RETN) increased susceptibility to type 2 diabetes by enhancing its promoter activity. Plasma resistin was highest in Japanese subjects with G/G genotype, followed by C/G, and C/C. In this study, we cross-sectionally analyzed plasma resistin and SNPs in the RETN region in 2,019 community-dwelling Japanese subjects. Plasma resistin was associated with SNP-638 (rs34861192), SNP-537 (rs34124816), SNP-420, SNP-358 (rs3219175), SNP+299 (rs3745367), and SNP+1263 (rs3745369) . SNP-638, SNP -420, SNP-358, and SNP+157 were in the same linkage disequilibrium (LD) block. SNP-358 and SNP-638 were nearly in complete LD (2r = 0.98), and were tightly correlated with SNP-420 . The correlation between either SNP-358 (or SNP-638) or SNP-420 and plasma resistin appeared to be strong . In haplotypes determined by SNP-420 and SNP-358, the estimated frequencies for C-G, G-A, and G-G were 0.6700, 0.2005, and 0.1284, respectively, and C-A was rare (0.0011), suggesting that subjects with A at −358, generally had G at −420. This G-A haplotype conferred the highest plasma resistin . In THP-1 cells, the RETN promoter with the G-A haplotype showed the highest activity. Nuclear proteins specifically recognized one base difference at SNP-358, but not at SNP-638. Therefore, A at -358 is required for G at −420 to confer the highest plasma resistin in the general Japanese population. In Caucasians, the association between SNP-420 and plasma resistin is not strong, and A at −358 may not exist, suggesting that SNP-358 could explain this ethnic difference.Insulin resistance is a feature of type 2 diabetes. Resistin, secreted from adipocytes, causes insulin resistance in mice. We previously reported that the G/G genotype of single nucleotide polymorphism (SNP) at −420 (rs1862513) in the human resistin gene ( In the general Japanese population, subjects with the G/G genotype had the highest plasma resistin levels, followed by C/G and C/C RETN, remains to be elucidated.We previously carried out systematic analyses of SNPs in the human resistin gene  analysis RETN mRNA is localized on chromosome 19p, where the strongest positional candidate is RETN itself RETN mRNA was positively correlated with its simultaneous serum level RETN would be of special interest.Melzer et al. reported that a SNP rs2431866, located at a distance of ∼100 Kb from RETN, we cross-sectionally analyzed 2,019 Japanese subjects. Plasma resistin was found to be most strongly associated with SNP-358 and SNP-420, where the relevant allele, A at-358, was tightly linked to G at −420.In view of this, to determine the SNPs responsible for circulating resistin around P = 0.049 for SNP+157, and P = 0.037 for SNP+1263 did not remain significant after Bonferroni's correction. Based on a confidence interval analysis, SNP-638, SNP -420, SNP-358, and SNP+157 were determined to be in the same LD block (2r = 0.98), and were tightly correlated with SNP-420 .We first typed the 8 SNPs listed in LD block . Four mahttp://www.genomatix.de/)) revealed the presence of transcription factor binding sites for SNP-358 but not for SNP-638. Therefore, we tentatively concluded that SNP-358 was a more functional SNP than SNP-638, and used SNP-358 when necessary in this study.To determine which SNP, i.e., SNP-358 or SNP-638, is potentially functional, we examined whether nuclear proteins specifically bound to DNA sequences using electrophoretic mobility shift assay (EMSA) . NuclearP = 2.75×10−329), SNP-537 (P = 4.63×10−14), SNP-420 (P = 1.71×10−133), SNP-358 (P = 4.94×10−324), SNP+299 (P = 5.24×10−85), and SNP+1263 (P = 2.33×10−16). These P values remained significant after Bonferroni's correction. A low P value and high 2rwere found for SNP-358 (or SNP-638), and SNP-420 (2r = 0.5224 (0.5275), and 0.2616, respectively). The difference in plasma resistin levels between each genotype appears to be highest for SNP-358 (or SNP-638), followed by SNP-420 . These findings remained unchanged when assessed using multiple regression analyses adjusted for age, gender, and BMI . Since oP = 0.2700), it was not possible to verify an effect of A at −358 itself, independent of G at −420, due to its low frequency (0.0011). The G-G haplotype, with G at −420, but not A at −358, showed an effect similar to that for C-G . Therefore, G at −420 was not sufficient to confer the highest plasma resistin, and A at −358 was required in the general Japanese population. It is difficult to determine whether A at −358 alone or in combination with G at −420 is more responsible for this effect, based on only statistical analyses of genetic data.To determine which combination of these potentially functional SNPs is most responsible for plasma resistin, we examined the relation between plasma resistin and haplotypes defined by SNP-420 and SNP-358 in this order . The estRETN promoter activity with G at −420 and A at−358 showed the higher activity than the reference control promoter with C at −420 and G at−358   . The proRETN in the general Japanese population. The lowest P value was observed for SNP-358 (or SNP-638), followed by SNP-420. SNP-420, and SNP-358 were in the same LD block, and a risk allele, A of SNP-358, was tightly linked with a risk allele, G of SNP-420. The G-A haplotype defined by SNP-420, and SNP-358, which included these risk alleles, conferred the highest plasma resistin. Functionally, the RETN promoter with this haplotype showed the highest activity in THP-1 cells. Nuclear proteins specifically recognized a difference in one base at SNP-358, but not at SNP-638.These findings herein indicate that plasma resistin levels are associated with SNP-638, SNP-537, SNP-420, SNP-358, SNP+299, and SNP+1263 around RETN promoter including G at −420 was higher than that of the wild type including C at −420 in vitroRETN promoter activity with G at −420 and G at−358 in the absence of SNP-638 in SL2 cells RETN promoter activity with G at −420 and A at−358 without SNP-638 was ∼60% higher than that with the other combinations. Therefore, both the A of SNP-358 and the G of SNP-420 appear to be required for the highest RETN promoter activity.We found that SNP-358 was associated with plasma resistin in the order of A/A >G/A>G/G in a large number of Japanese subjects. Subjects having the relevant allele for high plasma resistin, i.e. an A at−358, generally had a G at −420. In four independent studies, the activity of the mutant During the preparation of this manuscript, Asano et al. reported on-line that rs34861192 (SNP-638G>A) and rs3745268 (3′ UTR) are significant determinants of plasma resistin in an aged Japanese population http://www.genomatix.de/). The induction of RETN expression by lipopolysaccharides is inhibited by an NF-κB inhibitor in human macrophages P values clearly merit further investigation.Since the A at −358 was tightly linked with the G at−420, the issue of whether the A of SNP-358 itself is largely responsible for high plasma resistin levels, or whether both the A of SNP-358 and the G of SNP-420 are required remains to be elucidated. Also, the possible relevance of SNP-358 is suggested, since the sequences around SNP-358 are similar to the NF-κB consensus sequences based on the MatInspector program (http://www.hapmap.org/index.html.en). Therefore, SNP-358 is likely to be monophormic in Caucasians, which could account for the ethnic differences in the association between plasma resistin and SNP-420.We previously reported that a functional promoter SNP-420 was tightly associated with plasma resistin, its final gene product, in Japanese subjects retn is disrupted, a high fat diet was found to induce insulin resistance with inflammation of adipose tissue Most recently, in “humanized resistin mice”, where human resistin is specifically expressed in macrophages and endogenous RETN promoter with this G-A haplotype showed the highest activity. It is not clear why A of SNP-358 is required for G of SNP-420 to confer the highest plasma resistin, and whether the A of SNP-358 indeed accounts for ethnic differences in the association between plasma resistin and SNP-420. Further studies will be required to clarify these points.In summary, of the potentially functional SNPs, SNP-358 appears to be most strongly associated with plasma resistin in the general Japanese population, followed by SNP-420. Subjects having the responsible allele for the highest plasma resistin, A at −358, generally had G at −420. The G-A haplotype defined by SNP-420 and SNP-358 conferred the highest plasma resistin. Functionally, the All subjects were informed of the purpose of the study and their written consent was obtained. The study was approved by the ethics committee of the Ehime University Graduate School of Medicine.This cross-sectional study included community-dwelling Japanese subjects who were attending an annual medical check-up in a rural town located in Ehime prefecture in Japan. We previously analyzed resistin SNP-420 and plasma resistin levels in 2,078 subjects in this population RETNhttp://www.hapmap.org/index.html.en). These SNPs included all of the 4SNPs reported in HapMap around RETN. SNPs were typed by TaqMan analysis using assay on demand probes and primers sets (Applied Biosystems) except rs3760678, rs34861192, rs34124816, and rs3219177 which were generated as assay by design. The rs1862513 was previously typed as described The seven SNPs were selected, based on our systemic analysis of SNPs in Fasting plasma resistin was determined using a human resistin ELISA kit , according to the manufacturer's protocol 2 incubator. Nuclear extracts of THP-1 cells were prepared with NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo SCIENTIFIC). The following oligonucleotides were used for EMSA as probes or competitors, sense hRE-358A, 5′-CACTGTCTGCTCAGGAGCTTCCTCTTGGCCA-3′; antisense hRE-358A, 5′-GATCTGGCCAAGAGGAAGCTCCTGAGCAGACAGTGGTAC-3′; sense hRE-358G, 5′-CACTGTCTGCTCAGGGGCTTCCTCTTGGCCA-3′; antisense hRE-358G, 5′-GATCTGGCCAAGAGGAAGCCCCTGAGCAGACAGTGGTAC-3′, sense hRE-638A, 5′-CGTCACTGTAGCTTCAAACTCCCGGGCTCAA-3′; antisense hRE-638A, 5′-GATCTTGAGCCCGGGAGTTTGAAGCTACAGTGACGGTAC-3′; sense hRE-638G, 5′-CGTCACTGTAGCTTCGAACTCCCGGGCTCAA-3′; antisense hRE-638G, 5′-GATCTTGAGCCCGGGAGTTCGAAGCTACAGTGACGGTAC-3′. Complementary oligonucleotides were annealed and labeled with 32P. A 10 µg of nuclear extract was incubated for 30 min with a 32P-labeled oligonucleotide (0.01 pmol) and 1 µg of poly(dAdT) in reaction buffer containing 25 mM HEPES (pH 7.9), 0.5 mM EDTA, 0.5 mM DTT, 40 mM KCl and 4% Ficoll (v/v). Nonidet P-40 (0.5%), and a 100-fold molar excess of the unlabeled single-stranded sense and antisense oligonucleotides, corresponding to each labeled probe were added to reduce nonspecific binding. Competition experiments were performed by the addition of a 200-fold molar excess of unlabelled double-stranded oligonucleotides to the reaction mixture. After the binding reaction, the mixture was subjected to 6% PAGE in 45 mM Tris, 45 mM boric acid, 1 mM EDTA at 100V for 2 h at 4°C. The gel was dried and exposed to a FUJIFILM imaging plate. Signals were detected with the FUJIFILM FLA-5100 image analyzing system.THP-1 cells were grown in RPM1 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CORETN was amplified by PCR using DNA from either a subject homozygous for a wild-type allele (−420C) or a subject homozygous for a mutant allele (−420G). These products were inserted into pGL3 basic vector  to generate pGL3(−420G) and pGL3(−420C), both of which harbor G at the −358 locus, as described previously5′-CTTACTGTCTGCTCAGGAGCTTCCTCTTGGCCCCG, and 5′- CGGGGCCAAGAGGAAGCTCCTGAGCAGACAGTAAG. The DNA fragments between −450 and −206 of RETN with or without this point mutation were subsequently cloned into a luciferase reporter vector pGL4.11 (Promega) at the site between Kpn I and Bgl II. The sequence of the generated four plasmids, pGL4(−420C/−358G), pGL4(−420C/−358A), pGL4(−420G/−358G) and pGL4(−420G/−358A), were confirmed