Novel mutations of the arylsulphatase B (ARSB) gene in Indian patients with mucopolysaccharidosis type VI.

BACKGROUND & OBJECTIVES: Mucopolysaccharidosis type VI (MPS VI) is a rare, autosomal recessive lysosomal storage disorder caused by deficient enzymatic activity of N-acetyl galactosamine-4-sulphatase resulting from mutations in the arylsulphatase B (ARSB) gene. The ARSB gene is located on chromosome 5q11-q13 and is composed of eight exons. More than hundred ARSB mutations have been reported so far, but the mutation spectrum of MPS VI in India is still unknown. Hence, the aim of the present study was to identify the mutational spectrum in patients with MPS VI in India and to study the genotype-phenotype association and functional outcomes of these mutations. METHODS: Molecular characterization of the ARSB gene by Sanger sequencing was done for 15 patients (aged 15 months to 11 yr) who were enzymatically confirmed to have MPS VI. Age of onset, clinical progression and enzyme activity levels in each patient were studied to look for genotype-phenotype association. Haplotype analysis performed for unrelated patients with the recurring mutation W450C, was suggestive of a founder effect. Sequence and structural analyses of the ARSB protein using standard software were carried out to determine the impact of detected mutations on the function of the ARSB protein. RESULTS: A total of 12 mutations were identified, of which nine were novel mutations namely, p.D53N, p.L98R, p.Y103SfsX9, p.W353X, p.H393R, p.F166fsX18, p.I220fsX5, p.W450L, and p.W450C, and three were known mutations (p.D54N, p.A237D and p.S320R). The nine novel sequence variants were confirmed not to be polymorphic variants by performing sequencing in 50 unaffected individuals from the same ethnic population. INTERPRETATION & CONCLUSIONS: Nine novel mutations were identified in MPS VI cases from India in the present study. The study also provides some insights into the genotype-phenotype association in MPS VI.

Mos 3' UTR regulatory differences underlie species-specific temporal patterns of Mos mRNA cytoplasmic polyadenylation and translational recruitment during oocyte maturation.

The Mos proto-oncogene is a critical regulator of vertebrate oocyte maturation. The maturation-dependent translation of Mos protein correlates with the cytoplasmic polyadenylation of the maternal Mos mRNA. However, the precise temporal requirements for Mos protein function differ between oocytes of model mammalian species and oocytes of the frog Xenopus laevis. Despite the advances in model organisms, it is not known if the translation of the human Mos mRNA is also regulated by cytoplasmic polyadenylation or what regulatory elements may be involved. We report that the human Mos 3' untranslated region (3' UTR) contains a functional cytoplasmic polyadenylation element (CPE) and demonstrate that the endogenous Mos mRNA undergoes maturation-dependent cytoplasmic polyadenylation in human oocytes. The human Mos 3' UTR interacts with the human CPE-binding protein and exerts translational control on a reporter mRNA in the heterologous Xenopus oocyte system. Unlike the Xenopus Mos mRNA, which is translationally activated by an early acting Musashi/polyadenylation response element (PRE)-directed control mechanism, the translational activation of the human Mos 3' UTR is dependent on a late acting CPE-dependent process. Taken together, our findings suggest a fundamental difference in the 3' UTR regulatory mechanisms controlling the temporal induction of maternal Mos mRNA polyadenylation and translational activation during Xenopus and mammalian oocyte maturation.

Bio-mimetic composite matrix that promotes endothelial cell growth for modification of biomaterial surface.

