CCR5-P1[CCR5-P1 ; HLA-B*2HLA-B*27 ; HLA-B*5HLA-B*57 ; HLA-B*3 B*5301) ; and HLAzygosity  for EA; 40 years .To evaluate which haplotype or combination of haplotypes predicted survival, the AIC generated in the Cox model was used as the model selection criterion . Among mq-value measuring FDR, which is defined as the proportion of statistical tests called significant that are actually false-positive [q-values, also known as FDR adjusted p-values, was considered more powerful than the Bonferroni procedure when there is high degree of LD among the markers [To account for the multiple comparison tests performed in our analysis, we estimated the y/qvalue). The app markers . FDR is  markers .+ T cell counts over time were modeled using the random effects linear models from HIV-1 SCs enrolled in the ALIVE study, adjusted for sex and age [+ T cell measurements over time while accounting for the correlation of repeated measurements within each individual. In all models absolute CD4+ T cell counts were square-root transformed, and plasma HIV-1RNA levels were log10 transformed to better comply with model assumptions. A likelihood ratio test was used as a test of significance. Two observation periods were used: (1) from seroconversion to the censoring date of July 31, 1997, before any impact of HAART was observed in the ALIVE cohort [+ T cell and HIV-1 RNA levels were available for analysis.The repeated measurements of HIV-1 RNA levels and CD4 and age . This moE cohort ; and (2)Pairwise LD was quantified using the absolute value of Lewontin's D′ . AbsolutCUL5 locus, a minimum mutation phylogenetic network was constructed by using NETWORK package, based on the Reduced Median (RM) network method (http://fluxus-technology.com/sharenet.htm) [To depict the relationships among inferred haplotypes and evolutionary history of the genetic variants at the net.htm) . A chimphttp://www.peprotech.com) or 100 nmol/l human rIL-2  at 37 °C for 10 min. Cell pellets were frozen at −70 °C. The probe sequences were 5′-CAGTTGAACATaCCTTGTTAGGA-3′ for SNP6 A allele and 5′-CAGTTGAACATgCCTTGTTAGGA-3′ for SNP6 G allele. In cold oligonucleotide competition assay, 100-fold excess of unlabeled probe was added as a competitor. The band density was measured by the software ImageJ (http://rsb.info.nih.gov/ij/index.html).Cell culture and EMSA were performed as described ,47. FresTable S1(18 KB XLS)Click here for additional data file.Table S2(22 KB XLS)Click here for additional data file.Table S3(15 KB XLS)Click here for additional data file.Table S4(17 KB XLS)Click here for additional data file.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) accession numbers for the genes discussed in this paper are Cullin 5 (8065), Elongin B (6923), Elongin C (6921), Rbx1 (9978), Apobec3G (60489), Apobec3F (200316), and HIV-1 Vif (155459).The Entrez Gene databank (http://www.ncbi.nlm.nih.gov) accession number for chimpanzee CUL5 sequence is NW_113990.The GenBank (


Ce(III), lanthanum La(III), and neodymiumNd(III) complexes with4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one (warfarin)(W) and 3,3′-benzylidenebis[4-hydroxycoumarin](1) were synthesized and studied for the first timefor cytotoxicity (on MT-2 cells) and as anti-HIV agents underacute and chronic infection. Thecomplexes were characterized by different physicochemical methods:mass spectrometry, 1H NMR, 13C NMR, and IRspectroscopy. The spectra of the complexes were interpreted on thebasis of comparison with the spectrum of the free ligands.Anti-HIV effect of the complexes/ligands was measured in MT-2cells by microtiter infection assay. Detection of endogenousreverse transcriptase (RT) activity and RT processivity by PCRindicative for proviral DNA synthesis demonstrated that anti-HIVactivity has not been linked to early stages of viral replication.No effect on late steps of viral replication has been found usingcells chronically producing HIV-1LAI virus.W)La( demonstrated anti-HIVactivity (IC50=21.4 μM) close to maximalnontoxic concentration. W)NdCeNdLa( is under progress.The cerium  The need for new HIV inhibitorsespecially directed to so far unexploited primary targets  isconsidered to be promising for the development of anti-HIVdrugs. Coumarins and bicoumarins arewidely spread in nature , 2. TheiW), displaycytotoxic effect against tumour cell growth and metastasesdissemination (1) in complexation reaction with Ce, La,and Nd. An increased anti-HIV activity ofW)La( (IC50=21.4 μM) as comparedto (W)  has been demonstrated. Further study hasshown limited anti-HIV potency of W)NdCeNd-2H-1-benzopyran-2-one (warfarin)(3)3 · 6H2OCe3 · 6H2OLa3 · 6H2ONd(NO.4-Hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one(W) and 3,3′-benzylidenebis[4-hydroxycoumarin](25H16O6)C(1) were synthesized by us andwere used to prepare the solutions.Merck products p.a.Ln(III)W) complexes of  were described previously [eviously .(1)The complexes of lanthanides(III) with  weresynthesized by reaction of lanthanide(III) salts and the ligandin amounts equal to metal: ligand molar ratio of 1 : 2. The complexes were prepared by adding an aqueous solution oflanthanide(III) salts to an aqueous solution of the ligandsubsequently raising the pH of the mixture gradually to ca 5.0 by adding dilute solution of sodium hydroxide. The reactionmixture was stirred with an electromagnetic stirrer at 25°C for an hour. The precipitate formed at the moment ofsolution mixing was filtered, washed several times with water, anddried in a dessicator to constant weight. The complexes wereinsoluble in water, slightly soluble in methanol and ethanol, andwell soluble in DMSO.C, H, Ln, and 2OH were performed according to standard microanalyticalprocedures. The IR spectra (Nujol and KBr) were recorded onIR-spectrometer FTIR-8101M Shimadzu (3800–400 cm−1)and on IR-spectrometer Perkin-Elmer GX Auto image system(700–200 cm−1). The 1H NMR spectra were recorded at room temperature on Brucker WP 250 (250 MHz) spectrometer in DMSO-d6. The 13C NMR spectra were recorded at ambient temperature on Brucker 250 WM (62.9 MHz)spectrometer in DMSO-d6. Chemical shifts are given in ppmdownfield from TMS. The mass spectra were recorded on a Jeol JMS D300 double-focusing mass spectrometer coupled to a JMA 2000 datasystem. The compounds were introduced by direct inlet probe,heated from 50°C to 400°C at a rate of100°C/min. The ionization current was 300 mA, theaccelerating voltage 3 kV, and the chamber temperature150°C.The elemental analyses for LAIcells, kindly provided by ProfessorR. Gallo, and MT-2 uninfected human lymphoblastoid cells, HTLV-Itransformed. The cells were maintained in RPMI 1640 plus 10%FCS.The following cell lines were used: H9/HTLV III B—chronically