Pilot study on evaluation and determination of the prevalence of Polycystic Ovarian Syndrome (PCOS) associated gene markers in the South Indian population.

Background: Polycystic ovarian syndrome (PCOS) is typically characterized by a spectrum of manifestations that include menstrual irregularities, anovulation, cysts, hyperandrogenic features like hirsutism, acne, alopecia, and various metabolic complications. The pathology of PCOS is complex and several mechanisms have been potentially involved in the genetic abnormalities/dysfunctions. Hence, the present study aims to examine the prevalence and association of polymorphisms in candidate genes (thyroid adenoma-associated gene [THADA], luteinizing hormone and human chorionic gonadotropin receptor [LHCGR], DENN domain containing 1A [DENND1A], follicle-stimulating hormone receptor [FSHR], Connexin37 [CX37], angiotensin-converting enzyme [ACE], insulin receptor [INSR] and calpain 10 [CAPN10]) in PCOS patients of the South Indian regional population. Methods: The study group included 20 PCOS cases and 10 controls, whose deoxyribonucleic acid (DNA) were genotyped by the polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (RFLP), and PCR product sequencing to determine the prevalence of the DENND1A (rs10818854), LHCGR (rs13405728), FSHR (rs2349415), THADA (rs13429458), CX37 (rs1764391), ACE (rs1799752), INSR (rs1799817), and CAPN10 (rs2975760) polymorphisms. Clinical examinations including anthropometric measurements, biochemical investigations relevant to glucose metabolism, and hormones were measured. Results: A significant difference was observed in the DENND1A (rs10818854) polymorphism between the control and PCOS patients (P = 0.001). The variants of LHCGR, FSHR, THADA, CX37, ACE, INSR, and CAPN10 were not statistically significant with PCOS. The body mass index (BMI) (P = 0.01), triglycerides (P = 0.01), and dehydroepiandrosterone sulfate (DHEAS) (P = 0.05) were significantly different between the PCOS patients and controls. Significant results were observed in rs1799817 single nucleotide polymorphisms (SNP) of INSR with elevated levels of triglycerides and rs10818854 of DENND1A, rs13429458 of THADA, rs2349415 of FSHR with the high levels of DHEAS. Conclusion: In the study population, the presence of rs10818854 of DENND1A polymorphism may be associated with the risk of PCOS and high levels of DHEAS.

Carriers of the TCF7L2 rs7903146, rs12255372 Risk Alleles in the South Tamil Nadu T2DM Patients Present with Early Incidence and Insulin Dependence.

BACKGROUND AND AIMS: Metabolic abnormalities in T2DM (Type 2 diabetes mellitus) include classic manifestations such as impaired insulin secretion, synthesis and peripheral insulin resistance. The intronic variants rs7903146 and rs12255372 of the TCF7L2 (transcription factor 7-like 2) gene are strongly associated with risk of incidence of T2DM and impaired ß-cell functions. Studies addressing the early T2DM onset, and early insulin dependence in T2DM patients of south Tamil Nadu are still lacking, and hence the present study focuses in determining the influence of the TCF7L2 polymorphisms in the incidence and disease course in the T2DM patients of south Tamil Nadu. METHODS: Anthropometric measurements and biochemical parameters were carried out in early onset (Group A), early onset insulin dependent T2DM patients (Group B) and non-insulin dependent T2DM patients (Group C). PCR, allele specific PCR (ASP), PCR product sequencing strategies were utilized to determine the genotype and the impact of the TCF7L2 SNPs in the T2DM disease course. RESULTS: Female T2DM patients with the CT/TT rs7903146 genotype (P = 0.005) and the rs12255372 GT/TT genotype (P = 0.036) exhibited a significantly low mean age for T2DM incidence. Correlation/regression analysis in the T2DM patients revealed that rs12255372 (P = 0.042) is associated with early onset in the Group C patients and the rs7903146 (P = 0.018), rs12255372 (P = 0.026) are associated with insulin dependence in the group B patients. CONCLUSION: Screening for the TCF7L2 polymorphisms will prevent T2DM incidence and enable life style changes, appropriate therapeutic strategies that would help combat the accelerated disease course in the T2DM patients.

