Highly pathogenic avian influenza (HPAI) H5N1 viruses have caused dramatic economic losses to the poultry industry of Vietnam and continue to pose a serious threat to public health. As of June 2008, Vietnam had reported nearly one third of worldwide laboratory confirmed human H5N1 infections. To better understand the emergence, spread and evolution of H5N1 in Vietnam we studied over 300 H5N1 avian influenza viruses isolated from Vietnam since their first detection in 2001. Our phylogenetic analyses indicated that six genetically distinct H5N1 viruses were introduced into Vietnam during the past seven years. The H5N1 lineage that evolved following the introduction in 2003 of the A/duck/Hong Kong/821/2002-like viruses, with clade 1 hemagglutinin (HA), continued to predominate in southern Vietnam as of May 2007. A virus with a clade 2.3.4 HA newly introduced into northern Vietnam in 2007, reassorted with pre-existing clade 1 viruses, resulting in the emergence of novel genotypes with neuraminidase (NA) and/or internal gene segments from clade 1 viruses. A total of nine distinct genotypes have been present in Vietnam since 2001, including five that were circulating in 2007. At least four of these genotypes appear to have originated in Vietnam and represent novel H5N1 viruses not reported elsewhere. Geographic and temporal analyses of H5N1 infection dynamics in poultry suggest that the majority of viruses containing new genes were first detected in northern Vietnam and subsequently spread to southern Vietnam after reassorting with pre-existing local viruses in northern Vietnam. Although the routes of entry and spread of H5N1 in Vietnam remain speculative, enhanced poultry import controls and virologic surveillance efforts may help curb the entry and spread of new HPAI viral genes. The emergence of highly pathogenic avian influenza (HPAI) H5N1 virus as a human pathogen occurred in 1997 in Hong Kong Special Administrative Region (SAR) A global effort is underway to control or eradicate H5N1 in poultry and prevent human exposure, both of which may also reduce the risk of pandemic emergence. The scientific rationale for these programs is provided by ecologic, virologic, epidemiologic, and immunologic studies. In particular, molecular and functional characterization of H5N1 viruses from poultry will help inform development and implementation of public health control measures involving diagnosis, immunization and antiviral drug therapy.The putative ancestor of the currently circulating H5N1 viruses is A/goose/Guangdong/1/96 (Gs/GD/1/96), named after the province in southern China which is also thought to be the epicenter of H5N1 In 2005, unprecedented outbreaks of H5N1 in wild migratory birds at Qinghai Lake in western China apparently facilitated its spread to more than 30 countries in Europe, the Middle East and Africa Although HA gene evolution merits much attention because of its central role in antigenic drift and interaction with the host, the remaining seven viral genes are also critical to inform disease control efforts as they contribute to antiviral drug susceptibility, host range and virulence, among other phenotypic properties Vietnam (VN) is one of the countries most affected by H5N1 outbreaks in poultry and has accumulated 106 laboratory-confirmed human cases since 2003. Recurring epizootics of H5N1 were reported in poultry from 2003 until 2005 The nationwide H5N1 surveillance program launched in 2004 by the Government of Vietnam and implemented by the National Centre for Veterinary Diagnostics (NCVD), in collaboration with the Vietnam Department of Animal Health (DAH) and DAH Regional Animal Health offices, involved the collection and analysis of 20,567 samples from 38 provinces between that year and May of 2007. A total of 2,111 positive specimens were detected by RT-PCR or real-time RT-PCR specific for H5 HA RNA . AlthougIn order to investigate the origin of the HA genes of H5N1 viruses isolated in Vietnam prior to May 2007, we first constructed their phylogeny. Six different HA clades were identified and designated according to the recently described nomenclature system for the highly pathogenic H5N1 viruses as well as by potential precursors: clade 0, HK97-like (HK/483/97); clade 1, HK821-like (Dk/HK/821/02); clade 2.3.2, E319-like (Dk/China/E319-2/03); clade 2.3.4, FJ584-like (Ck/Fujian/584/05); clade 3, GX22-like (Dk/GX/22/01); clade 5, F1-like (swine/Fujian/F1/01) ; Table 2The timing of H5N1 clade isolation from poultry indicated that some lineages were present for one year or less, while others continued to circulate in Vietnam for more than 3 years. For instance, following their first detection in 2003, clade 5 viruses circulated in Vietnam for approximately 1 year and were replaced by clade 1 viruses in 2004. The clade 1 viruses first detected in late 2003 continued to circulate until 2007. In addition, other viral clades co-circulated in the region in 2005; e.g., clade 0 and clade 2.3.2 .In 2006, there were very few outbreaks reported in Vietnam possibly due to increased use of poultry vaccines after August, 2005. Our surveillance detected two positive samples among 64 tested using real-time RT-PCR. However, no H5N1 viruses were isolated from these positive samples. A sharp decrease in poultry outbreaks throughout Vietnam during 2006 led to a transition into a passive mode of surveillance for H5N1 and accounted for the low number of samples collected for testing in comparison to other years . HoweverPhylogenetic analyses of the NA and the so-called “internal gene” segments  of all available H5N1 viruses from Vietnam (>300 viruses) were performed to establish the diversity of the influenza gene pool present in Vietnam. Phylogenetic trees for each gene segment revealed the potential precursor virus for each lineage of the NA and internal gene segments . Each liThe diverse gene segment lineages identified in H5N1 viruses isolated in Vietnam between 2001 and 2007 provide a large gene pool for reassortment. By combining the phylogenetic information from all the genes of each isolate, we were able to determine the number of viral genotypes present in Vietnam from 2001 to 2007 . As showIn order to trace the time and location of H5N1 genotype emergence and spread within Vietnam, we plotted the geographical location of each genotype onto a map of the country. While the specific collection sites of the VN1 genotype virus within Vietnam was not known, VN2 genotype virus, which contains all F1-like (clade 5) segments except M, was first detected in 2003. During 2003, both the VN2 and VN3 genotypes were detected only in northern Vietnam despite equal sampling of birds in southern Vietnam during the same time period. In 2004, however, VN3 genotype viruses were isolated in both southern and northern Vietnam Despite markedly lower levels of H5N1 detection in 2006, perhaps as a result of a successful vaccination campaign coupled with a decline in sampling due to a transition to passive surveillance in poultry, five new genotypes were detected in 2007 (VN6-VN9). Each of these genotypes contained the HA gene from the clade 2.3.4 viruses, which were introduced into Vietnam in 2007. In addition, they contained gene segments from other viruses isolated earlier in Vietnam, suggesting that these genotypes emerged locally from reassortment of the newly introduced clade 2.3.4 viruses with pre-existing ones. Reassortment yielding genotypes VN6, VN7, and VN9 appear to have occurred near the Hanoi area as the majority of viruses from these genotypes have been detected in this region of Vietnam . Of thesThe VN3 genotype was predominant throughout Vietnam and neighboring countries between 2003 and 2005. The large size of Vietnam's bird population and the high rates of infection in this period were conducive to short viral generation times and rapid evolution. This genotype continued to circulate in 2007, but it was isolated with lower frequency than the VN6 genotype in northern Vietnam . The phyTo complement the genetic analysis of HK821α and HK821β, we compared their amino acid sequences at positions with known functional roles. Interestingly, the receptor binding site of both HK821α and HK821β contained residue 123P (corresponding to residue 127 in H3 influenza A virus numbering) instead of 123S, which was found in the HK821P and FJ584-like virus sub-lineages A panel of ferret antisera to the WHO reference subtype H5N1 viruses  was used in hemagglutination inhibition assays with the 40 HPAI H5N1 viruses isolated during 2007. The homologous clade HI titers were at least four-fold higher than those in the heterologous reactions, indicating a strong correlation of genetic and antigenic characteristics. A/chicken/Fujian/584/2006-like (clade 2.3.4) and A/duck/Hong Kong/821/2002-like (clade 1) viruses had distinct properties, as illustrated in HPAI H5N1 viruses were first identified in Vietnam in 2001 and have since caused repeated poultry outbreaks throughout the country leading to dramatic economic losses to the nation's poultry industry. Early introductions of H5N1 in Vietnam (in 2001) led to only sporadic detection of the virus in domestic birds During the 2003 H5N1 avian influenza outbreaks in Vietnam, the predominant viruses identified belonged to the so-called Z genotype, which were first identified in China in 2002 (referred to as the VN3 genotype in this study) The VN3 genotype, which was predominate in northern and southern Vietnam throughout 2004 and 2005, has continued to circulate at least through the early part of 2007 despite introduction of other genotypes in 2005 and 2007 . The NA Four distinct genotypes containing clade 2.3.4 (FJ584-like) HA genes were identified in Vietnam after May 2007: VN6-VN9. Interestingly, these viruses were primarily collected in northern Vietnam and appear to be replacing the VN3 genotype in this part of the country . AdditioThe diversity of genotypes present in Vietnam also provides a pluripotent reservoir from which an epidemic or pandemic strain may arise. The discovery of four previously unidentified genotypes  confirms previous observations that reassortment between H5N1 viruses occurs frequently. Reassortment between clade 1 viruses (HK821-like) and clade 2.3.4 viruses (FJ584-like) and between clade 2.3.2 (GX4016-like) and clade 5 (F1-like) viruses suggests a high level of genetic compatibility between viruses with diverse parental genotypes. In addition, the emergence of three new genotypes  following the introduction of clade 2.3.4 viruses (VN6) in 2007, and the concurrent eclipse of VN3 in northern Vietnam suggests that these genotypes may have a selective advantage. One obvious possibility is the antigenic novelty of the clade 2.3.4 HA. Another example of the possible advantage of antigenic diversity is the expansion of HK821β-like viruses that emerged in southern Vietnam in 2007 . It remaAs noted in previous studies, some viruses may have spread bi-directionally between countries New genotypes were first identified in northern provinces of Vietnam and appear to have gradually spread to southern provinces via unknown mechanisms. The majority of isolates were collected from specific locations in Vietnam with high human population densities, especially in areas around Hanoi and Ho Chi Minh City, which also contain most of the country's industrial and backyard poultry flocks. Although sampling outside of these large cities was less intensive, it is possible that avian influenza transmission from northern to