The intrinsically disordered N-ter domain (NTD) of p53 encompasses approximately hundred amino acids that contain a transactivation domain (1–73) and a proline-rich domain (64–92) and is responsible for transactivation function and apoptosis. It also possesses an auto-inhibitory function as its removal results in remarkable reduction in dissociation of p53 from DNA.in vitro and in cellular systems. NTD125 prevented irreversible thermal aggregation of heat denatured p53, enhanced p21-5′-DBS binding and further restored DBS binding activity of heat-denatured p53, in vitro, in a dose-dependent manner. In vivo ELISA and immunoprecipitation analysis of NTD125-transfected cells revealed that NTD125 shifted equilibrium from p53 mutant to wild type under heat stress conditions. Further, NTD125 initiated nuclear translocation of cytoplasmic p53 in transcriptionally active state in order to activate p53 downstream genes such as p21, Bax, PUMA, Noxa and SUMO.In this report, we have discovered that p53-NTD spanning amino acid residues 1–125 (NTD125) interacted with WT p53 and stabilized its wild type conformation under physiological and elevated temperatures, both Here, we showed that a novel chaperone-like activity resides in p53-N-ter region. This study might have significance in understanding the role of p53-NTD in p53 stabilization, conformational activation and apoptosis under heat-stress conditions. The cryoelectron microscopy study of full length p53 protein, that claimed to represent the in vivo nature of p53 oligomerization, reveals that aa 1–100 of N-ter of one monomer appears to abut the last aa 323–393 of C-ter of the partner in the dimer forming N/C nodes Heat denaturation of WT p53 and a majority of amino acid substitutions in p53 that occur in tumor destabilize the native DNA-binding conformation of core domain in vitro both at physiological and elevated temperatures. In cells, NTD125 stabilized and activated cytoplasmic p53 in initiating its nuclear translocation that led to activation of p53 downstream genes. Exogenously supplied NTD125 thus possessed a chaperone-like activity that activated p53 and could be of significance in restoring p53 mutant phenotype in cells under stress.In this report, we have discovered a novel function of NTD125 that binds to WT p53, stabilizes and restores its wild type conformation Highly purified recombinant p53 and NTD125 proteins were utilized for studying their thermostability and interaction. The thermal melting curve, recorded at 280 nm in a UV-visible spectrophotometer, showed that p53 started to melt at ∼35°C whereas NTD125 did not exhibit distinct melting pattern of folded proteins . Far-UV-We monitored the transition of wild type p53 conformation in to the mutant phenotype at elevated temperatures utilizing p53 conformation specific antibodies (PAb1620 and PAb240) by IPP and ELISA. The recombinant p53 preparation contained both wild and mutant phenotype at ∼1∶1 ratio; shown by IPP with conformation-specific antibodies PAb1620 (wild specific) and PAb240 (mutant specific) at RT . When NTHSP90 +/+) nuclear extract (NE) to check the effect of NTD125 on native WT p53. NE was first heated to denaturation in order to lose DBS binding and addition of NTD125 post denaturation resulted in restoration of DBS binding  was employed with or without NTD125 (in an increasing concentration), both under normal and denaturing conditions. p21-5′-DBS was radio-labeled and was mixed with recombinant p53 and DNA competitor for EMSA analysis either in the presence or absence of PAb421 antibodies (for super shift) and/or purified recombinant NTD125. An enhancement of DBS binding was observed after addition of NTD125  in the reaction mixture , lane 4  binding . Further binding , thus suin vivo ELISA and IPP utilizing conformation specific antibodies (PAb1620/PAb240). In vivo ELISA, at 37°C, showed that the ratio of p53 wild type (PAb1620) and mutant (PAb240) was ∼1∶1 . A negatively charged peptide based chaperone strategy to rescue p53 mutant conformation was reported to raise and stabilize p53 level th hour cytoplasmic p53 started migrating in to the nucleus , TAD2 (aa residues 40–61) and a proline-rich domain (aa residues 64–93) Various synthetic compounds and small molecules have been identified that allowed mutant p53 to maintain active conformation and caused accumulation of active p53 in cells Nuclear translocation of p53 can result in a change in the conformation from mutant to wild type in vitro and in cells raises the possibility whether WT p53 might possess a self-chaperoning role as un-cleaved molecule; intra-molecular chaperone-like fragments occur frequently in proteins and such proteins would be prone to changing conditions and in particular, to mutations in the critical building block region in vivo function of yeast HSP90 The chaperone-like role of NTD125 both E. coli BL21 (DE3) cells and purified using the same protocol as His-p53. HA-tagged p53, NTD125 and deleted variants were expressed in mammalian system by cloning PCR amplified fragments in pNHA1 vector at XbaI/EcoRI site, generating, pNHA1-p53, pNHA1-NTD125, pNHA1-NTD93, pNHA1-NTD61, and pNHA1-NTD55 plasmids. Primers used for amplification are summarized in Vectors used and methods for purification of bacterially expressed His-p53, GST-p53 and GST-CHIP were described earlier 2 incubator and then processed for IPP/in vivo ELISA assay. For IPP using conformation specific antibodies, cells were transfected, treated and processed as described earlier KB cells were procured from NCCS, Pune, India, and maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) with 10% FCS. Transfections were carried out using either Effectene Transfection Reagent (Qiagen) or Escort™ IV Transfection Kit (Sigma) according to manufacturer's instructions. Cells were processed for sample preparation after 24 hours, 36 hours post transfection or as described in the figures. For heat shock experiment, 24 hours post-transfection, cells were grown at 42°C for 75 minute in COThe heat denaturation studies were carried out by recording the change absorbance of 100 µg of p53 or NTD125, diluted in 1 ml PBS at 280 nm wavelength in UV-visible spectrophotometer  attached to temperature controller. The variation in temperature was done at the rate of 1°C/minute. The aggregation assay was performed as follows: p53 (1.0 µM) was incubated at 37°C and 45°C with or without NTD125 (2.0 or 5.0 µM). Thermal aggregation kinetics was monitored by measuring light scattering, in a fluorescence spectrophotometer  attached to temperature controller, in the Peltier controlled thermostatted quartz cuvettes. All the measurements were done at the excitation and emission wavelengths of 340 nm with a spectral bandwidth of 5 nm.For CD spectroscopic analysis, all the measurements were done on J-815 Circular Dichroism System (Jasco), in the Far-UV range from 260 to 195 nm using the cuvette of 0.2 cm pathlength. 100 µg of p53 or NTD125 was diluted in 1 ml 10 mM phosphate buffer (pH 7.5) and the spectra were collected at different temperatures. For each sample, an average of the three measurements was taken at a scan rate of 50 nm/minute. For interaction studies, 100 µg each of p53 and NTD125 were mixed, diluted in 1 ml 10 mM phosphate buffer (pH 7.5) and then incubated for 1 hour at the temperature at which spectra had to be recorded. For all the measurements, the spectrum of the buffer was deduced from the sample. Molar ellipticity was calculated using [molar ellipticity (θ) = millidegrees/(pathlength in millimeter × the molar concentration of proteins × the number of residues)] formula in vitro protection assay, 2 µg of recombinant His-p53 was incubated with or without recombinant GST-CHIP (1∶2) or His-NTD125 in (1∶5) molar ratio. Protein mixture was then diluted to 100 µl in PBS (containing protease inhibitor cocktail) and incubated at different temperatures i.e. RT, 37°C, 40°C, 42°C and 45°C for 1 hour. Further, the sample was diluted to 500 µl and 1 µg of PAb C-19/PAb1620/PAb240 added to the mixtue and incubated on a rotatory shaker for 1 hour at 4°C. After that 50 µl of 10% protein-A agarose (pre-saturated with BSA) was added to the sample and incubated for 2 hours at 4°C, with continuous stirring. After pelleting, beads were washed thrice with NP-40 washing buffer (0.5% NP-40 in PBS + protease inhibitor cocktail) and finally the immunocomplex was released in 50 µl of NP-40 washing buffer by adding SDS loading dye and boiling for 3 minutes. For assessment of restoration activity of NTD125, p53 was first denatured and then NTD125 was added to the mixture and incubated at 4°C for another hour and finally immunoprecipitated using different antibodies. For co-immunoprecipitaion assay, the entire procedure was repeated as above, only His-p53 and His-NTD125 were taken in (1∶1) molar ratio. For immunoblotting 20 µl of eluted sample was resolved on 12% SDS PAGE and semi-dry blotted on to nitrocellulose membrane, western blot was developed using PAb1801.For The DNA binding activity of recombinant p53 was monitored by EMSA with or without NTD125. The EMSA reaction was set using 3 ng probe and 100 ng recombinant p53, as described previously 2CO3, 1 mM MgCl2, pH 9.8) and incubated at 37°C. The reaction was then stopped by adding 100 mM EDTA, and absorbance was taken at 405 nm in the ELISA reader . To study the DNA binding by heat denatured p53 protein, p53 was diluted in PBS, heat denatured at 37°C for 1 hour in water bath with or without NTD125 and added on to the PAb421 coated wells, DBS was then added and bound DBS was detected using AAP.PAb421 (0.5 µg) diluted in 50 µl PBS was coated per well and incubated at 4°C overnight, plate was then washed once with PBS and blocked using 200 µl of 1% BSA in PBS for 1 hour. Plate was then washed thrice, 5 minute each, with wash buffer (PBS + 0.05% Tween-20). After that WT p53 protein was bound to the antibody by adding 0.5 µg of p53 protein diluted in 50 µl PBS in the well and incubating it at 4°C for 1 hour. To remove the unbound protein, plate was washed thrice, 5 minute per wash, with wash buffer. Subsequently 0.5 µg of biotin labeled DBS diluted in 50 µl 1X GMS buffer was added in the well and incubated at 4°C for 1 hour. Plate was washed again three times with wash buffer, and then 50 µl of 1∶400,000 diluted Avidin Alkaline Phosphatase (AAP from Sigma) in PBS was added per well and incubated for 2 hours. Washing was again done thrice with wash buffer, and the color was developed by adding 100 µl of 1 mg/ml PNPP in AP buffer  conjugated with reporter enzyme HRP .An ELISA-based protein-protein interaction assay was utilized for p53- and NTD125 interaction studies. 