infectedwith HIV-12CO) and then grown parallely withuninfected ones for 72 hours.As a source of HIV-1, the supernatant of H9/HTLV III B cell line wasused. The supernatants were collected and centrifuged to remove the cells, and virus stocks were prepared with known p24 antigen content, RT activity and infectivity. MT-2 cells were infectedwith HIV-1 (multiplicity of infection ≥ 1) in suspension(37°C/5% The following parameters were studied: cytotoxicconcentration 50—CC50 (concentration preventing death of 50% of MT-2 cells), maximal nontoxic concentration—MNC (the highest concentration causing nocytotoxicity), and inhibitory concentration 50–IC50 . CC50 and MNC weredetected by neutral red uptake assay . IC50 waμL MT-2 cells (4–5 × 10 4 cells);100 μL HIV-1 (undiluted or in the appropriatedilution to obtain multiplicity of infection ≥ 1); contact (2hours at 37°C/5% CO2); when CC50 and MNC weredetected, growth medium instead of virus has been added;50 μL sample of substance tested starting from4× MNC (because this volume is 1/4 of the whole volume inthe well) and 2- or 10-fold dilutions in growth medium.50 μL ofthem were removed to another 96-well plate previously treated foran hour with 50 μg/mL poly-L-lysine (PLL) in volume100 μL. A solution of 100 μL per well of neutralred in growth medium (0.016%) was added and thecells were incubated for 2 hours at 37°C/5%CO2. Neutral red dye was extracted by acidified alcohol(50% ethanol in 1% acetic acid) and measuredcolorimetrically at 540 nm. Each plate contained 8 cellcontrol wells  for all kinds ofexperiments. When IC50 was detected, a row of 8 viral controls and rows of 8 experimental wells  were performed. Thevalues of optical density at 540 nm (OD540) were averaged foreach row and the mean values of experimental and control rows werecompared. As far as the ligands and their complexes were DMSOsoluble, the appropriate controls of DMSO and DMSO + HIV-1 ingrowth medium were always considered.The cells were incubated for 72 hours and then 100 The effect of the compounds was also studied under conditions ofchronic infection using productively HIV-infected cells (H9/HTLVIII B). The production of infectious virions was measured againusing the cytopathic effect of supernatants on MT-2 cells asdescribed above.Endogenous RT activity of supernatants of HIV-1 infectedMT-2 cells treated/untreated with complexes and ligands was testedby HS-Lenti Kit-RT assay . The kit containedrecombinant RT (rRT) as a standard which made possiblequantitation of the RT in the sample. The method isnonradioactive and colorimetrically detects RT activity product(DNA) at 405 nm (OD405) [ (OD405) .2MgCl—2.5 mM, NP40—0.5%,and between 0.5% and 20% [μL) containing 30 μL proteinase Kand 25 μL 10% SDS was added to the cell lysate. DNA wasextracted byphenol-chloroform-isoamyl alcohol method. The detection ofamplified product was done after 35 cycles in PCR machine and thenvisualized in 1.5% agarose gel.The idea was to demonstrate how far the proviral DNA synthesis hasprogressed and whether mRNA splicing has occurred withinHIV-infected cells. (PCRs) were performed using primers M661 andM667 detecting 200 bp product considered expressive fordouble-stranded (ds) proviral DNA . DNA for and 20% .NucleolW) were confirmedby elemental analysis, DTA and TGA, and mass-spectral analysis. Thebinding mode of (W) was further elucidated by IR and1H NMR spectra of the complexes as compared with this ofthe free ligand. Some of these data were presented by uspreviously [The compositions of the complexes of (eviously .Ln(III) complexes obtained are in agreement with the formulaLn(L)(OH) ·2OnH, whereL = 25H14O62−C. The results of theelemental analyses were within ±0.4% of the theoreticalvalues. The suggested formula was further confirmed bymass-spectral fragmentation analysis. As seen inLn(III)complexes spectra  corresponds to themass weight of the complex formation and the second one to thatof the ligand. The results thus obtained are in agreement withmetal:ligand ratio, 1 : 1.The elemental analysis data of the (1) to Ln(III) was further elucidated by analysis of the IR spectra of (1) and the complex formation. A broadband characteristicfor ν(O−H) of coordinated water was observed in thespectra of the complexes in the range of 3500–3450 cm−1.The most notable change in the ligand spectral features whencoordinated to Ln(III) is the observed C=O redshift. The ν(C=O) band at 1660 cm−1 in theligand spectrum exhibited a red shift of 40 cm−1 in thespectra of the complexes. This finding may be taken as evidencefor participation of the C=O group in coordination tothe metal ion. Further, a comparison between the ligand andcomplex IR spectra revealed that the absorption bands associatedwith the stretching ν(O−H) of the phenolic groups(observed at 3074 cm−1 and 3032 cm−1 in the free ligand) disappeared in the Ln(III) complex spectra, indicating a loss of phenolic protons on complexation and thusforming a metal-oxygen bonds. The δ(COH) IP modes, which appeared at1345 cm−1 and 1336 cm−1 in the spectrum of the ligand, were not observed in the spectra of the complexes withLn(III) and thus supported the suggestion that the ligandcoordinates to the metal through its deprotonated form,2−L. The Ln(III) complex spectra showed newbands, in comparison with that of the free ligand, at570 cm−1 and 410 cm−1, and they were assigned tometal-oxygen stretching vibrations, in agreement with literature.The binding mode of Ln(III) complexes and (1) were further studied by their 1H and 13C NMR spectra. The changes ofchemical shifts of the 1H NMR spectra were observed in thecomplexes (Ln(III). The chemical shiftsof the protons vary in the lanthanide complexes, because of theshift properties of these metals.The omplexes  and theyLn(III), chemical shifts to lower ppm wereobserved for the neighboring C-4 and C-2 carbon atoms of the complexes and they confirmed the expected coordination of theligand through both deprotonated hydroxyl and carbonyl oxygenatoms (Ln(III) complexformation.Due to electron transfer from the hydroxyl and carbonyl oxygenatoms to enatoms . The oth(1) demonstrated at least5 times lower cytotoxicity compared to the other lanthanides andthe ligand (1) itself. MNCs of the ligands differed inbetween the complexes but were maximally nontoxic at 10–50 timeshigher concentrations for (1)Nd and(1)Ce compared to La(1) and the ligand(1). The ligand (W) and its complexesdemonstrated both equal cytotoxicity and equal MNCs. As far asintensive research is often being done to reduce the toxicity of acompound, we demonstrate here that the complexation with rareearth metals possibly leads to increase of MNCs of somederivatives.In (1) and (W)and their lanthanide