Browsing by Author "Adiga, P R"
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Item Active immunization against riboflavin carrier protein results in peri-implantation embryonic loss leading to pregnancy termination in rats: use of alternate adjuvants.(2000-09-04) Rao, J; Seshagiri, P B; Shetty, G; Ramesh, G; Adiga, P RTo investigate the mechanism of pregnancy termination following immuno-neutralization of riboflavin carrier protein (RCP) and to use acceptable adjuvants, we actively immunized female rats with reduced and carboxymethylated RCP (RCM-RCP) using various adjuvants (during primary immunization) such as sodium phthalylated lipopolysaccharide (SPLPS), purified S. typhi outer membrane proteins (porins) and a combination of them. Rats (5-14 per group) were immunized with alugel adsorbed RCM-RCP (100 microg/dose) either alone or with SPLPS or porins or SPLPS+porins. Control animals received RCM-RCP emulsified with Fruend's completelincomplete adjuvants (FCA/FIA). All animals received five boosters at intervals of 21 days. The lowest (4 X 10(-3)) and the highest (> 70 X 10(-3)) anti-RCM-RCP antibody titers were observed in alugel adsorbed-RCM-RCP group and control groups, respectively. Immunized animals showed reduced fertility following 3rd, 4th and 5th boosters. Reduction in fertility was 30-60% in alugel adsorbed RCM-RCP group, 90-100% in FCA-RCM-RCP group and 80-90% in SPLPS+porins group. Fertility reduction was not strictly correlatable with the serum antibody titers. RCP-specific IgG could be localized in the uterine endometrial glands and luminal epithelial cells in the immunized animals. Animals in the FCA/FIA group showed abnormal implantation/resorption sites and their histological sections showed degenerated embryos. But, day 5 preimplantation embryos were normal. These results show that (a) SPLPS+porins can be used as adjuvants in place of FCA/FIA for active immunization against RCM-RCP and (b) early termination of pregnancy in the immunized animals is due largely to the failure of normal embryo implantation.Item Active immunization of rats with chicken egg thiamin carrier protein results in early embryonic loss at periimplantation stages.(1996-04-01) Subramanian, S; Rao, J; Adiga, P RActive immunization with chicken egg white thiamin carrier protein (TCP) was performed to assess the functional importance of the vitamin carrier during gestation in rats. Towards this, fertile female rats were immunized with heterologous TCP and when the circulatory titres of anti-TCP IgG were high, these animals were mated with fertile male rats. Progression of pregnancy was monitored by measuring circulatory progesterone levels. A sudden fall in the steroid hormonal levels around day 8 post coitus was observed. Histological examination of the uterine tissue sections on day 7 revealed that active immunization with TCP affected the blastocyst viability resulting in unsuccessful implantation and hence pregnancy termination.Item Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies.(1992-06) Velu, N Kuzhandhai; Karande, Anjali A; Adiga, P RMonoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.Item Arginine decarboxylase is a component activity of the multifunctional.(1987-03) Prasad, G L; Adiga, P RA homogenous preparation of putrescine synthase, the versatile multifunctional enzyme involved in agmatine→putrescine conversion in Cucumis sativus was found to catalyze enzymatic decarboxylation of arginine also. Similarly, the purified arginine decarboxylase mediated the component as well as the complete set of coupled reactions harboured by putrescine synthase. Both the enzyme preparations exhibited identical electrophoretic and chromatographic behaviour and were immunologically indistinguishable. All the enzymic activities are stabilized concurrently by feeding arginine to the intact seedlings. Therefore, it is concluded that the multifunctional putrescine synthase in Cucumis sativus seedlings also harbours arginine decarboxylase activity unlike its counterpart in Lathyrus sativus.Item Biochemical and immunological aspects of riboflavin carrier protein.(1988-03) Adiga, P R; Visweswariah, S S; Karande, A; Kuzhandhaivelu, NRiboflavin carrier protein which is obligatorily involved in yolk deposition of the vitamin in the chicken egg, is a unique glycophosphoprotein present in both the yolk and white compartments. The yolk and egg white proteins are products of a single estrogeninducible gene expressed in the liver and the oviduct respectively of egg laying birds. Despite the fact that the carbohydrate composition of the yolk and white riboflavin carrier proteins differ presumably due to differential post-translational modification, the proteins are immunologically similar and have identical amino acid sequence (including a cluster of 8 phosphoser residues towards the C-terminus) except at the carboxy terminus where the yolk riboflavin carrier protein lacks 13 amino acids as a consequence of proteolytic cleavage during uptake by oocytes. The protein is highly conserved throughout evolution all the way to humans in terms of