The incidence of thrombogenesis and occlusion of cardiovascular implants is likely to be reduced by endothelial cell (EC) growth promotion on biomaterials used for device fabrication. However, proper signaling between the matrix proteins deposited on the device surface and the cells grown on it is a prime requirement for growth and function. It was demonstrated earlier that a composition of matrix proteins that include fibrin, fibronectin, gelatin, and growth factors maintain a steady proliferation potential and prolong the survival of endothelial cells in vitro. In this study, assessment of the same matrix to prevent EC from dedifferentiation during in vitro culture and to promote endothelialization of biomaterials used for fabrication of cardiovascular implants is carried out. Up/down regulation of m-RNA expression for a prothrombotic molecule-plasminogen activator inhibitor (PAI), and two antithrombotic molecules- nitric oxide synthetase (eNOS) and tissue plasminogen activator (t-PA) are chosen as the indicators of cell dedifferentiation during cell culture and passaging. Immunostaining for vinculin and actin demonstrated that composite coating on biomaterials improves focal adhesion and cytoskeletal organization that increases the quality of EC grown on it. EC proliferation, measured by (3)H-thymidine uptake, on all bare materials was poor and high incidence of cell apoptosis was noticed within 72 h in culture, whereas once coated with composite all materials showed good proliferation and survival. The results suggest that the designed composition of biomimetic adhesive proteins and growth factors is suitable for EC growth, survival, and functional integrity, thus making it suitable for cardiovascular tissue engineering that requires in vitro EC culture.

Hyperhomocysteinemia and the compound heterozygous state for methylene tetrahydrofolate reductase are independent risk factors for deep vein thrombosis among South Indians.

To investigate the role of methylene tetrahydrofolate reductase (MTHFR) (677 C-->T and 1298 A-->C), factor V (1691 G-->A), factor II (20210 G-->A) genetic polymorphisms and hyperhomocysteinemia in the aetiology of deep vein thrombosis (DVT) in 163 cases and 163 controls. Polymerase chain reaction-restriction fragment length polymorphism was used for genotyping, reverse-phase high-performance liquid chromatography for plasma homocysteine, and Student's t-test and Fisher exact tests were used for statistical analysis. Elevated mean plasma homocysteine levels were observed in DVT cases irrespective of gender differences. Homocysteine elevation above the 95th percentile of the control group associated with 9.4-fold and 7.6-fold increased risk for DVT in men and women, respectively. Genotyping showed the MTHFR 677CT/1298AC genotype (i.e. compound heterozygosity) is associated with 3.5-fold risk for thrombosis. The factor V Leiden mutation frequency was higher in DVT cases, but not statistically significant; however, genetic predisposition to this mutation was associated with early age of DVT onset. Factor II mutation was absent in cases and controls. Co-segregation of two or more risk factors was associated with 11.7-fold increased risk for thrombosis. This study projects that hyperhomocysteinemia and compound heterozygous state for MTHFR are independent risk factors for DVT among South Indians.

Phenotype gradation of human saphenous vein endothelial cells from cardiovascular disease subjects.

The highly organized histological architecture of the vascular wall and the specialized cellular phenotypes are perturbed in conditions such as atherosclerosis, restenosis, and hypertension. Alterations of endothelial cell (EC) phenotype in cardiovascular diseases (CVDs) as an effect of alteration of extracellular matrix (ECM) composition have not been well understood. In vitro study of EC phenotype is limited because they tend to dedifferentiate in subcultures. The objective of this study was to use in vitro cell culture on a biomimetic matrix model to characterize phenotype of human saphenous vein endothelial cells (HSVECs) harvested from CVD patients. Parameters studied were mRNA expression and synthesis of von Willebrand factor (vWF), plasminogen activation inhibitor (PAI), tissue plasminogen activator (t-PA), and endothelial nitric oxide synthetase (eNOS). Proliferation and apoptosis of HSVEC cultures obtained from eight different patients were compared on two matrices until passage 12. In early passages, both the prothombotic molecules vWF and PAI were overexpressed, whereas the antithrombotic molecules t-PA and eNOS were underexpressed. With increase in passage number, low expression of prothrombotic molecules and high expression of antithrombotic molecules were seen in cells on the model matrix. But when cells were grown on conventional gelatin-coated polystyrene, expression of prothrombotic molecules amplified further and antithrombotic molecules lessened with the progression of passages. On the model matrix HSVECs showed good proliferation rate and survival through several passages. It is demonstrated that matrix composition can influence switching of EC phenotypes into pro/antithrombotic states. This matrix model may be suitable to study the effect of exogenous factors on EC dysfunction with respect to CVD.