complexes in HIV-1 infected MT-2 cellsaccording to microtiter infection assay [Dose-effect dependence of ligands on assay  are listW)LaLa( as well as of (W) andW)NdCeNdLaLa and (W) and their complexes having shownsome anti-HIV effect were provided by PCR. 1) and (W) compounds . Therefore, the results confirm that neither theligands nor their rare metal ions complexes have an influence onthe reverse transcription. So, once again, the discrepancy inanti-HIV activity measured by microtiter infection assay andendogenous RT detection is clearly demonstrated. The conclusion isthat the active W)LaLa wascarried out. To do this, we treated H9/HTLV III Bcells—chronically producing HIV-1—with W)LaLaLaLaLa(. HIV-1 replication is too complicated and anumber of targets and steps (except those studied here) should betaken into consideration . Although not especially being a subject ofattachment studies, the results of RT assays (W)La( first on integration and then further tospecify the mechanism of its action.The fact that no effect on RT and protease (expressing early andlate stages of HIV-1 replication) was registered; one can suggestthat RT and protease are not targets for the antiviral activity ofT assays and RT pT assays  clearly Here we show for the first time both complexation of rare earthmetal ions with selected coumarins and study of their effect onHIV replication in cell culture. Although the complexes are of lowpotency against HIV-1 and should not enter clinical trials, theexperience with them shows once more that the cytotoxicity couldbe reduced and the antiviral effect—highly expressedthroughcomplexation reactions. Lack of protease and RTactivity has been recently reported for a number of HIV-1integrase inhibitors  and on t


Glycaemic traits such as fasting and post-challenge glucose and insulin measures, as well as glycated haemoglobin (HbA1c), are used to diagnose and monitor diabetes. These traits are risk factors for cardiovascular disease even below the diabetic threshold, and their study can additionally yield insights into the pathophysiology of type 2 diabetes. To date, a diverse set of genetic approaches have led to the discovery of over 97 loci influencing glycaemic traits. In this review, we will focus on recent advances in the genetic aetiology of glycaemic traits, and the resulting biological insights. We will provide a brief overview of results ranging from common, to low- and rare-frequency variant-trait association studies, studies leveraging the diversity across populations, and studies harnessing the power of genetic and genomic approaches to gain insights into the biological underpinnings of these traits. PPARG, KCNJ11 and TCFL2  (GCK association (G6PC2 (GCKR (Genome-wide association studies (GWAS) have transformed the landscape of glycaemic trait genetics. Prior to GWAS FG was associated with genetic variants in okinase) . Subsequociation ,17 and in  (a dynamic measure of whole-body insulin sensitivity)  . These mitivity) , includiresponse . An alteresponse ,32, and  Koreans   and low frequency (1% ≤ MAF < 5%) variants, and their role as possible causal variants. Current strategies include: 1) genotyping arrays targeting the exons  or with combined common variant backbone and exonic content; 2) genome- and exome –wide sequencing and 3) combined genotyping arrays and dense imputation using sequence based reference panels such as 1000 genomes , UK10K ,36 and Het al.  and KANK1  associated with fasting proinsulin levels  and two variants with MAF = 5.3%, and in near-perfect LD (r2=0.997) at PAM (D563G) and PPIP5K2 (S1228G) associated with insulin secretion (insulinogenic index). Novel low frequency variants at previously identified GWAS loci, SGSM2  and MADD  associated with fasting proinsulin, and common variants associated with insulin secretion or beta-cell function at GPSM1 (S391L), HNF1A (intergenic), and ABO (intronic) were also identified. Gene-based tests (aggregating rare/low frequency variants at the locus) identified significant associations with fasting proinsulin at TBC1D30, SGSM2 and ATG13, although conditional analyses suggested the ATG13 signal was partially driven by variants in MADD. Wessel et al.  associated with lower FG, early insulin secretion and type 2 diabetes risk, but higher 2hG  and a stop variant (R283X). Further evidence of FG association at G6PC2 was provided by Mahajan et al. (G6PC2 as an effector transcript at the G6PC2/ABCB11 locus (GCKR (P446L), SLC30A8 (R325W), RREB1 (S1554Y), PCSK1 , COBLL1 (N939D), TOP1 (N310S) and PPARG (P12A)  were also identified. Despite this success only two association signals were low frequency variants, H177Y MAF 0.8% at G6PC2/ABCB11 and E594V MAF 0.1% at URB2,  . Two novt GPSM1 S1L, HNF1Aat URB2, , and theG6PC2-ABCB11 with FG. Subsequent fine-mapping efforts identified missense variant associations as the causal variant or within the credible set of causal variants at GCKR (L446P) and SLC30A8 (R325W) (The UK10K Consortium  performe (R325W) .Driven by the availability of large sample sizes, the majority of early GWAS studies were performed in populations of European ancestry. Since then, efforts have expanded to diverse populations, leveraging differences in allele frequency and linkage disequilibrium (LD) structure, to harness power for novel locus discovery and fine-mapping . While gSC4MOL and TCERG1L associated with FI and insulin resistance (HOMA-IR)  , and FAMom MAGIC . In East with FG ,48, MYL2or HbA1c ,50  compared to the much larger European ancestry efforts (N∼ 108-133 K for FI and FG) that have led to the discovery of the loci being assessed. Nonetheless they highlight the utility of diverse populations to refine association signals, to fewer probable casual variants. For example, inclusion of African American samples in a trans-ethnic fine-mapping approach reduced the credible set  at GCK and ADCY5 for FG, PPP1R3B for FI, and GCKR for FG and FI, to a single SNP  on FI, with MAF 1.1% in Finns, but virtually absent (MAF ≤0.2%) in the individuals from other ancestries  and RBMA6 (at the AMT locus) were also implicated as plausible effector transcripts but the mechanism through which they impact islet function, is as yet, unknown   . AnotherADIPOR1) . The imp)  , SLC30A8 (PPARG (e.g. the P50T variant at AKT2 associated with FI (PCSK1 associated with proinsulin and FG (SLC2A2 (encoding GLUT2), GCK, GCKR, FOXA2 and PDX1 are the likely effector transcripts at these loci , GCK (OMIM # 125851), PPARG (OMIM # 604367), PCSK1 (OMIM # 600955), PDX1 (OMIM # 606392), GLIS3 (OMIM # 610199), IGF1 (OMIM # 608747) and HNF1A (OMIM # 600496) providing additional biological support for their candidacy as effector transcripts at these loci, and