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The present study aims to evaluate the effect of Sepia pharaonis ink on insulin resistance in PCOS-induced mice. Treatment with sepia ink in dehydroepiandrosterone (DHEA)-induced PCOS mice at various doses of 50 mg/kg, 100 mg/kg, and 200 mg/kg body weight mitigated the insulin resistance in the study groups with decreased concentration of testosterone and increased concentrations of estrogen and progesterone compared to the PCOS group tested by ELISA. The histopathological analysis and restoration of glucose analysis showed a significant reduction in treatment groups. Reduced expression of insulin resistance genes like androgen receptor (AR), insulin receptor substrate 1 (IRS-1), and insulin-like growth factor1 (IGF-1) by qRT-PCR indicate a positive impact of sepia ink in alleviating the symptoms associated with PCOS. Taken together, the results of this study indicate sepia ink as a promising therapeutic intervention and a possible drug target for insulin resistance in diabetes and gynecological disorders like PCOS.

Objective and Design: Inflammatory bowel disease (IBD) is an idiopathic inflammatory condition of the digestive system marked by oxidative stress, leukocyte infiltration, and elevation of inflammatory mediators. In this study, we demonstrate the protective effect of ethyl gallate (EG), a phytochemical, and propyl gallate (PG), an anti-oxidant, given through normal drinking water (DW) and copper water (CW) in various combinations, which had a positive effect on the amelioration of DSS-induced ulcerative colitis in C57BL/6 J mice., Materials and Methods: We successfully determined the levels of proinflammatory cytokines and anti-oxidant enzymes by ELISA, tracked oxidative/nitrosative stress (RO/NS) by in vivo imaging (IVIS) using L-012 chemiluminescent probe, disease activity index (DAI), and histopathological and morphometric analysis of colon in DSS-induced colitis in a model., Results: The results revealed that oral administration of ethyl gallate and propyl gallate at a dose of 50 mg/kg considerably reduced the severity of colitis and improved both macroscopic and microscopic clinical symptoms. The level of proinflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ) in colonic tissue was considerably reduced in the DSS + EG-treated and DSS + PG-treated groups, compared to the DSS alone-treated group. IVIS imaging of animals from the DSS + EG and DSS + PG-treated groups showed a highly significant decrease in RO/NS species relative to the DSS control group, with the exception of the DSS + PG/CW and DSS + EG + PG/CW-treated groups. We also observed lower levels of myeloperoxidase (MPO), nitric oxide (NO), and lipid peroxidation (LPO), and restored levels of GST and superoxide dismutase (SOD) in DSS + EG-DW/CW, DSS + PG/DW, and DSS + EG + PG/DW groups compared to DSS alone-treated group. In addition, we showed that the EG, PG, and EG + PG treatment significantly reduced the DAI score, and counteracted the body weight loss and colon shortening in mice compared to DSS alone-treated group. In this 21-day study, mice were treated daily with test substances and were challenged to DSS from day-8 to 14., Conclusion: Our study highlights the protective effect of ethyl gallate and propyl gallate in various combinations which, in pre-clinical animals, serve as an anti-inflammatory drug against the severe form of colitis, indicating its potential for the treatment of IBD in humans. In addition, propyl gallate was investigated for the first time in this study for its anti-colitogenic effect with normal drinking water and reduced effect with copper water., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The thymus is a primary lymphoid organ, essential for the development of T-cells that will protect from invading pathogens, immune disorders, and cancer. The thymus decreases in size and cellularity with age referred to as thymus involution or atrophy. This involution causes decreased T-cell development and decreased naive T-cell emigration to the periphery, increased proportion of memory T cells, and a restricted, altered T-cell receptor (TCR) repertoire. The changes in composition and function of the circulating T cell pool as a result of thymic involution led to increased susceptibility to infectious diseases including the recent COVID and a higher risk for autoimmune disorders and cancers. Thymic involution consisting of both structural and functional loss of the thymus has a deleterious effect on T cell development, T cell selection, and tolerance. The mechanisms which act on the structural (cortex and medulla) matrix of the thymus, the gradual accumulation of genetic mutations, and altered gene expressions may lead to immunosenescence as a result of thymus involution. Understanding the molecular mechanisms behind thymic involution is critical for identifying diagnostic biomarkers and targets for treatment help to develop strategies to mitigate thymic involution-associated complications. This review is focused on the consequences of thymic involution in infections, immune disorders, and diseases, identifying potential checkpoints and potential approaches to sustain or restore the function of the thymus particularly in elderly and immune-compromised individuals.

An aberrant immunologic mechanism and mitochondrial biogenesis have been suggested to be involved in the pathogenesis of endometriosis. Genetic alterations in the vitamin D receptor ( VDR ) gene and peroxisome proliferator-activated receptor-gamma coactivator 1α ( PGC-1α ) may lead to important defects in gene activation, which principally affect immune function and normal mitochondrial function. Therefore, we hypothesized a possible role of VDR and PGC-1α genes in the pathogenesis of endometriosis and analyzed the association of genetic variants ApaI A/C (rs7975232) and TaqI T/C (rs731236) of VDR and rs8192678 (G/A), rs13131226 (T/C), and rs2970856 (T/C) of PGC-1α gene. This study included a total of 425 reproductive-age women (cases = 200 and controls = 225). Detection of VDR and PGC-1α gene polymorphism was performed using polymerase chain reaction-restriction fragment length polymorphism and sequencing analysis. The chi-square test was used to compare allele and genotype frequencies between groups, and a p -value of <0.05 was considered statistically significant. The genotype and allele distribution of both the gene polymorphisms did not show statistically significant association with endometriosis. Our result indicated ApaI A and TaqI T of VDR and GTT of PGC-1α gene as the most common haplotype in Indian women. The data suggest that VDR and PGC-1α gene polymorphisms did not play an important role in the pathogenesis of endometriosis in Indian women studied.