Isolation, biomimetic synthesis, and cytotoxic activity of bis(pseudopterane) amines.

LC-MS/MS-based screening of the dichloromethane extract of the gorgonian coral Pseudopterogorgia acerosa led to the isolation of a novel bis(pseudopterane) amine (1). The structural assignment of 1 was achieved by 1D and 2D NMR and mass spectrometry analysis. A biomimetic synthesis of 1 and the known symmetrical diterpene 2 from pseudopterolide (3) is described in this report. Bis(pseudopterane) amine showed selective growth inhibition activity against cancer cell lines with IC(50) values of 4.2 microM (HCT116) and 42 microM (HeLa).

Autophagy-dependent viral recognition by plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) detect viruses in the acidified endosomes by means of Toll-like receptors (TLRs). Yet, pDC responses to certain single-stranded RNA (ssRNA) viruses occur only after live viral infection. We present evidence here that the recognition of such viruses by TLR7 requires transport of cytosolic viral replication intermediates into the lysosome by the process of autophagy. In addition, autophagy was found to be required for the production of interferon-alpha by pDCs. These results support a key role for autophagy in mediating ssRNA virus detection and interferon-alpha secretion by pDCs and suggest that cytosolic replication intermediates of viruses serve as pathogen signatures recognized by TLR7.

Porcine liver-expressed antimicrobial peptides, hepcidin and LEAP-2: cloning and induction by bacterial infection.

Hepcidin is a liver-expressed iron-regulating hormone that also is an antimicrobial peptide. Here we report the full-length cDNA sequences of porcine hepcidin and liver-expressed antimicrobial peptide-2 (LEAP-2). Porcine hepcidin and LEAP-2 cDNA sequences contain 411 and 525 bp, and encode predicted peptides of 82 and 77 amino acid residues, respectively. Both porcine hepcidin and LEAP-2 are highly expressed in liver and LEAP-2 also is expressed in intestinal tissues and kidney. Pigs infected with Salmonella enterica serovar Typhimurium showed inducible but differential expression of hepcidin and LEAP-2 in bone marrow and intestinal tissues. Conversely, although highly expressed in liver, expression of hepcidin mRNA in liver was not influenced by Salmonella infection. These findings provide fundamental comparative data showing the relationship of porcine hepcidin and LEAP-2 to other mammalian orthologs and indicate that bacterial infections influence their expression.

Gene silencing and overexpression of porcine peptidoglycan recognition protein long isoforms: involvement in beta-defensin-1 expression.

Peptidoglycan recognition proteins (PGRPs) are a group of newly identified proteins with emerging functions in mammalian innate immunity. Here we report the identification and characterization of two long isoforms of porcine PGRP. Their complete cDNA sequences encode predicted peptides of 252 and 598 residues and are named pPGRP-L1 and pPGRP-L2, respectively. These porcine isoforms share identical PGRP domains at their C terminus, which are highly conserved with human and mouse orthologs. pPGRP-L1 is expressed constitutively in several tissues, including bone marrow, intestine, liver, spleen, kidney, and skin. pPGRP-L2 is highly expressed in the duodenum and liver, and expression in intestinal tissues is increased by Salmonella infection. In intestinal cells, expression of both pPGRP-L1 and pPGRP-L2 is increased by bacterial infection. Recombinant pPGRP-L1 and pPGRP-L2 have N-acetylmuramoyl-L-alanine amidase activity. Loss-of-function and gain-of-function experiments indicate that these two pPGRPs are involved in expression of the antimicrobial peptide beta-defensin-1. Silencing of pPGRP-L2 in intestinal cells challenged with Listeria monocytogenes results in downregulation of beta-defensin-1. Conversely, overexpression of pPGRP-L1 or pPGRP-L2 dramatically upregulates expression of beta-defensin-1. Collectively, these findings suggest that porcine PGRPs are involved in antimicrobial peptide expression.