suggesting a role for rare penetrant and common variants influencing familial or polygenic traits, respectively.Complementing functional regulatory associations, the identification of multiple rare missense variants shown to affect protein function, and that contribute to a gene-based association signal, is a strong indicator that the effector transcript has been identified [ SLC30A8  and PPAR8  those loci whose primary effect appears to be on insulin sensitivity ; b) loci associated with decreased insulin secretion and with fasting hyperglycaemia ; c) a single locus, ARAP1, associated with impaired proinsulin processing; d) a large cluster of loci influencing insulin processing and secretion with modest or no detected effects on fasting glucose levels ; and e) a large set of 20 loci that despite influencing T2D risk did not have clear associations with any of the available measures of glycaemia and which may correspond to novel mechanisms influencing diabetes by as yet not understood biology (Fasting glucose is used to diagnosis type 2 diabetes (T2D) however, GWAS studies have demonstrated that the genetic architecture of these two traits does not fully overlap ,80,86, s biology . Similar biology .G6PD gene (G202A) responsible for glucose-6-phosphate deficiency, accounted for a significant fraction of the variance in the trait (14.4%) and led to a substantial decrease in HbA1c values in hemizygous men (0.81%-units) and homozygous women (0.68%-units). This variant, if unaccounted for, could lead to up to 2% of African Americans with T2D to remain undiagnosed, highlighting the importance of studying glycaemic traits in diverse populations in order to avoid racial health disparities in the application of precision medicine . In African Americans, a single variant in the medicine .In conclusion, large-scale genetic association analyses, combined with information on genomic features  and high- throughput functional assays  have pro


Moreover, Prr7 does not affect the TCR signalling pathway as T cells derived from Prr7 knockout and wild-type animals and stimulated in vitro express the same levels of the activation marker CD69, and retain their ability to proliferate and activate induced cell death programs. Importantly, Prr7 knockout mice retained the capacity to mount a protective immune response when challenged with Listeria monocytogenes infection in vivo. In addition, T cell effector functions  were normal following induction of experimental autoimmune encephalomyelitis (EAE) in Prr7 knockout mice. Collectively, our work shows that loss of Prr7 does not result in a major immune system phenotype and suggests that Prr7 has a dispensable function for TCR signalling.Transmembrane adaptor proteins (TRAPs) are important organisers for the transduction of immunoreceptor-mediated signals. Prr7 is a TRAP that regulates T cell receptor (TCR) signalling and potently induces cell death when overexpressed in human Jurkat T cells. Whether endogenous Prr7 has a similar functional role is currently unknown. To address this issue, we analysed the development and function of the immune system in Prr7 knockout mice. We found that loss of Prr7 partially impairs development of single positive CD4 Activation of lymphocytes by antigens is the key process of adaptive immune responses. A complex interplay of proteins in the signalling pathways originating from activated immune receptors  is required at specific stages of lymphocyte activation. A group of proteins called transmembrane adaptor proteins (TRAPs) participate in immune receptor signalling by organizing membrane-proximal signalling complexes that are either constitutively or inducibly associated with immune receptors , 2. TRAPRecently, we discovered and characterized a new member of the TRAP family, Proline rich 7 (Prr7) . Like otSince the expression of Prr7 in mouse immune system has not been studied, we first analysed the mRNA levels of Prr7 in the thymus, spleen, lymph nodes, and T cells purified from lymph nodes of wild-type C57BL/6 mice. As shown in www.komp.org). The targeting strategy replaces the entire Prr7 coding region by a cassette containing the LacZ gene expressed under control of the endogenous Prr7 promoter and an independently expressed Neomycin resistance gene  T cells migrate to and populate secondary lymphatic organs. To analyse whether Prr7 deficiency might interfere with this process we measured the composition of T cell subpopulations in the spleen and lymph nodes. We found the same percentage of CD4+ or CD8+ cells in spleen and lymph nodes isolated from Prr7+/+ and Prr7-/- mice , activated T cells (CD62L-CD25+), and memory T cells (CD62-CD25-) was unaffected by the absence of Prr7 (+TCRγδ+), subpopulation of T cells enriched in regulatory T cells (CD4+CD25+), natural killer (NK) cells (NK1.1+TCRβ-), and NK T cells (NK1.1+TCRβ+) were identical in the spleen and lymph nodes of Prr7+/+ and Prr7-/- mice . Surprisingly, T cells from both wild-type and Prr7 knockout mice responded in the same way and the number of apoptotic and dead cells was not statistically significantly different in the restimulated cultures -expressing Listeria monocytogenes (Lm ova). After 9 days, i.e. at the peak of the primary Listeria-specific CD8+ T cell response thymidine [3H-TdR] incorporation. [3H]thymidine was added at 0.2 μCi/well for the last 16 h of the incubation. At the end of the incubation period, all cells were harvested and radioisotope incorporation was measured as an index of lymphocyte proliferation using a Betaplate liquid scintillation counter .Proliferation of mouse splenocytes was assessed by measuring [Infection. Prr7-/- and Prr7+/+ mice (8- to 12-week old) were intravenously infected with 1x104 ovalbumin-expressing Listeria monocytogenes (Lm ova). Animals were sacrificed on day 9 post infection. Colony forming units (CFU). Single cell suspensions from the spleen and liver were prepared by passing the organs through a 70 μM cell strainer. Ten-fold serial dilutions were plated on heart-brain agar and incubated for 24 h at 37°C. Bacterial colonies were counted microscopically.Peptides. Myelin oligodendrocyte glycoprotein peptide 35–55 (MOG p35-55), corresponding to mouse sequence (MEVGWYRSPFSRVVHLYRNGK), was synthesized on a peptide synthesizer by standard 9-fluorenylmethoxycarbonyl chemistry, and purified by high-performance liquid chromatography (HPLC). Induction, treatment and evaluation of active EAE. Female Prr7-/- and control Prr7+/+ mice (8- to 12-week old) were immunized subcutaneously (s.c.) in depots distributed over 4 spots across the flanks with 200 μg MOG (p)35-55 in 0.2 ml emulsion consisting of equal volumes of PBS and complete Freund's adjuvant , containing 4 mg/ml of mycobacterium tuberculosis H37Ra . 