by DNA sequencing. All plasmids used for the transfection were prepared by use of a Qiagen plasmid kit, followed by CsCl2 density-gradient ultracentrifugation as described previously The 5′ flanking region between −450 and −206 of 5 cells/well. Trans IT-Jurkat Reagent  was used for transient transfection following the manufacturer's protocol. Each of the pGL4(−420C/−358G), pGL4(−420C/−358A), pGL4(−420G/−358G) and pGL4(−420G/−358A) vectors was transfected into these cells along with pGL4.11 basic vector , pGL4.11 promoter vector (positive control), and pRL-SV40 internal control vector. The THP-1 cells were then incubated with RPMI1640 containing 10% FCS for 48 hours. Luciferase assay were performed using the Dual-LuciferaseTM Reporter Assay System according to the manufacturer's protocol (Promega).THP-1 human monocytes purchased from Human Science Research Resources Bank  were grown in RPMI1640 containing 10% FCS. One day before transfection, THP-1 cells were plated on a 6 well plate at 4.0×10P value ×8). To assess isolated effects of each SNP on plasma resistin, multiple regression analyses involving each SNP, age (year), gender , and BMI as independent variables, and plasma resistin (ng/ml) as a dependent variable were performed. In these analyses, we assume that the major and minor alleles based on the allele frequency are normal (N), and susceptibility (S) alleles, respectively. Each SNP genotype, N/N, N/S, and S/S, were denoted by two dummy variables   = , , and , respectively. This first dummy variable estimates the difference between N/S and N/N, and the second between S/S and N/N. These analyses were performed using a commercially available statistical software package . Linkage disequilibrium (LD) was assessed using the Haploview software P<0.05.In the human population study, values are expressed as the mean±standard deviation. Differences in plasma resistin levels among genotypes were assessed by analysis of variance (ANOVA). Bonferroni's correction was applied to


C. elegans. Transcription is blocked by the presence of an “off cassette” between the promoter and the coding region of the desired product. The “off cassette” is composed of a transcriptional terminator flanked by FLP recognition targets (FRT). This sequence can be excised by FLP recombinase to bring together the promoter and the coding region. We have introduced two fluorescent reporters into the system: a red reporter for promoter activity prior to FLP expression and a green reporter for expression of the gene of interest after FLP expression. The constructs are designed using the multisite Gateway system, so that promoters and coding regions can be quickly mixed and matched. We demonstrate that heat-shock-driven FLP recombinase adds temporal control on top of tissue specific expression provided by the transgene promoter. In addition, the temporal switch is permanent, rather than acute, as is usually the case for heat-shock driven transgenes. Finally, FLP expression can be driven by a tissue specific promoter to provide expression in a subset of cells that can only be addressed as the intersection of two available promoters. As a test of the system, we have driven the light chain of tetanus toxin, a protease that cleaves the synaptic vesicle protein synaptobrevin. We show that we can use this to inactivate synaptic transmission in all neurons or a subset of neurons in a FLP-dependent manner.The FLP enzyme catalyzes recombination between specific target sequences in DNA. Here we use FLP to temporally and spatially control gene expression in the nematode  C. elegans has been an important model system for studying the roles of genes in animal development and physiology. However, worm researchers have had a limited toolkit for controlling gene activation. These drawbacks have been particularly restrictive when studying the function of a gene that has different roles in several cell types or at different times in development. Here we describe a way to turn any gene on at a specific time in specific cells. We provide a set of mix-and-match reagents that give researchers a way to quickly build new combinations of regulatory elements. These reagents will allow researchers to express a single gene in a wide array of temporal or spatial patterns, or to serially express many genes in a single cell. As a proof of principle, we made an artificial worm gene composed of a neurotoxin that would block neurotransmission. When we activated the gene in a small number of neurons in adult animals, these cells ceased to function. We anticipate that this new technique will find a wide variety of uses by the C. elegans community.Genes turn on and off as a natural part of development. The nematode  C. elegans it is simple to create a transgene by injecting DNA containing a complete genomic region. In most cases the transgene will be expressed in its native temporal and spatial pattern and will rescue the mutant phenotype. However, if the researcher would like to test the function of a gene at a specific time or in a specific tissue of the worm, one would need a very specific promoter. Due to a limited set of promoters available, it is often impossible to express a gene of interest in a specific cell. More importantly, there is only one temporally inducible promoter available – the heat-shock promoter. This promoter has been a workhorse of the field, but has a major drawback because it is expressed ubiquitously. Three techniques have been developed that provide more precise temporal and spatial control by making gene expression dependent on two independently controllable events.Every widely-used genetic model organism can be manipulated to express genes introduced by the experimenter. In The Chalfie laboratory has developed one solution by expressing the gene product as two complementary halves. In this case, the full gene product is reconstituted only in the cells that express both promoters. When expressed under the control of two different overlapping promoters, the complete protein is only reconstituted in a small number of cells. They have demonstrated that a two-part GFP can be used to label specific cells A second combinatorial control technique relies on temperature-dependent degradation of the mRNA of the target gene. This method was independently proposed by several groups, but made practical by Getz, Xu and Fire hsf-1 mutant background and gene expression from the heatshock promoter is still acute. Ideally, a combinatorial expression system would be in the wild-type strain and provide a permanent change in the genotype of the cells of interest.Bacaj and Shaham have developed a method to add heat-shock control to a transgene expressed under a tissue-specific promoterC. elegans. In this configuration, the transgene is expressed at the combinatorial intersection of two different promoters: either two spatially restricted promoters, or a spatially restricted and temporally controlled promoter.Here we describe a method that uses FLP recombinase to control transgene expression in The site specific recombinases Cre and FLP have been used in many systems to control gene structure and expression let-858 gene. The red-fluorescent mCherry protein acts as a reporter for the promoter activity of the transgene, verifying that the transgene is present and expressing in the expected cell types prior to FLP-induced recombination. The let-858 3′ genomic region provides the poly-adenylation signal for mCherry mRNA as well as a putative transcriptional terminator, preventing expression of downstream sequences. GFP acts as a reporter for transcriptional read-through or reinitiation of transcription in the let-858 genomic region. Recombination of the FRT sequences removes the mCherry coding sequence and the terminator and brings a GFP coding region under control of the promoter, to indicate that the FLP reaction was successful. The coding region for any gene of interest can be fused in frame to the 3′ end of the GFP sequence, thus providing FLP-inducible expression of that protein.The constructs are designed to provide a fluorescent readout in either the ‘off’ or ‘on’ state. The FRT-flanked “off cassette” contains the mCherry coding sequence followed by the 3′ genomic region from the in vitro recombination system from Invitrogen C. elegans expression constructs using this system. The C. elegans promoterome consortium has cloned the 5′ regulatory regions of approximately 6,000 different C. elegans genes into Gateway ‘entry’ vectors compatible with the slot 1 entry vectors To speed assembly of constructs and to make use of genome reagents generated by other laboratories, we based our constructs on the Multisite Gateway™ Because the promoterome constructs are designed to recombine directly to ORFeome constructs, it was not possible to introduce the off-cassette between them. Instead, we built vectors that have the off-cassette in either the promoter slot (slot 1)  or in thThe ORFeome-compatible format requires that the “off-cassette” be cloned into entry vectors containing various promoters. We generated several potentially useful promoter-“off-cassette” entry clones , Table 1For the promoterome-compatible constructs, we placed the off-cassette into the position usually occupied by the ORF of interest. We then used standard cloning methods to place several ORFs of interest into slot 3 entry vectors , Table 2Pmyo-2) followed by the “off-cassette” in the first Gateway slot. We added the HIS-11 open reading frame in the second slot and an unc-54 3′ polyadenylation site in the third slot (hsp-16.48 heat shock promoter lin-15(+) co-injection marker to produce a line of transgenic worms. As expected, these worms expressed diffuse red fluorescence in their pharyngeal muscles with no apparent GFP fluorescence . Bright mCherry fluorescence obscured expression of the cassette in these cells before heatshock. Thus, we could not determine whether the myo-2 promoter was driving expression of the transgene. After heatshock we did not score GFP expression in any pm1 nuclei (0/15) and only identified weak expression in 7/15 pm2 nuclei. In summary, robust expression due to recombination was observed in all cells in which the transgene was unequivocally expressed.As a proof of principle, we produced two FLP-inducible constructs that would express GFP-tagged histone in different muscle cells. The first construct was in the ORFeome compatible configuration described in ird slot . We injerescence . The lacee hours  and was ee hours . From romyo-3 promoterome plasmid in the first position, the FRT cassette was placed into the second position and a HIS-11 ORF cloned in front of the unc-54 3′ genomic region in the third position. This construct should express the GFP::HIS-11 fusion in the body muscles of the worm after FLP expression. There was diffuse red but no green fluorescence in the body wall muscles prior to heat shock , we never saw substantial loss of mCherry expression even after induction of the GFP fusion protein. Although the half-life of mCherry in worm cells is not known, the half life of GFP in body muscle cells is greater than 24 hours If all of the copies of the transgene are recombined, mCherry expression should not occur after FLP expression. However, in all cells examined (using the cha-1 mutants) or all synaptic neurotransmission  are arrested in the first larval stage. Thus, loss of synaptic transmission in at least some neurons may lead to broad developmental defects that blind the investigator to functions for a neuron in the adult. Using the FLP-on method, expression of tetanus toxin can be activated after the developmental requirement for a neuron. Conversely, early loss of neuronal function can lead to developmental compensation in the nervous system. For example, when particular sensory neurons were ablated in males in the L3 stage the nervous system compensated for their loss; by contrast ablation of