The aim of the study was to investigate the association between Histone deacetylase 1 ( HDAC1 ), Sirtuin1 ( SIRT1 ), and Sirtuin3 ( SIRT3 ) single-nucleotide polymorphisms (SNPs) and risk of endometriosis in South Indian women. A total of 300 subjects were recruited in this case-control study comprising 150 affected women and 150 women with no evidence of disease. All the subjects were of South Indian origin. The genotyping of HDAC1 , SIRT1, and SIRT3 SNPs (rs1741981T/C, rs144124002A/G, and rs536715G/A) was carried out on DNA from subjects by PCR-RFLP and sequencing analysis. The genotype ( p =  .00782) and allele ( p =  .02561) frequencies of the HDAC1 rs1741981 polymorphism showed significant difference between cases and controls. In contrast, SIRT1 (rs144124002) and SIRT3 (rs536715) SNPs did not show significant association with the disease. The HDAC1 polymorphism may constitute a heritable risk factor for endometriosis in South Indian women. To date, there is no reported study on the association of polymorphisms in HDAC1 , SIRT1 , and SIRT3 with endometriosis risk. Impact Statement What is already known on this subject? Endometriosis is a benign gynaecological disease characterised by the implantation of functional endometrial tissue at ectopic positions, associated with an increased risk of malignant transformation. Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns. Histone modification, including deacetylation of lysine residues by HDACs , is a key epigenetic mechanism of gene expression regulation in endometriosis, therefore genetic variation in HDACs causing epigenetic control defects might lead to disease susceptibility. What do the results of this study add? Our study shows that the HDAC1 SNP is significantly associated with endometriosis in South Indian women, whereas the SNPs of SIRT1 and SIRT3 could not show any association with the disease. What are the implications of these findings for clinical practice and/or further research? The polymorphism of HDAC1 rs1741981 could be used as an important marker of genetic susceptibility to endometriosis development. Analysis of this SNP might help to identify patients at high risk for disease outcome.

Background: Endometriosis is a multifactorial estrogen dependent gynecological disease characterized by implantation of functional endometrial tissue at ectopic positions. Though this disease is benign, it is associated with an increased risk of malignant transformation. Epigenetic disruptions like aberrant DNA methylation, resulting changes in gene expression capacity, are important in tumor progression and malignant cellular transformation. Therefore, variation in genes involved in DNA methylation might lead to disease susceptibility., Purpose: To investigate the association between DNA methyl transferases (DNMT1 and DNMT3B) single nucleotide polymorphisms (SNPs) and the risk of endometriosis in South Indian women., Methods: In the present study, we examined the genotypic and allele distribution of DNMT1 (rs10423341C/A, rs2228611G/Aandrs4804490C/A) and DNMT3B (rs1569686G/T) among the endometriosis patients (n = 150) and controls (n = 150). The genotypes were analyzed by polymerase chain reaction (PCR) and sequencing methods. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were surveyed by Haploview Software., Result: Significant increase in the frequencies of DNMT1 rs10423341 (P = 0.04601), rs2228611 (P = 0.00175) and DNMT3B rs1569686 (P = 0.033) genotypes and alleles was observed in patients compared to controls. In addition, the frequency of A/A/C (P = 0.0065) haplotype was significantly high in patients. But the DNMT1 (rs4804490) SNP did not show significant association with the disease., Conclusion: The DNMT1 and DNMT3B polymorphism may constitute an inheritable risk factor for endometriosis in South Indian women. To the best of our knowledge there is no reported study on the association of polymorphisms in DNMT1 and DNMT3B with endometriosis risk., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Purpose: The purpose of the study was to investigate the association between gene phosphate and tensin homolog (PTEN) single nucleotide polymorphisms (SNPs) and risk of developing polycystic ovary syndrome (PCOS) in South Indian women. PTEN is one of the most important tumor suppressor genes that regulate cell proliferation, migration, and death. It is also involved in the maintenance of genome stability. PCOS is one of the most common endocrine disorders among women of reproductive age. It is a heterogeneous syndrome characterized by abnormal reproductive cycles, irregular ovulation, hormonal imbalance, hyperandrogenism, acne and hirsutism., Research Question: What is the association status of PTEN SNPs with PCOS?, Methods: A total of 240 subjects were recruited in this case-control study comprising 110 patients with PCOS and 130 individuals without PCOS. All the subjects were of South Indian origin. The genotyping of PTEN SNPs (rs1903858 A/G, rs185262832G/A and rs10490920T/C) was carried out on DNA from subjects by polymerase chain reaction (PCR) and sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were surveyed by Haploview Software., Results: Our results showed significant increase in the frequencies of rs1903858 A/G (P = 0.0016), rs185262832 G/A (P = 0.0122) and rs10490920 T/C (P = 0.0234) genotypes and alleles in cases compared to controls., Conclusion: The PTEN (rs1903858A/G, rs185262832G/A and rs10490920T/C) gene polymorphisms may constitute an inheritable risk factor for PCOS in South Indian women.

Sequencing of mitochondrial displacement-loop (D-loop) of polycystic ovary syndrome (PCOS) patients and (n=118) and controls (n=114) of south Indian origin showed significant association of D310 (P=0.042) and A189G (P=0.018) SNPs with PCOS. qRT-PCR analysis revealed significantly diminished mtDNA copy number in PCOS patients compared to controls (P=0.038). Furthermore, mtDNA copy number was significantly lower in PCOS cases carrying D310 and 189G alleles when compared to non-carriers (P=0.001 and 0.006 respectively). The D310 carriers also showed significantly elevated LH/FSH ratio (P=0.026). In conclusion, mtDNA D-loop and copy number alterations may constitute an inheritable risk factor for PCOS in south Indian women., (Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Objective: Polycystic Ovary Syndrome (PCOS) is a heterogeneous multifactorial endocrine metabolic disorder. In addition to hyperandrogenism, acne, hirsutism, obesity, oligoanovulation and infertility, insulin resistance is also a common feature in women of PCOS. Tumor suppressor genes (TSGs) perform essential function in the maintenance of genomic stability and regulatory pathways influencing the activity of several replication and transcription factors. The main aim of this study was to investigate the association of Single Nucleotide Polymorphisms of TP53, BRCA1and BRCA2 genes with the susceptibility to PCOS in South Indian women., Study Design: Present study investigated association between TP53 gene (rs1042522 G/C), BRCA1 (rs71361504 -/GTT, rs3092986 T/C) and BRCA2 (rs206118 A/G) and, SNPs and PCOS risk. Genotyping of TSGs was carried out on DNA from PCOS patients (n = 110) and controls (n = 130) of South Indian origin by polymerase chain reaction (PCR) and confirmed by sequencing analysis. The genotype frequency and allele distributions of cases and controls were analyzed using Fisher's exact test. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pair wise linkage disequilibrium (LD) were assessed by Haploview Software., Results: Significant increase in frequencies ofTP53 (rs1042522 G/C), BRCA1 (rs71361504 -/GTT, rs3092986 T/C) genotypes and alleles in patients compared to controls. In addition, the frequency of the C/T (P = 0.002) and A/C (P = 0.012) haplotype was also significantly elevated in patients. But BRCA2 (rs206118 A/G) did not show significant association with PCOS., Conclusion: The TP53 and BRCA1 may constitute an inheritable risk factor for PCOS in South Indian women., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of reproductive age women. Emerging evidence suggests that Vitamin D Receptor (VDR) might be a causal factor for characteristics associated with PCOS such as obesity and type 2 diabetes. Present study investigated association between VDR gene BsmI A/G (rs1544410), ApaI A/C (rs7975232) and TaqI T/C (rs731236) single nucleotide polymorphisms and PCOS risk in South Indian women. Genotyping of VDR gene SNPs was carried out in PCOS patients (n = 95) and controls (n = 130) by PCR-RFLP method and confirmed by sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were assessed by Haploview software. Results showed significantly increased frequencies of BsmI G/G (p = .0197), ApaI C/C (p = .048), TaqI C/C (p = .044) genotypes and BsmI G (p = .0181), ApaI C (p = .0092), TaqI C (p = .0066) alleles in patients compared to controls. In addition, the frequency of the 'BsmI G, ApaI C, TaqI C' haplotype was also significantly elevated in patients (p = .0087). In conclusion, the VDR gene BsmI A/G ApaI A/C TaqI T/C and haplotype may constitute an inheritable risk factor for PCOS in South Indian women.