PU.1-mediated transcriptional regulation of prophenin-2 in primary bone marrow cells.

Prophenin-2 (PF-2) is a cathelicidin, 97-amino-acid antimicrobial protein stored in neutrophil secondary granules. PF-2 is expressed specifically in porcine immature myeloid cells; however, little is known about its regulation. In this study, we characterized the 5' regulatory regions of the PF-2 gene to understand the molecular mechanisms regulating its expression. Using bioinformatic approaches, site-directed mutagenesis, and transactivation experiments, we found that the PF-2 gene was regulated by transcription factor PU.1. In addition, PF-2 expression also is regulated by the cytokines GM-CSF and IL-3. Taken together, these results identify cis- and trans-acting factors involved in the regulation of PF-2 and clarify mechanisms of cathelidicin gene regulation.

Cloning of porcine triggering receptor expressed on myeloid cells-1 (TREM-1) and its induction by lipopolysaccharide, peptidoglycan, and Salmonella enterica serovar Typhimurium infection.

Triggering receptors expressed on myeloid cells (TREM) are a family of activating receptors expressed on neutrophils and monocytes. These receptors are involved in regulation of immunity by inducing the expression of inflammatory cytokines and adhesion molecules, augmenting dendritic cell maturation, and are implicated in septic shock. Here we report the cloning of full-length TREM cDNA from porcine bone marrow cells, which predicts a 238 amino-acid peptide. Treating porcine bone marrow cells with lipopolysaccharide or peptidoglycan caused an increase in TREM-1 expression. Moreover, bone marrow cells derived from pigs that were orally challenged with Salmonella enterica serovar Typhimurium showed increases in TREM-1 at 8 and 24 h post-infection, respectively. Complete down-regulation of TREM-1 expression was observed at 48 h post-infection. These findings provide fundamental comparative data indicating that bacterial infection induces TREM-1 expression.

Characterization of bovine cDNA encoding triggering receptor expressed on myeloid cells 1 (TREM-1).

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a type I transmembrane receptor of the immunoglobulin superfamily expressed predominantly on neutrophils and monocytes. TREM-1 induces the expression of inflammatory cytokines and adhesion molecules, and augments osteoclast, microglia, oligodendrocyte, and dendritic cell differentiation. Here, we report the cloning of TREM-1 from bovine bone marrow cells. Full-length bovine TREM-1 cDNA is 1202 base pairs in length and encodes a predicted 232 amino acid peptide. Comparative analyses showed that bovine TREM-1 has 48 to 61% amino acid identity with other TREM-1 proteins, sharing the greatest identity with porcine TREM-1. Cysteine residues characteristic of the immunoglobulin superfamily were conserved in bovine TREM-1 and RT-PCR analysis revealed diverse mRNA tissue expression for bovine TREM-1. Molecular cloning of bovine TREM-1 extends the repertoire of bovine pattern recognition receptors and provides information important for investigating their role in bovine innate immunity.

PR-39, a porcine antimicrobial peptide, inhibits apoptosis: involvement of caspase-3.

The porcine antimicrobial peptide, PR-39, has several activities beyond its function of killing bacteria. Here we report that PR-39 alters macrophage viability by inhibiting apoptosis, which was induced by nutrient depletion, LPS stimulation or camptothecin treatment. This antiapoptotic effect was pronounced resulting in significant reductions in annexin-V binding to externalized phosphatidylserine and was associated with a decrease in caspase-3 activity. These findings suggest that PR-39, a porcine neutrophil-derived antimicrobial peptide, might function in the inflammatory milieu not only to kill bacteria, but also to aid in modulating the viability of inflammatory cells by regulating apoptosis.

Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity.

Antimicrobial peptides are important host-defense molecules of innate immunity. Cathelicidins are a diverse family of potent, rapidly acting and broadly effective antimicrobial peptides, which are produced by a variety of cells. This review examines the classification, antimicrobial spectrum, mechanism of action, and regulation of cathelicidins.