these same neurons in the L4 stage led to behavioral abnormalities in the adult One important application for the FLP-on method is to silence neurotransmission in specific neurons in a temporally-controlled manner. The tetanus toxin light chain is a highly specific protease that recognizes and cleaves synaptobrevin Punc-47. unc-47 encodes the vesicular GABA transporter required to fill synaptic vesicles with GABA, and is expressed in all GABA neurons. We chose this promoter because loss of GABA function produces two distinct phenotypes: a locomotory phenotype and a defecation motor program defect.To test if tetanus toxin induction could inactivate neurotransmission, we designed an ORFeome-compatible tetanus toxin expression construct. The tetanus toxin sequence was inserted into slot 2, and the FRT-flanked terminator was placed in slot 1 after the GABA-specific neuron promoter myo-2::GFP extrachromosomal array marker. The presence of nonshrinking animals in the population was due to loss of the extrachromosomal array: 29 of the 30 non-shrinker animals had lost the Pmyo-2::GFP transgene marker. The one non-shrinking animal carrying the array lacked the mCherry marker in the VD and DD motor neurons, demonstrating that this animal was a somatic mosaic which lacked the array in the motor neurons.Animals lacking GABA neurotransmission exhibit a distinctive locomotory defect GABA function is also required for the motor program of the defecation cycle C. elegans: to provide spatial specificity and temporal specificity for gene expression. Because it provides expression within the spatial overlap of two promoters, the method essentially squares the number of transgene expression patterns now available. In many cases, especially in the nervous system, this can restrict expression to single cells of the worm.FLP-dependent gene expression will have two general uses in hsf-1 method developed by the Shaham laboratory are similar. In the FLP-on method, expression is permanently activated, whereas in the hsf-1 rescue method, expression is acute and depends on the length of the heatshock response. Depending on the circumstances and the gene product being expressed, one method may be more advantageous than the other.FLP recombinase can also be used to provide temporal specificity for gene expression. For most purposes temporal control is best provided by activation of the heatshock promoter. Thus, heatshock-driven expression of FLP recombinase will confer temporal specificity to any promoter. This will be particularly useful for expression of dominant negative or constitutively activated gene products that may kill cells before their effects can be assayed. Moreover, acute expression can outflank the criticism most often thrown at genetic analysis –that homeostatic mechanisms will compensate for chronic genetic changes. In this way the heatshock FLP-on method and the cell-specific rescue of For the neuroscientist, FLP-on constructs provide a method for analyzing the role of a neuron in a circuit by killing a specific cell, inactivating the cell, or activating the cell. By combining our system with the Chalfie split caspase system , cells cmyo-2::GFP. FLP regulation will also be useful for analyzing promoter expression patterns by permanently marking descendents of cells that have expressed a promoter. Combined with the known cell lineage of C. elegans, the expression pattern can quickly pinpoint the expression pattern of a transgene that may come on very briefly and in only a few cells in the embryo. Using traditional GFP reporters, such expression might be missed entirely, or if it is detected it might be very difficult to unambiguously identify the expressing cell among the dividing cells of the embryo. This technique could be combined with forward genetic screens. A FLP-dependent histone GFP reporter  using the BP recombination reaction.pWD157 (slot 2 TeTx): Tetanus toxin light chain was PCR amplified from CMV-LC-Tx (Heiner Niemann) using primers containing attB1 and attB2 tails GTATGCCGATCACCATCAACAAC and TTAAGCGGTACGGTTGTACAGG and cloned as a blunt fragment into pMH472 using SrfI. pMH472 is a slot 3 entry vector containing a SrfI site followed by two stop codons and then the unc-54 3′ UTR.pWD170 (slot 3 TeTx): Tetanus toxin light chain was PCR amplified from CMV-LC-Tx using primers Pmyo-2::GFP plasmid (gift of Andrew Fire). Tandem FRT sites pPD119FRTRFPGFP: A fragment of mRFP1 was PCR amplified and cloned between the SalI and SmaI sites in pPD118.33. pPD118.33 is a Pmyo-2-FRT-mCherry-terminator-FRT-GFP-terminator): pPD119FRTRFPGFP was modified to replace mRFP with mCherry. An MluI-KpnI double-stranded oligonucleotide containing an FRT fragment in a different frame was used to replace the second FRT in pPD119FRTRFPGFP. The plasmid was then cut with BamHI and Mlu and a PCR fragment containing mCherry followed by the let-858 3′ end was ligated in as a BamHI-BssHII digested fragment. This produced a myo-2 promoter::FRT::mCherry terminator::FRT::GFP::unc-54 3′ UTR plasmid.pWD176 (Pmyo-2-FRT-mCherry-terminator-FRT): The Pmyo-2::mRFP FRT cassette::GFP fragment from pPD119FRTRFPGFP was PCR amplified using primers containing attB4 and attB1 tails GGGGACAACTTTGTATAGAAAAGTTGCTTGCATGCCTGCAGGTCGAGG and GGGGACTGCTTTTTTGTACAAACTTGTTTTGTATAGTTCGTCCATGCCATG and recombined with the attP4 and attP1 sites in the slot 1 donor vector pDONR P4-P1R (Invitrogen) using the BP recombination reaction to make pWD159. The mRFP cassette was replaced with mCherry using a BamHI-XhoI fragment from pWD176. This plasmid was sequenced with primers T7, M13fwd, mCh r1: CTTTCACTTGAAGCTTCCCATCCC, GFP-I-r2: CTCCAGTGAAAAGTTCTTCTCC, and GFP-I-r1: TTGTGCCCATTAACATCACC.pWD177 (slot 1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTACGAAGTTCCTATTCTCTAGA and GGGGACCACTTTGTACAAGAAAGCTGGGTTTTTGTATAGTTCGTCCATGCC and recombined with the attP1 and attP2 sites in pDONR221 (Invitrogen) using the BP recombination reaction. The mRFP cassette was replaced with mCherry using a BamHI-XhoI fragment from pWD176. This plasmid was sequenced with primers T7, M13fwd, GFP-I-r2: CTCCAGTGAAAAGTTCTTCTCC, and GFP-I-r1: TTGTGCCCATTAACATCACCpWD178 (slot 2 FRT-mCherry-terminator-FRT): The mRFP FRT cassette::GFP fragment from pPD119FRTRFPGFP was PCR amplified using primers containing attB1 and attB2 tails unc-47 promoter was PCR amplified from plasmid pKS4.1 (K. Schuske) using primers: CGAACGCATGCGGATCCCGGAACAGTCGAAAG and CGAACGTCGACGCATCTGTAATGAAATAAATGTGACGCTG. This sequence was inserted into pWD159 as an Sph-Sal fragment. The mRFP cassette was replaced with mCherry using a SalI-BstZ17I fragment from pWD176. This plasmid was sequenced with primers T7, M13fwd, mCh r1: CTTTCACTTGAAGCTTCCCATCCC, GFP-I-r2: CTCCAGTGAAAAGTTCTTCTCC, and GFP-I-r1: TTGTGCCCATTAACATCACC.pWD179: The 1.2 kb rab-3 promoter was PCR amplified from N2 genomic DNA using primers: CGAACGCATGCATCTTCAGATGGGAGCAGTGG and CGAACGTCGACGCATCTGAAAATAGGGCTACTGTAGAT. This DNA was inserted into pWD159 as an Sph-Sal fragment. The mRFP cassette was replaced with mCherry using a BamHI-XhoI fragment from pWD176. This plasmid was sequenced with primers T7, M13fwd, mCh r1: CTTTCACTTGAAGCTTCCCATCCC, GFP-I-r2: CTCCAGTGAAAAGTTCTTCTCC, and GFP-I-r1: TTGTGCCCATTAACATCACC.pWD180: The 1.2 kb his-11 ORF was PCR amplified using primers containing attB1 and attB2 tails GGGGACAAGTTTGTACAAAAAAGCAGGCTTACCACCAAAGCCATCTGCCAAGG and GGGGACCACTTTGTACAAGAAAGCTGGGTATTACTTGCTGGAAGTGTACTTGG using pGH42 (G. Hollopeter) as template. The DNA fragment was recombined with the attP1 and attP2 sites in pDONR221 (Invitrogen) using the BP recombination reaction.pWD195 (slot 2 Histone 2B): The Pmyo-3-FRT-mCherry-FRT-GFP-Histone): A multisite LR reaction was performed using pEntry[4-1] Pmyo-3 (Open Biosystems), pWD178, pGH42 (G. Hollopeter), and pDEST R4-R3 (Invitrogen).pWD198 (Punc-47-FRT-mCherry-FRT-GFP-TeTx): A multisite LR reaction was performed using pWD179, pWD157, pMH473 (M. Hammarlund), and pDEST R4-R3 (Invitrogen). pMH473 is an attP2-attP3 entry clone carrying the unc-54 3′ polyadenylation site.pWD199 (Pmyo-2-FRT-mCherry-FRT-GFP-Histone): A multisite LR reaction was performed using pWD177, pWD195, pMH473 and pDEST R4-R3 (Invitrogen).pWD200 (unc-54 3′ UTR from TU#813 GGGGACAGCTTTCTTGTACAAAGTGGGAAGTGGTGTTGATGATGACATGGCG and GGGGACAACTTTGTATAATAAAGTTGCCATAGACACTACTCCACTTTC and BP cloned into pDONR P2R-P3 (Invitrogen).pWD203 (slot 3 Caspase C-terminus): The caspase 3 C-terminal fragment, leucine zipper and unc-54 3′ UTR from TU#814 GGGGACAGCTTTCTTGTACAAAGTGGGAATGGCTAGCGCACAGCTGGAGAAG and GGGGACAACTTTGTATAATAAAGTTGCCATAGACACTACTCCACTTTC and BP cloned into pDONR P2R-P3 (Invitrogen).pWD204 (slot 3 Caspase N-terminus) The caspase 3 N-terminal fragment, leucine zipper and Phsp-16-48:FLP): A PCR fragment containing the FLP coding sequence from pOG44 (Stratagene) was cloned as an MluI-NheI fragment into pJL44 (J.L. Bessereau). pJL44 contains the Phsp-16-48 heat-shock promoter and the glh-2 3′ UTR. The FLP coding sequence in pOG44 contains a point mutation which was repaired using a PCR fragment from the FLP coding sequence cloned into pBR322 (Makkuni Jayaram). An artificial intron was introduced into the FLP coding sequence at the EcoRV site using a double-stranded oligo: GTAAGTTTAAACATATATACTAACTAACCCTGATTATTTAAATTTTCAG.pWD79-2RV (GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCCACAATTTGGTATATTATGT and GGGGACCACTTTGTACAAGAAAGCTGGGTATTATATGCGTCTATTTATGTAGGATG and BP cloned into pDONR221 (Invitrogen). This version of FLP contains a stop codon at the native C terminus.pWD172 (slot 2 FLP-stop): The FLP ORF was PCR amplified from pWD79-2RV using primers containing attB1 and attB2 tails: GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCCACAATTTGGTATATTATGT and GGGGACCACTTTGTACAAGAAAGCTGGGTATATGCGTCTATTTATGTAGGATG and BP cloned into pDONR221 (Invitrogen). This version of FLP does not contain a stop codon, and can be used to make C-terminally tagged proteins.pWD173 (slot 2 FLP-no stop): The FLP ORF was PCR amplified from pWD79-2RV using primers containing attB2 and attB3 tails: Gateway BP and LR in vitro recombination reactions were carried out according to manufacturer instructions.Strains used in this study: The wild type is Bristol N2.unc-25(e156) III.EG3251 oxEx1099 was made by injecting: pWD198 (Pmyo-3-FTF-GFP-Histone) at 5 ng/ul, pWD79-2RV (Phsp-16-48:FLP) at 45 ng/ul, pPD118.33 (Pmyo-2::GFP)(A. Fire) at 1 ng/ul, and pL15EK (lin-15(+))EG4859 oxEx1100 was made by injecting: pWD199 (Punc-47-FTF-GFP-TeTx) at 5 ng/ul, pWD79-2RV (Phsp-16-48:FLP) at 45 ng/ul, pPD118.33 (Pmyo-2::GFP)(A. Fire) at 1 ng/ul, pL15EK (lin-15(+))EG4860 lin-15(n765ts) X oxEx1101 was made by injecting: pWD200 (Pmyo-2-FTF-GFP-Histone)at 2 ng/ul, pWD79-2RV (Phsp-16-48:FLP) at 45 ng/ul, pL15EK (lin-15(+))lin-15(n765ts).EG4866 ‘FTF’ symbolizes the off cassette composed of FRT-mCherry-terminator-FRT.Figure S1FLP-Dependent GFP-Histone Expression. Before heat induction, the myo-2 promoter drives mCherry expression in the pharyngeal muscle. FLP recombinase is induced by a 34° heat shock for one hour. Three hours after heat induction, the transgene produces nuclear-localized GFP-histone fusion protein.(0.18 MB TIF)Click here for additional data file.Figure S2The GABA Nervous System 23 Hours after Heatshock. A transgenic strain carrying FLP inducible expression of tetanus toxin (EG4860) was heat shocked one hour at 34°. 23 hours later, mCherry in the GABA nervous system was imaged. The architecture of the GABA nervous system is normal; thus heat shock, FLP expression, and tetanus toxin does not affect the anatomy of the neurons. Anterior is to the left, dorsal is at the top.(0.07 MB TIF)Click here for additional data file.Video S1A transgenic strain carrying FLP inducible expression of tetanus toxin before and after heat shock. An unc-25 mutant that lacks GABA synthesis is shown for comparison.(0.62 MB MPG)Click here for additional data file.