96-well Maxisorp plates (Nunc) were coated with 50 µl of a 10 µg/ml (NTD125/CHIP/BSA) protein in PBS at 4°C over night. The wells were rinsed with cold PBS at 4°C three times. Blocking was done with 2% BSA (Sigma) in PBS at 4°C for 4 hours. Following the blocking step, the wells were washed three times with PBS containing 0.01% (v/v) Tween-20 (Sigma). p53 protein (0.5 µg) was diluted in 50 µl PBS, 0.05% (v/v) Tween-20, 0.2% (w/v) BSA and (in increasing concentration) added into NTD125/CHIP/BSA coated wells. After an incubation period of 90 minute at 4°C, the ELISA plates were washed with PBS containing 0.01% Tween-20 three times. The p53 protein was detected using 0.2 µg of mouse monoclonal antibody PAb C-19 in 50 µl PBS, 0.05% (v/v) Tween-20, 0.2% (w/v) BSA and then AP-conjugated anti-mouse secondary antibody. Finally, 100 µl of alkaline phosphate substrate i.e. 1 mg/ml PNPP in AP-buffer (pH 9.6) was added and the enzymatic reaction was allowed to take place for 30 minutes at room temperature. The reaction was terminated by adding 50 µl of 0.1 M EDTA (pH 8.0). The optical density was determined at 405 nm using Microplate reader (Bio-Rad). Total p53 concentration in cells was measured using PathScanin vitro was carried out using two-site ELISA. Firstly the wells were coated with p53 conformation specific monoclonal antibody PAb1620 or PAb240 at concentration of 50 ng/100 µl per well in 0.1 M carbonate buffer (pH 9.2) at 4°C for 16 h. The wells were rinsed with PBS three times. Blocking was done with 2% BSA (Sigma) in PBS at 4°C for 2 hours. Following the blocking step, the wells were washed three times with PBS containing 0.05% (v/v) Tween-20 (Sigma). p53 protein (100 ng) was diluted in 100 µl of PBS, 0.05% (v/v) Tween-20, 0.2% (w/v) BSA with NTD125 (1∶5) or without NTD125 were incubated at different temperatures for 1 hour, added in the wells and incubated for 90 minute at 4°C. The ELISA plates were then washed with PBS containing 0.05% Tween-20 three times. p53 protein was detected using 50 ng of goat monoclonal antibody PAb C-19 in 50 µl PBS, 0.05% (v/v) Tween-20, 0.2% (w/v) BSA and then AP-conjugated anti-goat secondary antibody. Finally detection was done as described earlier. Sandwich ELISA (in vivo ELISA) from cell lysate was done as follows; wells were coated with 100 µl of 5 µg/ml anti-p53 antibodies  overnight at 4°C. After washing thrice with TBS buffer (0.05% Tween-20 in PBS), blocking was done using 5% skimmed milk in TBS for 2 hours at 4°C. After washing the wells thrice with TBS, 200 µg cell lysate in NP-40 buffer  1∶1 v/v diluted in 5% skimmed milk in TBS was added to each well and incubated at 4°C for 2 hours. Subsequently 100 µl of anti-p53 polyclonal antibody Fl-393 (1∶1000 diluted) was added to each well and incubated at 4°C for 2 hours. Again after three quick washes, 100 µl of AP-conjugated anti-rabbit secondary antibody (1∶1000) was added to each well and kept at RT for another 2 hours. ELISA was developed using 100 µl of 1 mg/ml PNPP solution in AP- buffer for 30 minutes and after terminating the reaction O.D. was recorded at 405 nm on Microplate reader (Bio-Rad).Investigation of the p53 conformation KB cells were scraped with a rubber policeman, and pelleted. 200 µl of cytoplasmic extraction reagent CER I  was added per 20 µl of packed cell volume, and the cell pellet was vortexed for 15 seconds. Cells were incubated in presence of CER I for 10 minutes, followed by incubation with 11 µl of CER II for another minute. Lysed cells were centrifuged at 13,000 rpm for 5 minutes to pellet the intact nuclei. The supernatant containing the cytoplasmic fraction was carefully separated. The pelleted nuclei were resuspended in 100 µl of NER I, votexed for 15 seconds and incubated on ice for 45 minutes with periodic vortexing after every 10 minutes. After this, the suspension was centrifuged at 13,000 rpm and the supernatants containing the nuclear proteins were stored at -80°C. Nuclear and cytoplamic fractions were checked by western blotting using anti-actin and anti-RNA pol-II antibodies (data not shown).2 incubator, in serum free media for 6 hours and then media was replaced with fresh complete DMEM media. After 24 hours the cells were washed in cold PBS three times and lysed with 200 µl of the 1 X lysis buffer (Promega) for 20 minutes at 4°C, the lysate was then centrifuged at 14000 rpm for 5 minute at 4°C. Supernatant was collected and 20 µl supernatant was used for the assay of luciferase activity using Luciferase reporter gene assay kit (Promega) as per the manufacturer's instruction. The β-galactosidase activity was determined using the β-galactosidase Enzyme Assay Kit (Promega). Luciferase activity was normalized by β-galactosidase activity and the data from triplicate determinations were expressed as mean ± SD.KB cells were plated in six-well plate the day before transfection such that they become 60–80% confluent before transfection. Reporter plasmid (1.0 µg), full length promoter-constructs were transfected together with (1.0 µg) expression vectors (pNHA1-NTD125) and (0.5 µg) β-galactosidase-expression plasmid  per well as per the manufacturer's instructions. For each transfection, DNA content was kept uniform by using empty vector for relative plasmid type. The cells were incubated at 37°C, in COKB Cells were lysed in appropriate amount of Trizol (1 ml Trizol per well of a 6 well plate for cultured cells). Cells were repeatedly and vigorously pippetted. Cells were then kept at room temperature for 10 minutes, after which 200 µl of chloroform per 1 ml of Trizol was added and mixed thoroughly. The cells were again left at room temperature for 10 minutes. Cells were then centrifuged at 12,000 rpm at 4°C for 15 minutes and the upper aqueous colorless layer was transferred to a fresh eppendorf tube. To this eppendorf tube, 75 µl Lithium Chloride (LiCl) followed by 1 ml chilled Ethanol were added and kept at -20°C for 2-3 hours. The eppendorf tube was centrifuge at maximum speed for 15 minutes at 4°C. The supernatant was discarded, 250 µl of 70% Ethanol was added and the tube was kept at room temperature for 2 minutes. The tube was again centrifuged at 7500 rpm for 5 minutes at 4°C, the supernatant was then discarded and finally, the pellet was resuspended in RNA grade water till it was completely dissolved. Single Strand c-DNA was synthesized with sense and anti-sense primers using RevertAid ™ H Minus First Strand cDNA Synthesis Kit (Fermentas). The resulting cDNA was diluted (1∶10) before proceeding with the PCR reaction. PCR was conducted in Mastercycler gradient . Each 50 µl PCR reaction employed cDNA, 2.5 U Taq polymerase , 0.2 mM dNTPs and 0.5 µM primer. PCR products were resolved on 2% agarose gel. The size of the PCR amplicon was determined by comparison with 100-bp DNA ladder . Primers used for amplification of appropriate gene are summarized in Table S1Primers for PCR amplification of p53, NTD125 and NTD-variants; for cloning in pNHA1 plasmid vector.(0.06 MB RTF)Click here for additional data file.Table S2Primers for RT-PCR analysis.(0.06 MB RTF)Click here for additional data file.


Caulobacter crescentus must often face changes in its environment such as phosphate limitation. Evidence reported in this paper indicates that under phosphate starvation, C. crescentus produces a membrane surface-anchored lipoprotein named ElpS subsequently released into the extracellular medium. A complete set of 12 genes encoding a type II secretion system (T2SS) is located adjacent to the elpS locus in the C. crescentus genome. Deletion of this T2SS impairs release of ElpS in the environment, which surprisingly remains present at the cell surface, indicating that the T2SS is not involved in the translocation of ElpS to the outer membrane but rather in its release. Accordingly, treatment with protease inhibitors prevents release of ElpS in the extracellular medium suggesting that ElpS secretion relies on a T2SS-secreted protease. Finally, secretion of ElpS is associated with an increase in alkaline phosphatase activity in culture supernatants, suggesting a role of the secreted protein in inorganic phosphate mobilization. In conlusion, we have shown that upon phosphate starvation, C. crescentus produces an outer membrane bound lipoprotein, ElpS, which is further cleaved and released in the extracellular medium in a T2SS-dependent manner. Our data suggest that ElpS is associated with an alkaline phosphatase activity, thereby allowing the bacterium to gather inorganic phosphates from a poor environment.Life in oligotrophic environments necessitates quick adaptive responses to a sudden lack of nutrients. Secretion of specific degradative enzymes into the extracellular medium is a means to mobilize the required nutrient from nearby sources. The aquatic bacterium  Caulobacter species live in all kinds of aquatic environments where nutrients are often scarce. While some nutrients are directly usable by the bacteria, other organic substrates must be partially degraded or modified before being assimilated by the cell as a nutrient source. These processes often rely on degradative enzymes that are secreted at the cell surface or in the extracellular environment depending on specialized secretion systems. The Type II secretion system (T2SS) or main terminal branch of General secretion pathway (Gsp) is well known for its role in virulence of pathogenic species but is also involved in environmental adaptation. A T2SS is present in at least 16 environmental non-pathogenic bacteria Acinetobacter calcoceticus and Pseudomonas alcaligenesEscherichia coli K12Klebsiella oxytoca where it triggers export across the outer membrane of the starch-hydrolysing lipoprotein pullulanase Neisseria meningitides was responsible for the proteolytic cleavage of lactoferrin-binding protein LbpB from the cell surface Although T2SS-encoding genes are generally well conserved and display a similar genetic organization in many bacterial species, secreted proteins have a wide variety of functions or enzymatic activities Caulobacter survival, is present in soil and water in organic and inorganic forms. Inorganic phosphate (Pi) is the preferential source and is readily taken up by the cell when available. Since nutrient-poor environments can also lack Pi, many bacterial species have developed mechanisms to use organic phosphates as a source of phosphorus i release before being taken up C. crescentus strikingly increases stalk length, thereby optimizing the surface/volume ratio and in turn facilitating phosphate uptake pho regulon best characterized in Escherichia coli. The pho regulon phoA, encoding an alkaline phosphatase required for the utilization of organic phosphates C. crescentus harbors the pho regulon but lacks a phoA homolog.Phosphorus, which can be limiting for C. crescentus to trigger production and T2SS-dependent secretion of a lipoprotein, ElpS, under phosphate starvation conditions. We show that ElpS is an extracellular outer membrane-anchored protein subsequently released in the environment in a T2SS-dependent manner. Functional data suggest that ElpS is involved in phosphate mobilization and stimulates extracellular alkaline phosphatase activity in low-phosphate medium.In the present study, we report the ability of Bradyrhizobium, Gluconacetobacter, Mesorhizobium) In silico analysis of the C. crescentus (CB15N) genome reveals the presence of an entire set of T2SS genes, gspC (cc0172) to gspN (cc0184) annotated in alphabetical order  of bacteria living in a wild variety of environments such as Shewanella species living in ocean or Bacteroides species found in the gut. This high degree of conservation implies that cc0170 probably plays an important role in bacterial survival in different environments. Sequence analysis reveals that cc0170 is homologous to putative lipoproteins or secreted proteins, indicating that this gene likely encodes an extracellular protein. Interestingly, the tandem organization of cc0170 and T2SS genes found in C. crescentus is also conserved in others species such as the plant pathogen Xhanthomonas campestris and the marine species Sphingomonas alaskensis and Hyphomonas neptunium in a similar genetic organization . gspE and gspL encode a putative cytoplasmic ATPase and an inner membrane protein promoting secretion pore opening, respectively. gspE and gspL mRNA levels are represented as cycle threshold (CT) ratios calculated from qRT-PCR raw data . Remarkably, one protein of about 50 kDa is strongly produced and secreted from CB15N cells grown in poor medium  highlights a conserved lipoprotein signal at the N-terminus , suggesting that a functional interaction may exist between ElpS and the T2SS apparatus.The potential role of the T2SS was first determined by comparing protein secretion profiles between CB15N and CB15N Δr medium . The corarvation . As a lyarvation . Furtherr medium . The indpromoter . Moreoveterminus . The lipelpS is strongly upregulated in poor medium. ElpS-3flag accumulates in CB15N ΔgspC-N cells and is not released into the culture supernatant , and FeSO4. Complementation of poor medium with M2 salts completely inhibited the production and the secretion of ElpS-3Flag, while 1 mM FeSO4 had no effect (elpS is not upregulated in iron-depleted medium (data not shown). In order to test which M2 salts component specifically represses ElpS synthesis, cells carrying elpS-3flag were grown in poor medium supplemented with phosphate or ammonium salts. The addition of NH4Cl had no effect on ElpS-3Flag level whereas phosphate salts (Na2HPO4 and KH2PO4) significantly reduced the production of ElpS-3Flag in both CB15N and CB15N ΔgspC-N strains . TonB-dependent receptors are generally sensors of extracellular signals elpS. Nevertheless, deletion of cc0171 does not suppress the production of ElpS-3Flag in poor medium (data not shown). We also assume that Cc0171 does not act on elpS induction under phosphate starvation. On the contrary, the TonB-dependent receptor may be involved in phosphate uptake through the activation of ElpS. However, no significant change in intracellular phosphate concentration is detected when cells are switched from rich to poor medium which could be due to the low sensitivity of the method used (data not shown). ElpS is specifically produced under phosphate starvation and is associated with an increased secreted alkaline phosphatase activity when overproduced, Together, our data suggest that ElpS participates in phosphate mobilization.Phosphate is an important limiting nutrient in E. coli and C. crescentus were derivatives of DH10B and CB15N strains, respectively. DNA manipulations were performed according to standard techniques. Restriction enzymes were purchased from Roche, and primers were purchased from Eurogentec. Strains and plasmids are summarized in E. coli strains were grown in Luria Bertani broth medium  supplemented with 100 µg/ml ampicillin or 50 µg/ml kanamycin when necessary. C. crescentus strains were grown in Peptone Yeast extract (PYE) 4 and 0.5 mM CaCl2+0.2% glucose), M2G All strains of gspC-N and CB15N ΔelpS  as follows: 45 ml culture (OD660 nm = 0.5 at 30°C) were centrifuged for 15 min at 3500 rpm. Bacterial pellets were resuspended in 100 µl 10% SDS and 20 µl 20 mg/ml proteinase K and incubated for 1 h at 37°C with shaking. Five ml of TRIzol® Reagent (Invitrogen) were added and suspensions were vigorously shaken. After a 10 min incubation at 65°C, 1 ml chloroform was added to the suspensions and the mixtures were shaken and incubated for 5–10 min at room temperature. Samples were centrifuged for 15 min at 14,000 rpm at 4°C, 2.5 ml propan-2-ol were added to the aqueous phases and samples were stored overnight at -20°C. After centrifugation for 30 min at 14,000 rpm at 4°C, pellets were washed with 75% RNase-free ethanol. Supernatants were discarded and pellets were dried for 15 min at room temperature. Total RNA samples were resuspended in 100 µl RNase-free H2O, incubated for 10 min at 55°C and stored at −80°C. The integrity of the RNA and the absence of DNA were checked by gel electrophoresis. RNA was quantified with a NanoDrop .Total RNAs were extracted from CB15N, CB15N ΔRNA samples were obtained using DNA-free kit (Ambion) and reverse-transcription was performed with SuperScript II Reverse Transcriptase (Invitrogen). Primers listed in C. crescentus strains were grown in PYE and 1/5X PYE media for 6 h at 30°C. Cells were harvested by centrifugation for 10 min at 6000 rpm and resuspended in 1/2 volume of Z-Buffer  and 1/185 volume of β-mercaptoethanol. Cell lysis was obtained by adding 1/10 volume of glass beads and vortexing 6 cycles of 30 seconds spaced with 30 seconds cycles on ice. Membrane and beads were separated from cytoplasmic fractions by centrifugation for 10 min at 6000 rpm. Supernatants containing soluble proteins were collected, mixed with 1/5 volume of 4 mg/ml ortho-Nitrophenyl-β-galactoside (ONPG) and incubated for 1 min to 1 h at room temperature. ONPG degradation was monitored at OD420 nm by formation of an yellow product ortho-Nitrophenyl (ONP) and stopped by the addition of 0.3 M Na2CO3. Specific β-galactosidase activity was determined in nmol of degraded ONPG (ε420 nm = 4500 M−1) per min per mg of total protein. Total protein concentrations were determined using Bradford assay (Bio-Rad Protein Assay).C. crescentus was grown in PYE, 1/5X PYE, M2G, balanced or low phosphate HIGG to OD660 nm = 0.5 at 30°C. Culture supernatants were isolated by a 10 min centrifugation at 3500 rpm. Corresponding supernatants were filtered on 0.22 µM filters and proteins were precipitated using a pyrogallol red-molybdate-methanol (PRMM) protocol <Caldwell, 2004 #5>. Protein pellets were resuspended in 1/150 volume of SDS-PAGE loading buffer . Cellular extracts were obtained by resupending bacterial pellets in SDS-PAGE loading buffer to a final corresponding OD660 nm = 2. All protein samples were boiled for 5 min and stored at −20°C.C. crescentus was grown in 1/5X PYE to OD660 nm = 0.5 at 30°C except when notified. Culture supernatants were 100 fold-concentrated on Amicon (Amicon® Ultra-10k Millipore). Cellular extracts were obtained by resuspending bacterial pellets in PBS  to a final OD660 nm = 2 and sonicating 6 times for 30 s. Protein samples were stored at −20°C.2O. Gels were incubated in sensitizing solution containing 0.02% sodium thiosulfate and washed again twice for 1 min in H2O. Proteins were revealed by incubating the gels another 20 min in 0.1% silver nitrate followed by 2 washes for 1 min in H2O and one last incubation in 0.04% formalin, 2% sodium carbonate. The staining reaction was stopped in 5% acetic acid. ElpS-3Flag, DivK, MreB, mGFP-GspL, FliF and CcrM were immunodetected with 1∶1000 anti-Flag , 1∶5000 anti-DivK When prepared under non-denaturing conditions, samples were first mixed with 2X SDS-PAGE loading buffer and boiled for 5 min. Proteins were then separated on 12% polyacrylamide gels and detected by silver nitrate staining or transferred to a PVDF membrane for immunoblotting. For silver nitrate staining, gels were first fixed for 20 min in 50% methanol, 5% acetic acid, then washed for 10 min in 50% methanol and for 2 h in H660 nm = 0.1 were diluted in 1/5X PYE containing 50 mCi/ml 3H-palmitic acid and grown for 6 h at 30°C. Cellular extracts and supernatants were prepared under non-denaturing conditions as above. ElpS-3Flag was immunoprecipitated with 1∶1000 anti-Flag antibodies  and Protein A - Sepharose® 4B (Invitrogen). Immunoprecipitates were resuspended in 100 µl SDS-PAGE loading buffer and concentrated supernatants were mixed with 2X SDS-PAGE loading buffer. Proteins were boiled for 5 min and 10 µl of each sample were separated on 12% polyacrylamide gels. Radioactive signal was enhanced using fluorography PYE precultures at ODelpS::elpS-3flag and CB15N ΔelpS grown for 6 h in 1/5X PYE, separated on 12% polyacrylamide gel and transferred to PVDF membrane. Proteins were detected by Coomassie blue staining using the Pierce Coomassie Plus Protein Assay Reagent and Kit (Thermo Scientific) and ElpS-3Flag was immunodetected with anti-Flag antibodies. The band corresponding to ElpS-3Flag was cut through the simultaneous analysis of total proteins profiles and immunoblot. The N-terminal end sequence of ElpS was obtained from Pick-n-Post (http://www.pick-n-post.com).Proteins extracts were obtained from 10000-fold concentrated supernatants on Amicon from CB15N 660 nm = 0.5 in 1/5 X PYE at 30°C. Cells were harvested by centrifugation at 4°C, washed in 50 ml CBB buffer , and resuspended in 10 ml CBB buffer with a protease inhibitor cocktail . Cells were passed twice through a French pressure cell  and the lysate was cleared by centrifugation at 20,000× g for 15 min at 4°C. The cleared lysate was then centrifuged at 150,000× g for 2 h at 4°C, and a sample of the supernatant was taken for analysis. The pellet was resuspended in 1 ml CBB buffer and spun at 105,000× g for 1 h. The supernatant was discarded and the washed pellet was resuspended in 1 ml CBB with 0.5% SDS and used for analysis. The protein content of the cleared lysate was measured using a Bradford assay and was used to normalize load volumes between samples. Within a fractionation sample, equivalent volumes were loaded on gels. Samples were mixed with 5X SDS-PAGE loading buffer, boiled for 5 min, separated on 12% polyacrylamide gels, transferred to PVDF membranes, and blotted with anti-Flag, anti-FliF and anti-CcrM antibodies.Cells (400 ml) were grown to OD660 nm = 0.5 in 1/5 X PYE at 30°C. Cells were harvested by centrifugation at 4°C, washed 3 times in 50 ml 50 mM ammonium bicarbonate (AmBic) pH 8 and resuspended in 5 ml AmBic. Cells were sonicated at maximal intensity 20 times for 5 s and kept on ice between each cycle. The lysate was cleared by centrifugation for 10 min at 12,000× g at 4°C. The cleared lysate was then centrifuged for 40 min at 100,000× g at 4°C, and a sample of the supernatant was taken for analysis (cytoplasmic fraction). The pellet was resuspended in 1 ml 1% sodium lauryl sarkonisate and spun for 40 min at 100,000× g. The supernatant corresponding to solubilized inner membrane proteins was taken for analysis and the pellet was resuspended in 1 ml 2.5 M NaBr and incubated for 30 min at 4°C and then ultracentrifuged for 40 min at 100,000× g. The outer membrane pellet was resuspended in 1 ml 100 mM Na2CO3 and spun again for 40 min at 100,000 g ×. The OM proteins was resuspended in SDS-PAGE loading buffer. Within a fractionation sample, equivalent volumes were loaded on gels. Samples were mixed with 2X SDS-PAGE loading buffer, boiled for 5 min, separated on 12% polyacrylamide gels, transferred to nitrocellulose membrane, and blotted with anti-Flag antibodies.Cells (300 ml) were grown to OD660 nm = 0.5 in 1/5X PYE at 30°C. Cultures were treated or not with 2 µg/ml proteinase K for 30 min at 37°C. Cells were harvested by centrifugation for 10 min at 3500 rmp, the supernatant was discarded and the pellet washed in 1 volume of M2 salts . Cells were finally resuspended in 1/4 volume of M2 salt containing 10 mM phenylmethanesulphonylfluoride (PMSF). Samples were mixed with 2X SDS-PAGE loading buffer, separated on a 12% polyacrylamide gel and transferred to nitrocellulose membrane. ElpS-3Flag and MreB were immunodetected using anti-Flag and anti-MreB antibodies respectively.Cells were grown to ODelpS::elpS-3flag was grown in 1/5X PYE for 4.5 h at 30°C. Cells were then harvested by centrifugation for 10 min at 3500 rpm and resuspended in an equal volume of fresh 1/5X PYE containing or not a cocktail of protease inhibitors  and incubated for 90 min at 30°C. ElpS-3Flag was immunodetected using anti-Flag antibodies in cellular extracts and precipitated supernatant from resulting samples.CB15N Alkaline phosphatase assays were performed on 100 fold-concentrated supernatants obtained under non-denaturating conditions from cells grown for 4 h in 1/5X PYE. Specific alkaline phosphatase activity was measured by degradation of 1 mg/ml p-Nitrophenyl Phosphate (PNPP) in 100 mM pH 7.9 Tris-HCl, 2 mM MgCl2. P-Nitrophenol formation induced by PNPP cleavage was monitored at OD420 nm. Alkaline phosphatase activity was calculated in nmol of degradated PNPP (e420 nm = 18000 M-1)/min/mg of total protein. Total protein concentrations were determined using micro-Bradford assays (Bio-Rad Protein Assay).Table S1Strains and plasmids.(0.05 MB DOC)Click here for additional data file.Table S2Primers used for RT PCR assays.(0.03 MB DOC)Click here for additional data file.Text S1Strain and plasmid constructions.(0.04 MB DOC)Click here for additional data file.