We investigated the link between polymorphisms in genes involved in mitochondrial biogenesis, mitochondrial transcription factor A (TFAM) and Peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α) and further studied the role of these genes on the pathophysiology of polycystic ovary syndrome (PCOS). This case-control study was carried out in 118 PCOS cases and 110 controls. In the present study we genotyped three polymorphisms of PGC1-α gene (rs8192678-Gly482Ser, rs13131226 and rs2970856) and polymorphism of TFAM gene (rs1937-+35G/C) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. In addition, to better understand genetic contributions to the pathophysiology of PCOS, mtDNA copy number (MCN) was quantified using a qRT-PCR assay in the subjects. The results revealed that the distribution of genotypes and allele frequency of the PGC-1α Gly482Ser polymorphism in PCOS patients was statistically significant from those of the control group respectively (OR-2.488; 95% CI-1.0673 to 5.7998; P=0.047), (OR-1.6091; 95% CI-1.0955 to 2.3634; P=0.015) indicating that the presence of 'A' allele might confer risk to PCOS. Patients with the 'AA' genotype showed significantly lower levels of MCN compared with patients with other genotypes. In addition, patients carrying CT genotype of PGC1-α rs2970856 demonstrated significantly higher levels of LH (P=0.030) than TT and CC genotypes. In conclusion, our study indicates that carriers of the PGC-1α rs8192678 'Ser' allele have increased risk of developing PCOS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mitochondrial displacement loop (D-loop) is the hot spot for mitochondrial DNA (mtDNA) alterations which influence the generation of cellular reactive oxygen species. In the present study, we sequenced the entire mitochondrial D-loop region (1124 bp) of colorectal cancer (CRC) patients (n = 174) and controls (n = 170) of south Indian origin to identify significant mutations/polymorphisms. Our results showed 152 polymorphisms in the D-loop region of patients and/or controls. Polymorphisms were predominantly located in hypervariable region I (54.6%) than in II (45.4%) of D-loop region. The frequencies of 310'C' insertion (p = 0.0078), T16189C (p = 0.0097) variants and 310'C'ins/16189C haplotype (p = 0.0029) were significantly higher in cases than in controls. Furthermore, strong linkage disequilibrium was observed between nucleotide position 310 and 16189 in cases (D'=0.68) as compared with controls (D'=0.27). In conclusion, mitochondrial D-loop sequence alterations may constitute inherent risk factor for CRC.

The present study principally sought to investigate the effect of green tea extract (GTE) supplementation on hepatic mitochondrial DNA (mtDNA) damage in alcohol receiving rats. MtDNA was isolated from hepatic tissues of albino wistar rats after alcohol treatment with and without GTE supplementation. Entire displacement loop (D-loop) of mtDNA was screened by PCR-Sanger's sequencing method. In addition, mtDNA deletions and antioxidant activity were measured in hepatic tissue of all rats. Results showed increased frequency of D-loop mutations in alcoholic rats (ALC). DNA mfold analysis predicted higher free energy for 15507C and 16116C alleles compared to their corresponding wild alleles which represents less stable secondary structures with negative impact on overall mtDNA function. Interestingly, D-loop mutations observed in ALC rats were successfully restored on GTE supplementation. MtDNA deletions were observed in ALC rats, but intact native mtDNA was found in ALC + GTE group suggesting alcohol induced oxidative damage of mtDNA and ameliorative effect of GTE. Furthermore, markedly decreased activities of glutathione peroxidise, superoxide dismutase, catalase and glutathione content were identified in ALC rats; however, GTE supplementation significantly ( P  < 0.05) restored these levels close to normal. In conclusion, green tea could be used as an effective nutraceutical against alcohol induced mitochondrial DNA damage.

Mitochondrial displacement loop (D-loop) is a hot spot for mitochondrial DNA (mtDNA) alterations that effects cellular reactive oxygen species (ROS) generation. Manganese-superoxide dismutase (Mn-SOD) is a major antioxidant enzyme that protects cells from ROS-mediated damage. In the present study, we investigated the relationship between sequence alterations of mitochondrial D-loop and Mn-SOD expression in colorectal cancer (CRC). Genotyping of entire mitochondrial D-loop (1124 bp) was carried out on mtDNA of analogous tumor and normal tissues from 35 CRC patients of south Indian origin by PCR-sequencing analysis. Tumor-specific large-scale mtDNA deletions and Mn-SOD expression was analyzed by PCR and Western blot analysis, respectively. We identified 87 polymorphisms in the D-loop region of tumor and/or control tissues. Polymorphisms were predominantly located in hypervariable region I (67.9 %) than in II (32.1 %) of D-loop. Significantly increased mtDNA microsatellite instability (mtMSI) [310'C' insertion (P = 0.00001) and T16189C (P = 0.0007)] and elevated Mn-SOD expression was observed in tumor tissues compared with controls. Interestingly, mtMSI was significantly high in tumors with Mn-SOD overexpression. Tumor-specific large-scale mtDNA deletions were not observed in CRC tissues. In conclusion, mtMSI and Mn-SOD overexpression are a common event in CRC. The analysis of mtMSI and/or Mn-SOD expression might help to identify patients at high risk for disease outcome, thereby helping to refine therapeutic decisions in CRC.