gross molecular characteristics such as molecular weight and isoelectric point, and in immunological properties, preferential affinity for free riboflavin and estrogen inducibility at the biosynthetic locus viz., liver. Obligatory involvement of the mammalian riboflavin carrier protein in transplacental flavin transport to subserve fetal vitamin nutrition during gestation is revealed by experiments using pregnant rodent or subhuman primate models wherein immunoneutralisation of endogenous maternal riboflavin carrier protein results in fetal wastage followed by pregnancy termination due to selective yet drastic curtailment of vitamin efflux into the fetoplacental unit. Using monoclonal antibodies to chicken riboflavin carrier protein, it could be shown that all the major epitopes of the avian riboflavin carrier protein are highly conserved throughout evolution although the relative affinities of some of the epitopes for different monoclonal antibodies have undergone progressive changes during evolution. Using these monoclonal antibodies, an attempt is being made to map the different epitopes on the riboflavin carrier protein molecule with a view to delineate the immunodominant regions of the vitamin carrier to understand its structure-immunogenicity relationship.Item Comparison of biotin binding protein of pregnant rat serum with rat serum albumin.(1989-09) Seshagiri, P B; Adiga, P RThe purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [125I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10–100 nl) and purified rat serum albumin (0·1–10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [125I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0·5–10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In twodimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity.Item Correlation between riboflavin carrier protein induction and its mRNA activity in estrogen stimulated chicken liver and oviduct.(1986-06) Kumari, B Durga; Adiga, P RPoly A enriched RNA from either liver or oviduct of estradiol-17β treated immature chicks supported [3H]-leucine incorporation into immunoprecipitable riboflavin carrier protein in a dose-dependent manner when translated in the rabbit reticulocyte lysate system. Primary translation product of riboflavin carrier protein had a molecular weight of 38,000 which on incubation with a stripped hepatic microsomal preparation was processed to a product with a size comparable to native riboflavin carrier protein. Poly A enriched RNA from both the liver and the oviduct of estrogen-treated birds stimulated [3H]-leucine incorporation into riboflavin carrier protein and this was 2-3 fold higher during secondary stimulation vis-avis primary stimulation with the steroid. Poly A enriched RNA from the liver of progesteronetreated birds during secondary stimulation did not support riboflavin carrier protein synthesis. In contrast, poly A enriched RNA from the oviduct of the birds treated with progesterone during secondary (but not primary) stimulation did exhibit riboflavin carrier protein-mRNA activity which was comparable to that stimulated by estradiol- 17β.Item Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings.(1986-06) Prasad, G L; Adiga, P RA purified preparation of arginine decarboxylase from Cucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine and Pi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase, viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3-4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine and vice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.Item Diamine oxidase of Lathyrus sativus seedlings. Purification and properties.(1979-06) Suresh, M R; Adiga, P RDiamine oxidase (EC 1.4.3.6) was purified from 5-day-old etiolated seedlings of Lathyrus sativus by MnCl2 treatment, (NH4)2SO4 and acetone fractionations, DEAE-Sephadex chromatography followed by gel filtration on Sephadex G-200. A single step purification of the enzyme was achieved by using an immunoaffinity column, wherein rabbit antibodies to the homogeneous diamine oxidase were coupled to CNBr-activated Sepharose. The enzyme thus obtained was homogeneous by electrophoretic, immunological and ultracentrifugal criteria. It had an Mr of 148,000 (6·46S) and was a dimer with similar sub-units (Mr 75,000). Amino acid analysis showed the absence of cysteine residues although it contained five disulphide bonds. The enzyme had copper (2·7 g atom/mol enzyme) but was not a glycoprotein. No absorption maximum in the visible region was detectable. Ethylenediamine 1,3-diaminopropane and histamine were potent competitive inhibitors for the substrate putrescine. The addition of monospecific antibodies to the enzyme increased the Km for benzyl amine without any change in the Vmax. Diamine oxidase from pea seedling, partially purified, exhibited complete crossreactivity with the antibodies to the L. sativus enzyme.Item Early pregnancy termination in rats immunized with denatured chicken riboflavin carrier protein.