Use and specificity of breast cancer antigen/milk protein BA46 for generating anti-self-cytotoxic T lymphocytes by recombinant adeno-associated virus-based gene loading of dendritic cells.

Antigen-targeted immunotherapy is an emerging treatment for breast cancer. However, useful breast cancer antigens are only found in a subset of cancer patients. BA46, also known as lactadherin, is a membrane-associated glycoprotein that is expressed in most breast cancer cells but not in general hematopoietic cell populations. Moreover, it is much more difficult to generate CTLs against self-antigens. We wished to determine if the use of recombinant adeno-associated virus (rAAV) type 2 vectors for gene-loading of dendritic cells (DCs) could generate rapid, effective cytotoxic T lymphocytes (CTLs) against BA46. We were able to demonstrate that AAV/BA46/Neo-loading of DCs resulted in: (1) BA46 expression in DCs, (2) chromosomal integration of the AAV/BA46/Neo vector within DCs, (3) strong, rapid BA46-specific, MHC class I-restricted CTLs in only 1 week, (4) T-cell populations with significant interferon-gamma (IFN-gamma) expression but low IL-4 expression, (5) high CD80 and CD86 expression in DCs, and (6) high CD8:CD4 and CD8:CD56 T cell ratios. These data suggest that rAAV-loading of DCs may be useful for immunotherapeutic protocols against self-antigens in addition to viral antigens and that the BA46 antigen is potentially appropriate for cell-mediated immunotherapeutic protocols addressing ductal breast cancer.

Survival of endothelial cells in vitro on Paclitaxel-loaded coronary stents.

Coronary stents that are developed for use with balloon angioplasty are known to cause acute occlusion and long-term stenosis. It is likely that a controlled release of drugs at the site of stent implantation might inhibit the proliferation of vascular smooth muscle cells (VSMC) and reduce restenosis. However, if the drug is necrotic and affects cell survival near the implant, it may interrupt the local tissue regeneration. Different methods have been used for the immobilization of drugs with stents to get an effective concentration that inhibits cell proliferation. The objective of this study is to assess the effectiveness of Paclitaxel-loaded stents by immobilization with a biodegradable polymer, to inhibit cell proliferation. The cells used for the evaluation are human umbilical vein endothelial cells (HUVEC) and the proliferation rate of these cells on the drug-coated stent is compared against an uncoated stent for a 72-h period. Evaluations were also made to differentiate between cell apoptosis and necrosis to prove that the drug released is not deleterious to the surrounding tissue. While a similar initial cell adhesion is observed in bare and coated stents, the proliferation of HUVEC is negligible when grown on a drug-coated stent (p < 0.001). By specific staining techniques, the cells on the drug-coated stents are found to be apoptotic and not necrotic, throughout the evaluation period. In vitro leukocyte adhesion and platelet deposition on the drug-coated stents are found to be low when they are exposed to human blood and platelet-rich plasma (PRP), suggesting that the coated stents may not be thrombogenic in vivo. Therefore, drug coating of stents using the described technique may have a considerable promise for the prevention of neointimal proliferation, restenosis, and associated failure of angioplasty.

Symbiotic characteristics of cysteine and methionine auxotrophs of Sinorhizobium meliloti.

Twenty one cysteine and 13 methionine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5. The cysteine auxotrophs were sulfite reductase mutants and each of these auxotrophs had a mutation in cysI/cysJ gene. The methionine auxotrophs were metA/metZ, metE and metF mutants. One hundred per cent co-transfer of Tn5-induced kanamycin resistance and auxotrophy from each Tn5-induced auxotrophic mutant indicated that each mutant cell most likely had a single Tn5 insertion. However, the presence of more than one Tn5 insertions in the auxotrophs used in our study cannot be ruled out. All cysteine and methionine auxotrophs induced nodules on alfalfa plants. The nodules induced by cysteine auxotrophs were fully effective like those of the parental strain-induced nodules, whereas the nodules induced by methionine auxotrophs were completely ineffective. The supplementation of methionine to the plant nutrient medium completely restored symbiotic effectiveness to the methionine auxotrophs. These results indicated that the alfalfa host provides cysteine but not methionine to rhizobia during symbiosis. Histological studies showed that the defective symbiosis of methionine auxotrophs with alfalfa plants was due to reduced number of infected nodule cells and incomplete transformation of bacteroids.