Objective: Polycystic ovarian syndrome (PCOS) is a complex and multifactorial disorder believed to be the consequence of a complex interaction between genetic, immunological, and environmental factors. The main aim of this study was to investigate the association of Connexin37 (Cx37)/Gap junction alpha 4 (GJA4) gene C1019T single nucleotide polymorphism (SNP) with the susceptibility to polycystic ovarian syndrome (PCOS) in South Indian women., Study Design: This study comprises 98 PCOS patients and 100 healthy women without PCOS of South Indian origin. We genotyped total of seventeen selected Cx37 SNPs including C1019T (rs1764391) by polymerase chain reaction and sequencing analysis. The genotype frequency and allele distributions of cases and controls were analyzed using Fisher's exact test., Results: The genotype and allele frequencies of the C1019T polymorphism significantly differ between cases and controls. The frequencies of C/C genotype (P=0.009) and 'C' allele (P=0.002) of the C1019T polymorphism showed a significant prevalence in cases compared to controls., Conclusion: Our findings suggest that the Cx37 C1019T variation may contribute to the risk of PCOS in the South Indian women., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Purpose: To investigate the role of genetic variations and expression alterations of BRCA1 and BRCA2 genes in the pathophysiology of endometriosis., Methods: A genetic association study was conducted in 573 endometriosis cases and 490 controls of Indian origin. We genotyped 13 selected promoter SNPs of BRCA1 gene and 2 selected promoter SNPs of BRCA2 gene by PCR-sequencing analysis. In addition, to better understand genetic contributions to the pathophysiology of endometriosis, the expression pattern of BRCA1 & 2 was analyzed in the eutopic endometria of endometriosis cases and controls by western-blot and immunohistochemical analysis., Results: Our results revealed significant association between BRCA1 rs71361504 (-/GTT) SNP and endometriosis risk in Indian women (P < 0.0001), while the remaining SNPs of both BRCA1 & 2 genes showed no difference between cases and controls. Western-blot and immunohistochemical analysis revealed significantly decreased BRCA1 expression levels in eutopic endometria of patients compared with controls (P < 0.05). Furthermore, nuclear BRCA1 was frequently lost compared with cytoplasmic BRCA1 in eutopic endometria of patients. Expression of BRCA2 did not differ between patients and controls., Conclusions: BRCA1 rs71361504 SNP may modify the endometriosis risk in Indian women. In addition, decreased expression of BRCA1 may play an important role in the pathophysiology of endometriosis. The analysis of BRCA1 genetic variants and/or expression might help to identify patients at high risk for disease outcome.

Cyclin D1 (CCND1) and E-cadherin (CDH1) are two important genes of the β-catenin/LEF pathway that is involved in tumorigenesis of various cancers including colorectal cancer (CRC). However, studies of the association between genetic variants of these two genes and CRC have shown conflicting results. We conducted a genetic association study in South Indian population (cases, 103; controls, 107) to assess the association of CCND1 870G/A and CDH1 -160C/A single nucleotide polymorphisms (SNPs) with CRC risk. Genotyping of SNPs was performed by PCR sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software. In addition, to better understand the role of CCND1 and CDH1 in the pathophysiology of CRC, the expression pattern was evaluated in analogous tumor and adjacent normal tissues from 23 CRC patients by Western blot analysis. The frequencies of CCND1 870A/A (P = 0.045) genotype, CDH1 -160A allele (P = 0.042), and 870A/-160A haplotype (P = 0.002) were significantly higher in patients as compared with controls. Strong LD was observed between 870G/A and -160C/A SNPs in cases (D' = 0.76) as compared to controls (D' = 0.32). Furthermore, elevated CCND1 and diminished CDH1 expression was observed in tumor tissue as compared with analogous normal tissue of CRC patients. Interestingly, advanced-stage tumors showed wider expression alterations than in early-stage tumors. In conclusion, CCND1 870G/A and CDH1 -160C/A SNPs may modify the risk of CRC susceptibility in South Indian population. In addition, elevated CCND1 and diminished CDH1 expression appears to be useful prognostic markers for CRC.

Purpose: The aim of this study was to investigate the association between two common single nucleotide polymorphisms (SNPs) in the vascular endothelial growth factor (VEGF) gene (-460C/T and +405G/C) and polycystic ovary syndrome (PCOS) risk in south Indian women., Methods: This study involves clinically confirmed PCOS patients (n = 126) and non-PCOS controls (n = 130) of south Indian origin (Dravidian linguistic group). Genotyping of the VEGF gene -460C/T and +405G/C SNPs were performed by PCR and sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were assessed by Haploview Software., Results: The frequencies of +405G/G genotype (P = 0.03) and +405G alleles (P = 0.006) were significantly higher in patients compared to controls. Whereas the genotype and allele frequencies of -460C/T SNP were not significantly different between patients and controls. In addition, LD analysis revealed no significant difference between patients and controls., Conclusion: Our findings suggest that the VEGF +405G/C polymorphism may constitute an inheritable risk factor for PCOS in south Indian women.

The objective of the present study was to investigate the association between TP53 gene single nucleotide polymorphisms (SNPs) and colorectal cancer (CRC) predisposition in south Indian population and to evaluate the role of TP53 expression in the pathophysiology of CRC. A genetic association study was conducted in 103 CRC cases and 107 controls of south Indian origin. We genotyped ten selected TP53 SNPs by polymerase chain reaction-sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were assessed by Haploview Software. In addition, to better understand the role of TP53 in the pathophysiology of CRC, the expression pattern was evaluated in analogous tumor and normal tissues from 23 CRC patients by Western blot analysis. The frequencies of Pro72Pro (P = 0.0033) genotype and Ser47/Pro72 (P = 0.00171) haplotype were significantly higher in patients as compared to controls. Strong LD was observed between codon 47 and 72 in cases (D' = 0.32) as compared to controls (D' = 0.21). The polymorphism was not observe at the remaining eight SNPs loci analyzed. Furthermore, increased TP53 expression was observed in tumor tissue than in analogous normal tissue of CRC patients. Interestingly, advanced stage tumors showed more elevated TP53 expression compared to early stage tumors. In conclusion, the TP53 Pro72Pro genotype and Ser47/Pro72 haplotype has an increased risk for CRC predisposition in south Indian population. In addition, elevated TP53 expression appears to be useful prognostic marker for CRC.

