Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.     

Cellular affinity is the only deciding factor for microcarrier selection for the cultivation of anchorage dependent cells

Summary Vero and BHK-21 cells are not attached and spreaded equally on the same microcarrier. BHK-21 cells require a microenvironment similar to the mammalian tissue for attachment and spreading. Cellular affinity, which control adhesion to the surface, varied with the growth phase, membrane integrity and the type of microcarrier.     

Role of ascorbic acid in metabolism of rat testis and epididymis in relation to the onset of puberty

The metabolism of ascorbic acid, cholesterol, serum testosterone level and activities of 3Β and 17Β hydroxysteroid dehydrogenases were studied in testis and Cauda epididymis of prepubertal, pubertal and postpubertal (5, 15, 30, 45, 55 and 60 day old) rats. The data showed that serum testosterone levels and 3Β and 17Β hydroxysteroid dehydrogenases were increased with the age. The ascorbic acid metabolism was found to be stabilized in testis at day 30 being comparable with the adult, whereas a spurt in its metabolism occurred by day 45 and a significant depletion in ascorbic acid content in relation to the passage of the first wave of spermatozoa through cauda epididymis. The results of this study clearly elucidate that ascorbic acid is involved in metabolism of testis and epididymis in developing postnatal rats, in relation to the increasing demands for attaining a stable hormonal milieu, and the onset of puberty and the passage of the first wave of spermatozoa,via the formation of its free radical monodehydroascorbic acid and charge transfer complex mechanism     

Isopongaglabol and 6-methoxyisopongaglabol,two new hydroxyfuranoflavones from Pongamia glabra

Isopongaglabol and 6-methoxyisopongaglabol, two new hydroxyfuranoflavones, together with two furanoflavones 5-methoxyfurano(8,7-4″,5″)flavone and 5-methoxy-3′,4′-methylenedioxyfurano(8,7-4″,5″)flavone, two simple flavones, desmethoxykanugin and fisetin tetramethyl ether, a chromenoflavanone, ovalichromene B, two triterpenes, cycloart-23-ene-3β,25-diol and friedelin, and β-sitosterol-β-d-glucoside were isolated from the petrol and CHCl₃ extracts of the flowers of Pongamia glabra. The structures of isopongaglabol and 6-methoxyisopongaglabol have been established as 4′-hydroxyfurano(8,7-4″,5″)flavone and 4′-hydroxy-6-methoxyfurano(8,7-4″,5″)flavone, respectively, on the basis of the spectral evidence and they have been confirmed by synthesis.     

Current trends in evaluation of sperm function: in vitro selection and manipulation of male gametes for assisted conception

With increasing medical utilization of assisted reproductive technology (ART), scientists and clinicians have been able to study extensively multiple cell functions operating synchronously and flawlessly during the events preceding, before and after fertilization. Critical evaluation of the functional status of spermatozoa for in vitro techniques such as sperm-mucus interaction, acrosome reaction status, sperm-zona pellucida binding and penetration tests, hyaluronic acid binding assay, and computer assisted semen analysis etc. can direct a male partner of an infertile couple to more aggressive forms of treatments. In vitro selection of functionally competent sperm cells is a pre-requisite for successful outcome in in vitro fertilization or in intracytoplasmic sperm injection (ICSI). Direct injections of acrosome-intact spermatozoa into oocyte during ICSI bypassing the normal events of sperm oocyte interaction and fusion events have raised concerns with regard to fertilization abnormalities and genetic issues. The present communication briefly reviews the sperm function tests with emphasis on its correlation with fertility outcome, and the currently employed sperm selection and manipulation procedures which may have implications in assisted conception programs.     

Biphasic kinetics of the colchicine-tubulin interaction: role of amino acids surrounding the A ring of bound colchicine molecule

Isotypes of vertebrate tubulin have variable amino acid sequences, which are clustered at their C-terminal ends. Isotypes bind colchicine at different on-rates and affinity constants. The kinetics of colchicine binding to purified (unfractionated) brain tubulin have been reported to be biphasic under pseudo-first-order conditions. Experiments with individual isotypes established that the presence of beta(III) in the purified tubulin is responsible for the biphasic kinetics. Because the isotypes mainly differ at the C termini, the colchicine-binding kinetics of unfractionated tubulin and the beta(III) isotype, cleaved at the C termini, have been tested under pseudo-first-order conditions. Removal of the C termini made no difference to the nature of the kinetics. Sequence alignment of different beta isotypes of tubulin showed that besides the C-terminal region, there are differences in the main body as well. To establish whether these differences lie at the colchicine-binding site or not, homology modeling of all beta-tubulin isotypes was done. We found that the isotypes differed from each other in the amino acids located near the A ring of colchicine at the colchicine-binding site on beta tubulin. While the beta(III) isotype has two hydrophilic residues (serine(242) and threonine(317)), both beta(II) and beta(IV) have two hydrophobic residues (leucine(242) and alanine(317)). beta(II) has isoleucine at position 318, while beta(III) and beta(IV) have valine at that position. Thus, these alterations in the nature of the amino acids surrounding the colchicine site could be responsible for the different colchicine-binding kinetics of the different isotypes of tubulin.     

Fibronectin and laminin enhance engraftibility of cultured hematopoietic stem cells

To test the hypothesis that extracellular matrix (ECM) components maintain stem cell property, murine bone marrow (BM) cells were expanded in fibronectin and laminin coated plate in the presence of cytokines. We observed significant phenotypic and functional improvement of expanded cells. In 10 days, 800-fold expansion of colony-forming unit-granulocyte erythrocyte monocyte megakaryocyte (CFU-GEMM) was observed in the cultured cells. No apparent activation of cell cycle was observed, but CD29 and very late antigen-4 (VLA-4) expression was increased, as compared to the normal BM cells. A fraction of the expanded cells became verapamil sensitive, suggesting upregulation of multi-drug resistant gene(s), as found in the primitive hematopoietic stem cells (HSCs). Competitive repopulation assay confirmed that HSCs compartment was amplified during culture. Overall, our study clearly demonstrated that ex vivo culture of murine HSCs in the presence of fibronectin and laminin resulted in expansion of primitive stem cells and improvement in the marrow engraftibility.     

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.