Several techniques are available to study gene function, but many are less than ideal for soybean. Reverse genetics, a relatively new approach, can be utilized to identify novel mutations in candidate genes; this technique has not produced an allelic variant with a confirmed phenotype in soybean. Soybean raffinose synthase genes and microsomal omega-6 fatty acid desaturase genes were screened for novel alleles in mutagenized soybean populations.RS2; two mutations resulted in amino acid mutations and one resulted in an altered seed oligosaccharide phenotype. The resulting phenotype was an increase in seed sucrose levels as well as a decrease in both raffinose and stachyose seed oligosaccharide levels. Three mutations in independent lines were identified in the omega-6 fatty acid desaturase gene FAD2-1A; all three mutations resulted in missense amino acid mutations and one resulted in an altered seed fatty acid profile that led to an increase in oleic acid and a decrease in linoleic acid in the seed oil.Four mutations in independent lines were identified in the raffinose synthase gene RS2 allele is similar to previously observed seed oligosaccharide phenotypes in RS2 mutant (PI 200508) allele-containing lines. Due to the anti-nutritional characteristics of raffinose and stachyose, this represents a positive change in seed composition. The fatty acid phenotype controlled by the novel FAD2-1A allele controls an increase in oleic acid in the seed oil, a phenotype also observed in a line previously characterized to have a null allele of the FAD2-1A gene. Molecular marker assays were developed to reliably detect the inheritance of the mutant alleles and can be used in efficient breeding for these desired seed phenotypes. Our results serve as the first demonstration of the identification of soybean mutants controlling seed phenotypes discovered through the reverse genetics technique TILLING.The oligosaccharide phenotype controlled by the novel  Reverse genetics is potentially a powerful strategy used to identify novel, induced mutations in candidate genes. Utilizing reverse genetics allows us to take advantage of genes characterized in other plant genomes and use this knowledge to create a pool of candidate genes in soybean which can then be screened for genotypic variants. The function of the gene is further characterized by identifying predicted mutant phenotypes. Alternatively this technique can be applied to genes with known function to create an allelic series to avoid possible lethality issues in genes essential for plant growth or development. The recent release of the soybean genome sequence is an especially valuable asset for soybean reverse genetics.Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics technique that serves as a high throughput method to identify unique, chemically induced mutations within target genes which have potential to change gene expression and/or function -3. We deAgrobacterium tumefaciens or A. rhizogenes has been effective in studying gene function, although it is not very efficient  was screened for mutations utilizing the TILLING strategy [RS2 mutations. The four identified lines all contained a SNP typical of EMS mutagenesis, G/C to A/T transitions. Two of the mutations did not result in amino acid changes and therefore were not considered candidates for phenotypic characterization. The other two lines, designated 165 and 397, contained mutations which resulted in missense amino acid changes  resulting in S150F amino acid change. DNA from M2 tissue of line 397 contained a heterozygous SNP (c319t in the coding sequence) resulting in a T107I amino acid change. The induced mutations in both line 165 and 397 lie in semi-conserved regions of plant raffinose synthase gene sequences  resulting in the amino acid change S117N. Line 615 contained a SNP (c713t in the coding sequence) resulting in the amino acid change S238F. Line 743 contained a SNP (g1121a in the coding sequence) resulting in the amino acid change G374E. The missense mutation for line 17D was in a highly conserved region of the protein sequence, while the missense mutations in lines 615 and 743 were in less conserved regions . However, the line 397 harboring the T107I RS2 allele displayed a phenotype predicted for mutations in the soybean raffinose synthase gene RS2.Seeds from the homozygous S150F line 165 did not have an obvious oligosaccharide phenotype as determined by quantitatively measuring sucrose, raffinose and stachyose of M3 seeds for both oligosaccharide phenotype and RS2 genotype  or homozygous mutant alleles at the RS2 locus in order to further characterize the phenotype resulting from the novel allele. Seven independent wild-type RS2 and nine independent mutant RS2 plants were selected to negate the action of unidentified genes that may contribute to the oligosaccharide content; the mutation density was previously determined to average 1/550 kilobases [RS2 mutation, the average seed sucrose was increased by 28%, raffinose was reduced to 37% and stachyose was reduced to approximately 24% of 397-derived seeds which carried the wild-type allele of RS2  which has been shown to be expressed in soybean seeds or the FAD2-2 gene family members members [Convincing evidence exists that points to genetic redundancy playing a role in oleic acid accumulation. Although the identification of independent  members ,19,26-28The use of TILLING to identify novel sources of important soybean seed composition traits confirms the utility of this reverse genetics technology as a route for both gene function analysis and direct applicability for soybean improvement. Soybean now joins wheat, sorghum, pea, and rapeseed as crops that have demonstrated success in identifying traits using TILLING for reverse genetics -32.1 plants were advanced to M2 families, leaf tissue was collected and DNA prepared from a single M2 plant from each family. M3 seeds from each M2 plant were catalogued for storage.The 'Williams 82'  EMS mutaRS2 were transplanted to 3-gallon pots; 1–3 plants per pot. Seven homozygous wild-type and nine homozygous mutant RS2 M2:3 plants from the mutagenized line 397 were grown to maturity in a growth chamber with 13 hour day length. The dark temperature was 22°C and the light temperature was 28°C. Plants were grown, three per 3-gallon pot, in PRO-MIX (Premier Horticulture) medium and fertilized with Osmocote Plus (Scotts) per manufacturer's instructions.Thirty-nine seeds were planted in packets , allowed to germinate, and transferred to soil in flats. Plants were sampled for genotypic determination by allele-specific molecular marker assay described below. A population of only the plants homozygous for either the wild-type or mutant allele of FAD2-1A, FAD3A, and FAD3C genes: M23 contains a deletion of FAD2-1A [FAD3A and FAD3C [FAD2-1A, FAD3A, and FAD3C alleles from M23 and 10–73 .Plants for seed fatty acid phenotype determination were grown at the Bradford Research and Extension Center (BREC) located near Columbia, MO in the summer of 2007 and 2008 with irrigation as needed. Williams 82 was the control line since it was the progenitor of line 17D. Several other lines were grown for comparison because of their known mutant alleles in the  FAD2-1A ; 10–73 cnd FAD3C ; KB05-7 1 seeds were advanced to F2:3 lines in a Costa Rica winter nursery. Two F3 seeds from each line were germinated and evaluated for their FAD2-1A genotype using the molecular marker assay. Homozygous wild-type or mutant S117N FAD2-1A lines were confirmed by analyzing three additional seedling genotypes from each line. Six individual homozygous mutant FAD2-1A F3 plants representing two independent F2 individuals and five individual homozygous wild-type FAD2-1A F3 plants representing two independent F2 individuals were germinated in germination packets and transferred to the field at BREC in 2008 for production of F4 seeds. Five seeds from each plant were sampled for individual fatty acid analysis.Seeds of a Williams 82 × 17D cross were produced at BREC in 2007. The FRS2 was screened for EMS induced mutations. Exon 1 of RS2 was screened using IR 700 and IR 800 labeled primers: 5'-GAGTCTCATATTGTACATGGTAG-3' and 5'-GCAATTCGATGCTTCTTATGAG-3'. A portion of FAD2-1A was first amplified with unlabeled primers (to overcome poor amplification of DNA directly with the labeled primers) followed by amplification with IR 700 and IR 800 labeled primers: 5'GTAGAGGTCGTGTGGCCAAAGTGGAAG-3' and 5'AACCATGATCGCAACAAGCTGTTTCAC-3'. Standard TILLING PCR parameters were as follows: One cycle of 95°C for 2 minutes and 94°C for 20 seconds followed by 56 cycles of 94°C for 20 seconds, 56°C for 30 seconds, and 72°C for 1 minute. The next step in the PCR was 72°C for 5 minutes, then a 99°C step for 10 minutes followed by a 70°C to 0°C melt. The reactions were then held at 10°C. Cel I based cleavage of PCR products and detection with polyacrylamide gels was essentially as described [A portion of RS2 mutations were confirmed by PCR amplification of a portion of the gene followed by sequence analysis. Primers used were 5' CCCACCATGTCACCACACC-3' and 5'-GGTGATGAATTTTTAGCGGCG-3'. PCR parameters were 95°C for 10 minutes, followed by 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and then 5 minutes at 72°C; the reaction was held at 4°C. Screening for the RS2 candidate gene was carried out at Purdue University in West Lafayette, IN and screening for the FAD2-1A candidate gene was carried out at the Fred Hutchinson Cancer Research Center.Pools containing cleaved products indicating an induced mutation or heteroduplex mismatch were deconvoluted by separating the pools into individual plant DNA samples for sequencing in order to identify the line containing the mutation -3. The lRS2 gene. The assay was designed as described [An allele specific molecular marker assay was developed for the mutation identified in line 397 to discriminate between wild-type Williams 82 or mutant alleles of the escribed . In orde2, 3.75 μg ml-1 BSA, 200 μM dNTPs), 5% DMSO, 0.25× SYBR Green I, and 0.2× Titanium Taq polymerase .Reactions were carried out in 20 μl; each primer was at 0.5 μM final concentration in reactions containing template, buffer (40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgClRS2 assay were as follows: 95°C for 12 minutes followed by 35 cycles of 95°C for 20 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and then a melting curve from 72°C to 90°C. The fluorescence was read after each cycle and every 0.2°C with a one second hold during the melt. Each genotype produced a product with a characteristic melting profile, as measured by Tm of the negative first derivative of the disappearance of fluorescent signal. The Williams 82 homozygous genotype gave a peak at 83.6°C, mutant homozygous genotype gave a peak at 79.2°C, and a heterozygous genotype gave a peak at 83.6°C with a shoulder at 79.0°C. Templates