Study Question: Are mutations in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene associated with endometriosis?, Summary Answer: Loss of heterozygosity (LOH) at the 10q23.3 locus, PTEN somatic mutations and changes in the levels and distribution of proteins in the PTEN-PI3K/Akt signal transduction pathway are associated with endometriosis., What Is Known Already: Endometriosis has a strong genetic basis. Recent genome-wide association and linkage studies have reported a significant association of endometriosis with 7p15.2, 9p21 and 10q23-26 loci. PTEN, which maps to 10q23.3, acts as a tumor suppressor gene through the action of its phosphatase protein product, phosphatase and tensin homolog (PTEN). This phosphatase is involved in the regulation of the cell cycle, and mutations of PTEN are a step in the development of many cancers., Study Design, Size, Duration: A total of 1252 subjects of Indian origin (endometriosis patients = 752; controls = 500) were recruited to participate in this case-control study. Recruitment took place from 2001 to 2009 at Institute of Reproductive Medicine (IRM), Kolkata, India; Infertility Institute and Research Centre (IIRC), Secundrabad, India and Vasavi Medical and Research Centre, Hyderabad, India., Participants/materials, Setting, Methods: LOH on 10q, 9p and 7p was analyzed in analogous ectopic-eutopic endometria along with blood samples from 32 advanced stage endometriosis patients by PCR-GeneScan analysis. Genotyping of PTEN was carried out on genomic DNA of analogous ectopic-eutopic endometria (n = 32) as well as blood samples from 720 patients and 500 controls by PCR-sequencing analysis to explore somatic and germ-line mutations, respectively. The levels and distribution of PTEN, p-Akt, p-Bad and p27 were analyzed in the eutopic endometria of patients (n = 5) and controls (n = 5) using western-blot and immunohistochemistry., Main Results and the Role of Chance: PCR-GeneScan analysis revealed a higher LOH frequency at 10q23.3 (84.4%) compared with other loci analyzed, hence we focused our attention on PTEN. PCR-sequencing analysis revealed seven novel somatic mutations and 23 germ-line polymorphisms in patients. Among somatic mutations, a frame-shift insertion at 10:89692992-89692993 (in the functionally important N-terminal phosphatase domain of PTEN) occurred in 11 of the 32 ectopic endometria. Western-blot and immunohistochemical analysis revealed decreased PTEN and increased p-Akt and p-Bad levels in eutopic endometria of patients compared with controls (all comparisons, P < 0.0001). Furthermore, PTEN loss was more frequent in the nucleus than in the cytoplasm. Expression of p27 did not differ between patients and controls., Limitations, Reasons for Caution: Protein analysis was performed in eutopic endometrial samples from only a small number of patients and controls. In future investigations, a larger sample size should be used and the role of the other genes involved in the PTEN-PI3K/Akt signal transduction pathway should be analyzed., Wider Implications of the Findings: Our findings revealed a possible involvement of the PTEN-PI3K/Akt-Bad axis in the pathogenesis of endometriosis, which may facilitate the discovery of suitable pathway inhibitors for disease treatment., Study Funding/competing Interest(s): This study was supported by grants from the Science & Engineering Research Board (SERB), India (Lr No: SR/FT/LS-188/2009) to BM. The authors have no competing interests to declare.

Background: Mitochondrial displacement loop (D-loop) is the hot spot for mitochondrial DNA (mtDNA) alterations which influence the generation of cellular reactive oxygen species (ROS). Association of D-loop alterations with breast cancer has been reported in few ethnic groups; however none of the reports were documented from Indian subcontinent., Methodology: We screened the entire mitochondrial D-loop region (1124 bp) of breast cancer patients (n = 213) and controls (n = 207) of south Indian origin by PCR-sequencing analysis. Haplotype frequencies for significant loci, the standardized disequilibrium coefficient (D') for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software., Principal Findings: We identified 7 novel mutations and 170 reported polymorphisms in the D-loop region of patients and/or controls. Polymorphisms were predominantly located in hypervariable region I (60%) than in II (30%) of D-loop region. The frequencies of 310'C' insertion (P = 0.018), T16189C (P = 0.0019) variants and 310'C'ins/16189C (P = 0.00019) haplotype were significantly higher in cases than in controls. Furthermore, strong LD was observed between nucleotide position 310 and 16189 in controls (D' = 0.49) as compared to patients (D' = 0.14)., Conclusions: Mitochondrial D-loop alterations may constitute inherent risk factors for breast cancer development. The analysis of genetic alterations in the D-loop region might help to identify patients at high risk for bad progression, thereby helping to refine therapeutic decisions in breast cancer.

Purpose: Polycystic ovary syndrome (PCOS) is a most common endocrine disorder of reproductive age women. Interleukin-6 is involved in the pathophysiological characteristics associated with polycystic ovary syndrome (PCOS). The-174 G/C IL-6 gene promoter region single nucleotide polymorphism (SNP) may influence or modulate gene function and/or transcriptional efficiency. The current study was aimed to evaluate the association between IL-6 gene -174 G/C promoter polymorphism and Polycystic Ovary Syndrome in South Indian women., Methods: In the present study, we examined the genotypic and allele distribution among the PCOS patients (n = 104) and controls (n = 156). The genotypes of IL-6 -174 G/C SNP were analyzed by polymerase chain reaction (PCR) and sequencing analysis. The allele frequency and genotype distributions of cases and controls were analyzed using Fisher's exact test., Results: The genotype frequencies observed among the 104 cases and 156 controls were G/G 66.3 % and 49.4 %, G/C 29.8 % and 46.8 %, and C/C 3.8 % and 3.8 % (OR: 1.6226, CI: 1.0574-2.4899). The G and C allele frequencies were 81.25 % and 72.8 %, and 18.75 % and 27.2 %, respectively. The genotype and allele distribution revealed significant differences between PCOS patients and controls (all P values < 0.05)., Conclusion: Our findings showed a significant statistical association between IL-6 -174 G/C SNP and PCOS risk in South Indian women. The 'G' allele frequency influences significantly higher in PCOS patients than controls. However, the exact mechanism by which 'G' allele frequency influence PCOS patients is yet to be determined.

Genetic alterations and aberrant expression of 'mitochondrial membrane complex I' (MMC-I) underlie several complex human disorders, but no reports are documented to date in endometriosis. Sequencing of mitochondrially encoded MMC-I subunits revealed 72 mutations of which 2 missense (G10398A; A13603A/G) mutations and 1 synonymous (T10400C) mutation showed higher prevalence in patients. In silico functional analysis predicted A13603A/G, a novel heteroplasmy as a 'damaging variant'. Our results indicate higher endometriosis risk for haplotype '10398A/10400C/13603AG' and haplogroup 'N'. Immunohistochemical analysis revealed elevated MMC-I expression in eutopic endometria of patients compared to controls. In conclusion, MMC-I alterations may constitute an inheritable risk factor for endometriosis., (Copyright © 2013 Elsevier B.V. and Mitochondria Research Society. All rights reserved. All rights reserved.)

Entire mitochondrial DNA (mtDNA) sequencing was carried out in 101 primary breast cancer patients and 90 controls of south Indian origin. We identified 69 novel mutations in breast cancer patients and 637 reported polymorphisms in patients and/or controls. PolyPhen-2 analysis predicted 5 out of 14 novel missense mutations as 'probably damaging variants'. Haplogrouping analysis identified a significant association between haplogroup M5 and breast cancer risk. Microsatellite instability and tumor specific large scale mtDNA deletions were not observed in tumor tissues from the patients. In conclusion, mtDNA mutations and haplogroups may constitute an inheritable risk factor for pathogenesis of breast cancer., (Copyright © 2013 Elsevier B.V. and Mitochondria Research Society. All rights reserved. All rights reserved.)