D1 macrophagesinduced by LPS. The binding of these cytokines to high affinity receptors on target cells, e.g. L929fibroblasts and IL-2 in HuT78 T lymphoma cells, were also suppressed by the agents. These compoundsblocked the adhesion of leukocytes and macrophages to the plasma membranes of L929 fibroblasts.A series of polyphenolic-sulfonated compounds were observed to have potent anti-inflammatory activityand were protective against induced endotoxic shock in mice at 8 and 16 mg/kg, I.P. These agents provedto be potent elastase inhibitors in human leukocytes and J774-AI and IC-21 mouse macrophages as well asprostaglandin cyclo-oxygenase inhibitors in J774-AI macrophages. The compounds from 5 to 50 μMinhibited TNFα release from IC-21 macrophages and IL-1 release from mouse P388


Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools.Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their  In the adult mouse, about 2×10− CD23− IgMhigh and IgDlow, T2 are CD21+ CD23+ IgMhigh and IgDhigh, and T3 are CD21+ CD23+ IgMlow and IgDhigh cells Surface expression of CD93 is a hallmark for immature B cells and on splenic B cells is a phenotypic characteristic for so called transitional B cells BAFF (B cell activating factor), a member of the TNF family  and BAFF receptor (BAFF-R) play a fundamental role during the transition from immature T1 to T2 B cells and therefore for the generation of mature B cells in the spleen. This was clearly demonstrated by an almost complete lack of follicular and marginal zone B cells and by a block at the T1 cell stage in BAFF as well as in BAFF-R deficient mice κB pathway and ultimately to the transcription of the anti-apoptotic factor Bcl-2 The binding of BAFF to the BAFF-R leads to the activation of the NF-in vitro survival of immature as well as mature B-2 cells, we hypothesised that BAFF-BAFF-R signaling was also playing a central role in the in vivo maintenance of the peripheral mature B cell pool. However, the potential survival role of BAFF in the mature B cell pool is masked in both BAFF and BAFF-R-deficient animals due to the associated developmental block at the T1 stage. Therefore, to address this question, we generated a collection of anti BAFF-R mAbs some of which blocked and others failed to block BAFF binding.Since BAFF-R is expressed on all mature peripheral B cells and its signaling promotes Administration of these blocking antibodies to wild-type mice resulted in an almost complete depletion of follicular B cells and a reduction of about 50% in the MZB cell compartment. Non-blocking antibodies had no, or only minor effects on the mature B cell pool. Moreover, by using FcRγ-deficient or Bcl-2-transgenic mice, we could show that this depletion was Fc-Receptor (FcR) and complement independent. Taken together, beyond its essential role in allowing the developmental progression from immature T1 cells into T2–T3 and mature B cells, we formally demonstrate the essential role of the BAFF-BAFF-R signaling in the long-term survival and homeostasis of mature B-2 and marginal zone B cells.A mixture of un-transfected and mouse BAFF-R-expressing Y3 rat myeloma cells was used to screen supernatants of individual hybridomas generated as described in All BAFF-blocking and non-blocking mAbs were used to reveal the expression of BAFF-R on ex vivo isolated spleen, lymph-node, bone marrow, peripheral blood and peritoneal cells. All mature B cells, irrespective of their localization within the lymphatic compartments, namely B-2, MZB, B-1a and B-1b B cells as well as the three immature transitional splenic B cell subsets  expressed similar levels of BAFF-R . Bone main vitro, we reasoned that the in vivo use of blocking anti-BAFF-R mAbs would affect the B cell pool. Therefore wild-type C57BL/6 mice were treated with two BAFF-blocking and two BAFF non-blocking anti-BAFF-R mAbs; all four of the same isotype. At day 14 after treatment, the percentage of mature circulating peripheral blood B cells, characterized as CD19+ CD93− cells, was determined by flow cytometry. Mature B cells in the control group of PBS treated mice represent about 40% of the circulating leukocytes. Similar percentages were obtained with the non-blocking 5A12 mAb  were used as negative and positive controls, respectively. Results are summarized in + T cells had disappeared and as a consequence, the other lymphocyte sub-populations had proportionately increased. Treatment with 9B9 resulted in a 40% depletion of mature B cells at day 4, which increased to 70% by day 7 and reached its maximal level of 80% by day 10. In accordance with the in vivo expected half-life of IgG antibodies, B cell numbers started to recover by day 25–30 after antibody treatment (data not shown).In order to determine the kinetics of mature B cells depletion, C57BL/6 mice were injected with the BAFF-blocking mAb 9B9 and at days 4, 7 and 10 blood lymphocyte subpopulations were analyzed. Mice injected with either PBS or the +CD93−), was determined in the blood and compared to untreated mice. In untreated or 5A12-treated mice, a similar percentage of circulating mature B cells was detected, namely 35% and 25–38%, respectively. In contrast, there were only 7–12% circulating B cells in FcR γ-deficient mice treated with the BAFF-blocking mAb 9B9  nor to complement-mediated depletion. In order to test this hypothesis, FcR common γ chain-deficient mice were injected with the BAFF-blocking mAb 9B9 or the non blocking mAb 5A12. At day 14 after injection, the percentage of circulating mature B cells  B cells, whereas MZB (CD21highCD23low) cells were only decreased by 50%. The number of CD21−CD23− mature splenic B cells, which to a large extent comprises B-1 B cells, was not affected at all. CD4 and CD8 T cells also remained unchanged , MZB and B-1 B cells. In order to determine the effect of a blocking anti-BAFF-R mAb on the different B cell subsets, we injected C57BL/6 mice with the mAb 9B9 and analyzed the bone marrow, spleen, lymph nodes and peritoneal cavity lymphocytes at days 14 to 21. In the spleen, the immature transitional B cell subpopulations were only slightly affected with no reduction of T1 and a two fold reduction in T2/T3 subsets . Mirrorinchanged .Mature B cells in the bone marrow or lymph nodes, both of which consist almost entirely of B-2 B cells, were also reduced by 80–90% upon 9B9 treatment (data not shown). Bone marrow B cell progenitors were not affected by the treatment (data not shown).+CD23+), B-1a (CD19+CD23−CD11b+CD5+) and B-1b (CD19+CD23−CD11b+CD5−) B cells , MZB cells were reduced by half and 10–20% of the B-2 B cell compartment was still present . Thus, tlow IgDhigh follicular B (B-2) cells are clustered in B cell follicles and are surrounded by a rim of IgMhigh IgDlow marginal zone B cells , whereas, a 5 to 10 fold reduction in the titers was observed in 9B9 treated compared to control animals, following a T dependent NIP-ovalbumin (NIP-OVA) immunization . HoweverTo address the role of BAFF-BAFF-R signaling on the maintenance of memory B cells, NIP-OVA primed mice were injected with 9B9 mAb at day 60 and 74 following immunization. At day 80 after priming mice were boosted and the IgG anti-NIP titer was determined at day 94. As shown in in vivo could not be addressed using these mice, since their precursors depended on BAFF for their generation and development.The role of BAFF in the development of mouse B cells was most clearly demonstrated by the generation of two different BAFF deficient strains and by the characterization of BAFF-R deficient mice in vivo survival of almost all follicular and half of the marginal zone B cells is dependent upon BAFF-BAFF-R signaling. Injection of mice with a blocking anti-BAFF-R antibody induced a profound depletion of the mature B cell compartment, whereas a non-blocking antibody had only a minimal or no effect. The possible scenarios which could explain this phenomenon include: an antibody-dependent cellular cytotoxicity (ADCC), a complement-mediated lysis, an impairment of the survival through prevention of BAFF binding or a limited generation of newly formed mature B cells as a consequence of the depletion of transitional B cells. The fact that an isotype-matched non-blocking anti-BAFF-R monoclonal antibody is not affecting the peripheral mature B cell pool and that in addition, the detectable presence of the non blocking anti-BAFF-R antibody on the surface of non-depleted B cells (data not shown), taken together would indicate that an ADCC mechanism is unlikely. Moreover, that B cell depletion is still occurring in FcR-γ-chain By the generation and administration of anti-BAFF-R monoclonal antibodies capable of preventing BAFF binding, we show in this report, that the in vivo labeling studies performed by us and others this seems to be an unlikely scenario Yet an alternative explanation for the observed depletion of mature B cells upon treatment would be that the numbers of differentiating T2/T3 immature B cells into mature cells decrease with time. However based on BrdU Different experimental approaches, performed by other groups, were also suggestive of a survival role of BAFF-BAFF-R signaling in mature B cells. Treatment of mice with a TACI-Fc fusion protein was shown to lead to a reduction of B cells B-1 B cells express relatively high amounts of BAFF-R on their surface. However, as shown in BAFF as well as BAFF-R deficient mice their generation and maintenance is not affected Following anti-BAFF-R treatment, we observed that the B-2 B cell compartment was the most affected B cell subset, indicating that the majority of B-2 cells rely on BAFF signaling for their survival. Marginal zone B cells were reduced only by half upon treatment, which compared to their almost complete absence observed in BAFF and BAFF-R deficient mice, is indicative for a crucial role of BAFF signaling during marginal zone B cell development or survival of their progeny, but dispensable for their survival subsequent to maturation. In BAFF-R deficient mice, over-expression of Bcl-2 could not overcome the marginal zone B cell defect By BrdU labeling experiments and FACS analysis we show that the survival of a small subset of B-2 B cells seemed to be BAFF-independent and not reflecting newly formed mature B cells. A similar result was shown in BAFF as well as BAFF-R deficient mice, where the mature follicular B cell compartment was drastically reduced but still present in small numbers Prolonged (5 months) treatment with the 9B9 mAb did not improve the depletion of these mature B cell subsets. FACS analysis with an anti-rat IgG mAb revealed the presence of 9B9 on the surface of B-2 and MZB cells surviving the treatment (data not shown). Moreover, the surface available BAFF-R seemed to be saturated by the injected 9B9, as anti-BAFF-R mAbs were undetectable by FACS analysis (data not shown). Therefore, the survival of these mature B cell subsets seems to be BAFF–BAFF-R signaling independent.It has been shown that surface BCR expression is mandatory for mature B cell survival, since conditional