for PCR were 1.2 mm washed FTA  card punches prepared from leaves according to the manufacturer's instructionsPCR parameters on a DNA Engine Opticon 2  for the FAD2-1A mutation. Re-isolation of DNA from line 18D as well as line 16D and 17D M2 tissue and FAD2-1A sequence characterization revealed that line 18D (or 16D) did not contain the variant allele of FAD2-1A, but line 17D was homozygous for the S117N mutation. FAD2-1A specific gene amplification and sequencing was performed with the primers: 5'-accacctacttccacctccttcctcaa-3' and 5'-TATATGGGAGCATAAGGGTGGTAGTGGCTT-3'. The homozygous state of the S117N FAD2-1A allele in M3 seedlings from line 17D was also confirmed by sequencing.The TILLING screen originally identified line 18D as heterozygous for the S117N FAD2-1A allele identified in line 17D [RS2 allele-specific assay. PCR parameters on a DNA Engine Opticon 2 (MJ Research/Bio-Rad) for the FAD2-1A GC Tail assay were as follows: 95°C for 5 minutes followed by 35 cycles of 95°C for 20 seconds, 65°C for 20 seconds, 72°C for 20 seconds, and then a melting curve from 75°C to 85°C. The fluorescence was read after each cycle and every 0.2°C with a one second hold during the melt with excitation at 470–505 nm and detection at 523–543 nm.. Each genotype produced a product with a characteristic melting profile, as measured by Tm of the negative first derivative of the disappearance of fluorescent signal. Homozygous wild-type FAD2-1A alleles produced a peak at 83°C, homozygous mutant alleles produced a peak at 81°C, and heterozygous samples produced both peaks. Templates for PCR were either genomic DNA samples isolated using the DNeasy Plant Mini Kit  or 1.2 mm washed FTA (Whatman) card punches prepared from leaves according to the manufacturer's instructions.An allele specific GC-tail molecular marker assay was developed for the S117N line 17D . The prig. The supernatant was passed through a 0.2 μm filter. Sugars were separated on a Dionex Carbo Pac PA 10 analytical column  connected to a Carbo Pac PA 10 guard column (50 mm × 4 mm). The mobile phase was 90 mM NaOH with flow rate of 1.5 ml min-1, maintained at 30°C. Detection settings were: time 0, 0.1 v, time 0.41, -2.0 v, time 0.42, 0.6 v, and time 0.44, -0.1 v.Oligosaccharides were determined by high performance ion chromatography with pulsed amperometric detection (PAD) employing an Agilent 1100 series HPLC and an ESA Coulochem III detector . A 12.5 mg ground seed sample from either a whole seed or a chipped seed  was extracted with 0.5 ml 50% ethanol at 70°C, 30 min. Samples were then centrifuged 15 min at 16,000 The fatty acid profiles of individual whole crushed seeds was determined by lipid gas chromatography of total fatty acid methyl esters of extracted oil, as previously described . Five inTILLING: Targeting induced local lesions in genomes; RS: raffinose synthase; FAD: fatty-acid desaturase; EMS: ethyl-methanesulfonate; NMU: N-nitroso-N-methylurea; SNP: single nucleotide polymorphism; PCR: polymerase chain reaction; NCBI: National Center for Biotechnology Information.ED designed and performed the raffinose synthase experiments and co-authored the manuscript. KB designed and performed the fatty acid desaturase experiments, guided the raffinose synthase experiments, and co-authored the manuscript. Both authors approved the final manuscript.


M). In each experiment, cells were irradiated using a continuous wave (cw)-diode laser . After laser irradiation, cell viability of dark control and of cells incubated with 500 μM ICG was 1.27 ± 0.11 or 0.28 ± 0.05 respectively. Using 100 μM ICG and D2O, cell viability was further decreased from 0.46 ± 0.03 (H2O) to 0.11 ± 0.01 (D2O). Using D2O and 100 μM ICG, the concentration of malondialdehyde, a marker of lipid peroxidation, increased from 0.89 ± 0.10 nmol 10−6 cells to 11.14 ± 0.11 nmol 10−6 cells. Using 100 μM ICG and laser irradiation sodium azide or histidine (50 mM), quenchers of singlet oxygen reduced the cell killing significantly. In contrast, when using mannitol, a quencher of superoxide anion and hydroxyl radical, cell killing was not inhibited. According to the present results, photoactivated ICG seems to kill colonic cancer cells due to the generation of singlet oxygen and the subsequent formation of lipid peroxides. Therefore, ICG might present a promising photosensitizer for PDT; first clinical results confirm these findings. © 1999 Cancer Research CampaignDespite of the approval of Photofrin® in various countries, chemically defined sensitizers for photodynamic therapy (PDT) are still needed for the absorption of light in the infrared spectrum, which provides a maximal penetration of light into tissue. Therefore, both the efficacy and the mechanism of action of the clinically approved dye indocyanine green (ICG) and laser irradiation were investigated in vitro. For the investigation of phototoxic effects, HT-29 cells were incubated 24 h prior to irradiation by using different concentrations of ICG (10–500 μ


The high occurrence of basic residues that characterize this isoform suggests however that it could feature specific regulation by HS.CXCL12α, a chemokine that importantly promotes the oriented cell migration and tissue homing of many cell types, regulates key homeostatic functions and pathological processes through interactions with its cognate receptor (CXCR4) and heparan sulfate (HS). The alternative splicing of the Using surface plasmon resonance and NMR spectroscopy, as well as chemically and recombinantly produced chemokines, we show here that CXCL12γ first 68 amino acids adopt a structure closely related to the well described α isoform, followed by an unfolded C-terminal extension of 30 amino acids. Remarkably, 60 % of these residues are either lysine or arginine, and most of them are clustered in typical HS binding sites. This provides the chemokine with the highest affinity for HP ever observed (Kd = 0.9 nM), and ensures a strong retention of the chemokine at the cell surface. This was due to the unique combination of two cooperative binding sites, one strictly required, found in the structured domain of the protein, the other one being the C-terminus which essentially functions by enhancing the half life of the complexes. Importantly, this peculiar C-terminus also regulates the balance between HS and CXCR4 binding, and consequently the biological activity of the chemokine.Together these data describe an unusual binding process that gives rise to an unprecedented high affinity between a chemokine and HS. This shows that the γ isoform of CXCL12, which features unique structural and functional properties, is optimized to ensure its strong retention at the cell surface. Thus, depending on the chemokine isoform to which it binds, HS could differentially orchestrate the CXCL12 mediated directional cell kinesis. CXCL12, also known as SDF-1 , belongs to the growing family of chemokines, a group comprising some fifty low molecular weight proteins, best known to mediate leukocyte trafficking and activation 10 helix, a three stranded β-sheet and a C-terminal α-helix. Up to recently, two CXCL12 isoforms, arising from the alternative splicing of a single gene From a structural view point, CXCL12 has a typical chemokine fold stabilized by two disulfide bonds: it consists of a poorly structured N-terminus of 10 residues, followed by a long loop, a 3In vivo data support the view that, within tissues, CXCL12 is sequestered by HS It has become clear that biological information required to run the chemokine system is not only stored in the sequences of the proteins involved, but also in the structure of a class of polysaccharide called glycosaminoglycans (GAGs), in particular heparan sulfate (HS), to which most chemokines bind CXCL12α binding to HS critically involves amino acids K24 and K27, which together with R41 form the essential part of the HS-binding site CXCL12γ cDNA, obtained from Balb/C mouse brain mRNA was cloned and over expressed in E. coli, purified to homogeneity, and characterized by mass spectrometry, NMR and amino acid analysis. The preparation routinely yielded 4–5 mg of purified protein per liter of bacterial culture. Wild type and mutants CXCL12α, β and γ, ) of partially aligned molecules in dilute liquid crystal CXCL12α structure has been solved both by X ray crystallography es TALOS . The folN RDCs, in the 10–64 domain of CXCL12γ were analyzed against CXCL12α, 33 of them showed an overall good correlation (15N-1H NOes) were highly similar to those observed for monomeric CXCL12α 1H dimension, suggesting they were poorly ordered in solution  real time monitoring was exploited to measure changes in refractive index caused by the binding of chemokines to each of the immobilized GAGs.eq/C against Req for different concentrations of chemokine , straight lines were obtained (data not shown) which slopes, corresponding to the equilibrium constant Kd, are reported in Binding curves, obtained when the CXCL12 isoforms were flowed over the HP, HS and DS surfaces, showed marked differences . These e15N-CXCL12γ with different HP derived di-(dp2), tetra-(dp4) and octa-(dp8) saccharides. The CXCL12γ/oligosaccharide interactions were in fast exchange regime compared to NMR chemical shift timescale, typical of interactions in the µM-mM Kd range. Interaction with dp4 reached saturation, with an apparent Kd of about 250 µM. Several resonances in the γ extension were highly perturbed upon interaction  indicated however a significant decrease in mobility upon dp4 binding for the γ extension with positive NOe values for residues 82 to 89. A maximum NOe value around 0.2 for Q87 (data not shown) suggested nevertheless that, even in complex with HP derived oligosaccharides, the γ extension still exhibits important flexibility.Interactions of dp2, dp4 and dp8 with CXCL12γ revealed two binding domains on the protein . On the off) were thus measured at the beginning of the dissociation phase (where rebinding is limited because the number of free immobilized GAGs remains low) and the on rates (kon) were then calculated using the equilibrium dissociation constant (kon = koff/Kd). Results are indicated in off of 0.0019 M−1s−1 compared to 0.111 M−1s−1 for CXCL12α and 0.0204 M−1s−1 for CXCL12β. In agreement with these observations, mutations of the 3 basic residues present at the C-terminus of the β isoform (β-m1) did not change the on rate, but increased the off rate to a value of 0.098 M−1s−1, thus resulting in a behavior very close to that of CXCL12α  of 6 seconds, while CXCL12γ/HP complex was characterized by a half life of 350 seconds , which do not express detectable amount of GAGs (data not shown), enabling the strict analysis of CXCL12/CXCR4 interaction. Results showed that CXCL12α and γ, although featuring identical receptor binding domain , behaved differently, the latter showing a reduced ability to bind to CXCR4, with an IC50 of 