southern Vietnam occurs via direct poultry trade routes between major population centers in each region Inactivated H5N1 and H5N2 vaccines have been used in Vietnam since August of 2005 to control H5N1 viruses In summary, our studies of viruses isolated in Vietnam during 2001–2007 demonstrate that multiple viral introductions and reassortment events have occurred frequently in Vietnam leading to the emergence of at least four novel genotypes. Thus, disease prevention and control must be applied inside the country and at the borders, to prevent introduction of new HPAI H5N1 strains, which expand the H5N1 viral gene pool. Strengthening vaccination and associated surveillance programs in poultry would help decrease the current avian influenza virus genetic reservoir in Vietnam, and H5N1 viruses in particular, thus reducing the threat to public health.The National Centre for Veterinary Diagnostics  surveillance program was initiated in 2004 in collaboration with the Vietnam Department of Animal Health (DAH) and DAH Regional Animal Health offices to detect H5N1 infections in poultry. Collection sites included poultry farms, backyard flocks, and live bird markets in 38 provinces representing regions in both northern and southern Vietnam. Cloacal swab specimens were collected in tubes with virus transport medium. Swab samples, as well as sick or dead bird specimens, were submitted to NCVD and regional laboratories. Tissue samples collected from slaughtered sick birds included the bronchus, lung, kidney, spleen, and brain. Tissue specimens were placed immediately on ice in sterile plastic bags and later stored at −80°C. From 2001–2003, samples were screened for H5N1 virus by inoculation in 10–11 day old embryonated chicken eggs (ECEs) for virus amplification. Allantoic fluid from ECEs was tested for the presence of virus by performing a hemagglutination assay using chicken red blood cells. In 2004 and 2005, an influenza subtype H5 specific RT-PCR test was used to identify samples containing H5N1 viral sequences. Since 2006, real-time RT-PCR has been used to identify samples containing H5N1 RNA, which were then selected for inoculation into ECEs for isolation.http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm).Following transfer of swab samples and/or viral isolates to the Centers for Disease Control and Prevention, Atlanta, GA, highly pathogenic H5N1 viruses were isolated from positive samples by infection of either 10–11 day old ECEs or MDCK cells. A total of 158 H5N1 viruses were isolated from 2001 to May of 2007. RNA isolation and sequencing procedures were described previously In addition to the 158 isolates collected by NCVD and sequenced for this study , for theTo identify the potential precursor viruses for each Vietnam isolate, we applied our newly developed Gene In Network (GIN) method Phylogenetic inferences relied on Maximum Parsimony (MP) and Neighbor-Joining (NJ) methods using PAUP* 4.0 Beta Euclidean distances between antigens were calculated using these normalized HI data. Within the multidimensional scaling analysis, the s-stress convergence value was set to the default value 0.001, the minimum s-stress value was set to the default value 0.05, and the maximum iteration was set to 100.Antigenic characterization of HPAI H5N1 isolated in Vietnam during 2007 was performed using the hemagglutinin inhibition (HI) assay with ferret antisera to the WHO reference H5 subtype viruses, including A/Turkey/65-596/06, A/whooper swan/Mongolina/244/05, A/Indonesia/5/05×PR8, A/Indonesia/5/05, A/Anhui/1/05×PR8, A/chicken/Malaysia/935/06, A/Anhui/1/05, A/Japanese white-eye/Hong Kong/1038/06, A/Anhui/2/05, A/Vietnam/1203/04×PR8, A/Vietnam/1203/04, A/Vietnam/HN30408/05, and A/Thailand/16/04. The HI assay was performed with a starting serum dilution of 1∶5. The multidimensional scaling analysis was performed using SPSS 15.0  to analyze HI titers in order to correlate data between 2007 Vietnam H5N1 viruses and WHO H5N1 reference antigens. The HI data for each antigen-antiserum pair was normalized by dividing the maximum HI value for that antiserum. The pairwise Gene sequences generated during this study have been deposited into the GenBank database, and accession numbers can be found in Table S1Summary of the HPAI H5N1 viruses analyzed in this study.(0.86 MB PDF)Click here for additional data file.Table S2Number of H5N1 isolates collected and percentage of each identified genotype in Vietnam per year.(0.01 MB PDF)Click here for additional data file.Table S3Geographic origin of HPAI H5N1 viruses from Vietnam.(0.01 MB PDF)Click here for additional data file.Figure S1Phylogenetic analysis of the H5N1 highly pathogenic avian influenza viruses isolated in Vietnam. Figures A-H represent segments HA, NA, PB2, PB1, PA, NP, MP, and NS, respectively. Posterior probabilities and bootstrap values are given above and below branches. A red dot was marked beside each predicted precursor virus. Previously reported genotypes, such as A, B, C, D, E, G, X0, X1, X2, X3, Y, V, Z, and Z+ (3.38 MB PDF)Click here for additional data file.Figure S2Phylogenetic tree of the HA gene of HK821-like avian influenza viruses isolated from Vietnam. HK821-like viruses formed three sub-lineages: HK821P, HK821α, and HK821β. The tree was rooted by Dk/HK/821/02. The phylogenetic tree was constructed by Maximum Likelihood using GARLI version 0.951 by selecting GTR+I+G model from Modeltest 3.7. Posterior probabilities and bootstrap values were given above and below branches, respectively.(0.11 MB PDF)Click here for additional data file.


CYP1B1) in Chinese patients with primary congenital glaucoma (PCG).This study was conducted to investigate the mutation spectrum of the cytochrome P450 gene  and heteroduplex analysis-single strand conformation polymorphism (HA-SSCP) followed by subsequent cloning and bidirectional sequencing. New variants were confirmed by restriction fragment length polymorphism (RFLP) analysis in 80 normal Chinese controls.The coding regions of CYP1B1 mutations in two patients were homozygous, and the other four patients were compound heterozygous. Beyond the four novel mutations , two reported missense mutations (R469W and R390H) were also identified. The missense mutation, R390H, was involved in 9.8% (4/41) of patients in our study. None of the novel mutations was observed in any of the 80 controls.Six distinct mutations, four of which are novel, were identified in 14.6% (6/41) of all patients. The CYP1B1 is a major gene for PCG, appearing to be responsible for the disease in roughly one in six Chinese PCG patients. The R390H mutation was identified as a predominant CYP1B1 allele among the Chinese PCG patients in our study. This observation emphasizes the importance of mutational screening of CYP1B1, especially for the R390H mutation in Chinese patients.Our results support the premise that  The promolecule .CYP1B1 is the predominant cause of autosomal recessive inherited PCG that has been reported in various ethnic backgrounds [CYP1B1 mutations causing PCG in the Chinese population is not yet well understood. Herein, we investigated CYP1B1 mutations in 41 unrelated Chinese patients with PCG.Mutations in kgrounds ,10-13. SThe study followed the tenets of the Declaration of Helsinki with written informed consent obtained from all patients or from their parents if their age was less than 18. Forty-one patients with PCG were recruited from the glaucoma clinics at Zhongshan Ophthalmic Center at Sun Yat-sen University . After undergoing a complete eye examination including slit-lamp biomicroscopy, optic nerve examination, and measurement of IOP by Perkins or Goldmann tonometry, all enrolled patients were diagnosed with PCG. Subjects with secondary causes of glaucoma  were excluded. PCG was defined according to the following criteria: age of onset less than three years; IOP greater than 21 mmHg without any treatment; optic nerve cupping, and moreover, rupture of Descemet’s membrane, or horizontal corneal diameter greater than 12 mm with or without corneal edema. Patients older than three years without secondary causes but with buphthalmos were considered to have PCG.In our study, six (14.6%) patients had unilateral PCG, and neither a family history of PCG nor parental consanguinity was established in any of the 41 unrelated Chinese patients. Eighty ethnically matched normal individuals without any ocular disorders served as controls.CYP1B1 (U56438). Polymerase chain reaction (PCR) amplification was performed in a 25 l volume by means of a touchdown PCR program with an annealing temperature decreasing from 64 °C to 58 °C or 56 °C over 10 cycles followed by 30 cycles with an annealing temperature of 58 °C or 56 °C. Subsequently, the PCR products were analyzed by heteroduplex analysis-single strand conformation polymorphism (HA-SSCP) as described previously [U56438) was performed using the SeqManII program of the Lasergene package . Heterozygous deletions (g.4531_4552del22bp and g.4633delC) were identified using a monoclone followed by direct sequencing.Blood samples were collected from 41 patients and 80 controls after informed consent was received. Genomic DNA was extracted from leucocytes by proteinase K-phenol-chloroform extraction. Ten sets of primers  were useeviously . PCR proFour mutations were confirmed by restriction fragment length polymorphism (RFLP), I471S by BtsI, S336Y by AhdI, g.4531_4552del22bp by TfiI, and g.4633delC by SmlI. Digestion products were loaded onto an 8% (49:1) polyacrylamide/0.5X TBE gel and resolved at 18 W for about 2 h. Subsequently, the gel was silver stained as previously described, similar to how it was used in SSCP analysis . The twoCYP1B1 from all 41 PCG patients was screened using HA-SSCP followed by direct sequencing. We identified six different mutations, four of which are novel, in six patients with bilateral PCG. We also identified five reported polymorphisms, g.3793 T>C, g.3947 C>G (R48G), g.4160 G>T (A119S), g.8131 C>G (L432V), and g.8184 C>T (D449D) [The complete coding region of U56438) and 1 bp (g.4633del C), and two missense mutations (S336Y and I471S). Except for the homozygous I471S, each of the other three new mutations was in the compound heterozygous condition with another mutation. All four mutations changed one restriction site, g.4531_4552del22bp (TfiI) and I471S (BtsI) created new restriction sites, and g.4633delC (SmlI) and S336Y (AhdI) resulted in the loss of restriction sites. Using special restriction endonuclease digestion , which affects the third nucleotide of codon 242, introduces a premature stop codon at codon 270, and g.4633delC (p.F276FfsX1) creates a premature stop codon at codon 277. Because the COOH-terminal half of the CYP1B1 protein is expected to be involved in heme-binding and proper folding of the molecule [Because of the creation of premature stop codons, both deletions were predicted to truncate the Because the novel missense mutations altered hydrophobic properties at their sites I471S) or consisted of replacement of the micromolecular Ser side chain of by the large hydrophilic Tyr (S336Y), the I471S and the S336Y could directly affect the helix L and the helix I of the conserved core structures (CCS) of CYP1B1 protein, respectively. These mutations could interfere with heme incorporation, by affecting the CCS that controls the proper folding and heme-binding ability of P450 molecules 1S or con,17. CompApart from these four novel mutations, two reported missense mutations were also found in our