ablation of BCR expression subsequent to the establishment of steady-state B cell numbers, resulted in rapid death of most peripheral B cells The survival of mature B cells is dependent on signaling processes that use the NF-κB signal transduction pathway in vitro studies have demonstrated a survival role of BAFF on transitional as well as mature B cells Several It is believed that formation of the different mature B cell compartments is influenced by specific BCR-ligand interactions 1 response to T-dependent antigen was significantly reduced As a consequence for the observed B cell depletion in anti-BAFF-R treated mice, we show that splenic follicles are greatly reduced in size. Moreover, the primary immune response to T dependent antigen was impaired, with IgG titers reduced by a factor of 5 to 10 following treatment with an anti-BAFF-R blocking antibody, whereas total immunoglobulin levels were not affected. This result is in line with what was shown for BAFF-R deficient mice, where the IgGThese findings enlighten a new role of BAFF-BAFF-R signaling as a crucial factor for the formation of memory or the survival of developing memory B cells, while confirming its dispensable role in the maintenance of memory B cells. Therefore the question whether therapies, based on BAFF as well as BAFF-R neutralization in B cell mediated autoimmune diseases, could be successful remains uncertain. On the other hand, since elevated BAFF serum levels and deregulated BAFF-R signaling were shown to contribute to the pathogenic B survival in oncological as immunological disorders −/− mice were obtained from Taconic . Bcl-2 transgenic mice Female C57BL/6 and Lewis rats were purchased from RCC Ltd., . FcRγcIn vivo BrdU labeling and subsequent analysis was performed as described FITC-, PE-, APC-Cy7-, PE-Cy7- or biotin-conjugated mAbs specific for CD4 (RM4-5), CD5 (53-7.3), CD8α (53-6.7), CD11b (M1/70), CD19 (1D3), CD21 , CD23 (B3B4) and CD45R  were purchased from BD Biosciences (BD Pharmingen™). Biotin-labeled Mouse anti-Rat IgG was purchased from Jackson ImmunoResearch Laboratories, Europe LtD. Antibodies specific for IgD (1.19), IgM (M41), CD90 , CD93  and HA-peptide (12CA5) were purified from hybridoma supernatants and labeled with FITC or biotin using standard procedures. Biotin-labeled antibodies were revealed by PE- or PE-Cy7-Streptavidin (BD Biosciences). Staining of cells was performed as described previously A soluble form of human BAFF was expressed as a HA-tagged molecule from Drosophila SL-3 cells as previously described 5′-ATT AGA TCT GAA ATG GGC GCC AGG AGA CTC C-3′) and mBAFF-R5r (5′-GAT GAA TTC CTA TTG CTC TGG GCC AGC TG-3′). The PCR fragment was digested with BglII and EcoRI and cloned into the BglII and EcoRI opened pMIG plasmid (Addgene plasmid 9044), allowing the bi-cistronic expression of BAFF-R together with green fluorescent protein (GFP). The retroviral vectors were transfected into the Phoenix retroviral packaging cell line (ATCC® Number: SD 3443); according to the manufacturer's instructions. The rat myeloma cell line Y3 (ATCC® Number: CRL-1631) and the mouse pre B cell line 40E1  were retrovirally transduced by spin-infection using standard procedures. Transduced cells were sorted by FACS ARIA (BD Biosciences) on the basis of high GFP expression.Full-length mouse BAFF-R cDNA (GenBank accession number: NM_028075) was amplified from mouse spleen cDNA using primers mBAFF-R4 (7 Y3-mBAFF-R cells to generate anti-mBAFF-R mAbs as described earlier Lewis rats were immunized subcutaneously with 10in vivo depleting Mice were injected intravenously (i.v.) with 0.5 mg mAb in PBS. Lymphocyte subpopulations in the blood, spleen, lymph nodes, bone marrow and peritoneal cavity were analyzed by FACS at various time points after injection. Mice treated with the Mice were immunized i.p. with 100 µg NIP-OVA  in alum (T dependent immune response) or i.v. with 50 µg NIP-FICOLL  in PBS (T independent immune response). At day 14 after immunization, the mice were bled and the serum IgG anti-NIP titer was determined by ELISA as previously described FITC (M41) and anti-IgDBiotin (1.19) or anti-Thy1FITC (T24) and anti-IgMBiotin (M41). Biotin-labeled antibodies were visualized using streptavidin Texas red (BD Biosciences). Stained sections were analyzed using an Axioskop Immunofluorescence  equipped with a Nikon digital camera.Spleens were snap frozen and embedded into OCT compound . Cryostat sections of 5 µm were prepared and fixed for 10 minutes in acetone. Sections were stained with anti-IgMDifferences between groups were evaluated for statistical significance using the two-tailed paired student's t test, assuming equal variances.


EFNB1 cause the X-linked malformation syndrome craniofrontonasal syndrome (CFNS). CFNS is characterized by an unusual phenotypic pattern of inheritance, because it affects heterozygous females more severely than hemizygous males. This sex-dependent inheritance has been explained by random X-inactivation in heterozygous females and the consequences of cellular interference of wild type and mutant EFNB1-expressing cell populations. EFNB1 encodes the transmembrane protein ephrin-B1, that forms bi-directional signalling complexes with Eph receptor tyrosine kinases expressed on complementary cells. Here, we studied the effects of patient-derived EFNB1 mutations predicted to give rise to truncated ephrin-B1 protein or to disturb Eph/ephrin-B1 reverse ephrin-B1 signalling. Five mutations are investigated in this work: nonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT, splice-site mutation c.406 + 2T > C and two missense mutations p.P54L and p.T111I. Both missense mutations are located in the extracellular ephrin domain involved in Eph-ephrin-B1 recognition and higher order complex formation.Mutations of Nonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT and splice-site mutation c.406+2T > C were detected in the primary patient fibroblasts by direct sequencing of the DNA and were further analysed by RT-PCR and Western blot analyses.in vitro site-directed mutagenesis. Investigation of missense mutations was performed using the Western blot analysis and time-lapse microscopy.The impact of missense mutations p.P54L and p.T111I on cell behaviour and reverse ephrin-B1 cell signalling was analysed in a cell culture model using NIH 3T3 fibroblasts. These cells were transfected with the constructs generated by Nonsense mutation c.196C > T/p.R66X and frameshift mutation c.614_615delCT escape nonsense-mediated RNA decay (NMD), splice-site mutation c.406+2T > C results in either retention of intron 2 or activation of a cryptic splice site in exon 2. However, c.614_615delCT and c.406+2T > C mutations were found to be not compatible with production of a soluble ephrin-B1 protein. Protein expression of the p.R66X mutation was predicted unlikely but has not been investigated.Ectopic expression of p.P54L ephrin-B1 resists Eph-receptor mediated cell cluster formation in tissue culture and intracellular ephrin-B1 Tyr324 and Tyr329 phosphorylation. Cells expressing p.T111I protein show similar responses as wild type expressing cells, however, phosphorylation of Tyr324 and Tyr329 is reduced.Pathogenic mechanisms in CFNS manifestation include impaired ephrin-B1 signalling combined with cellular interference. EFNB1  are frameshift, nonsense, and splice site mutations that lead to premature termination codons (PTCs). Missense mutations constitute about 42% of all EFNB1 mutations [utations . Most ofutations ,2,25.PCDH19 gene encoding the cell-cell adhesion molecule protocadherin 19. Like in CFNS, somatic mosaicism may cause cellular interference leading to malformations in the brain and development of epilepsy [PCDH19 mutation, who was identified in a cohort of patients with Dravet syndrome-like epileptic encephalopathy [The concept of cellular interference appears to be not unique to CFNS. Dibbens et al.  describeepilepsy . This paalopathy .EFNB1 on ephrin-B1 reverse signalling in vitro and in a cell culture model.Here we analysed the impact of patient-derived Genetic testing of the patients was performed after written informed consent from the patients' parents and complies with the tenets of the declaration of Helsinki. Biopsies of CFNS patients were obtained from skin (c.196C > T/p.R66X and c.406+2T > C) or following surgical therapeutic interventions (c.614_615delCT). Patient fibroblast cultures were established and maintained according to standard cell culture conditions and harvested for genomic DNA and total RNA or protein isolation.®) with Dulbecco's modified Eagle's medium  containing 15% fetal calf serum  in a 5% CO2 atmosphere at 37°C.NIH 3T3 cells were cultivated in tissue culture flasks .Genomic DNA and RNA from cultured cells were isolated using standard protocols. Mutations were detected by direct sequencing with the DYEnamic ET terminator cycle sequencing kit , and run on a MegaBace sequence analyser . Sequences were processed by DNASIS software . The sequencing data were compared with 08887.71 . Mutatio08887.71 . The PCREFNB1. RT-PCR products from cells carrying nonsense mutation c.196C > T/p.R66X and frameshift mutation c.614_615delCT were cleaved with AvaI and HinfI, respectively. RT-PCR products obtained from the c.406+2T > C EFNB1 allele were cloned into pGEM-Teasy vector (Promega) and sequenced as described [For expression analysis, total RNA was reverse transcribed using SuperScript™ One-Step™ RT-PCR System (Life Technologies) as recommended by the supplier. Primer pair 5'-CAAGTTCCTGAGTGGGAAGG-3' and 5'-GTGTGGCCATCTTGACAGTG-3' was used to amplify a 455 bp product from exons 2-4, and primer pair 5'-ATCATGAAGGTTGGGCAAGA-3' and 5'-TGGGGGCAGTAGTTGTTCTC-3' was used to amplify a 467 bp product from exons 4 and 5 of escribed .EFNB1 RNA was prepared from placenta and reverse transcribed as described above. Amplification of EFNB1 cDNA was performed using primer pair 5'-GGCAGAGGAAGGCGAGGCGA-3' and 5'-GCAAGGGGAGGGGGTGTG-3' that generates an 1.2 Kb product. This RT-PCR product was cloned into the pCR 2.1 vector (Invitrogen). Mutant EFNB1 cDNA containing c.161C > T/p.P54L and c.332C > T/p.T111I mutations were generated using QuikChange® II Site-Directed Mutagenesis Kit (Stratagene) according to the kit's protocol and EFNB1-specific primers: 5'-GGGCTTGGTGATCTATCTGAAAATTGGAGACAAGC-3' and 5'-GCTTGTCTCCAATTTTCAGATAGATCACCAAGCCC-3' for c.161C > T, 5'-CAGAGCAGGAAATACGCTTTATAATCAAGTTCCAGGAGTTCA-3' and 5'-CTGAACTCCTGGAACTTGATTATAAAGCGTATTTCCTGCTCTG-3' for c.332C > T with nucleotide exchanges underlined. Primers were designed using the web-based primer software program (Stratagene) [EFNB1 cDNA inserts were recloned in pcDNA 3.1(+) vector (BD Biosciences) using EcoRI restriction (MBI Fermentas).The presence of mutations was confirmed by sequencing with the AutoRead™ Sequencing Kit (Amersham Biosciences) according to the kit's protocol. Wild type and mutant 5 cells/well, Greiner Labortechnik). Cells were cultivated until they reached 70% confluence . Plated cells were co-transfected with