Biomedical subjects

P R Adiga

Publications and source records attributed to P R Adiga.

At least 19 recordsLinked to original sources

PubMed2001

Riboflavin carrier protein: a serum and tissue marker for breast carcinoma.

We have earlier shown that the estrogen-modulated riboflavin carrier protein (RCP) first isolated from the chicken egg is evolutionarily conserved in mammals and is elaborated by lactating mammary gland as demonstrated with rat mammary epithelial cells in culture and confirmed by isolation of the vitamin carrier from bovine milk. In view of several earlier reports that many milk proteins as well as other estrogen-inducible proteins are up-regulated and secreted into circulation in animal models and in women with neoplastic breast disease, we analyzed serum RCP levels in a double-blind study using a specific radioimmunoassay in pre- and post-menopausal women with clinically diagnosed breast cancer at early and advanced stages of the disease and compared these levels with those in normal age-matched control volunteers. Our data reveal that the serum RCP levels in cycling breast cancer patients are 3- to 4-fold higher (p < 0.01) than those in their normal counterparts. This difference in circulatory RCP levels between cancer patients and their age-matched normal counterparts is further magnified to 9- to 11-fold (p < 0.005) at the post-menopausal stage. In addition, there seems to be a good correlation between rising RCP levels and disease progression, since significantly higher RCP concentrations (p < 0.005) are encountered in patients with advanced metastasizing breast cancer versus those with early disease. Using specific monoclonal antibodies, RCP could be localized immunohistochemically in the cytoplasm of invading neoplastic cells of lobular and ductal carcinomas of the breast, indicating that the malignant cells are probably the source of the elevated serum RCP levels in breast cancer. These findings suggest that measurement of circulatory RCP and the immunohistochemical staining pattern of RCP in biopsy specimens could be exploited as an additional marker in diagnosis/prognosis of breast cancer in women.

Animals↗

PubMed2001

Helix stabilization in the C-terminal peptide of chicken riboflavin carrier protein enhances immunogenicity and prolongs contraceptive potential as an epitope-based vaccine in female rats.

Earlier investigations have shown that (a) antibodies against a carrier-coupled 20-residue synthetic peptide (C-20), (200)HACQKKLLKFEALQQEEGEE(219), corresponding to the C-terminal partially helical sequence of chicken riboflavin carrier protein (RCP; 219 AA) curtail pregnancy in mammals and (b) helix stabilization by introducing appropriately spaced salt bridges in the flanking sequences of its B-cell epitopic structure enhances RCP antigenicity to peptide antibodies. Among such engineered C-20 analogs, HE-20 (HAEQKKLLKFEALEQEKGKE) exhibited maximum helical propensity. Since C-20 per se, i.e., without carrier conjugation, elicits RCP-reactive neutralizing antibodies in rodents, we mapped its T-cell epitope which overlaps its B-cell epitope, both of which remain unmodified in HE-20. Comparative evaluation of immunogenicity of the two epitope-based peptide vaccines showed that HE-20 was far superior to C-20 in generating RCP-reactive antibodies in terms of both affinity and titer. With regard to bioefficacy, passive immunoneutralization of RCP in pregnant rats by administering purified IgG from either of the antipeptide sera terminated pregnancy. Similarly, active immunization of fertile female rats with the individual peptide analogs curtailed pregnancy. However, HE-20 was more efficient in eliciting higher affinity, longer-lasting, RCP-crossreactive antibodies with consequently more prolonged immunocontraceptive efficacy.

Amino Acid Sequence↗

PubMed2000

Immunocontraceptive potential of major antigenic determinants of chicken riboflavin carrier protein in the female rat.

Earlier investigations have demonstrated that antibodies generated against the N-terminal (10-17) and C-terminal (200-207) ends of chicken riboflavin carrier protein (RCP; 219 AA), but not towards the internal region (172-179), curtail pregnancy establishment in rodents and sub-human primates. In those studies, epitope peptides conjugated to diphtheria toxoid were used as immunogens. In the present study, linkage of these sequences to extraneous carriers was avoided to rule out the possibility of carrier-mediated suppression of hapten-specific antibody production in long-term immune response. The ability of these three free peptides to function as minivaccines was examined and the functional importance of these sequences in pregnancy establishment in rodents were evaluated. The results obtained reveal that the peptides YGC (residues 3-24) and HAC (residues 200-219) serve as immunocontraceptive vaccines.

