A mouse protein that localizes to acrosome and sperm tail is regulated by Y-chromosome.

BACKGROUND: Acrosomal proteins play crucial roles in the physiology of fertilization. Identification of proteins localizing to the acrosome is fundamental to the understanding of its contribution to fertilization. Novel proteins are still being reported from acrosome. In order to capture yet unreported proteins localizing to acrosome in particular and sperm in general, 2D-PAGE and mass spectrometry analysis of mouse sperm proteins was done. RESULTS: One of the protein spots identified in the above study was reported in the NCBI database as a hypothetical protein from Riken cDNA 1700026L06 that localizes to chromosome number 2. Immunofluorescence studies using the antibody raised in rabbit against the recombinant protein showed that it localized to mouse acrosome and sperm tail. Based on the localization of this protein, it has been named mouse acrosome and sperm tail protein (MAST, [Q7TPM5 (http://www.ncbi.nlm.nih.gov/protein/Q7TPM5)]). This protein shows 96% identity to the rat spermatid specific protein RSB66. Western blotting showed that MAST is expressed testis-specifically. Co-immunoprecipitation studies using the MAST antibody identified two calcium-binding proteins, caldendrin and calreticulin as interacting partners of MAST. Caldendrin and calreticulin genes localize to mouse chromosomes 5 and 8 respectively. In a Yq-deletion mutant mouse, that is subfertile and has a deletion of 2/3rd of the long arm of the Y chromosome, MAST failed to localize to the acrosome. Western blot analysis however, revealed equal expression of MAST in the testes of wild type and mutant mice. The acrosomal calcium-binding proteins present in the MAST IP-complex were upregulated in sperms of Yq-del mice. CONCLUSIONS: We have identified a mouse acrosomal protein, MAST, that is expressed testis specifically. MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome. The misexpression of all the proteins identified in a complex in the Yq-del mice invokes the hypothesis of a putative pathway regulated by the Y chromosome. The role of Y chromosome in the regulation of this complex is however not clear from the current study.

Angiogenic response of advanced glycation end products (AGEs) involves PPARgamma.

Diabetes is associated with increased formation of advanced glycation end products (AGEs), which have been implicated in micro and macrovascular complications of diabetes. Our earlier reports showed proangiogenic effect of AGE-bovine serum albumin (BSA). In order to understand the mechanism of AGE-mediated angiogenesis, the possibility of involvement of peroxisome prolifeator activated receptor (PPAR) gamma, a ligand activated transcription factor was examined. The angiogenic effect was studied in chick chorio allantoic membrane (CAM) and by analyzing angiogenic markers in human umbilical vein endothelial cells (HUVECs) in culture. The involvement of PPAR y was investigated using synthetic PPAR gamma agonist GW 1929 and antagonist GW 9662 and by RT-PCR. In CAM assay, PPAR gamma antagonist GW 9662 reversed the AGE-induced effect on vascularity. In HUVECs in culture, GW 9662 reversed the effect of AGE-BSA and decreased the expression of CD 31, E-Selectin and VEGF. RT-PCR analysis showed that treatment with AGE-BSA caused upregulation of PPAR gamma mRNA levels. The reversal of the effect of AGE on angiogenesis by treatment with PPAR gamma antagonists and up-regulation of PPAR gamma gene in HUVECs treated with AGE-BSA suggested the possible involvement of PPAR gamma-dependent downstream pathway in mediating the angiogenic effect of AGE.

Antiaflatoxigenic effects of selected antifungal peptides.

Aflatoxins are potent carcinogenic mycotoxins produced as secondary metabolites mainly by the fungi Aspergillus flavus and Aspergillus parasiticus. Control measures to curtail the contamination of aflatoxin in food products is still a challenge. Although there are several reports on the antifungal peptides, there is no specific study on the action of antifungal peptides on aflatoxin synthesis. This work details the effect of four antimicrobial peptides (AMPs) - PPD1 (FRLHF), 66-10 (FRLKFH), 77-3 (FRLKFHF) and D4E1 (FKLRAKIKVRLRAKIKL) on the aflatoxin production by A. flavus and A. parasiticus. Results of the investigations suggests that AMPs at near minimum inhibitory concentrations (MIC) were effectively inhibiting aflatoxins, without hindering the growth of the fungi. These AMPs, at concentrations near MIC, induced membrane permeabilisation, without inducing cellular leakage. The involvement of oxidative stress for the aflatoxin synthesis was reversed by the antioxidant nature of the peptides as evidenced by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay, reactive oxygen species production, malondialdehyde and antioxidant enzymes analysis. Quantitative real time polymerase chain reaction (RT-qPCR) analysis of the aflatoxin gene cluster showed that 'aflR' and its downstream genes expressions were significantly down regulated. Conidiation of the fungi were negatively influenced by the peptides as evidenced by scanning electron microscopy analysis and RT-qPCR. mRNA levels of Manganese-superoxide dismutase (Mn-SOD) showed a decrease in the expression in RT-qPCR. The effect of these peptides on aflatoxin inhibition provides insight into their use as novel antiaflatoxigenic molecules.

Differential modulation of angiogenesis by advanced glycation end products.

Divergent angiogenic responses occur in different organs in a diabetic state. Many of the pathological effects were mediated by the advanced glycation end products (AGEs) of non-enzymatically glycated molecules. Investigations were carried out using different angiogenic model systems to examine whether the angiogenic response to AGEs is influenced by the cellular microenvironment. AGE-albumin increased angiogenesis in chick chorioallantoic membrane (CAM). It also increased sprouting in rat aortic rings and the expression of angiogenic markers CD31 and E-selectin and the angiogenic growth factor, vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs) in culture, suggesting a proangiogenic effect. But in a serum-supplemented condition, AGE-albumin inhibited aortic sprouting and expression of angiogenic markers and VEGF production by HUVECs, suggesting an antiangiogenic effect in the presence of serum. Blocking of the AGE effect by the antioxidants, N-acetyl cysteine and ascorbic acid, suggested that the AGE effect involved oxidant stress. Reversal of the AGE effect by LY 294 002, an inhibitor of the Akt pathway and increased phosphorylation of Akt in cells maintained in serum-free medium, suggested the involvement of the Akt pathway in mediating the AGE effect; such an effect was absent in a serum-supplemented condition. These opposing effects of AGE-albumin on angiogenesis in the presence and absence of serum suggested that the AGE accumulated in a hyperglycemic condition can affect angiogenesis depending on the microenvironment of the cells.