study. The R390H mutation previously reported in Pakistani and Indian patients ,18 was tCYP1B1 mutations varies in different patient populations from 100% in Saudi Arabians with PCG to 20% among the Japanese with PCG [CYP1B1 mutations were found in 6 out of our 41 Chinese PCG patients (14.6%).Because of ethnic differences and geographical variations, the prevalence of with PCG ,10-13. ICYP1B1 is a major gene involved in PCG, though the mutation rate in our study was less than the 20% level reported in sporadic Japanese PCG patients [CYP1B1 mutations among PCG patients than ethnically diverse populations (like the Japanese and Chinese) [CYP1B1 mutations among different populations may be due to founder effects [CYP1B1 mutations are responsible for only about 14.6% of Chinese PCG patients, it is imperative that other causes of PCG be investigated in future studies.Our study supports the contention that patients ,10-13. AChinese) ,12. The  effects . BecauseCYP1B1 mutation spectrums have been discovered in different populations with PCG, certain mutations occur repeatedly in ethnically distinct populations. Analysis of the constructed haplotype for CYP1B1 was used to study these mutations [CYP1B1 allele in Indian PCG patients (17.8%) [Although different utations -22. If g 17.8%) , and G61% , and G (17.8%) , were idThe R390H mutation, which has been reported in 4.7% of Indian  and 3.6%CYP1B1 mutations in the Western world [CYP1B1 mutations in Chinese patients, and g.8131G not g.8131C was found to be associated with all the patients with CYP1B1 mutations , g.4160 G>T(A119S), g.8131C>G (L432V), and g.8184C>T (D449D), construct the 5′-CCGGTA-3′ haplotype associated with most rn world . The g.8utations . The mutCYP1B1 mutations and that R390H appears to be the predominant mutant allele causing PCG in Chinese patients. In light of these findings, we suggest that screening for this mutation should be a priority for the scientific community.In summary, this study shows that 14.6% of Chinese patients studied carried


Several subtypes of HIV-1 circulate in infected people worldwide, including subtype B in the United States and subtype C in Africa and India. To understand the biological properties of HIV-1 subtype C, including cellular tropism, virus entry, replication efficiency and cytopathic effects, we reciprocally inserted our previously characterized envelope V3–V5 regions derived from 9 subtype C infected patients from India into a subtype B molecular clone, pNL4-3. Equal amounts of the chimeric viruses were used to infect T-lymphocyte cell lines (A3.01 and MT-2), coreceptor cell lines (U373-MAGI-CCR5/CXCR4), primary blood T-lymphocytes (PBL) and monocyte-derived macrophages (MDM).+-CCR5+ but not U373MAGI-CD4+-CXCR4+ cell line, suggesting CCR5 coreceptor utilization and R5 phenotypes. These subtype C chimeras were unable to induce syncytia in MT-2 cells, indicative of non-syncytium inducing (NSI) phenotypes. More importantly, the subtype C envelope chimeras replicated at higher levels in PBL and MDM compared with subtype B chimeras and isolates. Furthermore, the higher levels subtype C chimeras replication in PBL and MDM correlated with increased virus entry in U373MAGI-CD4+-CCR5+.We found that subtype C envelope V3–V5 region chimeras failed to replicate in T-lymphocyte cell lines but replicated in PBL and MDM. In addition, these chimeras were able to infect U373MAGI-CD4Taken together, these results suggest that the envelope V3 to V5 regions of subtype C contributed to higher levels of HIV-1 replication compared with subtype B chimeras, which may contribute to higher viral loads and faster disease progression in subtype C infected individuals than other subtypes as well as rapid HIV-1 subtype C spread in India. The steepest increase in new cases of human immunodeficiency virus type 1 (HIV-1) infection has taken place in South America , of whicenv gp120 sequences from a large number of HIV-1 isolates shows that gp120 is made up of five variable regions (V1 to V5) that are interspersed with conserved regions [env glycoprotein control cell tropism were described by identifying two distinct co-receptors, fusin (CXR4) and CCR5, for the entry of T-lymphotropic and macrophage-tropic HIV-1, respectively [HIV-1 envelope gp120 interacts with CD4 receptor and CXCR4 or CCR5 coreceptor -13 on T  regions . The pot regions . The hyp regions -19, viru regions , replica regions ,23, wher regions ,25. In a regions ,27. The ectively ,15. The ectively -30. HoweIn this study, we have characterized the biological properties of HIV-1 subtype C envelope V3 to V5 regions by constructing chimeric recombinant viruses containing subtype C envelope V3–V5 regions from nine infected patients from India [env V3 to V5 regions chimeras with HIV-1NL4-3, HIV-1BaL and a known env V3–V5 region sequence of subtype C . The amino acid sequences between positions 310–315 and 350–373 were significantly different from HIV-1NL4-3 clone but were very similar to the previously known sequence of subtype C envelope region. It is interesting to note that a critical glycosylation site, which includes the first cysteine of the V3 loop, was mutated in all the clones except those obtained from patient 17 and 5 . The sequence analysis of all the clones indicate that these clones are different from HIV-1NL4-3 envelope sequences but very similar to envelope sequences from known subtype C clones and to R5 tropic viruses such as HIV-1BaL.We confirmed the reciprocal insertion of the sixteen envelope V3–V5 region sequences Fig.  from ninenv V3–V5 region sequences [We also used the Position Specific Scoring Matrix (PSSM) bioinformatics tool (X4/R5) to predict coreceptor usage by the Subtype C patient equences  and calcenv chimeras retained the lymphotropic properties of the parental clone HIV-1NL4-3 by infecting the T cell line A3.01 with the subtype C chimeric viruses and parental virus, HIV-1NL4-3. The T-cell line A3.01 expresses CD4 and CXCR4 but no CCR5. Our results showed that the parental X4 tropic HIV-1NL4-3 productively infected and replicated in A3.01 over a 27-day infection period. However, the subtype C chimeras did not replicate in A3.01 cell line  demonstrated remarkably high levels of infectivity when compared to other chimeras and primary isolate controls indicating an increased rate of entry for these chimeras. The levels of entry using increasing virus counts  during infection showed increased level of entry for the same chimeras. This shows that the envelope region of subtype C HIV-1 obtained from the patient samples shows an R5 phenotype for the virus infecting the patient. These results also suggest that all the chimeric viruses obtained could have had different rates of entry when infecting target cells. This can be attributed to the differences in the V3 region of the subtype C chimeric DNA, reflecting the differences found in patient samples. This also adds to previous work done by others [We then determined the coreceptor utilization of the subtype C y others ,33 showienv chimeras by infecting MT-2 cell lines with the chimeric viruses. Viruses that produce a greater than four syncytia per field were denoted as syncytium inducing (SI) phenotype and the viruses that did not induce any syncytia were called as non-syncytium inducing (NSI) phenotype. As shown in the Table BaL). The control parental virus HIV-1NL4-3 that has a known SI phenotype produced significant levels of syncytia (at least 10 per field of view). As expected, the R5 viruses used as control also did not produce syncytia in culture. These data suggest that the envelope sequences from subtype C infected patient samples render them less cytopathic as compared to HIV-1NL4-3. We also confirmed virus production in MT-2 cells and the syncytium formation correlated to virus production in our partial env (V3–V5) region chimeras (NSI) and R5 isolates as measured by RT assay in the culture medium (not shown).We examined the syncytium-inducing ability of the subtype C env chimeras, which did not replicate in A3.01, were able to replicate in PBL showing that there was no inherent defect in their replication as a results of the reciprocal insertion of the V3–V5 region from patient samples  and subWhile primary monocyte derived macrophages (MDM) express CD4 and CCR5 and a low level of CXCR4, they support productive infection of R5 but not X4 viruses. As the subtype C chimeras showed a R5 phenotype Table , replicaWe have provided evidence regarding the role of the HIV-1 envelope V3–V5 regions from subtype C infected patients from India in virus entry, coreceptor utilization and replication efficiency in primary T-lymphocytes and macrophages in comparison with those of subtype B viruses. Our data suggest that the reciprocal insertion of HIV-1 subtype C infected patients' envelope V3 to V5 regions ,36.NL4-3, the subtype C env V3–V5 region chimeras were unable to replicate in T lymphocyte cell lines A3.01  parental clone HIV-1.01 Fig.  and MT-2Our in-silico analysis of the V3 sequences of the subtype C isolates from India predicted R5 tropism Table . This alNL4-3 and subtype B primary isolates. In general, there was a correlation between higher virus entry, earlier replication peaks, and increased replication efficiencies in primary T-lymphocytes and MDM. Examination of sequence data published earlier on these patients [BAL (R5), as shown in Fig. BAL in Fig. Our data showed that subtype C V3–V5 region chimeras replicated better in primary blood T-lymphocytes Fig.  comparedpatients , whereasin vitro study with more fit subtype B and less fit subtype C viruses indicates more fitness of subtype B over subtype C viruses [in-vivo infection studies on rhesus macaques have shown that HIV-1 subtype C env chimeric viruses demonstrate greatly enhanced infectivity [in vivo studies [While a co-infection  viruses , severalectivity -43 and rectivity ,45. Our  studies -43. Incr studies ,47. Howe studies . During  studies ,48,49. TWhile various HIV-1 subtypes prevalent in different regions of the world show variability in their replication kinetics and disease progression in infected individuals , HIV-1 sBglII-BglII sites flanking the region. The 650 bp fragment encompassing the V3–V5 region corresponds to amino acids 240–439 of gp120 of the HIV-INL4-3 sequence with the V3-loop positioned between amino acids 267 and 300. PCR primers were designed to amplify the V3–V5 region and to replace a similar region in pNL4-3 by engineering a BglII site at the 3' end at position 7611 corresponding to all isolates. PCR amplification was carried out using Pfu polymerase. The ~600 bp PCR product was digested with BglII and cloned into pGEM (NL4-3) using the BglII sites [BglII-BglII fragment was reciprocally exchanged into the pGEM plasmid containing the EcoRI-BamHI fragment as there were no EcoRI or BamHI sites in this a region in any Indian isolate except AP.5 which contained an EcoRI site in the sequence. The recombinant clones were checked by digestion with BglII and the orientation was confirmed by DNA sequencing using primer (5'TCAACTGCTGTTAAATGGC3'). Finally, the modified EcoRI-BamHI fragment containing the sporadic subtype C Indian isolate V3 to V5 region was reciprocally substituted into pNL4-3. Two clones were obtained from each patient sample and these are numbered with numerals of patient identification number followed by clone number. All the clones were again checked by digestion with EcoRI/SalI and BamHI and confirmed by sequencing.Sixteen previously characterized