Objective: To evaluate the association of mitochondrial displacement (D-) loop alterations with endometriosis in south Indian women., Design: Case-control study., Setting: Biochemistry and molecular biology laboratories., Patient(s): Women with (n = 152) and without (n = 150) endometriosis of south Indian origin., Intervention(s): All women had a transvaginal ultrasound scan at screening followed by a laparoscopy to confirm the diagnosis (revised American Fertility Society stage III = 54; stage IV = 98)., Main Outcome Measure(s): Genotyping of entire D-loop (1,124 bp) was carried out on genomic DNA of blood from cases and controls by polymerase chain reaction sequencing analysis., Result(s): Twelve novel mutations and 187 reported polymorphisms were identified in the D-loop region of cases and/or controls. The A189G, 310 C insertion, T16189C polymorphisms, and 189G/310TC/16189C haplotype have significantly higher frequency in cases compared with controls., Conclusion(s): Mitochondrial D-loop alterations may constitute an inheritable risk factor for endometriosis. The analysis of D-loop alterations may help to identify patients at high risk for disease outcome., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: E-cadherin (CDH1) plays an important role in intercellular adhesion, cell signaling, and cellular differentiation. Association of single-nucleotide polymorphisms (SNPs) of CDH1 has been identified in a number of epithelial malignancies; however, studies related to breast cancer are very few., Aim: To investigate the association between CDH1 SNPs and breast cancer risk in south Indian women., Methods: Genotyping of CDH1 functional SNPs (-347G/GA, -160C/A, and +54C/T) was carried out on genomic DNA of blood from breast cancer patients (n=202) and controls (n=250) of south Indian origin by PCR-sequencing and PCR-restriction fragment length polymorphism techniques. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were assessed by Haploview Software., Results: The frequencies of -160A/A genotypes (p=0.038) and -160A alleles (p=0.046) were significantly higher in patients compared to controls. In addition, the frequency of the -347GA/-160A/+54C haplotype was also significantly elevated in patients (p=0.0238). Strong LD was observed between -347G/GA and +54C/T loci (D'=0.44) in patients compared to controls., Conclusion: The CDH1 -160C/A polymorphism may constitute an inheritable risk factor for breast cancer in south Indian women.

Aim: To investigate the role of loss of heterozygosity (LOH), single nucleotide polymorphisms (SNPs), and the expression of gene p53 in the pathogenesis of endometriosis., Methods: LOH at the p53 gene locus (17p13.1) was examined in matched ectopic and eutopic endometrial tissues from 31 endometriosis patients by polymerase chain reaction (PCR)-GeneScan analysis. The genotyping of selected p53 SNPs (n=10) was carried out on genomic DNA of blood from endometriosis patients (n=720) and controls (n=500) by PCR sequencing. The p53 expression levels were analyzed in the endometrial tissues from endometriosis patients (n=5) and controls (n=4) by Western blot and immunohistochemical analysis., Results: LOH was observed at the 17p13.1 locus (38.7%) in the ectopic endometrium but not in the eutopic endometrium of patients. The genotype (p=0.909) and allele (p=0.729) distribution of the p53 codon Arg72Pro polymorphism was not significantly different between patients and controls. The polymorphism was not observed at codon 47 along the other SNPs studied. There was no preferential loss of either "Arg72" or "Pro72" alleles among the LOH-positive heterozygous cases. In addition, decreased p53 expression was observed more often in the endometrium of patients than in controls., Conclusions: p53 SNPs are not associated with endometriosis in Indian women. However, LOH and reduced expression of p53 are related with the risk of endometriosis in Indian women.

The objective of the present study was to investigate the association between gene E-cadherin single nucleotide polymorphisms (SNPs) and risk of developing endometriosis in Indian women and to evaluate the role of E-cadherin expression in the pathophysiology of endometriosis. A genetic association study was conducted in 715 endometriosis cases and 500 controls of Indian origin. We genotyped -160 C/A, +54 C/T and -347 G/GA SNPs of gene E-cadherin by PCR-sequencing and PCR-restriction fragment length polymorphism techniques. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software. In addition, to better understand genetic contributions to the pathophysiology of endometriosis, the expression pattern of E-cadherin in the endometrium of women with and without endometriosis was analyzed by western blot and immunohistochemical analysis. The frequencies of -347GA/GA (P = 0.026) and -160A/A (P = 0.0019) genotypes and -347G/-160A/+54C (P = 0.007) and -347GA/-160A/+54C (P < 0.0001) haplotypes were significantly different between patients and controls. Strong LD was observed between -347G/GA and -160C/A loci (D' = 0.64) when compared with -347G/GA and +54C/T (D' = 0.585) or -160C/A and +54C/T (D' = 0.05) loci in cases. Furthermore, increased membranous E-cadherin expression was observed in cases than in controls. The expression seems to be genotype dependent. In conclusion, the E-cadherin -347GA/GA and -160A/A genotypes and -347GA/-160A/+54C and -347G/-160A/+54C haplotypes may jointly modify the risk of endometriosis in Indian women. In addition, the differential expression of E-cadherin may play an important role in pathogenesis of endometriosis.

Background: Endometriosis is a chronic gynecological benign disease that shares several features similar to malignancy. Mitochondrial DNA (mtDNA) mutations have been reported in all most all types of tumors. However, it is not known as to whether mtDNA mutations are associated with endometriosis., Methodology: We sequenced the entire mitochondrial genome of analogous ectopic and eutopic endometrial tissues along with blood samples from 32 advanced stage endometriosis patients to analyze the role of somatic and germ-line mtDNA variations in pathogenesis of endometriosis. All ectopic tissues were screened for tumor-specific mtDNA deletions and microsatellite instability (MSI). We also performed mtDNA haplogrouping in 128 patients and 90 controls to identify its possible association with endometriosis risk., Principal Findings: We identified 51 somatic (novel: 31; reported: 20) and 583 germ-line mtDNA variations (novel: 53; reported: 530) in endometriosis patients. The A13603G, a novel missense mutation which leads to a substitution from serine to glycine at the codon 423 of ND5 gene showed 100% incidence in ectopic tissues. Interestingly, eutopic endometrium and peripheral leukocytes of all the patients showed heteroplasmy (A/G; 40-80%) at this locus, while their ectopic endometrium showed homoplasmic mutant allele (G/G). Superimposition of native and mutant structures of ND5 generated by homology modeling revealed no structural differences. Tumor-specific deletions and MSI were not observed in any of the ectopic tissues. Haplogrouping analysis showed a significant association between haplogroup M5 and endometriosis risk (P: 0.00069) after bonferroni correction., Conclusions: Our findings substantiate the rationale for exploring the mitochondrial genome as a biomarker for the diagnosis of endometriosis.