200 ng pertussis toxin  was administered intraperitoneally (i.p.) at days 0 and 2. The mice were scored daily for clinical signs of EAE according to the following increasing severity scale: 0: no disease; 1: tail weakness (tail plegia); 2: hindlimb paraparesis and/or weak rightning-reflex; 3: hindlimb paraplegia; 4: paraplegia with forelimb weakness or paralysis; 5: moribund animals. Mice with intermediate clinical signs were scored in 0.5 increments. Daily clinical scores were calculated as the average of all individual disease scores of each group.For experiments with statistical analysis, a two-tailed Student’s t test was performed, and p values less than or equal to 0.05 were considered significant. Statistical comparison of EAE disease severity was accomplished by a non-parametric Mann-Whitney test as described previously . The datS1 Fig+CD25+) subpopulations in the lymph nodes. (B) Flow cytometry analysis of NK (NK1.1+TCRβ-) and NK T (NK1.1+TCRβ+) cells in the spleen stained with anti-NK1.1 and anti-TCRβ. (C) No gross defects were found in B cell development in Prr7 deficient animal. The upper schema depicts the main developmental stages of B cells in the bone marrow and in the spleen. MZ, marginal zone B cells. The middle and lower panels are the results of flow cytometry analysis of B cell developmental stages in the bone marrow and in the spleen of Prr7-/- mice and control Prr7+/+ mice defined as follow: Immature B cells (B220loIgM+), Mature B cells (B220hiIgM+), Pro & Pre B cells (B220+IgM-), T1 B cells (CD23+CD21+IgM+), T2 B cells (CD23+CD21+IgM+), MZ B cells (CD23-CD21+B220+), Immature B cells (IgMhiIgDlo), Mature B cells (IgMloIgDhi). Data in  represent the mean + SEM of at least three animals per group. n.s., not significant.(A) Flow cytometry analysis of γ/δ T cell and enriched in regulatory T cells (CD4(TIF)Click here for additional data file.S2 FigData in represent the mean + SEM of minimum three animals per group. n.s., not significant.(TIF)Click here for additional data file.S3 Fig+ and CD8+ T cells in liver of Lm ova-infected mice. (B) Frequency of CD4+ and CD8+ T cells in liver of infected mice. (C) Absolute number of CD4+ and CD8+ T cells in liver of infected mice. (D) Frequency of CD8+ naive (CD62L+CD44-), activated (CD62L-CD44+) and memory (CD62L+CD44+) T cells in liver of infected mice. (E) Absolute number of CD8+ naive, activated and memory T cells in liver of infected mice. (F-I) Hepatic leukocytes of infected mice were restimulated with Ova257-264-peptide  for 12 h in the presence of Brefeldin A. (F) Dot plot of TNF-producing CD8+ T cells. (G) Dot plot of IFN-γ producing CD8+ T cells. (H) Frequency of IFN-γ and TNF-producing CD8+ T cells in spleen of infected mice. (I) Absolute number of IFN-γ and TNF-producing CD8+ T cells in spleen of infected mice. Data are represented as mean + SEM of 3–4 mice per group. n.s. not significant.(A) Representative dot plot of CD4(TIF)Click here for additional data file.


SCN5A mutation, P1332L, is linked to a malignant form of congenital long QT syndrome, type 3 (LQT3), and affected patients are highly responsive to the Na+ channel blocking drug, mexiletine. In contrast, A647D is an atypical SCN5A mutation causing Brugada syndrome. An asymptomatic male with both P1332L and A647D presented with varying P wave/QRS aberrancy and mild QTc prolongation which did not shorten measurably with mexiletine.The + channels as well as their combinations in order to understand our proband’s phenotype and to guide mexilitine therapy.We characterized the biophysical properties of P1332L, A647D and wild-type (WT) Na+ channel biophysics and mexilitine-binding kinetics were assessed using heterologous expression studies in CHO-K1 cells and human ventricular myocyte modeling.Na+ currents (INa). While A647D had no effect on the biophysical properties of INa, it reduced peak and late INa density when co-expressed with either WT or P1332L. Additionally, while P1332L channels had greater sensitivity to block by mexiletine compared to WT, this was reduced in the presence of A647D. Modelling studies revealed that mixing P1332L with A647D channels, action potential durations were shortened compared to P1332L, while peak INa was reduced compared to either A647D coexpressing with WT or WT alone.Compared to WT, P1332L channels displayed a hyperpolarizing shift in inactivation, slower inactivation and prominent late NaNa density. These results explain our proband’s mild repolarization abnormality and prominent conduction defect in the atria and ventricles, but also suggest that expression of P1332L with A647D yields a novel disease phenotype for which mexiletine pharmacotherapy is no longer suitable.While A647D mitigates the lethal LQT3 phenotype seen with P1332L, it also reduces mexilitine sensitivity and decreases I SCN5A can alter the biophysical properties of the cardiac Na+ channel and lead to several different hereditary arrhythmias, including long QT syndrome (LQTS type 3 or LQT3), Brugada syndrome, sick sinus syndrome, heart block and atrial fibrillation (AF) [SCN5A mutations, Na+ channel blockers, such as mexiletine, can shorten the QTc interval and reduce the risk of cardiac events. The P1332L LQT3 mutation is particularly virulent causing marked QTc prolongation (>500 ms), AV block and juvenile sudden cardiac death [SCN5A mutation is reported to cause Brugada syndrome without appreciably altering the Na+ channel biophysical profile compared to WT [Mutations in ion (AF) . Althougac death . This muac death . In conted to WT .SCN5A mutations causing LQT3 are uncommon, and in some instances one mutation rescues the Na+ channel abnormalities of the other [+ channel blocking pharmacotherapy have not been investigated. We present a case of a compound SCN5A mutation, P1332L and A647D, found in an asymptomatic adult male, who manifested a novel pleiotropic phenotype of mild QTc prolongation, atypical ST elevation and abnormal conduction in both the atrium and ventricle. While mexiletine was initially indicated to treat the P1332L-related LQT3 phenotype, the proband’s resting conduction defects and Brugada-associated A647D mutation caused us to reconsider this treatment strategy [SCN5A channels with computer modeling of ventricular myocyte electrophysiology. In the absence of genetic validation of whether the P1332L and A647D mutations were oriented as cis versus trans, our expression studies considered both possibilities.Compound he other –9. In thstrategy . In ordeThe proband’s clinical evaluation included a standard 12-lead electrocardiogram (ECG), which was recorded at baseline and after intravenous procainamide challenge (10mg/kg infused over 15 minutes). The latter was performed to assess Brugada syndrome. In addition, an invasive electrophysiology study was performed in the fasting state, whereby multielectrode recording catheters were placed percutaneously in the right ventricular apex, high right atrium, His bundle region and coronary sinus. Intracardiac bipolar electrograms  were recording from these sites during baseline sinus rhythm. The clinical diagnosis was based on this evaluation and performed at University Health Network, Toronto, Canada. The study was approved by the Institutional Review Board of University Health Network and written informed consent was obtained.