Symbiotic characterization of isoleucine+valine and leucine auxotrophs of Sinorhizobium meliloti.

Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by screening of Tn5 derivatives on minimal medium supplemented with modified Holliday pools. Based on intermediate feeding, intermediate accumulation and cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively. Symbiotic properties of all ilvD mutants with alfalfa plants were similar to those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-. Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair curling and infection thread formation. The ilvC mutants were capable of curling root hairs but did not induce infection thread formation. All leucine auxotrophs were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not restore symbiotic effectiveness to the auxotrophs. Histological studies revealed that the nodules induced by the leucine auxotrophs did not develop fully like those induced by the parental strain. The nodules induced by leuB mutants were structurally more advanced than the leuC/leuD mutant induced nodules. These results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may have a role in symbiosis. The position of ilv genes on the chromosomal map of S. meliloti was found to be near ade-15 marker.

Multiple human papillomavirus genes affect the adeno-associated virus life cycle.

The risk of cervical cancer, one of the most prevalent cancers in the world, is determined by two viruses. Human papillomavirus (HPV) is the main risk factor for developing cervical cancer. However, although little known, it is well substantiated that the human Parvovirus adeno-associated virus type 2 (AAV), and its encoded Rep78 protein, interacts with HPV and lowers the risk of cervical cancer. HPV also contributes to AAV inhibition by serving as a helper virus for AAV and stimulating higher AAV replication levels. Here we surveyed four HPV-16 early genes, E1, E2, E6 and E7, for their ability to increase/decrease the basal level of AAV replication in stratifying squamous epithelium (the epithelial raft culture system). It was found that the HPV-16 E1, E2 and E6 genes were able to help/enhance AAV-2 replication in epithelial raft cultures. Under these conditions, with all the HPV genes being expressed from the AAV p5 promoter, E1 appeared to have the strongest enhancing effect on AAV DNA replication (Southern blot), RNA expression (RT-PCR), protein expression (Western blot) and AAV virion production (2 plate-Southern blot). Further study of E1 mutants showed that the carboxy-half of E1, the putative helicase/ATPase domain, was the main contributor of helper activity. These data are important for understanding the HPV-AAV interaction and its effect on modifying cervical cancer risk. These data also suggest the possibility that the identified HPV helper genes may be useful in the generation of recombinant (r)AAV virions for gene therapy, as rAAV is increasing in popularity for such purposes.

Infection, replication, and cytopathology of human papillomavirus type 31 in trophoblasts.

Human papillomavirus (HPV) DNA is preferentially found in spontaneous abortions, specifically residing in trophoblasts, and transfected HPV-16 DNA replicates and produces progeny in 3A trophoblasts in culture. In this study 3A trophoblasts were shown to display both HPV receptors and infection by HPV-31b and HPV-6 virus resulted in de novo (increasing) HPV DNA replication in these cells (inhibited by neutralizing anti-HPV31b antibodies). Reverse transcription-polymerase chain reaction analysis revealed that E1;E4, E6, and L1 were significantly expressed at days 5 (early) and 10 (late), respectively, and in situ immunocytochemistry verified L1 protein expression. Perhaps most important, HPV 31b virus infection caused both a decrease in 3A trophoblast cell numbers in a dose-dependent manner and a low trophoblast-endometrial cell adhesion (both inhibited by neutralizing anti-HPV-31 antibodies). These data further support the hypothesis that HPVs are fully active in trophoblasts and may cause some spontaneous abortions.

The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity.

Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process.