pcDNA 3.1(+) vector containing the mutant or the wild type EFNB1 cDNA (4 μg/well) and the pEGFP-N3 vector . Transfections were done using PerFectin™ Transfection reagent (PeqLab) according to the supplier's recommendations. Transfection efficiency was measured using fluorescent cytometry cell sorting (FACS) 24 h post transfection as described below.NIH 3T3 cells were placed on a 6-well plate (1 × 104 events were counted. Untransfected NIH 3T3 cells served as controls.Transfected cells were washed in PBS (Sigma) and treated with trypsine (Sigma) 24 h post-transfection. After the treatment, PBS was added and part of the cells was taken for the FACS analysis using the ectopic GFP-fluorescence and BD FACSCanto™ Flow Cytometer (BD Bioscience), 3 × 10Stimulation of NIH3T3 cells with EphB2-receptor was performed according to a modified protocol of Davy et al. . Briefly®) and anti-ephrin-B1 antibody (A-20). According to the manufacturer (Santa Cruz), rabbit polyclonal antibody A-20 was raised against a 20 amino acid peptide encoded by exon 3 that maps in the extracellular ephrin-B1 domain.Patient fibroblasts were lysed with RIPA buffer  and usedAll the experiments were performed at least twice.EFNB1 nonsense mutation c.196C > T/p.R66X that is located in exon 2, frameshift mutation c.614_615delCT in exon 4 and splice-site mutation c.406+2T > C at the junction of exons 2 and 3 was analysed, respectively. Two missense mutations located in exon 2 of EFNB1 were functionally studied . Escape from NMD, however, has been observed for the c.196C > T/p.R66X mutation that is located in exon 2. In patient fibroblast cultures, a mutant EFNB1 transcript was detected by RT-PCR in addition to the wild type transcript . This could lead to the synthesis of a truncated ephrin-B1 that lacks the signal peptide and almost the entire extracellular domain. However, such a protein will not enter the endoplasmatic reticulum and consequently will not appear on the cell surface.Another possible explanation for the NMD escape was raised by Zhang and Maquat . This grEFNB1 and generates a PTC but the transcripts escape NMD. Expression analysis of patient fibroblasts revealed transcripts from mutant and wild type alleles at similar amounts, whereas only the wild type but no truncated ephrin-B1 was detected by Western blot analysis. This rather suggests that truncated ephrin-B1 proteins are unstable and do not contribute substantially to the CFNS phenotype. In another female patient with classical CFNS phenotype, splice site mutation c.406+2T > C was detected. In the patient examined here c.406+2T > C occurred de novo. Expression analysis of the patient's fibroblasts revealed retention of intron 2 as has been previously detected for the splice acceptor "AG" mutation c.407-2A > T at the same exon junction [EFNB1, in fact, only a single EFNB1 transcript has been reported [Escape from NMD also could give rise to truncated, soluble ephrin-B1 polypeptides that lack the transmembrane and intracellular domain. Such polypeptides could exhibit dominant-negative or gain-of function effects. Frameshift mutation c.614_615delCT occurred in exon 4 of junction . In addijunction ,42. Crypreported . Both, iEFNB1 mutation in patient fibroblasts. Mutation c.196C > T/p.R66X generates a PTC just 36 amino acids following the signal peptide. Presumably, this will not allow production of a functional polypeptide, however, we were not able to further analyse it because the polyclonal anti-ephrin-B1 antibody we used does not recognize the N-terminal part of ephrin-B1.Like c.614_615delCT no truncated soluble ephrin-B1 polypeptide was generated from c.406+2T > C The impact of two missense mutations on ephrin-B1 signalling and cell behaviour was studied in a cell culture model using NIH 3T3 fibroblasts. Reverse signalling influences actin cytoskeletal rearrangement and may result in transcriptional regulation of different genes involved in extracellular matrix reorganization ,43. We pAltogether, eight different mutations were functionally analysed at the mRNA and protein level, respectively Table . Most ofNIH3T3 cells expressing either wild type or mutant ephrin-B1 exhibited differences in cluster formation after EphB2-Fc stimulation. Cells expressing wild type or mutant ephrin-B1 were visualized by the green fluorescent protein and showed a scattered distribution in tissue culture dishes before stimulation. EphB2-Fc stimulation induced formation of clusters in the wild type and p.T111I protein expressing cells, whereas no cell clusters were found in p.P54L expressing cells. This suggests that phosphorylation of Tyr324/329 is closely linked with the cluster formation, however, the mechanism for this is unclear. Possible reasons are that wild type and p.T111I expressing cells experience a proliferative signal upon EphB2 stimulation. After division daughter cells do not move apart but rather stay close together. Alternatively, cells respond to EphB2 stimulation with increased motility and migrate into clusters. Embryonic mouse cells expressing an ephrin-B1 lacking the most C-terminal PDZ binding domain do not sort-out from wild type cells, whereas ephrin-B1 null cells do ,45,9. InEFNB1 mutations have neither dominant-negative, nor gain-of-function effects but rather loss-of-function effect.In this work three PTC-causing mutations were analysed: nonsense c.196C > T/p.R66X, frameshift c.614_615delCT and splice-site c.406+2T > C mutation. These mutations give rise to the mutant RNA, but no mutant protein was detected. According to these results and previously published data, it can be concluded, that the majority of PTC-causing The analysis of missense mutations p.P54L and p.T111I revealed that both cause CFNS but have different mechanisms of ephrin-B1 disturbance of signalling. Mutation p.P54L seems to have loss-of-function effect since no Tyr324/329 phosphorylation of the p.P54L ephrin-B1 and no cluster formation of the p.P54L expressing cells were shown, whereas p.T111I ephrin-B1 differs slightly from the wild type in phosphorylation timing. Therefore, additional mechanisms involved in phenotypic manifestation need to be postulated. This may include other tyrosine residues of ephrin-B1 are more important for reverse signalling. Another possibility could be impaired forward signalling of Eph receptor expressing cells. Combined with cellular interference this may be the main pathogenic mechanism in CFNS manifestation in female patients.The authors declare that they have no competing interests.RM and IW designed and performed the experiments; patients material and information was provided by BS, ZG, VT and PW. The manuscript was written by RM and IW and read and approved by all authors.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/98/prepub


Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.tarbp2-/- cells, which do not express TRBP, do not support RNA interference (RNAi) mediated by short hairpin or micro RNAs against EGFP. Both TRBPs, but not TRBPsΔC4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPsΔC4, rescued RNAi function.We show that the TRBP binding site in Dicer is a 165 amino acid (aa) region located between the ATPase and the helicase domains. The binding site in TRBP is a 69 aa domain, called C4, located at the C-terminal end of TRBP. The TRBP1 and TRBP2 isoforms, but not TRBPs lacking the C4 site (TRBPsΔC4), co-immunoprecipitated with Dicer. The C4 domain is therefore necessary to bind Dicer, irrespective of the presence of RNA. Immunofluorescence shows that while full-length TRBPs colocalize with Dicer, TRBPsΔC4 do not. The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPsΔC4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPsΔC4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function. Drosophila melanogaster. This is an enzymatic process that involves RNAse III-like proteins (Dicer and Drosha) and a dsRNA binding protein  [RNA interference (RNAi) is a natural mechanism used by eukaryotes for gene silencing. This mechanism uses small double-stranded (ds)RNA, named micro (mi) or small interfering (si) RNAs, which are complementary to a target gene to degrade the corresponding mRNA or block its translation. The dsRNA triggers the assembly of a ribonucleoprotein complex called the RNA-induced silencing complex (RISC) . The mecuacious) . The secuacious) ,6.-/-) mice and cells are not viable indicating a major function for this protein during development and normal cell function [in vitro reconstitution of RNAi activity [Mammalian cells have a single Dicer protein, with a molecular weight of ~200 kDa. Dicer contains an ATPase/RNA helicase domain, a DUF domain, a PAZ domain, two RNase III domains and a dsRNA binding domain (dsRBD) . The Dicfunction . In humafunction -12, wherfunction ,14. Furtactivity .tarbp2 gene. Two adjacent promoters initiate transcription of alternative first exons for TRBP1 and TRBP2 mRNAs and as a consequence, in comparison to TRBP1, TRBP2 has 21 additional amino acids (aa) [TRBP1 and TRBP2 are isoforms of the cellular protein TRBP which was isolated by its ability to bind the human immunodeficiency (HIV)-1 TAR RNA and characterized for its stimulation of the expression of the HIV long terminal repeat in human and murine cells -20. TRBPids (aa) ,29. TRBPids (aa) ,30-32. AThe TRBP-Dicer interaction was found by immunoprecipitation (IP) with a Dicer antibody followed by mass spectrometry analysis of the interacting proteins ,11. We iThe Medipal domain was previously defined as a protein-protein interacting domain in TRBP (aa 228–366 or TRBP-C) that binds Merlin, Dicer and PACT . TRBP inThe cellular distribution of Dicer and TRBP has not yet been fully elucidated. We next examined their localization in the cell using immunofluorescence with antibodies directed against endogenous TRBP and Dicer. Interestingly, TRBP localized mainly to the cytoplasm, while Dicer was distributed throughout the cytoplasm and the nucleus. We found significant colocalization between Dicer and TRBP in HeLa cells predominantly in the perinuclear space Fig. . These dtarbp2 knock-out cells [To determine the role of the C4 domain in the protein context, we used a complete TRBP knock-down environment. Using murine tail embryonic fibroblasts (TEFs) ut cells ,32, we eut cells , and Mycut cells . The losTo set up conditions to compare the cellular distribution of wild-type and truncated TRBPs, we first expressed TRBP1 and TRBP2 Myc-tagged proteins in HeLa cells. The cellular distribution of the Myc-tagged proteins, as well as their colocalization with Dicer was consistent with the endogenous localization of TRBP Fig. . ImportaTo further define the importance of the C4 subdomain within TRBP Medipal, we expressed Myc-TRBP1ΔC4 and Myc-TRBP2ΔC4 in the same conditions as the wild type proteins Fig. . With thTo further verify the distributions of full-length TRBP2 as compared to TRBP2ΔC4, their localization was examined in relation to an EGFP-tagged TRBP2 construct. EGFP-TRBP2 and Myc-TRBP2 or Myc-TRBP2ΔC4 were coexpressed as previously