envelope V3–V5 region sequences Fig.  from nin6 cells/well in RPMI 1640 with 15% human AB serum (Gemini biotech) and MCSF (Sigma) for 7 days and were allowed to differentiate into macrophages in this media. The cells were fed every two days during differentiation. The cells collected as unbound flow through from the CD14 bead isolation protocol were used as PBL. PBL were cultured in RPMI 1640 with 10% FBS and penicillin-streptomycin. PBL were stimulated with 5 μg/ml of PHA for 24–48 h. The stimulated cells were washed with PBS and resuspended in RPMI 1640 with 10% FBS and 20 U/ml of recombinant human IL-2 (Invitrogen).HeLa cells, U373-MAGI-CXCR4 and U373-MAGI-CCR5 cell lines were cultured in DMEM with 10% fetal bovine serum (FBS) and penicillin-streptomycin (Invitrogen). The MAGI cell media was also supplemented with 0.2 mg/ml of G418, 0.1 mg/ml of hygromycin B and 1 μg/ml of puromycin. T-lymphocyte cell lines, A3.01 and MT-2 were cultured in RPMI 1640 supplemented with 10% FBS and penicillin-streptomycin. Peripheral blood mononuclear cells (PBMC) were obtained by single step density gradient centrifugation with Ficoll-Hypaque (Amersham) from whole blood of normal donors. The blood collections were made after informed consent, and were approved by the Human Subject Ethical committees of International Center for Genetic Engineering and Biotechnology (ICGEB) and the Human Subjects Committee of the University of Arizona  and were based on Indian Council of Medical Research (ICMR) and National Institutes of Health (NIH) guidelines, respectively. PBMC were collected, washed twice with cold PBS and centrifuged at 1000 rpm for 10 min to avoid collecting platelets. Primary monocyte/macrophages and peripheral blood lymphocytes (PBL) were separated from PBMC using magnetic bead isolation protocols. Monocytes were isolated by CD14 magnetic bead isolation (Miltenyi biotech) according to the manufacturer's protocols. The cells bound to the CD14 bead were used as primary monocytes. The primary monocytes eluted from the CD14 isolation columns were counted and plated in 48 well plates at 1 × 105 cells/well in DMEM with 10% FBS without antibiotics. The cells were transfected the next day with 3 μg DNA in DNA-lipofectamine complexes as per manufacturer's procedure. Chimeric viruses were harvested by collecting culture supernatant from the wells 72 hrs post-transfections. Virus production was measured by a reverse transcriptase (RT) assay [Subtype C chimeric proviral DNAs were transfected in HeLa cells by electroporation as described before  or by LiT) assay ,51.6), MT-2 (2 × 106), PBL (1 × 106) and MDM (0.5 × 106) per well were cultured and infected with equal amounts of subtype C env region chimeras, subtype B V3 region chimeras  [2. After incubation, 500 μl of appropriate media containing serum and antibiotics were added. MDM were infected in media containing serum and polybrene (8 μg/ml) incubated at 37°C and 5% CO2 for 16 hrs. After incubation, the macrophages were washed in PBS to remove polybrene and resuspended in macrophage culture media.A3.01 cells  ,50, subt4 cells/well in 24 well plate in complete DMEM with G418-hygromycin-puromycin. Both cells lines were infected with 5,000 and 10,000 cpm (RT assay counts) of chimeric subtype C virus and primary isolate controls diluted in a total volume of 300 μl of complete DMEM (without antibiotics) with DEAE-dextran . Two hours post-adsorbtion, 1.5 ml of fresh MAGI media was added. To assess the rate of entry, 40 h post-infection, the medium was removed and cells were fixed in 1% formaldehyde and 0.2% glutaraldehyde for 5 min. Then, the cells were washed with PBS and stained for 2 hrs in staining solution containing 0.2 M potassium ferricyanide, 0.2 M potassium ferrocyanide, 2 M MgCl2 and 40 μg/ml X-Gal. After staining, the cells were washed in PBS and resuspended in PBS with Sodium Azide. Cells stained blue were counted immediately and counts were estimated as number of blue cells per well of infected cells.U373-MAGI-CXCR4 and U373-MAGI-CCR5 cell lines were plated at ~6 × 10NL4-3) and a non-syncytium-inducing (NSI) viruses (HIV-1BaL and HIV-1Ada-M) by infecting the MT-2 cell line with equal amounts of these viruses. Syncytium formation was monitored every day in the cultures up to 30 days post-infection. The viruses that formed more than four syncytia per field of view in the culture were designated as SI viruses (ACTG protocol), whereas viruses that failed to induce this number of syncytia in the culture were termed NSI viruses.The syncytium-inducing ability of the subtype C envelope chimeras was determined and compared to a known syncytium-inducing (SI) virus (HIV-1The author(s) declare that they have no competing interests.VS and SRD contributed equally to this work. SRD carried out the PCR, cloning, and sequencing. VS performed all biological experiments, including T-cells lines and primary cells infection experiments. RR contributed in transfections and infection experiments. SS and SG contributed to the collection of patients' samples and clinical data. VS, SRD, RR, NA and SJ participated in the experimental design, data interpretation and writing of the manuscript. All the authors read and approved the final manuscript.


Cellular contractility, essential for cell movement and proliferation, is regulated by microtubules, RhoA and actomyosin. The RhoA dependent kinase ROCK ensures the phosphorylation of the regulatory Myosin II Light Chain (MLC) Ser19, thereby activating actomyosin contractions. Microtubules are upstream inhibitors of contractility and their depolymerization or depletion cause cells to contract by activating RhoA. How microtubule dynamics regulates RhoA remains, a major missing link in understanding contractility.We observed that contractility is inhibited by microtubules not only, as previously reported, in adherent cells, but also in non-adhering interphase and mitotic cells. Strikingly we observed that contractility requires ubiquitin mediated proteolysis by a Cullin-RING ubiquitin ligase. Inhibition of proteolysis, ubiquitination and neddylation all led to complete cessation of contractility and considerably reduced MLC Ser19 phosphorylation.Our results imply that cells express a contractility inhibitor that is degraded by ubiquitin mediated proteolysis, either constitutively or in response to microtubule depolymerization. This degradation seems to depend on a Cullin-RING ubiquitin ligase and is required for cellular contractions. Cellular contractility and the ability of cells to change their shape are prerequisites for many biological phenomena such as cytokinesis, movement, differentiation and substrate adherence. These changes in cell shape are achieved by modulation of the cytoskeleton, most importantly the actin cytoskeleton, through forces generated by the actomyosin network. This network is regulated mainly through the activity of proteins from the Rho-GTPase family that regulate both actin nucleation and myosin activity through downstream effectors such as mDia and ROCK (Rho kinase), respectively . To our surprise we observed that ubiquitination and proteasomal degradation are required not for the activation but for the inhibition of contractility. The largest group of ubiquitin ligases in the cell constitutes of the Cullin-RING type. This vast group comprises among others the seventy different SCF  complexes encoded by the human genome, and possibly more then a hundred Cul3-BTB based ligases The microtubule cytoskeleton inhibits contractility of adherent cells in interphase Nocodazole impairs the capacity of α-tubulin to polymerize and rapidly shifts the dynamic equilibrium of microtubules to a de-polymerized state . We chosWe wondered how adherent cells that become detached behave upon microtubule depolymerization. We therefore detached NIH3T3 cells from tissue culture dishes and observed their attachment to glass cover slips in the presence or absence of nocodazole. Without nocodazole the cells attached to the glass within one hour while in the presence of nocodazole they failed to attach for more than two hours and underwent vigorous contractions .During mitosis the microtubule cytoskeleton undergoes a radical change of its shape, composition and dynamics. We thus wondered if it retains its capacity to inhibit cellular contractions. Such an observation would be of significance for the capacity of cells to undergo furrowing in cytokinesis. To follow the behavior of the spindle we generated a NIH3T3 mouse fibroblast cell line that stably expresses α-tubulin fused to the mCherry We next asked whether microtubule stabilization would induce an inhibitory effect on contractility. This was achieved by inhibiting α-tubulin depolymerization with Paclitaxel (taxol). Treated cells exhibited thick and rigid microtubule bundles. Like in the case of nocodazole, taxol-treated cells arrested in prometaphase by the SAC. This arrest had either of two phenotypes. The majority of the cells exhibited rigid spindles, did not contract but arrested as motionless round mitotic cells for several hours until they flattened, presumably due to mitotic slippage The cells discussed so far were treated for several hours in interphase with the indicated drugs until they reached prometaphase. Since these drugs arrest the cells at the SAC, we sought an alternative method to arrest cells later in mitosis, allowing the spindles to form normally and test the instant effect of their de-polymerization. To this end we transiently transfected cells with an expression vector for a destruction box mutant of full length Cyclin B1-GFP (Cyclin B1-DM-GFP), which is not ubiquitinated by the APC/C and is thus not degraded. These cells arrested in mitosis either before metaphase or in anaphase We further used monastrol, which arrest the cells in mitosis with mono-astral spindles without interfering with microtubule dynamics The results we report here show that cells that are not attached to a matrix will undergo contractions upon microtubule depletion whether they grow constitutively in suspension, are artificially detached in interphase, or physiologically in mitosis. We thus conclude that both the interphase microtubule cytoskeleton and the mitotic spindle inhibit cellular contractility. A detailed breakdown of these results and quantitative data is presented in Cellular contractility by actomyosin depends on the RhoA signaling pathway. We wanted to verify that the contractions induced by microtuble depolymerization also depend on this pathway. Cells were immuno-stained for RhoA and Myosin. Ubiquitin mediated proteolysis plays a role in most cellular events. We observed that ubiquitin is localized to the furrows formed in the absence of microtubules as well as in the cleavage furrow during cytokinesis (our unpublished data) and we wondered whether ubiquitin mediated proteolysis is also involved in regulation of contractility. We therefore treated cells with nocodazole together with the proteasome inhibitor MG132. Proteasomal inhibition leads to cell cycle arrest at various phases. The requirement for proteasomal activity for contractions could thus be due to cell cycle effects. To address this possibility, we treated unsynchronized DT40 cells with nocodazole together with MG132. This double treatment completely abolished contractions regardless of cell cycle stage . The reqTo verify the need for ubiquitin-mediated proteolysis in a non-drug dependent manner we used the balb/c 3T3 derived mouse fibroblast cell lines