Amino Acid Sequence↗

PubMed2000

Immunocontraceptive efficacy of synthetic peptides corresponding to major antigenic determinants of chicken riboflavin carrier protein in the female rats.

PROBLEM: Earlier studies have demonstrated that antibodies directed towards the N-terminal (residues 10-17) and C-terminal (residues 200-207) regions on chicken riboflavin carrier protein (RCP; 219 AA) are effective in pregnancy termination in rodents and sub-human primates. In the present study, the immunocontraceptive potential of three additional immunodominant sequences comprising of residues 33-49, 64 83 and 130-147 (CYA, CED and CGE peptides, respectively) of chicken RCP was investigated. METHOD OF STUDY: The three antigenic peptides were synthesized by using Fmoc chemistry. Oligoclonal antibodies were generated in rabbits. Bioneutralizing capacity of these peptides was assessed by passive and active immunoneutralization studies. RESULTS: All the three peptides-specific antisera recognized their cognate epitopes on native RCP. When the affinity purified peptide IgG were administered on three consecutive days to pregnant rats (on days 10, 11 and 12), it was observed that the rats injected with CED and CGE-IgG failed to deliver any pups whereas the animals which received CYA IgG delivered normal pups. Active immunization of fertile female rats with CED or CGE peptide conferred protection from pregnancy. CONCLUSIONS: These results demonstrate the presence of two additional stretches in chicken RCP which can serve as mini-vaccines.

Amino Acid Sequence↗

PubMed2000

Strain-dependent variability in immune response to chicken riboflavin carrier protein in mice with different haplotypes.

Active immunization of fertile female mice, rats and sub-human primates with linearized chicken riboflavin carrier protein (RCP) curtailed pregnancy suggesting that sequence-specific RCP antibodies interfere with fertilization/early embryo development. To investigate the genetic basis of variations in immunogenecity, antibody response to reduced and carboxymethylated RCP (RCM-RCP) was studied in different strains of mice of independent H-2 haplotypes. Among these, AKR (H-2k) were low or non-responders. Measurement of antibody titers in hyperimmune sera showed that among responder strains of mice, C57BL/6 (H-2b) and BALB/c (H-2d) generated higher levels of antibody compared to mice of SJLJ (H-2S). The relative affinities of these antibodies also varied depending upon the strain, with BALB/c mice showing highest affinity. Epitope mapping by pepscan ELISA revealed significant variability in determinant-specific antibody populations, with SJLJ strain lacking antibodies to N-terminal half of RCP sequence. However, four immunodominant sequential epitopes (residues 100-107, 134-141, 174-181 and 200-207) common to all the three strains of mice have been identified. Binding to these regions was not haplotype restricted although there were qualitative differences in recognition patterns. Present investigations have shown that site-specific antibodies directed towards any one of the four epitopic regions comprising of residues 3-24, 64-83, 130-147 and 200-219 in chicken RCP sequence effectively interfered with pregnancy establishment in female BALB/c mice. This implies the propensity of RCP antibodies to curtail pregnancy in the other two responder mouse strains also.

Amino Acid Sequence↗

PubMed2000

Active immunization against riboflavin carrier protein results in peri-implantation embryonic loss leading to pregnancy termination in rats: use of alternate adjuvants.

To 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.

Abortifacient Agents↗

PubMed1999

Immunological relatedness of chicken and human riboflavin carrier protein.