350 nM versus 15 nM for CXCL12α. This difference clearly relied on the C-terminal domain of CXCL12γ, since specific mutations within this domain (γ-m1) restored binding to a level comparable to that of CXCL12α  expression vector between NdeI and SpeI restriction sites, and checked by DNA sequencing. CXCL12γ was overexpressed overnight in E. coli BL21 (DE3) cells, with 0.4 mM IPTG, either in LB or M9 minimal medium supplemented with 15NH4Cl and 12C or 13C- glucose for isotopic enrichment. After 30 minutes of sonication at 4°C in 50 mM Tris pH 8.0 (buffer A), inclusion bodies were pelleted (20000g for 15 minutes) and washed with buffer A supplemented with 2M urea and 5 % Triton X100, then with 2 M urea and finally with buffer A. Inclusion bodies were solubilised for 15 min at 50°C in buffer A with 7.5 M GdCl2 and 100 mM DTT. Refolding was performed by rapid dilution with buffer A up to 1 M GdCl2. The mixture was gently stirred overnight at 4°C after addition of Complete protease inhibitors (Roche), then diluted 4 times with buffer A and loaded onto a 3 ml Source S column (Amersham) equilibrated in 20 mM Na2HPO4 pH 6.0. CXCL12γ was eluted with a NaCl gradient, concentrated and further purified on a G75 gel filtration column (Amersham) in 20 mM Na2HPO4, 150 mM NaCl pH 6.0. Purified material was analyzed by MALDI mass spectrometry and quantified by amino acids analysis. Wild type and mutants CXCL12α, β and γ were also produced by chemical synthesis, using the Merrifield solid-phase method and fluorenylmethyloxycarbonyl chemistry, as described Murin Porcine mucosal HP was depolymerized with heparinase I. The digestion mixture was resolved from di-(dp2) to octa-(dp18) decasaccharide, and dp2 to dp8 were further purified by strong-anion-exchange HPLC as described 15N-13C sample in 20 mM NaH2PO4 pH 5.7, 10% D2O, 0.01% NaN3 with protease inhibitors at 600 MHz. HNCACB, CBCA(CO)NH and HNCO, 15N-1H NOes and T2 experiments were from Varian Biopack and T1 experiment from 2 and 10 and 180 ms for T1. RDCs were measured as the difference between isotropic (25°C) and anisotropic (34°C) IPAP experiments 15N-CXCL12γ in the NMR buffer.NMR experiments were recorded at 30°C on Varian spectrometers , processed with NMRpipe and analyzed with NMRview. CXCL12γ backbone assignment and relaxation experiments were recorded on 1 mM Size defined HP (6 kDa), HS and DS were biotinylated at their reducing end, and immobilized on a Biacore sensorchip. For this purpose, flow cells of a CM4 sensorchip were functionalized with 2500 to 2800 resonance units (RU) of streptavidin as described 7 cells/ml) were incubated with 0.25 nM of 125I-CXCL12α  and a range of concentrations of unlabelled CXCL12  in 100 µl of PBS for 1h at 4°C. Incubations were stopped by centrifugation at 4°C. Cell pellets were washed twice in ice-cold PBS, and the associated radioactivity was counted. For measuring the ability of CXCL12 to interact with cellular HS, the CXCR4 negative CHO-K1 or HS-deficient CHO-pgsD677 (ATCC) were incubated with the ckemokine and after removal of unbound proteins, were labelled with an anti-CXCL12 mAb (clone K15C) and a PE-conjugated secondary antibody. Immunolabelled cells were analysed by flow cytometry using a FacsCalibur (BD Biosciences).CEM cells  with 0.1% of pluronic acid and 0.5 mM of Fluo4-AM (106 cells/mL). After a washing step, cells were suspended in load buffer at a final concentration of 2×106 cells/mL and stored at 4°C. For intracellular calcium measurements, aliquots of cells (2×105 cells) were preincubated at 37°C for 1 min, then placed in a 96-well flat bottom plate. Fluorescence emission was recorded at 535 nM (excitation at 485 nM) every second before  and after programmed injection of different concentration of the ligands. Maximum and minimum fluorescence values were determined after addition of Triton X-100 and EDTA, respectively. Data are expressed as fluorescence increment rate after ligand addition.Intracellular calcium measured in CXCR4-expressing cells loaded with fluo-4-AM (Interchim) was conducted in a Mithras LB 940 counter (Berthold Technologies). Briefly, A3.01 cells were incubated for 45 min at 37°C in the load buffer (10 mM Hepes, 137.5 mM NaCl, 1.25 mM CaCl


Etanercept, a TNF receptor 2-Fc fusion protein, is currently being used for the treatment of rheumatoid arthritis (RA). However, 25% to 38% of patients show no response which is suspected to be partially due to insufficient affinity of this protein to TNFα. By using computational protein design, we found that residue W89 and E92 of TNFR2 were critical for ligand binding. Among several mutants tested, W89Y/E92N displayed 1.49-fold higher neutralizing activity to TNFα, as compared to that of Etanercept. Surface plasmon resonance (SPR) based binding assay revealed that the equilibrium dissociation constant of W89Y/E92N to TNFα was 3.65-fold higher than that of Etanercept. In a rat model of collagen-induced arthritis (CIA), W89Y/E92N showed a significantly better ability than Etanercept in reducing paw swelling and improvement of arthritic joint histopathologically. These data demonstrate that W89Y/E92N is potentially a better candidate with improved efficacy in treating RA and other autoimmune diseases. Rheumatoid arthritis (RA) is a frequently occurring, chronic, debilitating disease. TNFα plays a pivotal role in regulating its inflammatory response. TNFα inhibition with TNF receptor 2-Fc fusion protein (TNFR2-Fc) was effective in the treatment of RA. However, quite a few patients may not achieve good clinical outcomes after TNFR2-Fc therapy, and the high dosage of TNFR2-Fc in clinical treatment usually causes some side effects, such as injection site reactions. Enhancing the affinity of TNFR2-Fc to TNFα would be of benefit to its therapeutic effect on RA, and may reduce the clinical dosage. We modeled the interactions of TNFα and TNFR2, and figured out the sites that may be critical for ligand-receptor binding. A high affinity TNFR2-Fc variant (E92N/W89Y) was obtained by rational mutagenesis at residue 89 and 92. It shows significant improvements compared with wild type TNFR2-Fc in suppressing rat arthritis induced by collagen. This variant is more potent in neutralizing TNFα, and thereafter may offer a higher degree of RA symptom relief, and be in a much lower dosage. Overproduction of TNFα is an underlying mechanism of autoimmune diseases, including RA, ankylosing spondylitis and psoriatic arthritis in vitro and in vivo assays. Here we report the design and characterization of this high affinity TNFR2-Fc variant, and focus on evaluating its therapeutic effect of neutralizing activity of TNFα on RA.We developed some higher affinity TNFR2-Fc variants by computational protein design method. Since the structures of TNFR2-TNFα complex and TNFR2 were absent, we chose 1a8m and 1tnr, the crystal structure of a TNFα variant and a complex of TNFR1 and TNFβ in protein data bank, as templates to model the interactions of TNFR2-TNFα. TNFα and TNFβ share high sequence identity and similar binding characters to two common receptors, TNFR1 and TNFR2, containing 4 highly conserved cysteine rich domains within the extracellular region We modeled the TNFR2-TNFα complex with the template 1a8m and 1tnr by the computer program Homology, a module within InsightII, the backbone of the TNFR1 in 1tnr was substituted by the corresponding residues of TNFR2, the construction of disulfide bonds was referenced to Banner's models Site-directed mutagenesis at amino acids 89 and 92 of TNFR2 was performed to determine the contribution of these residues to the ligand binding properties, and the effect of these variants on TNFα mediated cytotoxicity was characterized by the neutralizing activity assay. As shown in DK) of W89Y/E92N for TNFα showed a 3.65-fold decrease, which meant the affinity of W89Y/E92N to TNFα was enhanced. The binding kinetics indicates that the ligand binding mode of W89Y/E92N might differ from that of Etanercept. Specifically, the dissociation kinetic constant (dk) of W89Y/E92N for TNFα (1.29×10−6 s−1) was markedly lower than that of Etanercept (4.1×10−6 s−1); while the association kinetic constant (ak) of W89Y/E92N (9.72×103 M−1s−1) for TNFα was only a little higher than that of Etanercept (8.45×103 M−1s−1). Therefore, we identify W89Y/E92N a higher affinity TNFR2-Fc variant, and take notice of its lowered dissociation rate to TNFα compared to Etanercept, which may improve its pathologic effect in vivo.The binding kinetics data of the W89Y/E92N and TNFR2 was obtained using the SPR technique . Comparein vivo effect of W89Y/E92N and Etanercept was evaluated by employing a rat model of CIA, and the paw swelling of rats was monitored throughout the period of arthritis by drainage method The The histopathologic changes of paw joints following W89Y/E92N and Etanercept treatment in rats suffering from CIA was shown in Computational protein design method has been demonstrated to represent a valuable tool for the improvement and modification of protein-protein interactions in vivo.The extracellular part of TNFR2 shared about 30% sequence identity with TNFR1 and the alignment contains a long insertion and some deletions between the CRD3 to CRD4 (data not shown), it was difficult to model the whole structure of TNFR2 with TNFR1 accurately. However, The CRD2 and CDR3 of TNFR2 showed a considerable degree of sequence identity with TNFR1 (∼50%), consequently it was decided to model TNFR2 with the backbone of TNFR1 in 1tnr, and focus on the specific interactions of CDR2 and CDR3 of TNFR2 with its ligands. The TNFR2-TNFα model showed that residue 89 and 92 of TNFR2 may affect its binding to TNFα. The following site-directed mutagenesis found that residue substitution at these points could obtain potential candidates. We selected the variant W89Y/E92N for further evaluation, which displayed improved anti-TNFα activity in vitro. Consistent with results from neutralizing activity assays, we found that W89Y/E92N variant had higher affinity to TNFα than Etanercept, and had better pathologic improvement on experimental RA in vivo for its lowered dissociation rate compared with Etanercept (1.29×10−6 s−1 versus 4.10×10−6 s−1).Etanercept is known to have fast rates of association and dissociation with TNFα. It releases TNFα within approximately 10 minutes, and again binds TNFα immediately in vivo improvement of W89Y/E92N may considerably ascribe to its high affinity, as the pharmacokinetics profile of W89Y/E92N was comparable to that of Etanercept (data not shown). The fact that only some RA patients (about 40% Etanercept patients receiving 25 mg Etanercept twice weekly for 24 weeks and 38.9% of patients receiving MTX-6 mg/kg-Infliximab for 54 weeks) W89Y/E92N exhibited significant improvement in suppressing rat arthritis induced by collagen, presented lower paw swelling after two weeks treatment than the same dosage of