™, San Francisco, CA) on a panel of 57 genes implicated in inherited arrhythmias, including LQTS and Brugada syndrome. Genomic DNA was extracted from white blood cells using standard protocols. Next generation sequencing was used to evaluate exons and splice junctions for sequence variants and large deletions or duplications. The following genes were evaluated: ABCC9, ACTN2, AKAP9, ANK2, ANKRD1, CACNA1C, CACNA2D1, CACNB2, CALM1, CALM2, CALM3, CASQ2, CAV3, CTNNA3, DES, DSC2, DSG2, DSP, EMD, GPD1L, HCN4, JUP, KCND3, KCNE1, KCNE2, KCNE3, KCNE5, KCNH2, KCNJ2, KCHJ5, KCNJ8, KCNQ1, LDB3, LMNA, NKX2-5, PDLIM3, PKP2, PLN, PRKAG2, RANGRF, RBM20, RYR2, SCN10A, SCN1B, SCN2B, SCN3B, SCN4B, SCN5A, SLMAP, SNTA1, TGFB3, TMEM43, TNNI3, TNNT2, TRDN, TRPM4, TTN. Control data on variants were obtained from population databases (ExAC).Genetic testing was performed by an accredited commercial laboratory  according to the manufacturer’s protocol. The SCN5A cDNA plasmids (1μg) were coexpressed with plasmids containing the GFP-marker plus the β1-subunit cDNA (1μg). When combinations of two SCN5A genes were expressed, the total SCN5A cDNA contained 0.5μg of each channels type. For example, when wild-type (WT) SCN5A was coexpressed with the A647D SCN5A mutant (i.e. WT+A647D), we mixed 0.5μg of WT SCN5A cDNA plasmid with 0.5μg of A647D SCN5A cDNA plasmid.Mutations were introduced into the human + currents (INa) in green fluorescent protein-positive CHO-K1 cells, showing green fluorescence under mercury light, were recorded at room temperature using an Axopatch 200B amplifier  under whole-cell patch clamp mode with 80–85% series resistance compensation. The bath solution contained (in mM): 140 NaCl, 4 CsCl, 1 MgCl2, 1.2 CaCl2, 10 HEPES, 10 D-glucose, pH 7.35 with NaOH. Internal solution contained (in mM): 10 NaCl, 1 CaCl2, 135 CsCl, 1 MgCl2, 4 MgATP, 10 HEPES, 10 EGTA, pH 7.2 with CsOH. Different mexiletine concentration solutions were prepared by diluting freshly made mexiletine methanol stock (200mM) .Whole cell NaNa was measured as a function of voltage in response to 500ms depolarizing pulses (Vm) ranging between -80 to +50mV from a holding potential at -110mV. To assess the activation properties and densities of Na+ channel expression, we estimated the whole-cell Na+ channel conductance (GNa) at various depolarizing voltages (Vm) by dividing the measured peak INa (INapeak) by the electrochemical driving force Vm-ENa, where ENa is the equilibrium voltage (estimated to be +55mV). To assess the steady state activation and the densities of Na+ expression, we plotted GNa as a function of Vm and fitted to the Boltzmann equation:NaMax is the maximal conductance, V1/2act is the voltage (Vm) at which the Na+ channels are activated by 50% and k is the slope factor.INa measured during the test pulse to 0mV was normalized to the maximum peak INa (INaMax) and plotted against the conditioning voltage. These data were fit to a Boltzmann equation:1/2inact is the voltage (Vm) at which the Na+ channels are inactivated by 50% and k is the slope factor.Steady-state inactivation was determined by applying a family of 500ms conditioning pulses to voltages ranging from -100 to -10mV followed by a 50ms test pulse to 0mV. The holding potential was -110mV which was applied for 4 seconds between each conditioning pulse. To assess steady-state inactivation properties, peak ISCN5A channels, we measured the time for 50% decay of INa from its peak. We also fit the time course of INa decay to multi-exponential functions using algorithms previously reported [Na was best fit statistically with a tri-exponential function:SCN5A channels by applying ramp protocols which involved initially activating the INa by a 5ms step to +20mV from a holding potential of -100mV followed by a 150ms ramp back to -100mV. To eliminate the background leak currents, we applied a P/4 protocol from -120mV 5 seconds prior to the voltage protocol.To assess the inactivation kinetics of reported . We founNa was assessed by applying the standard double pulse protocol wherein channels were held at -100mV after which a depolarizing pulse to -20mV was applied for 50ms (i.e. the conditioning pulse) which ensures complete inactivation. The membrane was then repolarized back to -100mV for variable periods of time ranging from 10ms to several seconds, followed by a second step to -20mV (i.e. the test pulse). Peak INa measured in the test pulse was normalized to peak INa during the conditioning pulse and plotted as a function of the period between the conditioning and test pulses. These data (i.e. recovery ratio versus time) were fit with a mono-exponential function:Recovery from inactivation of I+ channel blockers cause the recovery from inactivation kinetics to display a bi-exponential time-course, with the slow recovering component arising from unblock of the channels and the faster component mapping the time course of recovery from inactivation of the unblocked Na+ channels. It is noted that the slow time constant estimated from the bi-exponential fit of the recovery from inactivation does not depend on the blocker concentration while the amplitude of the slow recovering component is a direct measure of the number of Na+ channels that are blocked by the drug [EC50 is the estimate of mexiletine blocking effectiveness.It has previously been established  that mosthe drug , 14. UsiSCN5A mutations on human ventricular myocyte electrophysiology, we modeled their Na+ channel behavior using the O’Hara-Virag-Varro-Rudy model (OVVR) [+ channel model parameters were iteratively adjusted one-by-one until the average absolute difference between experimental and simulation data was <0.1%. The parameters used for the simulation study can be found in the Results and were determined in the following order: Maximal GNa scaled according to the ratios in experimental data  since human atrial and CHO-K1 cells do not have equivalent GNa; V1/2 and k for m∞ adjusted to match steady-state activation in experimental data; V1/2 and k for h∞ adjusted to match