Polyclonal antibodies (pAbs) to riboflavin carrier protein (RCP) purified from human amniotic fluid were raised in two rabbits. These pAbs apart from recognizing the parent antigen, reacted significantly with chicken RCP as well. When all the 212 octapeptides overlapping by one amino acid residue and covering the entire primary amino acid sequence of chicken RCP immobilized on polyethylene rods were probed with rabbit anti-human RCP antibodies, it was found that these antibodies recognize five epitopic sequences (residues 37-42, 73-76, 133-140, 174-176 and 200-207) in chicken RCP primary structure common to both the rabbits. In addition, the antisera from the two individual animals also recognized additional epitopes characteristic to that particular animal. The reactivities of these antigenic determinants have been confirmed with polyclonal anti-human RCP sera by using synthetic peptides harbouring these chicken RCP sequences in inhibition ELISA. This high level of immunological similarities among linear epitopes observed may reflect the degree of conservation of local conformations among linear antigenic determinants shared by the two evolutionarily distant homologous proteins.

Amino Acid Sequence↗

PubMed1999

Characterization and hormonal modulation of immunoreactive thiamin carrier protein in immature rat Sertoli cells in culture.

Immature rat Sertoli cells synthesize and secrete a protein species which has immunological similarity with chicken egg thiamin carrier protein (TCP) as assessed by immunocytochemical localization, liquid phase radioimmunoassay (RIA), immunoprecipitation of [35S]-methionine incorporated newly synthesized proteins by polyclonal antibodies (pAbs) to chicken TCP and tryptic peptide mapping of iodinated immunoprecipitated proteins. FSH and testosterone together bring about 4-fold induction of Sertoli cell TCP over the control levels which is inhibitable upto 75% by an aromatase inhibitor. Addition of optimal concentrations of exogenous estradiol-17beta to the cultures causes 2-fold enhancement of secretion of TCP which can significantly be inhibited by tamoxifen, when added along with estradiol-17beta. These results show that Sertoli cells produce estrogen-inducible TCP, presumably to transport the vitamin to the developing germ cells.

Animals↗

PubMed1999

Characterization and hormonal modulation of immunoreactive thiamin carrier protein secreted by adult rat Leydig cells in vitro.

Leydig cells isolated from adult rats and maintained under defined conditions in culture secrete a protein of molecular weight (Mr) 70 000 which is immunologically similar to chicken thiamin carrier protein (TCP). Synthesis of immunoreactive TCP by these cells is demonstrated by immunoprecipitation of [35S]methionine incorporated, newly synthesized proteins with monoclonal and polyclonal antibodies to chicken TCP. The amount of immunoreactive TCP secreted into the culture supernatant is quantitated by using a specific radioimmunoassay. Under the influence of LH, secretion of immunoreactive TCP is enhanced 3-fold and can be inhibited by up to 70% with aromatase inhibitor (1,4,6-androstatrien-3,17-dione). Cyclic AMP acts as a second messenger in the sequence of events involved in LH-induced elevation of immunoreactive TCP in Leydig cells. The effects of exogenous estradiol-17beta and diethylstilbestrol are comparable in terms of stimulation of secretion of immunoreactive TCP by these cells. Tamoxifen brought about a 70% decrease in the elevated levels of immunoreactive TCP. These results suggest that estrogen mediates immunoreactive TCP induction in hormonally stimulated adult rat Leydig cells.

Animals↗

PubMed1998

Identification and mapping of linear antigenic determinants of chicken riboflavin carrier protein.

All of the possible 212 overlapping octapeptides covering the amino acid sequence of chicken egg white riboflavin carrier protein (RCP) was synthesized on polyethylene rods. To identify the peptides constituting the sequential antigenic epitopes on chicken RCP, a PEPSCAN ELISA was employed where antibodies raised against SDS-treated, reduced and carboxymethylated RCP (RCM-RCP) in mice, rats (n = 4), rabbits and monkeys (n = 3) were used. By this approach, we could identify five sequences on chicken RCP commonly recognized by all the antisera from these species which were spanning the residues 10-17, 42-49, 134-141, 172-179 and 200-207. Analysis of the data for each individual antiserum indicated inter- and intra-species variations with N- and C-terminal shifts in peak reactivity. The present study offers clues for development of potential immunocontraceptive vaccines.