(1991-10-01) Karande, A A; Adiga, P RImmunoneutralization of the maternal riboflavin carrier protein in the pregnant rat with antibodies to chicken egg vitamin carrier has earlier been shown to terminate their pregnancies. In order to understand the nature of the epitopic conformations capable of eliciting antibodies bioneutralizing the endogenous riboflavin carrier protein in the pregnant rat, we compared pregnancy progression in the fertile rodents following active immunization with either the native, SDS-denatured, reduced-carboxymethylated or SDS-treated reduced carboxymethylated avian egg white riboflavin carrier protein. The data revealed that despite the total antibody titers being higher in the animals immunized with the native protein, the antibodies elicited against the denatured avian vitamin carrier exhibited relatively better potencies to bioneutralize the endogenous maternal protein as evidenced by higher rates of early fetal resorption.Item Establishment of the functional importance of thiamin carrier protein in pregnant rats by using monoclonal antibodies.(1996-04-01) Subramanian, S; Karande, A A; Adiga, P RMonoclonal antibodies (mAbs) to chicken thiamin carrier protein (TCP) have been produced by hybridoma technology to identify the crucial epitopes involved in bioneutralization of the vitamin carrier. The monoclonality of these mAbs (A4C4, F3H6, H8H3, C8C1 and G7H10) was sought to be confirmed by sub-class isotyping; they all belong to IgG1, k type. The epitopes recognized by all the five mAbs are conserved in TCP from the chicken to the rat as assessed by liquid phase RIA and immunoprecipitation of 125I-labelled proteins from pregnant rat serum. Among these mAbs. passive immunization of pregnant rats with the mAb C8C1 only on three consecutive days (day 10, 11 and 12) resulted in embryonic resorption. These results demonstrate the importance of epitopic structure specified by the mAb C8C1 on TCP during pregnancy in rats.Item Estrogen stimulation of chicken liver ornithine decarboxylase: relationship between the levels of enzyme and poly A-enriched RNA.(1977-12-01) Murthy, U S; Suresh, M R; Prasad, M S; Adiga, P RItem Hormonal modulation of reproduction–specific thiamin carrier protein in the rat.(1985-03) Malathy, P V; Adiga, P RThe hormonal modulation of thiamin carrier protein in the plasma and uterine luminal secretion during the normal reproductive phases of the animal (estrous cycle and pregnancy) as well as during experimental estrogenisation was investigated in the rat using a specific and sensitive homologous radioimmunoassay procedure developed for this purpose. Following a single injection of estrogen to immature male rats, thiamin carrier protein rapidly accumulated in plasma attaining peak concentration at 48 h and declining thereafter. A 1·5- fold amplification of the inductive response was observed on secondary stimulation with the hormone. The magnitude of the response exhibited a clear dependency on the dose of the steroid hormone, whereas the time at which peak levels of thiamin carrier protein production was remained unaltered in the concentration range of the steroid tested. The inductive effect of estrogen was severely curtailed by the antiestrogens, viz., En- and Zu-clomiphene citrates, while progesterone was incapable of either modulating the estrogen-induced response or eliciting an induction by itself. Cycloheximide drastically blocked the response to estrogen. Evidence for the ability of uterus to serve as yet another independent site of thiamin carrier protein synthesis was obtained by in vitro incorporation of radioactive amino acids into immunoprecipitable thiamin carrier protein in the tissue explants of estrogenised female rats. The levels of thiamin carrier protein in uterine luminal fluid measured during estrous cycle, pregnancy and experimental estrogenisation exhibited remarkable similarity to the plasma thiamin carrier protein profiles.Item Hormonal modulation of secretion of immunoreactive riboflavin carrier protein by adult rat Leydig cells in vitro.(1996-08-01) Subramanian, S; Adiga, P RAdult rat Leydig cells in culture synthesize and secrete riboflavin carrier protein (RCP) as demonstrated by [35S]-methionine incorporation into newly synthesized proteins followed by immunoprecipitation as well as specific radioimmunoassay. LH stimulates the secretion of RCP 4-fold which could be inhibited upto 75% by an aromatase inhibitor. 8-bromo-cyclic AMP and cholera toxin could mimic the LH stimulated secretion of the carrier protein. The extent of stimulation of RCP secretion brought about by exogenous estradiol-17 beta is comparable to that of LH. The antiestrogen tamoxifen, when added along with either LH or estrogen, inhibited the stimulated levels significantly. These results show that the estrogen-inducible riboflavin carrier is secreted by Leydig cells under positive regulation of LH.Item Immunohistochemical localization of riboflavin carrier protein in testicular cells of mammals.