Etanercept and better joint histopathology amelioration. The The crystal structure of TNFβ in complex with the TNFR1 is known as 1tnr in protein data bank (PDB) 5′- aagcttATGGCTCCCGTCGCCGTCTGGG) comprising Hind III restriction site prior to the ATG start codon and a 24- to 36-mer reverse primer within TNFR2-Fc coding region containing the desired mutation, another pair was the forward primer complementary to the above reverse primer and the reverse primer (5′-gaattcctatttacccggagacaggg) comprising EcoRI restriction site at the end of Fc coding sequence. The PCR products of TNFR2-Fc mutants were digested with the Hind III and EcoRI, and inserted into the pcDNA3 (Invitrogen).Amino acid substitutions of TNFR2-Fc were generated by PCR-based site-directed mutagenesis E. coli strain Top10F' and extracted by a commercial purification kit (Qiagen) following the manufacturer's instructions. CHO-K1 cells were transfected with a linear vector encoding a TNFR2-Fc variant using lipofectamine 2000 based protocol (Invitrogen). Stable transfectants were selected by culturing in a medium containing Geneticin  and selected clones were used for TNFR2-Fc protein production. The proteins were purified by Protein A affinity chromatography. The purity of recombinant proteins was analyzed by SDS-PAGE, and the protein concentrations were determined by Bradford method Plasmids were amplified in the 4 cells/well in 100 µL DMEM supplemented with 10% FBS. The cells were preserved at 37°C for 18h. 0.1ml 20units/ml TNFα (Sigma) in present of 2 µg/ml actinomycin D (Sigma) was added per well, and then the cells was incubated with gradient concentrations of Etanercept (Amgen), TNFR2-Fc mutants or medium control for 24h at 37°C. Cell viability was measured by using a crystal violet staining method Mouse L929 cells were seeded into 96-well microtiter plates at a density of 1.5×10The binding kinetics of the Etanercept and W89Y/E92N for TNFα were analyzed by the surface plasmon resonance technique . Etanercept and W89Y/E92N were immobilized onto a CM5 sensor chip (G.E. Inc.), which resulted in an increase of 5000–5500 resonance units (RU). During the association phase, TNFα diluted in running buffer  surfactant P20, pH 7.4, Sigma) at 20.0, 10.0, 5.0, 2.5, 1.25, 0.625, 0.3125 nM, was allowed to pass over the chips immobilized with Etanercept and W89Y/E92N, respectively, at a flow rate of 40 µl/min for 1 min. During the dissociation phase, HBS-EP buffer was applied to the sensor chip at a flow rate of 40 µl/min for 2 min. The data were analyzed globally with the BIAEVALUATION 3.0 software (BIAcore®) using a 1∶1 Langmuir binding model in vivo, a CIA assay was performed as described To assay for anti-TNFα inflammation activity


To the Editor: Recently, Longtin et al. .2, 250 nmol/L primers, double-distilled H2O, and 2 μL of DNA (50–200 ng) cycled on an ABI PRISM 7900HT  instrument at 94oC for 2 min, followed by 40 cycles of 94°C for 20 s, 60°C (NP1 primers) or 68°C (VP1/VP2 primers) for 20 s, and 72°C for 14 s. Amplification and melting curves were analyzed with 7900HT 2.2.1 software (Applied Biosystems); positive samples were verified by sequence analysis. Sequenced VP- and NP1-generated amplicons were 99%–100% identical to HBoV strain ST1  of the 91 children who underwent elective tonsillectomy/adenoidectomy. Ages ranged from 1.9–4.6 years, with a median age of 3.4 years. The reason for the much lower HBoV prevalence in this group of children, compared with prevalences found in studies by Longtin et al.  than the Lu group  or our group . Preliminary seroepidemiology reports indicate the presence of HBoV antibodies in >50% of children by 2–3 years of age (The detection of HBoV in the tonsillar tissues we tested showed a higher rate of infection than would be expected in an asymptomatic population. However, the rate was far lower than that previously reported for tonsillar tissues (


CRYGC in a Chinese family with autosomal dominant congenital nuclear cataracts and microcornea.To report the identification of a novel nonsense mutation in We investigated a four-generation Chinese family with six members affected with nuclear cataracts and microcornea. The family resides in a relatively isolated region of northern China. Genomic DNA was isolated from blood leucocytes, genotyping was performed using more than 100 microsatellite markers for the known cataract candidate gene loci, and LOD scores were calculated using the LINKAGE programs. Mutations were detected by DNA sequence analysis of the candidate genes.CRYGA-CRYGD) on chromosome 2q32.3-q35. Direct sequencing of the candidate CRYGA-CRYGD gene cluster revealed a c.470G>A transversion in exon 3 of CRYGC, which cosegregated with cataracts in the family and was not observed in 100 normal controls. This single nucleotide change was predicted to introduce a translation stop codon at tryptophan 157 (W157X).Evidence for linkage was detected at marker D2S325 , which closely flanks the γ-crystallin gene cluster (CRYGC associated with autosomal dominant cataracts and microcornea in a Chinese family. Our finding expands the spectrum of CRYGC mutations associated with congenital cataract and confirms the role of γ-crystallin in the pathogenesis of congenital nuclear cataracts.The present study has identified a novel nonsense mutation in  Congenital cataract is a clinically and genetically heterogeneous lens disorder that typically appears as a sight-threatening trait in childhood and accounts for one-tenth of the cases of childhood blindness . The preCRYAA [CRYAB [CRYBB1 [CRYBB2 [CRYBB3 [CRYBA1/A3 [CRYBA4 [CRYGC [CRYGD [CRYGS [GJA8 [GJA3 [MIP [LIM2 [BFSP1 [BFSP2 [PITX3 [MAF [HSF4 [FOXE3 [EYA1 [CHX10 [PAX6 [There are currently 34 loci implicated in nonsyndromic congenital cataract, and of these, over 20 have been associated with mutations in specific genes [CRYAA ,6, CRYABA [CRYAB ,8, CRYBB [CRYBB1 ,10, CRYB [CRYBB2 -14, CRYB [CRYBB3 , CRYBA1/RYBA1/A3 ,17, CRYB [CRYBA4 , CRYGC [4 [CRYGC -23, CRYGC [CRYGD ,21,24-26D [CRYGS ; (2) twoGS [GJA8 ,29, and A8 [GJA3 ,31; (3) JA3 [MIP  and LIM2IP [LIM2 ,34; (4) 2 [BFSP1  and BFSP1 [BFSP2 -38; and 2 [PITX3 , MAF [40TX3 [MAF , HSF4 [4AF [HSF4 , FOXE3 [4 [FOXE3 , EYA1 [4E3 [EYA1 , CHX10 [1 [CHX10 , and PAX10 [PAX6 .Crystallins are the dominant structural components of the vertebrate eye lens. At least 11 crystallin genes encode over 95% of the water-soluble structural proteins present in the crystallin lens, representing more than 30% of its mass and accounting for its optical transparency and high refractive index . TherefoCRYGC on 2q.In this study, we performed linkage analysis of a four-generation Chinese family with autosomal dominant nuclear cataract and microcornea, and we identified a novel nonsense mutation in We identified a four-generation Chinese family with autosomal dominant nuclear cataracts and microcornea in the absence of other ocular or systemic abnormalities. The family resides in a relatively isolated region of northern China. Informed consent in accordance with the Declaration of Helsinki was obtained from all participants. Thirteen members participated in the study, six affected and seven unaffected family members . Affecte2, 0.2 mM dNTPs, 0.3 U HotStar Taq DNA polymerase, and 0.05 µM microsatellite markers. After an initial denaturation period of 12 min at 95 °C, 14 cycles were performed at 95 °C for 30 s, 63–56 °C for 30 s (with a 0.5 °C decrease at each step), and 72 °C for 1 min. Thirty cycles were performed at 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min followed by an extension at 72 °C for 10 min and a final hold at 4 °C. The PCR products were pooled on the basis of size , denatured at 95 °C for 1 min, and electrophoresed in a 96 capillary automated DNA analysis system . The results were analyzed by Genetic Profiler version 1.5 (Amersham). Two point LOD scores (Z) were calculated using the LINKAGE software package (version 5.1). A gene frequency of 0.0001 and a penetrance of 100% were assumed for the cataract locus. LOD scores were calculated at recombination fractions (θ) of 0.00, 0.05, 0.1, 0.2, 0.3, and 0.4.We conducted a genome wide linkage scan based on a set of dinucleotide repeat microsatellite markers spaced at approximately 10 cM intervals using an ABI PRISM Linkage Mapping Set version 2.5 . “Touchdown” polymerase chain reaction (PCR) was performed in a 5 μl reaction volume containing 20 ng of genomic DNA, 1 µl of 10X PCR buffer, 7 mM MgClCRYGA-CRYGD) and two pseudogenes (CRYGE-CRYGF) with in-frame translation stop codons are clustered within the physical region defined for the cataract on 2q33–35. We systematically sequenced all four functional CRYG genes, each comprising three exons and two introns, in two affected and two unaffected members of the family using specific primers  and 100 unrelated normal individuals were screened.Four functional candidate genes  of 2.29 was obtained at marker D2S325 with recombination θ=0.00. The adjacent markers  also showed LOD scores greater than 1.0. The results of the two-point LOD scores are summarized  on 2q33.3. Further analysis of the marker showed a positive LOD score at the recombination fraction of 0.00 and strongly supported this candidate region. The maximum two-point LOD score . The cosegregation of the c.470G>A transition in only affected members of the pedigree and its absence in 100 normal control individuals strongly suggested that the W157X substitution was a causative mutation rather than a benign single nucleotide polymorphism (SNP) in linkage disequilibrium with the cataract  makes up a distinct phenotype within the group of autosomal dominant congenital cataracts. CCMC has been considered a rare phenotype. The frequency of CCMC may be underestimated because the focus is often on the sight-threatening cataract and corneal diameters may pass unnoticed. Hansen et al. [The association of developmental cataract and microcornea  [CRYGC-related opacities involve the nucleus of the lens. This clinical manifestation is in agreement with the function of CRYGC, which is abundantly expressed at an early developmental stage in elongating fiber cells. CRYGC is expressed primarily in the lens nucleus, which is consistent with the location of the opacity in the nucleus.Herein, we report the identification of a novel nonsense mutation (W157X) in processed ts (T5P) , variablts (T5P) , lamellats (T5P) , nuclearts (T5P) , and nucts (T5P) . Therefo2-terminal extensions, and COOH-terminal extensions. γ-Crystallins are a homogeneous group of highly symmetric, monomeric proteins. They are differentially regulated after early development. Four γ-crystallin genes  and two pseudogenes (CRYGE and CRYGF) are tandemly organized in a genomic segment as a gene cluster.Crystallins constitute the major proteins of the vertebrate eye lens, and they maintain the transparency and refractive index of the lens. Because lens central