Amino Acid Sequence↗

PubMed1998

Refolding of native and recombinant chicken riboflavin carrier (or binding) protein: evidence for the formation of non-native intermediates during the generation of active protein.

Riboflavin carrier (or binding) protein (RCP) is a phosphoglycoprotein originally purified from the egg white, yolk and serum of laying hens. The 18 cysteine residues present in RCP form nine disulfide bridges, allowing the protein to form a compact structure to generate a hydrophobic pocket in which riboflavin sits. We studied the refolding of totally reduced and denatured egg white RCP and found that the protein initially folded to generate a molecule that did not possess riboflavin-binding activity, despite near-complete oxidation of the cysteine residues. Riboflavin-binding activity was then slowly regained, but the final refolded form of the protein was less compact in structure than the native molecule, due to incomplete oxidation of all the cysteine residues. Denatured and reduced dephosphorylated RCP refolded as efficiently as the native protein, with similar rates of disulfide-bond oxidation and generation of riboflavin binding, showing that the phosphoserine stretch of RCP has little role to play during refolding. In order to study the role of glycosylation in the refolding process, the cDNA for full-length RCP was expressed in Escherichia coli and purified. Recombinant RCP refolded only in the presence of redox buffers, demonstrating that glycosylation of RCP could allow the formation of high yields of productive intermediates in the folding pathway. Using a panel of conformation-specific monoclonal antibodies to RCP, it appeared that the folding intermediates of RCP possessed a structure distinctly different to the native protein, indicating that the correct folding pathway of RCP passed through conformation(s) generated by non-native disulfide bridges.

Animals↗

PubMed1998

Identification and characterization of receptors for riboflavin carrier protein in the chicken oocyte. Role of the phosphopeptide in mediating receptor interaction.

Riboflavin carrier protein (RCP) is a phosphoglycoprotein found in the egg and the serum of laying birds and other animals. We have investigated the binding of chicken RCP (cRCP) to membranes prepared from the whole chicken oocytes. RCP binding had an absolute requirement for calcium, with an affinity (Kd 10(-8) M) high enough to be physiologically relevant. Ligand blotting experiments using labeled RCP and vitellogenin, with proteins solubilized from oocyte membranes, indicated that RCP and vitellogenin bound specifically to three proteins of Mr 380, 260 and 110 kDa. Vitellogenin also bound to proteins of Mr 515 kDa and 97 kDa, similar in size to those identified by receptor associated protein of RAP. Reduced and carboxyamidated RCP inhibited the binding of 125I-labeled RCP to chicken oocyte membranes, but recombinant RCP expressed in E. coli, and dephosphorylated RCP, failed to interact with the receptors, indicating that post-translational modifications were necessary for ligand-receptor interaction. The purified phosphopeptide, prepared from tryptic digests of egg white RCP, was able to inhibit the binding of RCP to the receptor proteins, with an affinity comparable to native RCP indicating that the phosphopeptide sequence present in RCP serves as the focal point for RCP-receptor interactions.

Amino Acid Sequence↗

PubMed1998

Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone.

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CalphaH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides.

Acetone↗

PubMed1998

A comparison of two dissimilar monoclonal antibodies that are directed to a common epitope in the reduced and carboxymethylated chicken riboflavin carrier protein.

Two monoclonal antibodies, D2A1 and D5G3 were elicited by immunization with a preparation of chicken egg riboflavin carrier protein which had been reduced and carboxymethylated. Epitopes recognised by the monoclonal antibodies were mapped using the Pepscan method. Epitopic determinants for D2A1 as well as D5G3 were identified within a region spanning 13 amino acids (residues 170-182) in the primary sequence of riboflavin carrier protein. Interestingly, these monoclonal antibodies, despite sharing a common epitope exhibited a marked difference in their binding to native (folded) riboflavin carrier protein versus reduced carboxymethylated (unfolded) riboflavin carrier protein. Both monoclonal antibodies bound reduced carboxymethylated riboflavin carrier protein to comparable extents in solid phase (ELISA and immunoblots) and liquid phase (radioimmunoassay) assays. However, while D5G3 could bind native riboflavin carrier protein in solid and liquid phase assays, D2A1 showed negligible binding to the native structure. Alanine substituted peptide analogs of the epitope in question defined the crucial amino acids of the epitope needed for binding to the two antibodies.