in HeLa cells. The colocalization of the EGFP-tagged form of TRBP2 with the Myc-tagged form of TRBP2 is shown by an extensive yellow coloration in the cytoplasm Fig. . In comptarbp2-/- cells previously described [tarbp2+/- and wild type murine embryo fibroblasts (MEF). To have a qualitative and a quantitative assay, we designed shRNAs against EGFP, and exogenous miRNAs that have the siRNA sequence against EGFP in an miR30 backbone, as previously shown for another target [tarbp2+/- cells as shown by EGFP fluorescence, no significant activity could be detected in tarbp2-/- cells . Using an shRNA and an miRNA targeting EGFP, these cells displayed low or no RNAi activity as compared to murine cells expressing TRBP (tarbp2+/- and MEF)  Fig. , confirmEF) Fig. . In thisin vitro and in vivo with no requirement for ssRNA, dsRNA or another protein [The interaction between Dicer and TRBP has been shown to be direct  protein . Althoug protein . Two-hyb protein ,36, sugg protein ,37. Heretarbp2-/- cells  was generated from pBluescript-Dicer-His6  by PCR uEcoRI and BglII was inserted in frame into pCMV-Myc (BD biosciences). The BstEII-KpnI fragment from pBS-TRBP2 [XmaI and KpnI and inserted in frame into pCMV-Myc-TRBP1 or pCMV-Myc-TRBP2 cut with the same enzymes. The resulting proteins have 69 aa deleted in the terminal end of either TRBP1 or TRBP2. All inserts were verified by sequencing.EGFP-C1-TRBP2  and Myc-BS-TRBP2  was inseshRNA and miRNA expression cassettes against EGFP were constructed using a two step PCR method described previously . In the Non-silencing shRNA:5'-GTGGCTTCACAGACGTGACACGTTCGGAGAATCGCAGTATATGTGCTGCCGAAGCG-3' EGFP shRNA:Taq polymerase (Invitrogen). The second PCR was performed with the matching primer as follows: Non-silencing miRNA: 5'-CTAGTCTAGAAAAAAAGCAGTTCTCCGAACGTGTCACGTCCCATCTGTGGCTTCACAGACGTG-3' EGFP miRNA:5'-GTGGCTTCACAGCTCCTTGAAGTCGATGCCCTCGCAGTCTCTGTGCTGCCGAAGCG-3' With the following thermal profile: 95°C for 5 min, 30 cycles , 72°C for 7 min, using ®II-TOPO® (Invitrogen), and the resulting pCRII-U6+27 vectors confirmed by sequencing.5'-CTAGTCTAGAAAAAAAGCAGGGGCATCGACTTCAAGGAGCCCATCTGTGGCTTCACAGCTCCT-3' shRNA and miRNA expression vectors were generated by ligation of the final PCR product into pCRYeast expression plasmids were introduced into the yeast reporter strain SFY526. The double transformants were selected and screened for β-galactosidase activity as described .tarbp2+/- and tarbp2-/-, described in [Human HeLa and astrocytic cells U251MG, murine embryo fibroblasts (MEF) and primary tail embryonic fibroblasts ribed in , were maCells were transfected in 6-well plates with 4 μg sh- or mi-RNA constructs using TransIT Reagent (Mirus) at a 1:3 DNA:TransIT ratio prior to transfection with 1 μg pEGFP-C1 (Clontech). Cells were observed by fluorescence using an Axiovert 25 inverted microscope (Zeiss). EGFP expression was assed by calculating mean luminosity of individual fields, using Adobe Photoshop CS3 10.0.1 software. Results are expressed as a percent knock-down, as compared to non-silencing conditions. Cells were analyzed for EGFP expression by flow cytometry using a FACS Calibur cytometer (BD) and analyzed using Cellquest software (BD). Transfected cells were gated by green fluorescence greater than cells in a mock transfection. Relative fluorescence values were calculated as the product of the percentages of EGFP fluorescent cells gated and the mean fluorescence of cells gated positive.Cell lysates were prepared, separated and transferred for immunoblotting as previously described . The memtarbp2-/- cells were washed twice with PBS and lysed in cold lysis buffer [48 hrs post-transfection s buffer  with pros buffer . 1 mg ofHeLa cells were cultured on coverslips in 12-well dishes. Untransfected cells were incubated with mouse anti-Dicer mAb42  and rabbaa: amino acid; dsRNA: double-stranded RNA; dsRBP: dsRNA binding protein; dsRBD: dsRNA binding domain; EGFP: enhanced green fluorescent protein; FACS: fluorescence automated cell sorting; IF: immunofluorescence; IP: immunoprecipitation; Medipal: Merlin Dicer PACT liaison; MEF: murine embryonic fibroblast; miRNA: micro RNA; RISC: RNA-induced silencing complex; RNAi: RNA interference; shRNA: short hairpin RNA; siRNA: small interfering RNA; TEF: tail embryonic fibroblast; TRBP: TAR RNA binding proteinSD performed the coIPs Fig. , the flu


In silico models show Vpu to form pentamers with an ion channel activity.The human immunodeficiency virus type 1(HIV-1) is a complex retrovirus and the causative agent of acquired immunodeficiency syndrome (AIDS). The HIV-1 Vpu protein is an oligomeric integral membrane protein essential for particle release, viral load and CD4 degradation. in vitro. This was also true for the TM domain of Vpu. Confocal microscopy localized Vpu to the endoplasmic reticulum and Golgi regions of the cell, as well as to post-Golgi vesicles. In fluorescence resonance energy transfer (FRET) experiments in live cells we show that Vpu oligomerizes in what appears to be either the Golgi region or intracellular vesicles, but not in the ER.Using Vpu proteins from a primary subtype C and the pNL4-3 subtype B isolates of HIV-1, we show oligomerization of the full-length protein as well as its transmembrane (TM) domain by genetic, biochemical and biophysical methods. We also provide direct evidence of the presence of Vpu pentamers in a stable equilibrium with its monomers We provide here direct evidence that the TM domain, is critical for Vpu oligomerization and the most favourable channel assembly is a pentamer. The Vpu oligomerization appears to be either the Golgi region or intracellular vesicles, but not in the ER. Primate lentiviruses, including HIV, encode a number of accessory proteins that perform essential functions during the viral life cycle . One sucThe Vpu protein of HIV-1 is an 81-amino acid type I integral membrane protein in which a short extracellular N-terminus is followed by a transmembrane (TM) domain and a cytoplasmic domain, the latter with two prominent alpha helices ,5. The rLike all retroviruses, HIV-1 also interferes with the expression of its cellular receptor and uses redundant mechanisms to achieve this . The VpuIn silico modeling studies have predicted the same for the TM domain of Vpu [An earlier study has used chemical cross-linking to show that Vpu can form oligomers . In silin of Vpu . The olivpu genes, one from the HIV-1 Subtype B lab-adapted isolate NL4-3 and the other from a HIV-1 Subtype C primary isolate from India . The translated amino acid sequences . To ensure that the cloned vpu gene expressed a functional protein, a transfection-based HIV replication assay was set up. HeLa cells were transfected with wild type or vpu-deficient proviral DNA, the latter in the absence or presence of an R5-vpu expression vector. The virions released in the culture medium and those present within the cells were quantitated by western blotting with anti-Gag antibodies. As shown in Fig. vpu-deficient proviral DNA produced as much virions as wild type proviral DNA (lanes 4 and 5), the release of virions into the culture medium was compromised in the absence of vpu (lanes 1 and 2). However, this phenotype was rescued following cotransfection of the vpu-deficient proviral DNA with the R5-vpu expression vector (lane 3). The effects were clearly visible at the level pf p55/p41 precursors as well as the p24 capsid protein. Thus, the R5 Vpu protein was functional in promoting virus release from cells.We used PCR to clone two ces Fig.  showed tces Fig. . Phylogeces Fig. . Earliervpu genes were subcloned into the yeast two-hybrid expression vectors as fusions to the GAL4 DNA-binding domain (BD) and activation domain (AD). The expression of the full-length or truncated Vpu fusion proteins from these constructs was verified by T7 RNA polymerase mediated coupled in vitro transcription-translation (data not shown). The yeast two-hybrid assays were performed in S. cerevisiae AH109 cells as described in Materials and Methods. A representative set of plates is shown in Fig. - plates (panel 3); similarly, those containing BD-Vpu grew on SD/T- plates (panel 4) and cotransformants grew on SD/LT- plates (panel 5). Cotransformants that contain interacting protein pairs fused to AD and BD can transactivate the HIS3 gene resulting in growth on SD/LTH- plates. The growth of AD-Vpu/BD-Vpu cotransformants on a SD/LTH- plate (panel 6) showed homodimerization of the Vpu protein. Colonies were transferred to a nitrocellulose filter and a β-galactosidase filter assay was carried out. The presence of β-galactosidase activity only in the positive control and AD-Vpu/BD-Vpu cotransformants (panel 7) further confirmed the Vpu-Vpu interaction.To test for the interaction between Vpu monomers, the full-length and truncated - plates also grew on SD/LTH- 3AT plates. Compared to the positive control (BD/SNF1+AD/SNF4), the semi-quantitative liquid β-galactosidase assay showed reasonably strong interactions between the full-length Vpu proteins and slightly weaker interactions between their TM domains. The values were higher for homologous interactions (NL vs NL and R5 vs R5) as opposed to heterologous interactions (NL vs R5). A variety of negative controls showed no interaction and only background β-galactosidase activity.A more extensive screen, whose results are summarized in Fig. in vitro-coupled transcription-translation assay , in the presence of 35S-methionine. When these proteins were analyzed on native polyacrylamide gels . The 35S-labeled in vitro synthesized Vpu proteins were then passed through these beads. Both full-length Vpu and its TM domain were retained on amylose beads saturated with MBP-Vpu, but not with the MBP control  as well as its TM and cytoplasmic domains by polyacrylamide gel electrophoresis. The Vpu proteins were synthesized from the pGBK-Vpu and pGAD-Vpu plasmid templates using an els Fig. , oligomerol Fig. . The cyt35S-labeled full-length Vpu protein or its TM domain was carried out to characterize their oligomeric states. The proteins synthesized by in vitro coupled transcription-translation reactions were separated on a pre-calibrated Sephacryl S200HR column. Two prominent peaks of radioactivity were eluted for full-length as well as the TM domain proteins . This non-radiative energy transfer between donor and acceptor fluorophores is critically dependent upon the distance and dipole orientations of the two partners, and is taken as evidence of an interaction between them [To detect intimate protein-protein interactions een them . We cotreen them . To measin vivo evidence of Vpu-Vpu interaction in a live cell. Further, based on these results, Vpu appears to form oligomers in distinct subcellular locations, primarily in the Golgi and vesicular regions but not in the ER.We carried out FRET measurements in live cells showing various patterns of Vpu subcellular distribution. Two different areas within the same cell, one showing colocalization and another where no colocalization was observed were subjected