fiber cells lose their nuclei during development, these crystallins are made and then retained throughout life, requiring them to be extremely stable proteins. Mammalian lens crystallins are divided into α-, β-, and γ-crystallin families. β- and γ-crystallins are also considered a superfamily. Seven protein regions exist in β-crystallins: four Greek key motifs, a connecting peptide, NHCRYGC, the W157X mutation lies in the COOH-terminal domain at the end of the fourth Greek key motif  but also reduces protein solubility and stability [CRYGC-associated cataracts.Based on the crystal structure of human ey motif . Howevertability . Furthertability . How the2-terminal domain, and motifs 3 and 4 are located in the COOH-terminal domain [Alignment of the CRYGC protein sequence from three different species with three human γ-crystallins revealed that the tryptophan residue at position 157 is highly conserved . To furtl domain ,52. The CRYGC has also been reported in the mouse [Crygc) is causative for the cataract phenotype. The mutation is therefore designated CrygcChl3. The deletion of bases 420−425 leads to a loss of two amino acid residues, Gly and Arg, in the fourth Greek key motif.It is noteworthy that an autosomal dominant nuclear and radial cataract caused by a 6 bp deletion in the he mouse . A 6-bp CRYGC associated with autosomal dominant cataracts and microcornea in a four-generation Chinese family. The association between microcornea and congenital cataracts in all affected individuals indicate that mutations disrupting lens biochemistry and physiology early in development can result in microphakia and subsequent microcornea as a secondary effect of damage to the lens.In summary, the present study has identified a novel nonsense W157X mutation in


Cytochrome P450 2D6 (CYP2D6) has a crucial role in the metabolic conversion of tamoxifen into the active metabolite endoxifen. In this cohort study, the effect of CYP2D6-predicted phenotype, defined as the combined effect of CYP2D6 genetic variation and concomitant use of CYP2D6-inhibiting medication, on time to breast cancer progression (TTP) and overall survival (OS) in women who use tamoxifen for metastatic breast cancer (MBC) was examined.CYP2D6*3, *4, *5, *6, *10 and *41) was available. Patient charts (n=102) were reviewed to assess TTP and OS, and to determine whether CYP2D6 inhibitors were prescribed during tamoxifen treatment.We selected patients treated with tamoxifen (40 mg per day) for hormone receptor-positive MBC from whom a blood sample for pharmacogenetic analysis . Co-administration of CYP2D6 inhibitors alone was also associated with a worse OS  and TTP  compared with patients without CYP2D6 inhibitors.OS was significantly shorter in patients with a poor CYP2D6 metaboliser phenotype, compared with extensive metabolisers (HR=2.09; CYP2D6 phenotype is an important predictor of treatment outcome in women who are receiving tamoxifen for MBC. Co-administration of CYP2D6 inhibitors worsens treatment outcome of tamoxifen and should therefore be handled with care. Breast cancer is the most common type of cancer among women worldwide with nearly 1.2 million new diagnoses each year .N-desmethyl-tamoxifen (NDM-TAM), 4-hydroxy-tamoxifen (4-OH-TAM) and finally the more potent metabolite 4-hydroxy-NDM-TAM(endoxifen). Both 4-OH-TAM and endoxifen are known to have a 30–100 times higher anti-oestrogen activity than tamoxifen. However, on average, plasma concentrations of endoxifen are approximately 10 times higher than those of 4-OH-TAM, making endoxifen the most potent metabolite of tamoxifen . Patients with one functional and one non-functional allele or two decreased activity alleles are referred to as intermediate metabolisers (IMs). Individuals with two non-functional alleles of the gene lack CYP2D6 enzymatic activity and, therefore, are classified as poor metabolisers (PMs). In Caucasians, CYP2D6*4 allele is the most common non-functional allele, having an allele frequency of about 20%  using tamoxifen tend to have a higher risk of breast cancer recurrence than EMs. This finding was confirmed by *4, *5, *10 and *41 had an increased risk of breast cancer recurrence and worse relapse-free survival rates. More recently, a multicenter study including retrospectively and prospectively collected patient data also confirmed an association between CYP2D6 variation and clinical outcome in women receiving adjuvant tamoxifen , which may be used in breast cancer patients to treat depression or hot flashes; a common side effect of tamoxifen (CYP2D6*10/*10 (commonly occurring in the Eastern people in contrast to Caucasians) with clinical outcome , and receiving a tamoxifen dose of 40 mg per day. In The Netherlands, 40 mg per day is the standard tamoxifen dose for MBC patients compared with 20 mg per day in the adjuvant setting. As the terminal elimination half-life of tamoxifen for a single dose is 5–7 days, and the time to reach steady state plasma concentrations is 3–4 weeks, an observational period of 1 month may be necessary before the effect of hormonal therapy can be seen , whichever came first. Time to progression was defined as the time from first tamoxifen prescription for MBC to the documentation of progression, which was assessed by standard RECIST criteria , duloxetine and terbinafine (moderate inhibitors), amiodarone, cimetidine, citalopram and sertraline  .CYP2D6*3, *4, *5 and *6 polymorphisms, which will detect over 95% of CYP2D6 PMs and also for the CYP2D6 polymorphisms *10 and *41 that are associated with reduced enzyme activity. The source of genomic DNA was EDTA blood and the genotyping was done using Taqman allelic discrimination assays on the ABI Prism 7000  Sequence detection system. Primers and probes were designed by Applied Biosystems using their Assay-by-Design service. Polymerase chain reactions (PCR) were carried out in a reaction volume of 10.0 μl, containing assay-specific primers, allele-specific Taqman MGB probes (Applied Biosystems), Abgene Absolute QPCR Rox Mix (Applied Biosystems) and genomic DNA (1 ng). The thermal profile consisted of an initial denaturation step at 95°C for 15 min, followed by 40 cycles of denaturation at 92°C for 15 s and annealing and extension at 60°C for 1 min. The *5 was determined using long range PCR on 20 ng genomic DNA using primers 5′-CACACCGGGCACCTGTACTCCTCA-3′, 5′-CAGGCATGAGCTAAGGCACCCAGAC-3′, 5′GTTATCCCAGAAGGCTTTGCAGGCTTCA-3′ and 5′-GCCGACTGAGCCCTGGGAGGTAGGTA-3′ with PCR profile 7 min 94°C, followed by 35 cycles of 1 min 94°C, 1 min 65°C annealing and 5 min 68°C elongation using rTth DNA polymerase (Applied Biosystems). PCR products were analysed on an EtBr gel. Presence of a CYP2D6 gene deletion (*5) can be observed by the presence of a 3.5 kb fragment. A 5.1 kb fragment will always be amplified, and serves as an internal control. Genotypes were scored through measuring allele-specific fluorescence using the SDS 2.2.2 software for allelic discrimination (Applied Biosystems).All patients were genotyped for the CYP2D6*3, *4, *5 or *6) allele and who were not using a CYP2D6 inhibitor for at least 6 months (or until tamoxifen was stopped) were defined as EMs. Intermediate metabolisers (i) carry CYP2D6*10 or *41 alleles either homozygous or in combination with a dysfunctional allele or (ii) were heterozygous for the CYP2D6*3, *4, *5 or *6 allele  and did not use a CYP2D6 inhibitor or (iii) had no dysfunctional alleles but were using a weak or moderate CYP2D6 inhibitor. Women classified as PMs had (i) two dysfunctional alleles , or (ii) one dysfunctional allele  with concurrent use of a moderate CYP2D6 inhibitor or (iii) a functional genotype (wt/wt) with co-administration of a strong CYP2D6 inhibitor  on TTP and OS was assessed using Cox proportional hazards models. Analyses were adjusted for age. Confounders were adjusted for in the analysis if they caused a change in the point estimate of more than 10 percent. Kaplan–Meier estimates and log-rank test were used in univariate analysis of TTP and OS. The 5% cut-off level was chosen as significance level. All analyses were carried out using SPSS software .Deviations from Hardy–Weinberg equilibrium and differences in allele frequencies of the CYP2D6*3, *4, *5, *6, *10 and*41 alleles were 3.5, 21.7, 1.5, 1.0, 1.5 and 6.6%, respectively, which is in line with earlier published data . Co-administration of CYP2D6 inhibitors  occurred in 6.9% of patients .Of the 116 patients treated with tamoxifen for MBC enroled, 104 patients had a positive hormone receptor status. Two patients discontinued tamoxifen treatment within 30 days and were excluded from the analysis. The characteristics of the 102 patients treated with tamoxifen for MBC that met the inclusion criteria are described in hed data . GenotypOn the basis of CYP2D6-predicted phenotype definition, 48.5% of the patients were classified as EMs, 38.4% as IMs and 13.1% as PMs . PatientP=0.62; 95% CI: 0.50–1.50). However, OS was significantly shorter for the PMs compared with EMs . Although not significantly different, PMs tended to have a worse TTP  compared with EMs.In P=0.012) (vs 1.8 years (95% CI: 1.4–2.3), respectively, (P=0.089), as shown in As intermediate and EMs had a comparable TTP and OS, both were taken together in the Kaplan–Meier estimates. The median OS-time period for PMs was 5.0 years (95% CI: 4.1–5.9) compared with 7.9 years (95% CI: 6.2–9.5) for the other predicted phenotype groups, which is statistically significantly different (P=0.012) . For PMsP=0.49; 95% CI: 0.63–2.66 and HR=1.38; P=0.42; 95% CI: 0.63–2.99, respectively), the seven patients using tamoxifen together with a CYP2D6 inhibitor had a significantly worse TTP  and OS  compared with tamoxifen users without co-administration of CYP2D6 inhibitors  of genotype and/or co-medication on tamoxifen treatment outcome has been investigated in the specific group of hormone-sensitive MBC patients. Besides this, the FDA approved fixed dose of tamoxifen used (40 mg per day) contributed to a homogeneous selection of patients in this study. Most studies published focused at a tamoxifen dose of 20 mg per day. In our study, EM and IM are not significantly different in terms of OS or TTP, although differences between these groups have been found in other studies when the tamoxifen dose is 20 mg per day  have an increased risk of breast cancer recurrence and mortality , but for this study there is no reason to assume that these variables differ significantly between the three phenotype groups nor have influenced outcome . In addition, it would be of interest to further explore the role of plasma concentrations of tamoxifen metabolites  as predictors of therapeutic outcome  for the treatment of hormone receptor-positive MBC. As co-administration of CYP2D6-inhibiting medication seems to diminish the treatment effect of tamoxifen, this combination of drugs should be handled with care.