Membrane orientation of laminin binding protein.

Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.     

Role of protein phosphorylation in activation of interferon-stimulated gene factors.

Possible involvement of protein phosphorylation in interferon (IFN)-mediated activation of IFN-stimulated gene factor 3 (ISGF3) was investigated. For this purpose, in vivo experiments with specific inhibitors of protein kinases and in vitro experiments with protein phosphatases were carried out. In HeLaM cells, 2-aminopurine, an inhibitor of double-stranded RNA-dependent protein kinase, blocked the induction of ISGF3 gamma subunit but not the activation of ISGF3 alpha subunit. A series of experiments using combinations of protein and RNA synthesis inhibitors and 2-aminopurine indicated that the block elicited by 2-aminopurine was at the level of ISGF3 gamma mRNA synthesis. Activation of ISGF3 alpha, although insensitive to 2-aminopurine, was completely blocked by 10 nM staurosporine, an inhibitor of protein kinase C. On the other hand, even 500 nM staurosporine did not block the induction of ISGF3 gamma. Incubation of cytoplasmic or nuclear extracts of IFN-treated HeLaM cells in vitro with alkaline phosphatase completely eliminated their ability to form the ISGF3 complex but not the ISGF1 complex. Treatment with acid phosphatase, on the other hand, changed the electrophoretic mobility of the ISGF3 complex but did not obliterate it. Complementation experiments revealed that ISGF3 alpha was the alkaline phosphatase-sensitive component of the complex. These results suggest that a protein kinase C-mediated phosphorylation step is involved in ISGF3 alpha activation and a 2-aminopurine-sensitive component is involved in ISGF3 gamma mRNA induction.     

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich’s ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Environmental distribution and genetic diversity of vegetative compatibility groups determine biocontrol strategies to mitigate aflatoxin contamination of maize by Aspergillus flavus

Maize infected by aflatoxin‐producing Aspergillus flavus may become contaminated with aflatoxins, and as a result, threaten human health, food security and farmers' income in developing countries where maize is a staple. Environmental distribution and genetic diversity of A. flavus can influence the effectiveness of atoxigenic isolates in mitigating aflatoxin contamination. However, such information has not been used to facilitate selection and deployment of atoxigenic isolates. A total of 35 isolates of A. flavus isolated from maize samples collected from three agro‐ecological zones of Nigeria were used in this study. Ecophysiological characteristics, distribution and genetic diversity of the isolates were determined to identify vegetative compatibility groups (VCGs). The generated data were used to inform selection and deployment of native atoxigenic isolates to mitigate aflatoxin contamination in maize. In co‐inoculation with toxigenic isolates, atoxigenic isolates reduced aflatoxin contamination in grain by > 96%. A total of 25 VCGs were inferred from the collected isolates based on complementation tests involving nitrate non‐utilizing (nit^?) mutants. To determine genetic diversity and distribution of VCGs across agro‐ecological zones, 832 nit^? mutants from 52 locations in 11 administrative districts were paired with one self‐complementary nitrate auxotroph tester‐pair for each VCG. Atoxigenic VCGs accounted for 81.1% of the 153 positive complementations recorded. Genetic diversity of VCGs was highest in the derived savannah agro‐ecological zone (H = 2.61) compared with the southern Guinea savannah (H = 1.90) and northern Guinea savannah (H = 0.94) zones. Genetic richness (H = 2.60) and evenness (E₅ = 0.96) of VCGs were high across all agro‐ecological zones. Ten VCGs (40%) had members restricted to the original location of isolation, whereas 15 VCGs (60%) had members located between the original source of isolation and a distance > 400 km away. The present study identified widely distributed VCGs in Nigeria such as AV0222, AV3279, AV3304 and AV16127, whose atoxigenic members can be deployed for a region‐wide biocontrol of toxigenic isolates to reduce aflatoxin contamination in maize.     

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.     

Genetic portrait of Lisboa immigrant population from Cabo Verde with mitochondrial DNA analysis

Journal of Genetics -     

Are “multiple cross-multiple pollen hybrids’ an answer for productive populations in Brassica campestris var. ‘brown sarson’?

Summary A set of complex crosses with multiple crosses as female parents were made using multiple pollen in turnip rape (Brassica campestris L.). These multiple cross — multiple pollen hybrids (mucromphs) were evaluated for a large number of quantitative characters including yield. New methods were proposed to study such genetic material in depth so as to formulate suitable strategies to breed for attractive seed yield.Part of the Ph. D. Thesis of junior author submitted to Indian Agricultural Research Institute, New Delhi