Our previous in silico studies identified a high-affinity nickel transporter, TNC, from the metal transportome of Neurospora crassa. A knockout mutant of the tnc gene in N. crassa failed to transport nickel, showed phenotypic growth defects and diminished urease activity under physiological levels of nickel. Transport assays conducted in wild type and knockout mutant strains showed that TNC transports nickel with high affinity but exhibits selectivity for other transition metal ions like cobalt. Heterologous complementation of Schizosaccharomyces pombe nickel uptake mutant by TNC further substantiates its nickel transport function. Transcriptional analysis of the nickel transporter encoding gene, tnc in N. crassa by qRT-PCR showed its constitutive expression in various phases of its life cycle. However, levels of the corresponding protein TNC were down-regulated only by increasing the nickel, but not cobalt concentration in the media. Immunolocalisation data suggested that TNC is distributed in the plasma membrane of N. crassa. Thus, the present study establishes TNC as a functional plasma membrane nickel transporter necessary for physiological acquisition of nickel in the multicellular fungi N. crassa., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Transcriptional analysis using qRT-PCR of 62 metal ion transporters during conidial germination of Neurospora crassa showed a significant up regulation of a hypothetical copper transporter gene, tcu-1, that belongs to the Ctr family. Herein we characterised the Ctr family genes (tcu-1, tcu-2 and tcu-3) and deciphered their role in various developmental phases of the N. crassa life cycle. Cross complementation assays in copper uptake mutant of Saccharomyces cerevisiae revealed that tcu-1, tcu-2 and tcu-3 are functional homologs of S. cerevisiae copper transporters. Expression studies of Ctr family members in various developmental phases of N. crassa showed differential expression pattern for high-affinity copper transporter, TCU1. Functional analysis of their gene knockout mutants showed that tcu-1 is essential for saprophytic conidial germination, vegetative growth and perithecia development under copper limited conditions while conidiation remained unaffected., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Objective: Migration of vascular smooth muscle cells (VSMCs) from media to intima is a key event in the pathophysiology of atherosclerosis and restenosis. The lipoxygenase products of polyunsaturated fatty acids (PUFA) were shown to play a role in these diseases. cAMP response element binding protein (CREB) has been implicated in the regulation of VSMC growth and motility in response to thrombin and angiotensin II. The aim of the present study was to test the role of CREB in an oxidized lipid molecule, 15(S)-HETE-induced VSMC migration and neointima formation., Methods and Results: 15(S)-HETE stimulated VSMC migration in CREB-dependent manner, as measured by the modified Boyden chamber method. Blockade of MEK1, JNK1, or p38MAPK inhibited 15(S)-HETE-induced CREB phosphorylation and VSMC migration. 15(S)-HETE induced expression and secretion of interleukin-6 (IL-6), as analyzed by RT-PCR and ELISA, respectively. Neutralizing anti-IL-6 antibodies blocked 15(S)-HETE-induced VSMC migration. Dominant-negative mutant-mediated blockade of ERK1/2, JNK1, p38MAPK, or CREB suppressed 15(S)-HETE-induced IL-6 expression in VSMCs. Serial 5' deletions and site-directed mutagenesis of IL-6 promoter along with chromatin immunoprecipitation using anti-CREB antibodies showed that cAMP response element is essential for 15(S)-HETE-induced IL-6 expression. Dominant-negative CREB also suppressed balloon injury-induced IL-6 expression, SMC migration from media to intimal region, and neointima formation. Adenovirus-mediated transduction of 15-lipoxygenase 2 (15-LOX2) caused increased production of 15-HETE in VSMCs and enhanced IL-6 expression, SMC migration from media to intimal region, and neointima formation in response to arterial injury., Conclusions: The above results suggest a role for 15-LOX2-15-HETE in the regulation of VSMC migration and neointima formation involving CREB-mediated IL-6 expression.

Objective: To investigate whether the eNOS gene influences the risk of developing endometriosis in south Indian women., Study Design: The single nucleotide polymorphism, Glu298Asp, in exon7 of the eNOS gene was tested for association in a case-control study of 232 affected women and 210 women with no evidence of disease. All the women were infertile, ascertained from the same infertility clinic. The genotype frequencies of the polymorphism were compared, using polymerase chain reaction and sequencing analysis. The localization and expression of eNOS in the eutopic endometrium of five cases and four controls was also analyzed using immunohistochemistry and western blotting., Results: No statistically significant differences were observed in the genotype distributions and allele frequencies (p=0.3) between the cases and controls according to codominant, dominant and recessive genotype models. The localization and expression of this protein were similar in the endometrium of cases and controls., Conclusion: In the present study we could neither observe a difference in the eNOS expression nor establish an association between the eNOS Glu298Asp exon 7 polymorphism in south Indian women with endometriosis.

Background: The aim of the study was to test whether the signal transducer and activator of transcription 6 (STAT6) gene influences the risk of developing endometriosis., Methods: The single-nucleotide polymorphism, G2964A, in the 3'-untranslated region (UTR) of the STAT6 gene was tested for association in a case-control study of 232 affected women and 210 women with no evidence of disease. All the women were infertile, ascertained from the same infertility clinic and of South Indian origin. The genotype frequencies of this polymorphism were compared using PCR and sequencing analysis., Results: There were statistically significant differences in the genotype distributions (P = 0.002) and allele frequencies (P = 0.0002) between the cases and controls, according to codominant, dominant and recessive genotype models., Conclusions: We report for the first time an association between the STAT6 G2964A 3'-UTR polymorphism and endometriosis in South Indian women. This finding suggests that STAT6 may contribute to disease susceptibility in endometriosis, which carries an extra interest as the gene lies in a region which has been implicated, albeit weakly, in a previous genomewide scan.

Objective: To investigate the association of the -174 G/C promoter polymorphism of the interleukin-6 (IL-6) gene with endometriosis in South Indian women., Methods: The genotype frequencies of the common IL-6 -174 G/C polymorphism were compared in infertility patients with (n = 232) and without (n = 210) endometriosis using polymerase chain reaction (PCR) and sequencing analysis., Results: The genotype frequencies among the cases and controls were G/G 62.9% and 71.9%, G/C 34.1% and 25.2%, and C/C 3.0% and 2.9%. The G and C allele frequencies were 80% and 84.6%, and 20% and 15.4%, respectively. There were no statistically significant differences in the genotype distributions or allele frequencies between the cases and controls (P = .12)., Conclusions: The present study demonstrates no significant association between the IL-6 -174 G/C promoter polymorphism and endometriosis in South Indian women.

