In vivo and in vitro evaluation for immunomodulatory activity of three marine animal extracts with reference to phagocytosis

The whole body ether extracts of a marine prawn Nematopaleamon tenuipes and two gastropods viz. Euchelus asper and Hemifusus pugilinus, obtained by Soxhlet extraction and cold percolation were tested for their effects on phagocytosis by in vitro (slide method) and by in vivo (carbon clearance) methods. Extract of E. asper exhibits immunostimulatory activity in vitro and immunosuppressant activity in vivo. The in vitro test for N. tenuipes and H. pugilinus shows biphasic activity, but the former shows immunostimulatory while the latter shows immunosuppresant activity in vivo test.     

Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimer formation and ligand binding

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3zeta. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3zeta in vitro and in HEK293 cells. The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated p38-MAPK-dependent phosphorylation of 14-3-3 proteins in human neutrophils. Mutation analysis showed that MAPKAPK2 phosphorylated 14-3-3zeta at Ser-58. Computational modeling and calculation of theoretical binding energies predicted that both phosphorylation at Ser-58 and mutation of Ser-58 to Asp (S58D) compromised the ability of 14-3-3zeta to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3zeta dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3zeta functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.     

Adrenomedullin expression in pathogen-challenged oral epithelial cells

Adrenomedullin, a multifunctional peptide, is expressed by many surface epithelial cells and, previously, we have demonstrated that adrenomedullin has antimicrobial activity. The oral cavity contains an epithelium that is permanently colonized by microflora, yet infections in a host are rare. We exposed oral keratinocytes to whole, live cells from four microorganisms commonly isolated from the oral cavity, Porphyromonas gingivalis, Streptococcus mutans, Candida albicans and Eikenella corrodens. There was upregulation of protein and gene expression in these cells in response to bacterial suspensions, but not with the yeast, Candida albicans. We propose there is a potential role for microbial products in enhancing mucosal defense mechanisms and that adrenomedullin participates in the prevention of local infection, thus contributing to host defense mechanisms.     

A functional proteomics screen of proteases in colorectal carcinoma

BACKGROUND: Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. MATERIALS AND METHODS: An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver metastases were screened for protease activity. RESULTS: The major proteases detected were matrix metalloproteases (MMP9, MMP2, and MMP1), cathepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metalloproteases were expressed at higher levels in the primary tumor than in adjacent normal tissue. The mast cell proteases, in contrast, were at very high levels in adjacent normal tissue, and not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the metastases. The major proteases detected by activity assays were then localized in biopsy sections by immunohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matrix metalloproteases were localized to the tumor cells themselves; whereas, cathepsin B was predominantly expressed by macrophages at the leading edge of invading tumors. Although only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor. CONCLUSIONS: This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low molecular weight protease inhibitors as potential chemotherapy.     

A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced levels of frataxin protein. We have previously reported the generation of human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing 90–190 GAA repeats, but the presence of multiple GAA repeats within these mice is considered suboptimal. We now describe the cellular, molecular and behavioural characterisation of a newly developed YAC transgenic FRDA mouse model, designated YG8sR, which we have shown by DNA sequencing to contain a single pure GAA repeat expansion. The founder YG8sR mouse contained 120 GAA repeats but, due to intergenerational expansion, we have now established a colony of YG8sR mice that contain ~200 GAA repeats. We show that YG8sR mice have a single copy of the FXN transgene, which is integrated at a single site as confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We have identified significant behavioural deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8sR FRDA mice compared with control Y47R and wild-type (WT) mice. We have also detected increased somatic GAA repeat instability in the brain and cerebellum of YG8sR mice, together with significantly reduced expression of FXN, FAST-1 and frataxin, and reduced aconitase activity, compared with Y47R mice. Furthermore, we have confirmed the presence of pathological vacuoles within neurons of the dorsal root ganglia (DRG) of YG8sR mice. These novel GAA-repeat-expansion-based YAC transgenic FRDA mice, which exhibit progressive FRDA-like pathology, represent an excellent model for the investigation of FRDA disease mechanisms and therapy.KEY WORDS: GAA repeat, Friedreich’s ataxia, FRDA, YG8sR, Mouse model     

Biometry of frozen-thawed sperm from eight breeds of Indian buffaloes (Bubalus bubalis)

Sperm morphometry, in combination with other objective traits, can be useful for developing a fertility index. The objective of the present study was to measure various biometric end points of frozen-thawed sperm from eight breeds of Indian buffaloes (Murrah, Surti, Tarai, Mehsana, Jaffrabadi, Bhadawari, Pandharpuri and Nili-Ravi). The sperm head of Pandharpuri buffaloes had the greatest length (10.21 microm), width (6.05 microm), area (52.31 microm(2)) and perimeter (31.86 microm). The ratio of sperm width to length was also greatest (0.61) in Pandharpuri as well as in two other breeds, viz. Nili-Ravi and Jaffrabadi. Murrah had the smallest sperm head width (4.75 microm), area (41.65 microm(2)) and perimeter (29.17 microm), but its sperm tail was longest (57.02 microm), along with that of Jaffrabadi buffaloes (56.96 microm). Based on mean values of sperm tail length, mid piece length and its width the eight buffalo breeds were categorized into three, four and five groups, respectively. Multivariate analysis and clustering put six breeds (Surti, Tarai, Mehsana, Jaffrabadi, Bhadawari and Nili-Ravi) in one cluster, whereas Murrah and Pandharpuri appeared as separate entities.     

Expression of a polyphemusin variant in transgenic tobacco confers resistance against plant pathogenic bacteria,fungi and a virus

Antimicrobial cationic peptides provide a promising means of engineering plant resistance to a range of plant pathogens, including viruses. PV5 is a synthetic structural variant of polyphemusin, a cationic peptide derived from the horseshoe crab-Limulus polyphemus. PV5 has been shown to be benign toward eukaryotic membranes but with enhanced antimicrobial activity against animal pathogens. In this work, the cytotoxicity of PV5 toward tobacco protoplasts and leaf discs was assessed using TTC (2,3,5-triphenyltetrazolium chloride) and Evans blue colorimetric assays. PV5 showed no measurable cytotoxic effects even at levels as high as 60 μg. As a possible approach to enhancing plant resistance, a gene encoding PV5 was fused to the signal sequence encoding the C-terminus portion of the BiP protein from Pseudotsuga menziesii, under the control of 2 × 35S CaMV promoter. When introduced into Nicotiana tabacum var Xanthi gene integration and expression was confirmed by both Southern and northern analyses. When transgenic plants were subsequently challenged with bacterial and fungal phytopathogens enhanced resistance was observed. Moreover, transgenic plants also displayed antiviral properties against Tobacco Mosaic Virus making PV5 an excellent candidate for conferring unusually broad spectrum resistance to plants and the first anti-plant virus antimicrobial peptide.     

Cation-aromatic database

Cation-aromatic database (CAD) is a publicly available web-based database that aims to provide further understanding of interaction between a cation and the pi interactions. A tool to identify the interactions in a user-given protein is also added to the database. CAD is freely accessible via the Internet at http://203.199.182.73/gnsmmg/databases/cad/.     

Entrainment during Ablation of Ischemic Ventricular Tachycardia. What is explanation for Post Pacing Interval shorter than the tachycardia cycle length?

Entrainment mapping of ischemic ventricular tachycardia at a site in the left ventricle where radiofrequency ablation was successful in terminating the tachycardia revealed a post-pacing interval shorter than the tachycardia cycle length. The reason for the same is explained in the current report.