steady-state inactivation in experimental data; Sτj adjusted to match recovery from inactivation in experimental data; Sτh adjusted to match INa decay at 50% in experimental data; and maximal GNaL scaled to match the ratios in experimental data (see To investigate the effects of l (OVVR)  and the SCN5A mutations on action potential dynamics. Each OVVR model was preconditioned by basal pacing at a cycle length (CL) of 700ms for 100 beats using a 2ms-long stimulus at twice capture amplitude. Single S2 stimuli were then applied beginning at a CL of 690ms, and decreased by 10ms decrements until loss of stimulus capture. Action potential duration (APD) at 90% repolarization was computed for each S2 beat according to Bayer et al [m/dt) during action potential upstroke, action potential activation times at the 0mV threshold crossing of the action potential upstroke, and peak whole-cell INa (early and late) by summing all Na+ dependent currents in the OVVR model. Each was plotted against the S2 diastolic interval (DI).All voltage clamping protocols applied to the modified OVVR models were identical to those in the experimental study. An S1S2 pacing protocol was applied to each OVVR model parameter set see  to inveser et al , in addiNa behavior.Single-cell voltage clamping and pacing simulations were performed using the Cardiac Arrhythmia Research Package CARP  running t test and one-way ANOVA followed by Tukey test were used to compare the means. Statistical significance was considered when p<0.05.Pclamp 8.2 and Clampfit 10 , Excel  and Prism 5.3  were used for data acquisition and analysis. Data are presented as mean±standard error. Unpaired student The proband is a 22-year old asymptomatic male, with a structurally normal heart and no family history of sudden death, who presented with an abnormal resting 12-lead ECG showing sinus tachycardia at 105bpm, mild QTc prolongation (420-470ms) and atypical ST elevation in V2 and V3. Striking beat-to-beat variation in P wave morphology and RBBB-aberrancy were seen in the absence of premature ectopy or heart rate acceleration . Invasivdistal→CSproximal transitioning to CSproximal→CSdistal) despite constant heart rate  (CSart rate . Spontanart rate .+ and K+ channel blockade with procainamide (10mg/kg infused at 50mg/min) did not provoke a Brugada type 1 ECG pattern, although interpretation was confounded by the baseline QRS aberrancy and atypical ST elevation in the anterior precordial leads. Beta-blocker challenge with nadolol (80mg daily) produced sinus slowing without marked QTc prolongation (i.e. >500 ms) (Mexiletine therapy (100mg twice daily) produced no change in P wave or QRS morphology, no appreciable change in the QTc interval or ST segments, and no effect on resting sinus rate. Mixed Na>500 ms) .SCN5A, A647D and P1332L. The P1332L mutation was de novo, but the A647D mutation was inherited from the father, who manifested an asymptomatic type 3 Brugada ECG pattern. No mutations were identified in the proband’s mother and only sibling, both of whom had normal ECGs  with the mixture of P1332L SCN5A channels and A647D SCN5A channels coexpressed .Cis/trans genotyping of the proband’s compound mutation was evaluated as follows: First, the family’s genotype was considered, but cis/trans could not be clarified because one mutation was od cells , 19. In trans configuration, thereby mitigating the QT prolongation induced by the P1332L mutation as a result of A647D-dependent reductions in the late INa currents generated by P1332L channels. SCN5A channels reduced peak INa densities in CHO-K1 cells compared to either WT channels or A647D SCN5A channels alone. Specifically, Na traces from WT, A647D alone and WT+A647D channels, recorded in response to depolarizing steps to -20mV from a holding potential of -110mV. Na densities as a function of voltage and establishes that peak INa is reduced when WT and A647D channels are co-expressed . To more accurately quantify the ability of A647D to reduce peak INa densities, we estimated the INa conductance (i.e. GNa(Vm)), normalized to cell capacitance, as a function of Vm. Fitting G(Vm) to the Boltzmann equation revealed that the Gmax of A647D alone (1.37±0.21pS/pF) was similar (P = 0.48) to WT (1.58±0.10pS/pF); but that Gmax was significantly reduced (P = 0.036) when A467D was co-expressed with WT Na+ channels (1.05±0.22pS/pF)  . Having Na at all the voltages studied compared to P1332L. Gmax calculated in CHO-K1 cells expressing P1332L channels alone was 1.64±0.21pS/pF and was reduced (P = 0.039) to 1.08±0.17pS/pF when A647D and P1332L were co-expressed. The magnitude of reduction was similar to that seen when A647D and WT channels were co-expressed. In contrast, Gmax was not decreased in the cis construct, A647D-P1332L, (1.65±0.28pS/pF) compared to WT. Taken together, these results are consistent with our hypothesis that A647D and P1332L, in the trans configuration, can lead to reduced peak INa; thereby explaining the conduction abnormalities seen in our patient.As summarized in Na generated by P1332L by considering both cis versus trans arrangement of the mutations, in order to explain the mild QTc prolongation in our patient. Consistent with previous studies [Na current decay compared to WT channels. Specifically, the time required for 50% decay of INa at -30mV was prolonged (P = 0.021) in P1332L channels  compared to WT channels  as summarized in Na decay . Indeed, late INa / peak INa was 0.32±0.03% (n = 14) in P1332L channels which was almost 3-fold larger (P<0.0001) than 0.12±0.02% (n = 8) in WT channels  for P1332L channels which was more than 2-fold greater (P<0.0001) than that for WT channels .To assess the potential impact of slowed inactivation rates of the I1/2) were shifted (P = 0.027) markedly toward negative voltages in P1332L (to -42.02±1.97mV) compared to WT channels (-33.67±2.32mV)  , Fig 6B.