Background: Vascular endothelial growth factor (VEGF), a major mediator of angiogenesis and vascular permeability, is known to play a key role in the pathophysiology of endometriosis., Methods and Results: The single nucleotide polymorphisms, -460C>T and +405G>C, in the 5'-untranslated region of the VEGF gene were tested for association in a case-control study of 215 affected women and 210 women with no evidence of disease. All the women were of South Indian origin and ascertained from the same infertility clinic. The genotype and allele frequencies of the -460C>T polymorphism did not differ significantly between cases and controls. In contrast, the genotype (P = 0.002) and allele (P = 0.001) frequencies of the +405G>C polymorphism showed a significant difference between cases and controls. The +405 GG genotype was found more often in patients with an endometrioma >3 cm compared to controls. The frequency of the -460T/+405C haplotype (P = 0.016) was significantly lower in affected women compared to controls., Conclusions: The -460T/+405C haplotype in the VEGF gene, which is associated with lower promoter activity, was significantly less common in women with endometriosis than in controls. These data suggest that the +405G allele may influence the likelihood of a woman developing the disease.

Thiols provide the major intracellular redox milieu and can undergo reversible oxidation and reduction. To understand the role of thiols in redox signaling events, we have studied the effect of N-ethylmaleimide, a specific thiol alkylating agent, on platelet-derived growth factor-BB (PDGF-BB)-induced mitogenesis in vascular smooth muscle cells (VSMC). Thiol alkylation inhibited PDGF-BB-induced expression of the Fos and Jun family proteins and AP-1 activity in VSMC. Thiol alkylation also inhibited PDGF-BB-induced expression of cyclin A and growth in these cells. In contrast, thiol alkylation enhanced and sustained the effect of PDGF-BB on the activation of the Jak STAT pathway, and this event was correlated with inhibition of protein tyrosine phosphatase lB activity. Thiol alkylation via inducing the expression of p21(waf1/cip1) in a STAT1- and p53-dependent manner antagonized the downregulation of this cell cycle inhibitory molecule by PDGF-BB. The inhibition of AP-1 and activation of STATs, particularly STAT1, by thiol alkylation correlated with increased production of active caspase 1 and apoptosis in VSMC. Together, these findings suggest a role for thiols in mediating mitogenic and/or apoptotic signaling events in VSMC. These results also show that a sustained change in the intracellular thiol redox state can convert a mitogen into a death promoter.

The redox state plays an important role in gene regulation. Thiols maintain the intracellular redox homeostasis. To understand the role of thiols in redox signaling, we have studied the effect of thiol alkylation on platelet-derived growth factor-BB (PDGF-BB)-induced cell survival events in vascular smooth muscle cells. PDGF-BB stimulated Akt phosphorylation predominantly at Ser-473. N-Ethylmaleimide (NEM), a thiol alkylating agent, blocked PDGF-BB-induced Akt phosphorylation without affecting its upstream phosphatidylinositol 3-kinase (PI3K). On the other hand, LY294002 and wortmannin, specific inhibitors of PI3K, prevented PDGF-BB-induced phosphorylation of Akt and its downstream effector molecules, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. NEM also abrogated the phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E induced by PDGF-BB, suggesting that thiol alkylation interferes with the PI3K/Akt pathway at the level of Akt. In addition, NEM blocked PDGF-BB-induced phosphorylation of BAD and forkhead transcription factor FKHR-L1, and these events correlated with increased apoptosis. NEM alone and in concert with PDGF-BB increased reactive oxygen species (ROS) production and protein phosphatase 2A (PP2A) activity in VSMC. The inhibition of PDGF-BB-induced Akt phosphorylation by NEM was completely reversed by PP2A inhibitors fostriecin and okadaic acid, ceramide synthase inhibitor fumonisin B1, and ROS scavenger N-acetylcysteine (NAC). NAC also attenuated the apoptosis induced by NEM, alone or in combination with PDGF-BB. Together, these findings demonstrate for the first time that PP2A mediates thiol alkylation-dependent redox regulation of Akt and cell survival.

Previously we have demonstrated that activation of p38 mitogen-activated protein kinase (MAPK) and induction of DNA synthesis in response to receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) agonists require NADH/NADPH-like oxidase activity in vascular smooth muscle cells (VSMC). Here we tested the role of p38 MAPK in RTK and GPCR agonist-induced DNA synthesis in VSMC. Platelet-derived growth factor (PDGF)-BB and thrombin (RTK and GPCR agonists, respectively) activated p38 MAPK in a time-dependent manner in VSMC. Inhibition of p38 MAPK led to a 50% decrease in the DNA synthesis induced by thrombin but not PDGF-BB. ATF-1 was found to be the predominant member of the cyclic AMP response element (CRE)-DNA complex formed in VSMC in response to PDGF-BB and thrombin, and both agonists induced its phosphorylation. Regardless of this, inhibition of p38 MAPK reduced only thrombin- but not PDGF-BB-induced ATF-1 phosphorylation. Similarly, inhibition of p38 MAPK caused a 50% decrease in thrombin- but not PDGF-BB-induced CRE promoter-dependent transcription. Ectopic expression of an inhibitory anti-ATF-1 single-chain antibody fragment, ScFv, significantly interfered with DNA synthesis induced by thrombin but not PDGF-BB. Together, these results suggest the following conclusions. 1) Both RTK and GPCR agonists activate p38 MAPK and induce CRE promoter-dependent transcription; 2) both RTK and GPCR agonists induce ATF-1 phosphorylation, and ATF-1 is a predominant member in the CRE-DNA complexes formed in response to these agents; and 3) p38 MAPK-dependent ATF-1 phosphorylation and CRE promoter-mediated transcription are associated with GPCR agonist-induced VSMC growth.

Fungal cell wall, mainly composed of chitin, an N-acetylglucosamine polymer, is known to participate in heavy metal detoxification. In the present study, an effort was made to elucidate the sites involved in complexation of cadmium by the chitin material of cell wall of Neurospora crassa. Based on the results of physical techniques, such as solid-state 13C-NMR, X-ray diffraction, IR and molecular modeling, a structure was proposed for the chitin-cadmium complex. The ring and C-3 hydroxyl oxygens of N-acetylglucosamine were implicated in the complexation of cadmium by the chitin of the fungal cell wall. The studies further revealed that the conformation of chitin did not alter after cadmium complexation.