to FRET analysis. As expected (and seen in the merged image), the Vpu-ECFP and Vpu-EYFP proteins colocalized in transfected cells Fig. , set A. Lentiviruses encode a number of unique accessory proteins that are important for viral replication and pathogenesis, but are not encoded by other retroviruses. The versatility of HIV accessory proteins arise from their ability to function as adaptor molecules that connect various viral and cellular proteins to pre-existing cellular pathways, modulate these pathways and control processes important for viral replication. Thus, protein-protein interactions are an important aspect of the functioning of these proteins. The Vpu protein of HIV-1 is known to bind CD4 and βTRCP, the latter being a component of the E3 ubiquitin ligase complex. This association is instrumental in dislocation of CD4 from the ER, its ubiquitination and subsequent degradation by the proteosome . The othin vitro and in vivo. Molecular dynamic simulations and conductance studies have shown that the Vpu TM domain is sufficient for its ion channel activity [in vitro synthesized Vpu proteins and their TM domains. Further, molecular sizing of in vitro synthesized Vpu by gel permeation chromatography clearly showed it to assemble into a pentameric species. Thus, while complementing earlier studies [In vitro synthesized Vpu proteins, in the absence of other cellular components, demonstrated a stable equilibrium between monomers and pentamers. This demonstrates the inherent ability of Vpu to oligomerize and pentamers appear to be the thermodynamically most stable form of these oligomers.The nature of the Vpu oligomer is important due to its functional significance in virion release. An earlier study used chemical cross-linking to demonstrate the oligomerization of Vpu . Here weactivity  and the activity . This suactivity ,28. Receactivity  have use studies ,28,29 we2+ release from its intracellular stores in the ER [Earlier studies have shown Vpu to be localized to the perinuclear region of the cell that includes the ER and Golgi ,20. Usinn the ER . The FREOligomerization of Vpu was observed by FRET in structures that were distal to the ER. While these structures could not be identified positively in the same cell due to the technical limitations, separate stainings in two different cell types (COS-1 and U2-OS) suggested that these structures might either be Golgi or vesicles associated with exocytic protein transport. The Vpu ion channel activity would be detrimental to ER function, inducing ER toxicity, stress and apoptosis of the infected cell. This would go against the plan of survival of productively infected cells followed by retroviruses and so nicely exemplified by HIV . It woulWe have used genetic, biochemical and biophysical methods to complement earlier studies on Vpu oligomerization and the role of its N-terminal transmembrane domain in this oligomerization. While theoretical modeling studies ,28 and svpu gene was PCR amplified with Pfu polymerase  using as template either the pNL4-3 plasmid DNA (NIH AIDS Research and Reference Reagent Program) or a 3.5 kb fragment encompassing the vpr to env region previously amplified and cloned from a primary isolate of HIV-1 subtype C . The PCR primers used were as follows : For the NL4-3 vpu gene, Vpu-NL-F, GGATCCATGCAACCTATAATAGTA GCAATA and Vpu-NL-R, GAATTCACTACAGATCATCAATATCCCAAG; for the R5 vpu gene, Vpu-R5-F, GGATCCATGTTAAATTTAGATTATAAATTAGGAGTA GG and Vpu-R5-R, GAATTCATTACAAATCATTAACATCCAAAAGCC. The amplified fragments designated as NL vpu and R5 vpu respectively, were cloned in plasmid pGEMT-Easy  and sequenced in both directions. The gene fragments corresponding to the transmembrane (TM) domains were assembled from the following synthetic oligonucleotides: For the NL4-3 vpu TM region, NL-TM-F, CATGGAGATGCAACCTATAATAGTAGCAATAGTAGCATT AGTAGTAGCAATAATAATAGCAATAGCTGTGTGGTCCATAGTAATCATAGAATAGG and NL-TM-R, AATTCCTATTCTATGATTACTATGGACCACACAGCTATTGCTATTATTATTGCTACTACTAATGCTACTATTGCTACTATTATAGGTTGCATCTC; for the R5 vpu TM region, R5-TM-F, CATGGAGATGTTAAATTTAGATTATAAATTAGGAGTAGGAGCATTGATAGTAGCACTAATCATAGCAATAGTCGTGTGGACCATAGTATATATAGAATAGG and R5-TM-R, AATT CCTATTCTATATATACTATGGTCCACACGACTATTGCTATGATTAGTGCTA CTATCAATGCTCCTACTCCTAATTTATAATCTAAATTTAACATCTC. The cytoplasmic domains were PCR amplified using specific primers for NL vpu and R5 vpu as follows: for the NL vpu cytoplasmic region, NL-Cyto-F, CCATGGAGTATAGGAAAATATTAAGA and Vpu-NL-R (shown above); for the R5 vpu cytoplasmic region, R5-Cyto-F, CCATGGAGTATAGGAAATTGGTACAAC and Vpu-R5-R (shown above).The 6 HeLa cells were plated per 60 mm dish. These were cotransfected with 2 μg of either wild type or vpu-deficient HIV-1 proviral DNA and 0.5 μg of the expression vector pEGFP/R5-Vpu using Lipofectin (Clontech). As a control, the empty vector pEGFP-N1 was used. Twenty-four hr post-transfection, the released virions in the culture medium were pelleted through a 20% sucrose cushion for 2 hr at 100,000 × g in a Beckman SW41 rotor. The pelleted virions and harvested cells were lysed in Laemmli sample buffer. Proteins were separated by electrophoresis on SDS-10% polyacrylamide gels and western blotted with an anti-p24 antibody.Eighteen to 24 hr prior to transfection, 0.3 × 10vpu genes were cloned into the pGBKT7 and pGADT7 vectors as EcoRI-BamHI fragments from the pGEMT-Easy clones. The nucleotide sequences corresponding to the TM and cytoplasmic domains of the Vpu proteins were similarly cloned in the two-hybrid vectors as NcoI-BamHI fragments, respectively. Expression of the relevant fusion proteins from each of the Vpu two-hybrid constructs was checked in a T7 polymerase based in vitro coupled transcription-translation system  followed by immunoprecipitation with anti-Vpu antibodies. The yeast two-hybrid analysis was essentially carried out as described earlier [Saccharomyces cerevisiae strain AH109  containing the HIS3 and lacZ reporter genes under the control of GAL4-binding sites. The host strain containing plasmids pAS2-SNF1 and pACT2-SNF4 was used as a positive control [The GAL4-based two-hybrid system contained the DNA binding domain vector pGBKT7 and the activation domain vector pGADT7. The NL and R5  earlier ,36. Plas control . Various control ,36. A se control .in vitro expression of full-length Vpu or its TM or cytoplasmic domains was carried out using a coupled transcription-translation system  as recommended by the supplier. The proteins were labeled with 35S-methionine in the same reaction and their authenticity was checked by immunoprecipitation with anti-Vpu antibodies. Five μl of the in vitro expression mix was analyzed by electrophoresis on 12% native polyacrylamide gels.The vpu gene was cloned as a BamHI-EcoRI fragment into the pMal-c2 vector  and the maltose-binding protein-Vpu fusion (MBP-Vpu) protein was expressed in BL21(DE3) cells. Following induction of a freshly diluted overnight culture with 1 mM IPTG for 4 hr at 37°C, the cells were harvested and resuspended in PBS containing 0.1% Triton-X100, 10 μg/ml lysozyme and 1 mM PMSF, and subjected to 5 cycles each of freeze-thaw and sonication. The sample was centrifuged at 12,000 rpm at 4°C in a microfuge . To the clarified lysate, amylose resin  was added and the MBP-Vpu protein was allowed to bind for 2 hr at 4°C. The resin was washed with 10 volumes of wash buffer containing 20 mM Tris-HCl, pH 7.4, 0.2 M NaCl, 10 mM β-mercaptoethanol, 1 mM EDTA to remove non-specifically bound proteins. To 50 μl of amylose beads saturated with either MBP or MBP-Vpu, 25 μl of 35S-labeled Vpu (prepared using a coupled in vitro transcription-translation system) diluted in 500 μl PBS was added and allowed to bind for 3 hr at 4°C with end-over mixing. The beads were then centrifuged down and washed five times with 500 μl each of PBS containing 0.1% Triton X-100, resuspended in SDS dye loading buffer and subjected to SDS-PAGE and fluorography.The in vitro expression and 35S labeling, 50 μl of the reaction mixture was loaded on a 36 ml column pre-equilibrated in PBS. The column was run at a flow rate of 0.4 ml/min and 0.25 ml fractions were collected. The elution of Vpu was estimated by liquid scintillation counting and SDS-PAGE analysis of the peak radiolabeled fractions. For molecular size estimations, the column was calibrated with lysozyme and BSA under the same run conditions.The oligomeric forms of Vpu were also analyzed by gel permeation chromatography on Sephacryl S200HR (Pharmacia-Amersham). Following vpu genes were cloned as EcoRI-BamHI fragments in the Living Colors™ vectors pEGFP-N3, pEYFP-N1 and pECFP-N1 (Clontech). Prior to this, the genes were first PCR amplified, cloned in the pGEMT-Easy vector and sequenced. The following PCR primers  were used: for NL vpu, NL-X-F, GAATTCATGCAACCTATAATAGTAGCAATA and NL-R-GFP, GGATCCGCG CAGATCATCAATATCC; for R5 vpu, R5-X-F, GAATTCATGTTAAATTTAG ATTATAAATTAGGAGTAGG and R5-R-GFP, GGATCCTGCCAAATCATT AACATCCAAAA. For colocalization experiments, COS-1 and U2-OS cells were seeded at about 50% confluency on coverslips in 12-well plates, grown for 18 hr, and then cotransfected with Living Colors™ vectors expressing Vpu and any of the subcellular markers. At 24 to 48 hr post-transfection, the PBS-washed cells were fixed with 2% paraformaldehyde in PBS at room temperature for 10 min. These were then mounted using Antifade  and sealed. Confocal images were collected sequentially using a 60 planapo NA 1.4 objective on a Radiance 2100 laser scanning system  equipped with a Nikon Eclipse TE2000-U microscope. For FRET analysis, COS-1 and U2-OS cells were similarly transfected with ECFP-vpu and EYFP-vpu expression plasmids. The ECFP-Vpu (FRET donor) and EYFP-Vpu (FRET acceptor) images were acquired sequentially in live cells using the Blue diode 405 nm and the Argon ion 514 nm laser lines, respectively. Images of the ECFP emission were collected using a 500 DCLPXR dichroic mirror with an HQ 485/30 emission filter. The EYFP emission images were collected using a 560 DCLPXR dichroic mirror with an HQ 545/40 emission filter. FRET was detected using the acceptor photobleaching approach. Two different regions within a cell expressing both proteins were selected as the region of interest (ROI), one for FRET and the other as a control. The ROI was zoomed in and bleached with the high intensity Argon 514 laser. The mean intensities of ECFP and EYFP were simultaneously recorded in the pre-bleach and post-bleach periods as a live graph. An increase in ECFP intensity following EYFP bleaching is indicative of FRET between the donor and acceptor fluorophores. The percent FRET efficiency was calculated using the formulaFor confocal microscopy and FRET, the NL and R5 The author(s) declare that they have no competing interests.AH, SRD and SJ conceived of the study. SRD made the clones and carried out the Vpu functional assay. AH carried out the yeast two-hybrid and biochemical assays. CT carried out the confocal microscopy and FRET assays. AH and SJ wrote the paper. All authors read and approved the final manuscript.