A31N-wt and A31N-ts20. The latter expresses a thermo-sensitive E1 ubiquitin activating enzyme that becomes inactive at the restrictive temperature of 39.5°C Cullin-RING complexes are the largest group of ubiquitin ligases and are involved in the regulation of many cellular pathways These mutant cells enabled us also to test the requirement for neddylation for contractility in detached interphase cells. We plated E36-wt and E36-ts41 cells on glass in the presence of nocodazole at the restrictive temperature of 39.5°C. E36-wt cells contracted vigorously like the NIH3T3 cells shown in The most downstream regulatory event of cellular contractility is the activating phosphorylation of Ser19 of MLC. The data presented so far suggested that microtubules inhibit contractility and upon relief the contractility requires ubiquitin mediated proteolysis by a Nedd8 dependent pathway. In order to assess whether this requirement directly impinges on the phosphorylation of this site we used anti-phospho Ser19 MLC antibodies. We addressed this issue with all three treatments. First we analyzed the effect of inhibition of proteolysis in human HeLa cells. Cells were detached from the tissue culture dishes and treated with nocodazole for five hours with or without MG132. Preliminary experiments have shown that detachment induces Ser19 phosphorylation, which gets further enhanced by nocodazole. caRhoA-GFP) and cultured them at 39.5°C in the presence of nocodazole. Under these conditions protein degradation, which is mediated by Cullin-RING ubiquitin ligases, is blocked while the RhoA pathway is active. caRhoA-GFP expressing cells vigorously contracted. This suggests that RhoA activity is downstream to the Cullin-RING E3 and might be dependent on it for contracting. This experiment also verifies that loss of contractions in response to neddylation inhibition is a rather specific event and not a result of ATP depletion in cells.We have shown that contractions depend both on the RhoA pathway and on the activity of a Cullin-RING E3. To establish the relationship of these two pathways, we transfected E36-ts41cells with an expression vector for a constitutively active form of RhoA (Q63L-hRhoA) fused to GFP  in 5% CO2.NIH-3T3, E36-ts41, E36, A31N and A31N-ts20 cell lines were maintained in Dulbecco's modified Eagle medium  containing 10% fetal calf serum, 10 u/ml Penicillin and 100 µg/ml Streptomycin . DT40 cells were maintained in RPMI-1640 (Gibco) containing 10% fetal calf serum, 3% chicken serum, 10 u/ml Penicillin, 100 µg/ml Streptomycin and 50 µM 2-mercaptoethanol (Merck). NIH3T3 and DT40 were grown at 37°C with 5% COThe reagents were added to the growing media to reach the indicated final concentration as follows: nocodazole (Sigma) 2 µM (0.2 µM for HeLa cells), Taxol (Sigma) 5 µM, MG132 (Sigma) 0.1 µM (0.05 µM for HeLa cells), Cell permeable exoenzyme C3 transferase (Cytoskeleton) 7.5 µg/ml, Y27632 (Sigma) 15 µM. Ect2 siRNA (Santa Cruz Biotechnology sc-35259), non-specific-siRNA and siRNA delivery control (Mirus) were transfected using Lipofectamine 2000 (Invitrogene) according to manufacturer guidelines.dnRhoA) and pEGFP-hRhoA Q63L (caRhoA) were a gift from S. Ravid. pEGFP-Ect2-N1 (dnEct2) The pEF-Cyclin B1DM-GFP vector expressing non degradable full-length mouse Cyclin B1-GFP was previously described α-tubulin downstream and in frame with the gene for the mCherry fluorescent protein 4 co-precipitation method pEF-mCherry-α-tubulin was made by Y. Oren by cloning http://rsb.info.nih.gov/ij/) and Photoshop 7 (Adobe) software. Movies were assembled by Premier-Pro (Adobe).Rabbit anti-pMLC  and Goat anti-actin (Santa Cruz Biotechnology sc-1616) were used for western blotting. Mouse anti-RhoA (26C4) (Santa Cruz Biotechnology sc-418) diluted 1∶200, Rabbit anti-mMHC-B (S. Ravid) diluted 1∶200, Donkey anti-Mouse IgG (H+L) Cy2 conjugated diluted 1∶500, Donkey anti-Rabbit IgG (H+L) Cy2 (Jackson Immunoresearch). RhoA immunofluorescent labeling was done as recently described. For mMHC-B labeling, cells were fixed in 4% formaldehyde in PBS for 20 minutes and blocked with PBST. The slides were visualized either on an inverted IX70 Olympus microscope and captured with a CoolSnap HQ (Photometrics) digital camera, or on an Olympus FV1000 confocal microscope. The images were analyzed and assembled with Image pro plus 5.0 (Media Cybernetic), ImageJ (2 and temperature-controlled incubator (EMBL GP168) and SM1 motorized stage (Luigs & Neumann). Images were captured with LD32X/0.4 air or 40X/1.3 oil objective lenses using a SensiCam QE (PCO) digital camera and CompiC Inject (Cell Biology Trading) software.Cells were seeded on 35 mm tissue culture plates (Nunc) a day prior of filming. The cells were observed by an Axiovert 200 M microscope (Zeiss) equipped with a COFigure S1Knockdown of Ect2 results in binucleated cells. A. Ect2 siRNA reduce Ect2 levels in NIH3T3 total protein extract. Extracts from mock, Ect2 siRNA and non-specific siRNA (NS-siRNA) transfected cells analyzed by Western blotting. Only Ect2 siRNA transfected cells show reduction in protein level. B–D, Immunostaning with anti-α-tubulin and anti ECT2 antibodies show that while in the mock and the siRNA non-specific transfected cells ECT2 level was not affected, ECT2 siRNA transfected cells had much lower levels of ECT2. Commonly, these cells were also bi-nucleated as they fail to undergo cytokinesis.(2.15 MB TIF)Click here for additional data file.Movie S1Nocodazole leads to almost instantaneous microtubule de-polymerization. NIH3T3 cells stably expressing mCherry-α-tubulin subjected to nocodazole treatment. Microtubule disruption occurs within 4–8 minutes of nocodazole addition (108–116 min).(1.29 MB AVI)Click here for additional data file.Movie S2Non adherent DT40 cells contract throughout the cell cycle in a microtubule-dependent manner. A. DT40 cell undergo mitosis. B. Asynchronous culture treated with nocodazole contracts vigorously.(3.49 MB AVI)Click here for additional data file.Movie S3Detached adherent cells contract and fail to re-attach in a microtubules- dependent manner. NIH3T3 cells detached by trypsinization and allowed to re-adhere on a glass cover slip. Control cells (left panel) flatten within 30–60 minutes. Nocodazole-treated cells (right panels) contract and fail to re-adhere for at least 2 hours and usually flatten only after 4 hours.(2.26 MB AVI)Click here for additional data file.Movie S4Microtubules inhibit contractility in NIH3T3 mitotic cells. A, NIH3T3 cells stably expressing mCherry-α-tubulin undergo normal mitosis within an hour. B, Nocodazole was added one hour after the beginning of the movie. Seven hours later the cell detached from the plate, entered mitosis and immediately started to contract for more than 7 hours.(8.07 MB AVI)Click here for additional data file.Movie S5The mitotic spindle in cells arrested by nondegradable Cyclin B1 inhibits contractions in a spatial dependent manner. A, NIH3T3 cells stably expressing mCherry-α-tubulin were transfected with Cyclin B1-DM-GFP. Following mitotic entry and spindle formation (arrow) a cell portion, which was distal to the spindles (arrowhead) started to contract. When the spindle approached the distal part the contractions stopped (224–280 min). Phase (left panel), mCherry-α-tubulin , Cyclin B1-DM-GFP (right panel). B, NIH3T3 cells stably expressing mCherry-α-tubulin were transfected with Cyclin B1-DM-GFP, as described in A. The part of the movie presented here starts 20 hours after mitotic entry. After an additional 8.5 hours the cells were treated with nocodazole, which led to prompt spindle depolymerization and to vigorous contractions.(5.97 MB AVI)Click here for additional data file.Movie S6Inhibition of the RhoA-ROCK signaling pathway stops nocodazole induced contractions. NIH3T3 cells stably expressing mCherry-α-tubulin were transfected with dnRhoA-GFP and treated with nocodazole. A dnRhoA expressing cell (arrow) entered mitosis as indicated by the nuclear envelope breakdown (620 min), remained arrested for 5 hours and did not contract. Note the non transfected adjacent cell that entered mitosis earlier (568 min) and vigorously contracted.(6.23 MB AVI)Click here for additional data file.Movie S7Nocodazole induced contractions depend on proteasome activity. A. NIH3T3 cells stably expressing mCherry-α-tubulin were treated with both nocodazole and the proteasome inhibitor MG132. Nocodazole was added 4 hours before the beginning of the movie. MG132 was added at the beginning of the movie while the cell was already arrested and contracting. The cell continued to contract for an additional 7.5 hours and then ceased and remained as an arrested round mitotic cell. B. NIH3T3 cells where arrested for 15 hours with MG132 and nocodazole. They were subsequently washed and released into fresh medium without inhibitors. The cells seem to recover well and attach within about 2 hours.(7.43 MB AVI)Click here for additional data file.Movie S8Nocodazole induced contractions depend on ubiquitination. A31N-wt and A31N-ts20 cells were subjected to nocodazole at the permissive and restrictive temperatures. A, A31N-wt control cells undergo normal mitosis at 39.5°. B, An A31N-wt cell grown at 39.5° was treated with nocodazole and contracted upon mitotic entry. C, An A31N-ts20 cell grown at 34°, was treated with nocodazole. The cell contracted for 7 hours before it flattened. D, An A31N-ts20 cell was grown at 39.5° in the presence of nocodazole for 13 hours before it entered mitosis (788 min). During the next 14 hours the cell remained arrested, did not contracted and did not flatten.(10.08 MB AVI)Click here for additional data file.Movie S9Nocodazole induced contractions depend on neddylation. E36-wt or E36-ts41 cells were treated with nocodazole at the permissive and the restrictive temperatures. A, E36-wt control cells undergo normal mitosis at 39.5°C. B, An E36-wt cell grown at 39.5°C was treated with nocodazole and contracted upon entry into mitosis. C, An E36-ts41 cell grown at 34°C was treated with nocodazole. The cell arrested and contracted for 4 hours before it flattened. D, E6-ts41 cells undergo cell cycle arrest at 39.5°C. The movie started at 34°C and the cell  underwent normal mitosis. Fifteen hours later the temperature was shifted to 39.5°C for an additional 11 hours. The cell was overall arrested for at least 26 hours. The cell cycle time of these cells at 34°C is about 16 hours. E, Temperature shift to 39.5°C of nocodazole-treated, mitotic E36-ts41 cells stopped their contractions. At the beginning of the movie the temperature was shifted to 39.5°C and nocodazole was added. Upon mitotic entry the cell  contracted for 14.5 hours (944 minutes), and then stopped for an additional 4 hours. F, Detached control E36-wt (left panel) and E36-ts41 (right panel) were allowed to re-adhere on a glass cover slip. Nocodazole was added and the temperature was shifted to 39.5°C at the beginning of the movie. While control cells contracted and re-adhered within 4–8 hours, E36-ts41 cells contracted for 10 hours and then ceased. These cells failed to adhere the matrix.(10.17 MB AVI)Click here for additional data file.Movie S10Neddylation is upstream of the RhoA pathway. E36-ts41 cells were transiently transfected with constitutively active form of RhoA Q63L fused to GFP(caRhoA-GFP), grown at 39.5°C and treated with nocodazole. While the non-transfected control cell on the left panel did not contract see also  the tran(5.42 MB AVI)Click here for additional data file.


Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation.Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in  cis-acting epigenetic mechanisms. Here we describe a case where the local character of chromatin is not only determined by cis-acting mechanisms but also negatively regulated in trans. The case involves loading of the histone H2A variant, H2A.Z, into chromatin. We show that H2A.Z in the yeast Schizosaccharomyces pombe is mainly found in genes at the first transcribed nucleosome and is inserted into this nucleosome by the Swr1C remodeling machine. However, Swr1C has a regulatory subunit, Msc1, which is not required for H2A.Z promoter loading but prevents H2A.Z occupancy in the inner centromere and subtelomeric regions. These two specialized regions are neither eu- nor heterochromatin and share certain characteristics, which may predispose them to the aberrant inclusion of H2A.Z and the requirement for trans regulation by Msc1.Chromatin is based on a repetitive structural unit called the nucleosome. However, the regulatory properties of chromatin are mediated by the differences between nucleosomes, due to post-translational modifications or the inclusion of histone variants. These differences are maintained by inheritance through  Chromatin is based on a repetitive structural unit called the nucleosome. However the regulatory properties of chromatin are mediated by the differences between nucleosomes, due to post-translational modifications or presence of histone variants. Cytologically, chromatin was initially divided into heterochromatin and euchromatin Another way to maintain nucleosomal differences and chromatin domains are boundary mechanisms. By blocking the spread of a self-reinforcing mechanism, boundaries such as those provided by insulators This question is especially relevant for the processes that exchange canonical histones for histone variants. After DNA replication, both daughter DNA molecules must be packaged in the same chromatin status as the parental molecule. Canonical histone deposition occurs in a replication-coupled (RC) manner. However, the deposition of certain histone variants occurs in a DNA replication-independent (RI) manner in vitroRI chaperones are particularly susceptible to mistargeting of histone variants. For example, the H3 variant CENP-A is enriched in the centromeric domain under guidance from neighbouring heterochromatin and epigenetic mechanisms in vitro and in vivo, but does not remove H2A.Z from chromatin In this paper we focus on the H2A variant, H2A.Z, which is incorporated into budding yeast chromatin by Swr1, the catalytic subunit of the Swr1 complex (Swr1C) and one of the SWI2/SNF2 superfamily of ATPase chromatin remodelers In budding yeast, H2A.Z is mainly positioned at the promoters of lowly expressed or inducible genes H2A.Z has also been implicated in centromere function and chromosome segregation in mammals Hence H2A.Z and Swr1C are involved in many aspects of chromatin regulation. Central to these processes is the incorporation of H2A.Z into specific nucleosomes. However the basis for this specificity is unclear. Here we report that this process is due to both positive and negative target selectivity by Swr1C, due in part to the JmjC-domain protein, Msc1, which is a stoichiometric subunit of the fission yeast Swr1C. Msc1 negatively regulates H2A.Z incorporation into specific chromatin regions at the inner centromere and sub-telomere.S. pombe complex with high subunit orthology to the S. cerevisiae Swr1 complex As part of a study to develop datasets for comparative proteomics, we purified a S. cerevisiae Swr1C. Msc1 is a member of the highly conserved Lid/Jarid1 family and has five zinc fingers, including one JmjN and three PHD fingers, an ARID/BRIGHT AT rich DNA binding domain and a Plu domain  of Msc1 in H2A.Z metabolism, we performed genome-wide chromatin immunoprecipitation (ChIP-chip) analyses using myc-tagged H2A.Z in WT, c region , H2A.Z pth of promoters in vegetative, exponentially growing, S. pombe contain strongly enriched H2A.Z  and the open reading frame (ORF). At a cutoff of >1.5×, 660 IGRs showed enrichment for H2A.Z, indicating that about 1/7ed H2A.Z . This isS. cerevisiaeDrosophilaWe further divided the occurrence of H2A.Z peaks into five categories with respect to mRNA expression level from very low to very high . In WT tWe further evaluated the relationship between H2A.Z promoter peaks and gene expression levels to observe a strong negative correlation . As expeS. pombe euchromatin, whereas the role(s) for Msc1 are more subtle. Msc1 is not required to specify the sites of H2A.Z loading, rather it contributes to H2A.Z occupancy either through loading efficiency or persistence.Furthermore we observed strong positive correlations between H2A.Z peaks and H4K16, H3K14 and other histone tail acetylations . These pNotably we also observed a strong inverse correlation between the genome wide distributions of H2A.Z and the nucleosome chaperones, Nap1, Hrp1 and 3 msc1Δ strain demonstrates that Msc1 acts as a negative regulator of H2A.Z inclusion or persistence at centromeres. Additionally, the presence of centromeric H2A.Z in swr1Δ implies that H2A.Z does not strictly rely on Swr1C for loading into chromatin.Normally H2A.Z is absent from all centromeric regions, including both the CENP-A containing inner centromere and the pericentric heterochromatin . HoweverSimilar to the centromeres, H2A.Z deposition at sub-telomeric domains was also affected by the losses of Msc1 and Swr1. In WT, H2A.Z is depleted from sub-telomeric domains (approximately 100 kb in size) at the left and right ends of chromosomes 1 and 2 . Loss ofThe sharp transition between euchromatin and sub-telomeric chromatin also corresponds to a transition of H3K4me2 levels swr1Δ and msc1Δ strains.Like ST-chromatin, the inner centromeric (IC) domain is depleted in H3K4me2 compared to levels typically found in euchromatin msc1Δ, very few genes were misregulated  compared to swr1Δ (265) or H2A.ZΔ (490). Genes up-regulated in all three mutant strains were lowly expressed in WT. However loss of Msc1 had virtually no effect on the expression of any other genes, whereas loss of either H2A.Z or Swr1 also affected highly expressed genes  or increased sub-telomeric deposition (msc1Δ and swr1Δ) of H2A.Z. The overlap between up-regulated genes in H2A.ZΔ, msc1Δ and swr1Δ was also higher at sub-telomeres than in the rest of the genome, indicating a similar loss of sub-telomeric transcriptional control in the three deletion strains (data not shown). In fact, more than 2/3rds of genes up-regulated in the absence of Msc1 lie in the sub-telomeric regions. Notably, up-regulation spreads beyond the ST-chromatin boundaries, suggesting that the loss of ST-chromatin and its boundaries caused neighbouring effects.The most striking observation from the expression profiling was increased expression in the mutant strains of many genes within approximately 160kb of the ends of chromosomes 1 and 2 . H2A.ZΔ,msc1Δ or swr1Δ strains with concomitant retrieval of associated H2B . The correct sequence is presented in S. pombe H2A.Z includes two N-terminal methionines, which are either both present or absent, resulting in two variations of the N-terminal peptide (named 1–22 or 3–22 respectively). A comparison of absolute levels of peptides 1–22 and 3–22 revealed that 1–22 is the major isoform. This isoform is always N-terminally acetylated. About 2/3rds of total H2A.Z also carries 2 or more lysine acetylations . The transition from the normal euchromatic H2A.Z pattern to the sub-telomeric region appears to be sharp and coincides with an altered profile of H3K4me2, the presence of retroviral insertions and also presumably the furthest limit of Swi6 binding and H3K9 methylation, which spread from the telomeres Because many meiotic specific genes are found in this domain, it appears that ST-chromatin is an example of regulation of a gene expression program by chromatin domain status. Msc1 is required to maintain this status. In its absence, many genes are derepressed. Notably this derepression extends beyond the ST/euchromatin boundary into euchromatin. Gene derepression in ST-chromatin was not only found in the absence of Msc1 or Swr1, which provoke ectopic H2A.Z deposition into ST-chromatin, but also paradoxically in the absence of H2A.Z, which is normally absent from this domain. This indicates that the maintenance of ST-chromatin requires euchromatic H2A.Z. Furthermore gene repression in ST-chromatin requires Clr3 and Hrp1/3 . This evSwr1C purifications from TAP-tagged baits and mass spectrometry identification of complex members were carried out as described elsewhere Histones were purified using a protocol adapted from budding yeast Arg-C digested samples were first treated with propionic anhydride or deuterated acetic anhydride S. pombe 1.0FR Arrays) at Pearson correlation coefficients of r>0.97. Probes are tiled for both strands of the genome at an average of 20 base pair resolution. Antibodies used were against H3  and H2A.Z-myc . Expression arrays were carried out as described ChIP was carried out as previously described S. pombe genome  coordinates in TAS. Visualization of data was performed using the Affymetrix Integrated Genome Browser (IGB). The resulting linear ratio was extracted for each probe position, defined as the center (13th) base coordinate for each 25-nucleotide probe.Raw data from Affymetrix (.CEL format) was analyzed by Affymetrix Tiling Analysis Software (TAS) v1.1 using quantile normalization plus scaling and assigned with a bandwidth of 100. The data was normalized with DNA input and each probe was assigned to the Data sets from ChIP on chip experiments were used to map all coding genes onto an average gene, using a similar method as previously described Figure S1S. pombe Lid2 and Msc1 using the colour coding of Gibson et al, TiBS, 19, 349–53 1994. Conserved protein domains are indicated.Msc1 is a conserved JmjC-domain containing Jarid family member. Multiple amino acid alignment of H. sapiens Jarid1A, Jarid1C, (3.77 MB PDF)Click here for additional data file.Figure S2Further analyses of euchromatic H2A.Z ChIP. (A) To validate the ChIP-chip, samples were amplified by semi-quantitative PCR using primers to IGR and ORF regions according to the scheme for the gene SPAC12G12.01c, which is also shown in (1.35 MB EPS)Click here for additional data file.Figure S3The inner centromeres of Chromosomes 2 and 3 also acquire H2A.Z and increased H3 levels in the absence of Swr1 or Msc1. (A) Chromosome 2 H2A.Z-myc ChIP. (B) The corresponding H3 ChIP. (C) Chromosome 3 H2A.Z-myc ChIP. (D) The corresponding H3 ChIP. The structural features are labeled below the panels.(4.97 MB EPS)Click here for additional data file.Figure S4H2A.Z ChIP on the sub-telomeres of all three chromosomes. ChIP-chip binding profiles for H2A.Z-myc at 180kb regions at both ends of chromosome 1 , 2 , and 3  in WT, msc1Δ, and swr1Δ. Open reading frames are represented by black boxes and LTR retrotransposon elements by orange boxes. The dotted lines demonstrate the approximate transition points between chromatin domains. The ribosomal gene repeats lie at the far left (E) and far right (F) of chromosome 3 and are represented by the large black boxes. The probe distribution in these regions is very sparse.(5.05 MB EPS)Click here for additional data file.Figure S5Purification of H2A.Z-TAP and H2A.Z-associated H2B for MS. (A) Coomassie stained SDS-PAGE gel and dot blot of fractions from the C4 RP-HPLC separation of H2A.Z-TAP in WT. The dot blot was probed with an antibody directed against the TAP-tag. Histones are indicated. (B) Chromatogram of RPHPLC separation (absorbance 214nm) in WT, msc1Δ, and swr1Δ.(5.95 MB EPS)Click here for additional data file.Figure S6H2A.Z is found in two isoforms, distinguished by two N-terminal methionines, and can be acetylated on all four lysines of the N-terminal tail. (A) Listed are H2A.Z peptides as detected by LC-MS/MS analysis. The observed and predicted masses are presented, and the difference between these values (delta) given in parts per million (ppm), as are the number of missed cleavages (from an Arg-C digest), the amino acid sequence, the detected modifications and Mascot scores for MSMS fragmentation spectra. Note that each acetylation isoform was detected as 2+ and 3+ charge states. (B) MSMS spectra for H2A.Z 1-22ac4 and (C) H2A.Z 3-22ac4 peptides (parent ions not shown). Fragmentation is also represented schematically.(0.82 MB EPS)Click here for additional data file.Figure S7Acetylation of H2A.Z-associated H2B is not strongly affected by the loss of either Swr1 or Msc1. Relative quantification of H2A.Z-associated H2B 1–18 acetylation from WT, msc1Δ, and swr1Δ, plus global H2B 1–18 acetylation levels in WT. H2B can be acetylated on three lysine residues of the N-terminal tail  and the N-terminus, and is predominantly found acetylated at all three potential sites (termed the 3ac isoform) in WT whether associated with H2A.Z or not. The number of acetyl marks are indicated either as acetylation of the N-terminus itself (“Nt Ac”) or of tail lysine residues (“Ac”).(0.39 MB EPS)Click here for additional data file.