(1995-01-01) Bhat, K G; Malhotra, P; Karande, A A; Adiga, P RUsing specific polyclonal antibodies against chicken riboflavin carrier protein (cRCP), immunocytochemical localization of riboflavin carrier protein was carried out in testicular sections and isolated cells of mammals. A positive reaction was observed in the developing germ cells of rat testis, especially in meiotic and post-meiotic germ cells such as pachytene spermatocytes, round spermatids and spermatozoa. In addition both the somatic cells of the testis, viz. Leydig and Sertoli cells with vital function in germ cell proliferation and differentiation, displayed a moderate to strong staining reaction. This was further confirmed using in utero X-irradiated rat testis devoid of germ cells. Different types of cells isolated from testis when subjected to immunostaining showed similar patterns of reaction as in the intact tissue. Mature spermatozoa from different mammals (rat, bull and monkey) exhibited strong staining reaction in their head regions localized mainly in acrosomal caps. It is suggested that the testicular riboflavin carrier protein has a role in cell to cell communication and may be crucial during development of germ cells especially at the meiotic and post-meiotic stages.Item Induction of riboflavin-carrier protein in the immature male rat by estrogen: kinetic and hormonal specificity.(1982-06) Murthy, C V Ramana; Adiga, P RThe kinetics of estrogen-induced accumulation of riboflavin-carrier protein in the plasma was investigated in immature male rats using a specific and sensitive homologous radioimmunoassay procedure developed for this purpose. Following a single injection of the steroid hormone, plasma riboflavin-carrier protein levels increased markedly after an initial lag period of approximately 24 h, reaching peak levels around 96 h and declining thereafter. A 1.5 fold amplification of the inductive response was evident on secondary stimulation with the hormone. The magnitude of the response was dependent on hormonal dose, whereas the initial lag phase and the time of peak riboflavin-carrier protein induction were unaltered within the range of the steroid doses (0.1-10 mg/ kg body wt.) tested. Simultaneous administration of progesterone did not affect either the kinetics or the maximum level of the protein induced. The hormonal specificity of this induction was further adduced by the effect of administration of antiestrogens viz., En and Zu chlomiphene citrates, which effectively curtailed hormonal induction of the protein. That the induction involved de novo-protein synthesis was evident from the complete inhibition obtained upon administration of cycloheximide. Passive immunoneutralization of endogenous riboflavin-carrier protein with antiserum to the homologous protein terminated pregnancy in rats confirming the earlier results with antiserum to chicken riboflavin-carrier protein.Item Isolation & characterization of aliphatic amines from Lathyrus sativus seedlings & the changes in their concentrations during development.(1974-06-01) Ramakrishna, S; Adiga, P RItem Isolation of sheep anterior pituitary messenger RNA and its translation in a heterologous cell-free system.(1979-09) Prasad, M S K; Adiga, P RA preparation rich in the specific messenger RNA involved in the synthesis of prolactin from sheep anterior pituitary glands was obtained by employing both the immunochemical and affinity techniques. A dose-dependent and efficient stimulation of protein synthesis by the isolated total pituitary RNA as well as poly (A) rich RNA were achieved with the reticulocyte system. The synthesis of prolactin as one of the translational products of this cell-free system was established by specific immunoprecipitation followed by resolution on sodium dodecyl sulphate polyacrylamide gel electrophoresis.Item Modulation of prolactin receptors in the male rat.(1979-11-01) Prasad, M S; Adiga, P RItem Modulation of testicular lutropin receptors in the developing male rat.(1980-03) M S K, Prasad; Adiga, P RIn the developing male rat around 40 days of age, the testis appears to contain the maximum amount of lutropin receptors per unit weight. During this period, circulating levels of testosterone markedly increase without the concomitant major surges in lutropin levels. The increased sensitivity and responsiveness of tests to basal levels of circulating lutropin during this period is accompanied by enhanced serum prolactin levels suggesting that this hormone may be involved in this process. The finding that prolactin treatment of pubertal rats for 3 days induced the formation of more testicular lutropin receptors supports the above premise. However, shortterm immunoneutralisation of endogenous prolactin did not significantly alter the specific binding of [ 125 I ]-labelled lutropin to testicular membranes. Interestingly, during development, a close correction exists between receptor occupancy and capacity of the tissue to bind labelled lutropin. The apparent dissociation between serum lutropin levels, on the one hand and tissue occupancy and free receptor contents on the other, suggests that factors other than lutropin (presumably prolactin) are involved in the modulation of the sensitivity and the responsiveness of the testis to lutropin during early development.