±2.32mV) , Fig 6C ±2.32mV) , Fig 6D.trans configuration of the A647D and P1332L mutations on late INa, the late INa/ peak INa ratio was measured at 150ms after depolarizing steps to 0mV. As shown in Na / peak INa ratio was smaller (P = 0.0003) when A647D and P1332L channels were coexpressed  compared to P1332L channels alone . These reductions were associated with less (P = 0.0099) net inward Na+ charge entry in co-expressed A647D+P1332L channels  versus P1332L channels alone  as summarized in Na generated by P1332L were accompanied by small changes in the rate inactivation  and the net inward Na+ charge entry (63.13±17.26mC/F)  in the late I.26mC/F)  in assoc.26mC/F)  compared+ channel blockers was also assessed by quantifying the recovery from inactivation kinetics of INa in the presence of mexiletine. As expected [Na recovery from inactivation with a time constant ranging from 301 to 635ms depending on the channel type (+ channels by mexiletine was lower (P<0.0001) for P1332L channels  compared to WT , indicating more potent block of P1332L channels relative to WT channels. On the other hand, the blocking efficacy of mexilitine was not different from WT when cells expressed either A647D  or WT+A647D channels . However, the EC50 was shifted to higher (P<0.05) mexilitine concentrations when A647D and P1332L channels were co-expressed (91.65±7.07μM n = 6) compared to P1332L alone.As described in the Methods section, the relative blocking efficacy  for Na+ expected , mexiletnel type . FittingNa blockade was also examined. As summarized in Na recorded at the 7th pulse to peak INa in pulse 1 (i.e. peak I7/ peak I1), recorded when cells were stimulated at 2Hz in the presence of 20μM mexiletine, was 0.75±0.01 (n = 7) in P1332L, which was smaller (P<0.05) than WT channels  as well as A647D channels  or WT+A647D co-expressed channels . Thus, mexilitine showed an increased blocking efficacy for P1332L channels compared to WT, A647D or WT+A647D co-expressed channels. Consistent with the increased EC50 for mexilitine blockade in P1332L+A647D co-expressed channels, the peak I7/ peak I1 was greater, although not statistically significant, when P1332L+A647D were co-expressed  compared to P1332L channels alone. Interestingly, A647D-P1332L channels also showed a slightly higher EC50  compared to P1332L alone and this was matched by a trend towards an increase in peak I7/ peak I1 . The implications of this are considered in the Discussion.To further characterize the blocking properties of mexiletine in our channels, use-dependence of INa kinetics for WT and mutant Na+ channels, P1332L, WT+A647D and P1332L+A647D, generated by the model are presented in cis construct, A647D-P1332L, because its biophysical properties were similar to the trans construct, P1332L+A647D, and WT. Moreover, A647D-P1332L did not manifest reduced peak INa, unlike P1332+A647D, which made it less likely to explain our patient’s conduction abnormalities. INa kinetics of delay, steady-state activation, steady-state inactivation, and recovery from inactivation were matched closely to the respective voltage-clamp data, as shown in The OVVR model was implemented using the parameters in SCN5A mutations on action potential properties were next examined by simulating pacing of ventricular myocytes in the OVVR model using the parameters in SCN5A mutants was less steep than WT (608 vs. 352-469mV/ms) with the lowest slope being observed in the P1332L+A647D model (352mV/ms), as summarized in SCN5A mutants.The dynamic effects of the SCN5A channels, we also quantified INa during action potentials generated from simulated pacing. During basal pacing at 700ms, peak INa was reduced for P1332, WT+A647D and P1332L+A647D compared to WT, with the greatest reduction seen in P1332L+A647D  as summarized in Na during basal pacing at 700ms, the reduction in peak INa was P1332L 23%, WT+A647D 34%, and P1332L+A647D 44%  was similar to that of WT . Thus, it is conceivable that our patient possesses the compound mutations in a cis configuation. Unfortunately, without the ability to perform direct cis/trans genotyping, the finding of reduced peak INa with the trans construct likely supports the trans genotype in our proband.While the P1332L and A647D cis or trans configuration. These results explain the mild LQT3 phenotype and the inability of mexiletine to appreciably shorten our probands’s QTc interval, despite the high sensitivity of P1332L channels to block by this drug [Na in P1332L+A647D than P1332L or WT+A647D, which explains our proband’s mild QTc prolongation and conduction defect, respectively. Thus, although mexiletine is reported to be therapeutic in LQT3 patients possessing the P1332L mutation, long-term treatment with this agent was not considered in our proband because it would be ineffective and potentially exacerbate the inherent conduction abnormalities [The presence of the A647D channels also reduced the mexiletine sensitivity observed in the P1332L channels, whether in the malities .Na, in association with hyperpolarizing shifts in steady-state activation and inactivation while also increasing block by mexiletine. We further established that P1332L channels have marked elevation in the net charge entry during voltage ramp protocols designed to mimic human ventricular action potentials, which helps explain the profound QT prolongation and the malignant clinical phenotype seen in patients with this mutation [Our studies confirmed that the P1332L LQT3 mutation, which is located in a highly conserved region of the S4-S5 cytoplasmic, slows inactivation of ISCN5A mutation, which is located in the intracellular linker between domains I and II  [SCN5A polymorphisms H558R [SCN5A mutation to mitigate a lethal phenotype (i.e. LQT3), while concurrently introducing a new phenotype  and modulating pharmacotherapy (i.e. Class IB agents like mexiletine for LQT3). The therapeutic benefit of these agents can be quite variable in single LQT3 mutations, in some cases due to differences in drug-binding affinity [+ channel blockers in LQT3 and our findings draw attention to the potential interaction of compound mutations on drug pharmacology.Compound atients)  and someatients)  or W156Xatients)  or reducms H558R , 8 or R1ms H558R . Our repaffinity . In one affinity . Hence, trans genotype was initially assumed and was able to account for the ECG phenotype and lack of response to therapy of our patient, we also noted for the first time that the presence of both the A647D and P1332L mutations in the same protein also altered the biophysical properties in a manner that might explain some of the features in our patient. It is also acknowledged that our proband’s resting sinus tachycardia could not be readily explained, and this is contrary to the sinoatrial nodal disease reported with other Na+ channel mutations or LQTS [Cis/trans genotyping was not possible. While the SCN5A mutation, P1332L and A647D. The lethal LQT3 phenotype of P1332L was mitigated with the Brugada- associated A647D, which negated the previously reported therapeutic benefit of mexiletine. Concurrently, the A647 mutant reduced INa sufficiently to produce a novel global cardiac conduction defect affecting the atria and ventricles, but not the sinoatrial or AV nodes. These finding highlight the complex, unpredictable biophysical interactions of LQT3-related compound mutations and the relevance of cellular expression studies in clarifying the role of Na+ channel blocking pharmacotherapy.We demonstrate a novel pleiotropic phenotype arising from the compound