We recently identified matrix extracellular phosphoglycoprotein/osteoblast factor 45 (MEPE/OF45) as a new cofactor of CHK1 in rat cells. The aim of this study was to determine the role of human MEPE/OF45 (hMEPE/OF45 has ∼50% homology with rat MEPE/OF45 (rMEPE/OF45)) in affecting the sensitivity of human tumour cells to DNA damage.2 accumulation, CHK1 half-life and the CHK1 level in ligase 3 complexes were examined.hMEPE/OF45 expression in different human tumour cell lines and its relevance to the resistance of cell lines to DNA damage inducers such as ionising radiation (IR) or camptothecin (CPT) were assessed. Cells lines stably expressing wild-type MEPE/OF45 or mutant MEPE/OF45 (with the CHK1 interactive key domain (amino acids 488–507) deleted) were established. Cell survival, G2 checkpoint response and increases the resistance of tumour cells to IR or CPT treatment.hMEPE/OF45 expression correlates with the resistance of cell lines to IR or CPT. Upregulating wild-type hMEPE/OF45 (but not mutant hMEPE/OF45) could stabilize CHK1 by reducing CHK1 interaction for its E3 ligases Cul1 or Cula4A; it increases the GhMEPE/OF45 could be a new target for sensitizing tumour cells to radiotherapy or chemotherapy. Matrix extracellular phosphoglycoprotein/osteoblast factor 45 (MEPE/OF45) was originally cloned from a human oncogenetic hypophosphataemia tumour , Cul4 (sc-10782) and ubquitin (Ub) (sc-9133) were purchased from Santa Cruz Biotechnology Inc . The antibody against Cul1 was purchased from Zymed Laboratories  and the antibody against H2A (07-146) was purchased from Upstate Biotechnology .All human tumour cell lines used in this study were derived from ATCC. These cell lines were grown in DMEM medium supplemented with 10% calf serum. The antibody against hMEPE/OF45 was as described . PurifiehMEPE/OF45 or mutant hMEPE/OF45 (with the sequence encoding 488–507 amino acids (aa) deleted) was ligated with HA tag at the C terminus by PCR. hMEPE/OF45 was then cloned into pLenti6/V5-D-TOPO vector (Invitrogen), according to the manufacturer's protocol. The HA tag was inserted into the plasmid. The primers used for wild-type hMEPE/OF45 are: forward, 5′-CACCATGGGAGTTTTCTGTGTGGGACTACTCCTTTTCAGTGTGACCTG-3′ and reverse, 5′-AGGTCCTCCCAGGCTGGCATAGTCAGGCACGTCATAAGGATAGTCACCATCGCTCTCACTTGAAC-3′. The procedure to construct mutant hMEPE/OF45 has been described in μM). Titres were determined by infecting 293 cells with a serial dilution of the concentrated virus. For a typical preparation, the titre was approximately 2- to 7 × 104 TU ml−1. For infection of 293 cells, 30–50% confluent cells were incubated with a suitable amount of virus particles and 10 μg ml−1 polybrene for 8–16 h at 37°C. A volume of 10 μg ml−1 of blasticidin (Invitrogen) was used to select stably expressed cells.The full-length cDNA of wild-type M Na3VO4, 5 mM NaF and 0.2 mM PMSF in PBS buffer) and boiled in 30 μl of protein loading buffer. They were loaded onto 10% Tris-PAGE gel, followed by the standard western blot.Immunoprecipitation was performed as described previously  at room temperature for 1 h. The distribution of cells in the cell cycle was measured using a flow cytometer .Cell sensitivity to IR or to CPT was determined by clonogenic assay as described previously (t-test). P-values <0.05 were regarded as significant.Differences between treatment groups were analysed using statistical software (MEPE/OF45s. Our group recently identified a new exon of hMEPE/OF45 between formerly identified exons 3 and 4 (GenBank accession no. DQ854717). The functions of the different forms of MEPE/OF45 need to be investigated further. Interestingly, the levels of MEPE/OF45 in different human tumour cell lines broadly correlate with the resistance of cell lines to DNA damage inducers  is longer than that in parental cells (293), the 293 cells transfected with the vector alone (V1) and the 293 cells transfected with mutant hMEPE/OF45 (D1) . These r2 checkpoint response and the sensitivity of 293 cells to DNA damage inducers. The results showed that wild-type hMEPE/OF45 could increase the G2/M accumulation of cells after IR, but not mutant hMEPE/OF45  and other groups are <0.01. These results indicate that hMEPE/OF45 protects cells from DNA damage-induced killing depending on its interaction with CHK1. It is known that CHK1 promotes HRR through regulation of checkpoint activation (To further examine whether hMEPE/OF45 was similar to rMEPE/OF45, and could protect cells from DNA damage-induced killing through interaction with CHK1, we compared the effects of wild-type hMEPE/OF45 and mutant hMEPE/OF45 on the GEPE/OF45 . Similarmulation . These rependent . The P-vCHK1 is an essential gene (Taken together, the results of our study indicate that MEPE/OF45 protecting human cells from IR- or CPT-induced killing mainly depends on its interaction with CHK1. The specific molecular mechanism for this protective role of MEPE/OF45 could be linked to MEPE/OF45 stabilising CHK1 by reducing CHK1 degradation. Because


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS) and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD) imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS.G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage – dependent cytosolic Ca2+ elevations and mitochondria – controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5YWT) and G93A (SH-SY5YG93A) expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation.To gain insights into the molecular mechanisms of SOD12+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells an established cellular model of ALS. The primary goals of this paper are (1) method development, and (2) screening for deficits in mutant cells on the single cell level. On the technological level, our method promises to serve as a valuable tool to identify mitochondrial and Ca2+-related defects during G93A-mediated MN degeneration. In addition, our experiments support a model where a specialized interplay between cytosolic calcium profiles and mitochondrial mechanisms contribute to the selective degeneration of neurons in ALS.In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca SOD1 with high inter-subject variation of progression, including the point mutation G93A i) signaling in cells transfected with wild-type and G93A-mutant SOD1 varies at physiological levels, and if there are functional consequences of changes in mitochondrial function on Ca2+ homeostasis in the presence of mtSOD1G93A. To elucidate the underlying molecular events and cellular alterations involved in oxidative stress induced by the aberrant Cu-Zn chemistry and the roles of impaired Ca2+ handling and oxidative stress induced by ROS in fALS, we used SH-SY5Y neuroblastoma cells transfected with the G93A mutant form of SOD1 typical for fALS (SH-SY5YG93A or G93A) and SH-SY5Y cells transfected with wild-type (WT) human SOD1 (SH-SY5YWT or WT) and mimic the situation present in heterozygous patients, previously established as an in-vitro cell culture model of ALS, resembling the situation of heterozygous patients .2 i were measured in non-transfected SH-SY5Y cells  and SH-SY5Y cells expressing WT or G93A attached to glass cover slips after 2–5 days in culture. Cell layers were incubated with RPMI-1640  containing 10% FCS  and 0.846 mM Ca2+ (supplier's data). Cells were rinsed with RPMI -1640 and incubated for 20 min at 37°C to allow for complete de-esterification.The calcium-sensitive fluorescent dye, Fura-2, was introduced into cells by loading the membrane-permeable AM-ester forms .Changes in [Cantration . Therefo2+]i and was represented as F/F0. Further analysis was performed off-line with the IGOR software  and Origin software, version 7.5.Fura-2 (Kd ~ 0.2 μM) was monitored by a computer-controlled monochromator , which was connected to an Axioscope microscope  via quartz fiber optics and a minimum number of optical components for maximum fluorescence excitation . The apparatus was equipped with a CCD camera system ,52, whic2+]i and mitochondrial calcium ([Ca2+]mito) were performed utilizing calcium-sensitive fluorescent dyes . SH-SY5Y cells expressing either WT or G93A were attached to glass cover slips and loaded initially with 5 μM Fura-2 AM at 37°C for 30 min as described above. Subsequently, cells were washed with RPMI-1640 for 20 min and further incubated with 10 μM Rhod-2 AM for 20 min at 37°C (5% CO2 and 95% O2). The coverslips were rinsed with RPMI-1640 and further incubated for 20 min at 37°C to allow for complete de-esterification.Simultaneous fluorometric measurements of [Ca2+]i and [Ca2+]mito profiles with a temporal resolution in the millisecond time domain, we utilized a computer-controlled monochromator that rapidly switched between excitation wavelengths of 390 nm (Fura-2) and 550 nm (Rhod-2) and collected wavelengths above 565 nm for both excitation conditions. Schematic representation of the CCD-imaging setup used for conventional calcium imaging and simultaneous imaging of [Ca2+]i and [Ca2+]mito signals in the cell line preparation are illustrated in Additional file To clearly separate and simultaneously monitor the dynamic i and [Ca2+]mito signals to remove the noise.Each cover slip was used for a single experiment and included more than 4 cells for each imaging experiment. Unless otherwise indicated, values in the text are given as mean ± standard deviation (SD) and the error bars indicate SD too. All values represent at least three separate experiments. Significance was calculated using the two tailed unpaired Student t-test. A 2+ influx in WT and G93A transfected cells was done in the presence of FCCP. For these experiments the SH-SY5Y cells were loaded with 5 μM Fura-2 AM through the incubation method and [Ca2+]i transients were triggered once Fura-2 AM concentration was equilibrated after washing. Ca2+ release from intracellular stores by inhibition of the sarcoplasmic/endoplasmic reticulm Ca2+-dependent ATPase pump and inhibition of F1, F0-ATP Synthase was done by intervention of thapsi, and oligo, respectively.Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), thapsigargin, and oligomycin were purchased from Sigma-Aldrich Chemie . FCCP was bath applied at a dose of 2 μM. A comparative analysis of the Ca2+ release, caffeine has been established as a specific activator of Ca2+ influx from the RyR-dependent Ca2+ stores [2+]i regulation together with the association on MN degeneration in G93A transfected SH-SY5Y cells, we used pharmacological agents as a specific activator of the ER-dependent Ca2+ release response i measured for both WT and G93A-transfected SH-SY5Y cells , after peak FCCP challenge (3 min), and after FCCP wash in WT-SOD1 (a-c) and G93A (d-f) transfected cells are shown in Fig. 2+]i as cytoplasmic calcium versus time curves in 6 representative cells during FCCP challenge. The mitochondrial uncoupler induces a fast transient elevation in [Ca2+]i and a fast recovery to baseline in WT transfected cells .The transfected cells were treated with FCCP (3 min) and the [Calls Fig. . A CCD i2+ may have a perceptible influence on the existence of a [Ca2+]i transient. This effect was studied by following the Ca2+ signal under healthy and deleterious mitochondrial conditions in the presence of FCPP. We found that, in G93A transfected SH-SY5Y cells, the Ca2+ influx (peak fluorescence) was diminished compared to WT transfected SH-SY5Y cells, which, peaked within 2–3 min and was followed by a baseline recovery in approximately 2 min. The G93A transfected cells took almost 5–6 min to achieve a baseline recovery. However, the cells transfected with G93A suffered from reduced [Ca2+]mito after depletion of mitochondrial Ca2+ stores by FCCP  located in the inner mitochondrial membrane . The accCCP Fig. .2+ sequestering organelle, FCCP was applied to evacuate mitochondrial Ca2+after the WT and G93A transfected cells had been exposed to an evoked Ca2+ load through a depolarizing stimulus of K+. First, the SH-SY5Y cells transfected with WT or G93A were stained with Fura-2 AM and exposed to 30 mM K+ for 30 sec, which was followed by a 3 min challenge with FCCP  and 0.1497 ± 0.0362  for the WT and G93A transfected cells, respectively. Fig. To analyze the comparative efficiency of mitochondria as a Ca2+-ATPase inhibitor thapsi, an extremely tight-binding inhibitor of intracellular Ca2+ pumps which induces rapid Ca2+ release from intracellular stores by inhibition of the sarcoplasmic/endoplasmic reticulm Ca2+-dependent ATPase pump without inositol phosphate formation mito increase while measuring an increase in [Ca2+]i, we were able to efficiently separate cytosolic and mitochondrial Ca2+ at a temporal resolution in the millisecond time domain. As shown in Fig. 2+ release in G93A transfected cells is smaller in amplitude and exhibits slower kinetics compared to WT transfected cells. To confirm that there are separate Ca2+ uptake and release events from the cytosol and mitochondria, the transfected cells were treated with FCCP and the separate Ca2+ levels measured simultaneously  compared with mitochondria after Ca2+ elevation by FCCP (3 min) indicating its low efficiency to sequester Ca2+ in the WT and G93A transfected SH-SY5Y cells, which was slightly higher in WT  was added before FCCP application. Oligo does not influence [Ca2+]i heavily and left basal Ca2+ levels, as well as recovery times of Ca2+ transients, unaffected for upto several minutes  phenylhydrazone; FCS: fetal calf serum; FMNs: facial motoneurons; HMNs: hypoglossal motoneurons; hALS: human amyotrophic lateral sclerosis; MCU: mitochondrial calcium uniporter; MN: motoneuron; mtSOD1: mutant Cu/Zn superoxide dismutase1; oligo: oligomycin; ROI: regions of interest; ROS: reactive oxygen species; RPMI 1640: Roswell Park Memorial Institute medium; RyR: ryanodine receptor; SD: standard deviation; SOD1: Cu/Zn superoxide dismutase1; TPPThe authors declare that they have no competing interests.MKJ performed the experiments, analyzed the data, and wrote the initial version of the manuscript. WDZ, MG and RN established the primary cultured human SH-SH5Y cell line and maintained them during the course of the project. Cell lines expressing either wild-type (Wt) human SOD1 or the G93A mutants of this enzyme were produced by AF and MTC. CL, AZ, MTC and RN participated in the design of the study and participated in the organization of financial support. BUK is the principal investigator of the project. Besides providing laboratory support and supervision, he is primarily responsible for financial support and project management. All authors read and approved the final manuscript.Schematic representation of method and spectra. Schematic representation of the (A) CCD-imaging setup used for calcium imaging and (B) simultaneous measurements of [Ca2+]i and [Ca2+]mito in SH-SY5Y cell line preparations. C) Spectral view of calcium imaging. The fluorescence excitation at 510 nm and emission detected at 340 nm are shown for Ca2+-saturated (A) and Ca2+-free (B) Fura-2 in pH 7.2 buffer. D) Spectral view of simultaneous [Ca2+]i and [Ca2+]mito measurements generated by the Molecular Probes spectral view program. Fluorescence excitation of fura-2 was done at 390 nm and 550 nm of rhod-2, respectively; emission was at 510 nm  and at 600 nm (rhod-2) separated by a 565 nm dichroic mirror.Click here for fileMitochondria-dependent responses of cytosolic calcium in SH-SY5Y neuroblastoma cells  loaded with Fura-2 AM and superfused with DMEM medium. A) Representative CCD camera photomicrograph of Fura-2 AM loaded SH-SY5Y cells. Scale bar is 10 μm. B) FCCP-evoked mitochondria-dependent responses of cytosolic calcium.Click here for file


Gpnmb gene encodes a transmembrane protein whose function(s) remain largely unknown. Here, we assess if a mutant allele of Gpnmb confers susceptibility to glaucoma by altering immune functions. DBA/2J mice have a mutant Gpnmb gene and they develop a form of glaucoma preceded by a pigment dispersing iris disease and abnormalities of the immunosuppressive ocular microenvironment.The Gpnmb genotype of bone-marrow derived cell lineages significantly influences the iris disease and the elevation of intraocular pressure. GPNMB localizes to multiple cell types, including pigment producing cells, bone marrow derived F4/80 positive antigen-presenting cells (APCs) of the iris and dendritic cells. We show that APCs of DBA/2J mice fail to induce antigen induced immune deviation (a form of tolerance) when treated with TGFβ2. This demonstrates that some of the immune abnormalities previously identified in DBA/2J mice result from intrinsic defects in APCs. However, the tested APC defects are not dependent on a mutant Gpnmb gene. Finally, we show that the Gpnmb mediated iris disease does not require elevated IL18 or mature B or T lymphocytes.We find that the Gpnmb in bone marrow derived lineages. They suggest that affects of Gpnmb on innate immunity influence susceptibility to glaucoma in DBA/2J mice.These results establish a role for  The glaucomas are a common group of potentially blinding diseases that by 2010 will affect approximately 60 million people worldwide . This glExperiments with DBA/2J (D2) mice have suggested that immune abnormalities may contribute to some forms of glaucoma ,21. D2 mTyrp1 and Gpnmb . Immune deviation was detected as the suppression in DTH in the recipients of TGFβ2 treated APCs as compared to those infused with untreated APCs. A two-tailed Student's t-test was used with significance assumed at P < 0.05. DTH results were confirmed by repeating the experiments a second time.Peritoneal exudate cells (PECs) were obtained 3 days following 2 ml intra-peritoneal injections of a 3% thioglycollate solution . Plastic adherent macrophages from these cells were used as APCs (>95% F4/80+). Cells were cultured overnight (1 × 102Se and 0.2 μg/ml Fe(NO3)3] .Serum-free medium (SFM) was used for in vitro assays. The medium contained: RPMI-1640, 10 mM HEPES, 0.1 mM NEAA, 1 mM Sodium pyruvate, 100 U/ml Penicillin, 100 mg/ml Streptomycin , 0.1% BSA  and ITS+ culture supplement [1 μg/ml iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml NaGpnmb). The ipd allele of Gpnmb results from the R150X premature stop codon mutation, GpnmbR150X [Tyrp1). The b allele of Tyrp1 encodes two amino acid substitutions compared to the C57BL/6J-derived allele [isa in the mouse genome database and elsewhere [Rag1). The tm1Mom mutation is a targeted knock-out [Rag1tm1Mom stock utilized here was B6.129S7-Rag1tm1Mom/J (stock 002216 from The Jackson Laboratory). 4. Protein kinase, DNA activated, catalytic polypeptide (Prkdc). The scid mutation arose spontaneously in the CB17 strain and has since been backcrossed onto C57BL/6J genetic background [Prkdcscid stock utilized here was B6.CB17-Prkdcscid/SzJ (stock 001913 from The Jackson Laboratory).The official full names of genes (with abbreviations in parentheses), alleles, and stocks utilized in this study are as follows. 1. Glycoprotein (transmembrane) nmb . (A to D) Eyes of young mice of all genotypes are characterized by healthy irides. By 8–9 mo, Tyrp1 and Gpnmb mutant mice exhibited typical signs of early disease that did not differ whether the mice were (E to H) immune competent, or (I to L) immune deficient. At 14+ mo, Tyrp1 and Gpnmb mutant mice exhibited signs of late disease that did not differ whether the mice were (M to P) immune competent or (Q to T) immune deficient. Panels K and Q are based on observations of 2 eyes, panel I from 6 eyes, and all other panels from groups of 8 to 26 eyes.Click here for fileGpnmbR150X mutation does not directly elevate IOP. Mean IOP ± SEM is presented. For ease of comparison, some of the data are duplicated from Figure GpnmbR150X mutation contributes to the iris disease of DBA/2J mice and is a critical component of the glaucoma. Manipulations that prevent severe iris disease in DBA/2J mice prevent both anterior chamber enlargement (a marker of IOP elevation) and glaucoma development [GpnmbR150X mutation also influences IOP independently of the iris disease, we have assessed IOP in aged DBA/2J mice that have the Gpnmb mutation in all tissues but that do not develop the severe iris disease. These mice were homozygous for the wild-type C57BL/6J allele of Tyrp1  [Gpnmb (GpnmbD2 = GpnmbR150X). Since mutant DBA/2J alleles of both Tyrp1 and Gpnmb are necessary to develop severe iris disease, the Tyrp1B6GpnmbD2 mice did not do so. Importantly and despite the presence of the Gpnmb mutation in the iris and bone marrow, the Tyrp1B6 GpnmbD2 mice did not develop elevated IOP. Thus, the GpnmbR150X mutation does not induce high IOP directly but does so by inducing the iris disease.elopment ,22. Thusrations)  and the Click here for file