Amino Acid Sequence↗

PubMed1997

A monoclonal antibody to avidin dissociates quaternary structure and curtails biotin binding to avidin and streptavidin.

An anti-avidin mAb, viz., H12G4, is shown to release bound biotin in a dose-dependent manner from holoavidin and holostreptavidin and inhibit the binding of ligand to the two apoproteins. The release of biotin by this mAb is accompanied by quenching of ligand-induced enhanced fluorescence of the FITC-avidin conjugate. In terms of mechanism of release of bound biotin, we demonstrate that on binding to the Fab fragment of the mAb, the native tetrameric holoavidin undergoes dissociation progressively with time to monomers with no bound biotin associated with the latter. Based on the immunoreactivity associated with defined overlapping fragments of avidin obtained by chemical cleavage, the epitope recognized by mAb H12G4 has been localized to residues 58-96 of the primary sequence. By pepscan method of epitope mapping, this mAb is shown to identify a minimal core sequence of 87RNGK90 in avidin and a corresponding sequence of 84RNAH87 in streptavidin.

Amino Acid Sequence↗

PubMed1997

Mapping the common antigenic determinants in avidin and streptavidin.

An epitope scan analysis of the whole sequence of avidin and core streptavidin using polyclonal antibodies to these two antigens reveal the presence of multiple common epitopes in both the proteins. These antigenic determinants consist mostly of either identical or similar residues. The antibody recognition sites in both antigens are shown to be localized to homologous regions.

Amino Acid Sequence↗

PubMed1996

Hormonal modulation of riboflavin carrier protein secretion by immature rat Sertoli cells in culture.

We report here that a protein species with biochemical and immunological similarity with chicken egg riboflavin carrier protein (RCP) is synthesized and secreted by immature rat Sertoli cells in culture. When quantitated by a specific heterologous radioimmunoassay, optimal concentrations of FSH (25 ng/ml) brought about 3-fold stimulation of RCP secretion. FSH, in the presence of testosterone (10(-6) M) brought about 6-fold stimulation of secretion of RCP over the control cultures which were maintained in the absence of these two factors. The aromatase inhibitor (1,4,6-androstatrien-3,17-dione) curtailed 85% of the enhanced secretion of RCP, suggesting that the hormonal stimulation is mediated through in situ synthesized estrogen and this could be confirmed with exogenous estradiol-17 beta which brought about 3-fold enhancement of secretion of RCP at a concentration of 10(-6) M. When tamoxifen (10 microM) was added along with FSH and testosterone, there was 75% decrease in the enhanced secretion of RCP. Addition of this anti-estrogen together with exogenous estradiol resulted in 55% decrease in elevated levels of RCP. Cholera toxin (1 microgram/ml) and 8-bromo-cyclic AMP (0.5 mM) mimicked the action of FSH on the secretion of RCP thus suggesting that FSH stimulation of RCP production may be mediated through cyclic AMP. These findings suggest that estrogen mediates RCP induction in hormonally stimulated Sertoli cells presumably to function as the carrier of riboflavin to the developing germ cells through blood-testis barrier in rodents.

Animals↗

PubMed1996

Refolding of riboflavin carrier protein as probed by biochemical and immunological parameters.

The unfolding of the chicken egg white riboflavin carrier protein by disulfide reduction with dithiothreitol led to aggregation with concomitant loss of ligand binding characteristics and the capacity to interact with six monoclonal antibodies directed against surface-exposed discontinuous epitopes. The reduced protein could, however, bind to a monoclonal antibody recognizing sequential epitope. Under optimal conditions of protein refolding, the vitamin carrier protein regained its folded structure with high efficiency with simultaneous complete restoration of hydrophobic flavin binding site as well as the epitopic conformations exposed at the surface in a manner comparable to its native form.

Animals↗