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Monoclonal antibodies have attracted wide attention in therapeutics owing to their high efficacy, low toxicity, and specific targeting. However, antibodies cannot cross the cell membrane barrier. Therefore, their therapeutic potential is limited to surface-exposed antigens or secreted proteins. In the present investigation, we have developed a chimeric virus-like particle (VLP) of pepper vein banding virus (PVBV) and explored the possibility of using it as a delivery vehicle for antibodies against intracellular antigens as well as for future applications in immunodiagnostics. The chimeric PVBV particles were generated by genetically engineering the B domain of Staphylococcus aureus protein A (SpA) at the N-terminus of the PVBV coat protein (CP). The chimeric VLPs purified by sucrose density gradient centrifugation had ~440-fold higher affinity towards IgG antibody when compared to SpA. Interestingly, the unassembled chimeric CP with the B-domain at the N-terminus (BCP) purified by Ni-NTA chromatography was a monomer, and it had ~45-fold higher affinity towards antibodies compared to SpA. Additionally, the chimeric particles were able to bind and deliver antibodies against both intracellular (α-tubulin) and surface-exposed antigens (CD 20). However, the BCP monomer failed to enter mammalian cells. Thus, for the first time, we have demonstrated that the assembled VLPs are essential for internalization. These results demonstrate the potential of the use of chimeric PVBV VLPs in diagnostics and, more importantly, as nanocarriers for intracellular delivery of antibodies.

The capsids of viruses have a high degree of symmetry. Therefore, virus nanoparticles (VNPs) can be programmed to display many imaging agents precisely. Plant VNPs are biocompatible, biodegradable and non-infectious to mammals. We have carried out bioconjugation of sesbania mosaic virus (SeMV), a well characterized plant virus, with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries. Monitoring of cellular internalization of labelled SeMV nanoparticles (NPs) by confocal microscopy and flow cytometry showed that the particles have a natural preference for entry into MDA-MB-231 (breast cancer) cells, although they could also enter various other cell lines. The fluorescence of SeMV NPs labelled via the cysteines with Cy5.5 dye was found to be more stable and was detectable with greater sensitivity than that of particles labelled via the lysines with Alexa Fluor. Live-cell imaging using SeMV internally labelled with Cy5.5 showed that it could bind to MDA-MB-231 cells in less than 5 minutes and enter the cells within 15 minutes. The particles undergo endolysosomal degradation by 6 h as evidenced by their co-localization with LAMP-1. Far-western blot analysis with a HeLa cell membrane protein fraction showed that SeMV interacts with 54-, 35- and 33-kDa proteins, which were identified by mass spectrometry as vimentin, voltage-dependent anion-selective channel protein (VDAC1), and annexin A2 isoform 2 (ANXA2), respectively, suggesting that the particles may bind and enter the cell through these proteins. The results presented here demonstrate that the SeMV NPs provide a new platform technology that could be used to develop in vivo imaging and targeted drug delivery agents for cancer diagnosis and therapy.

Aim: Plant virus-like particles (VLPs) have emerged as a novel platform for delivery of drugs/antibodies. The aim of the present investigation is to establish the entry mechanism of flexuous rod-shaped virus particles into mammalian cells. Methods: Far-Western blot analysis, pull-down and ELISA were used to characterize vimentin and Hsp60 interaction with VLPs. The mode/kinetics of internalization of VLPs was deciphered using pharmacological inhibitors/endosomal markers. Results & discussion: The flexuous rod-shaped VLPs of Pepper vein banding virus (PVBV) enter HeLa and HepG2 cells via cell-surface proteins: vimentin and Hsp60, respectively. VLPs internalize via different modes of endocytosis in HeLa, HepG2 cells and are biodegradable. Vimentin and Hsp60 could be potential epithelial ligands that facilitate targeting of nanoparticles to tumor cells.

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the βH-βI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies–D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

As riboflavin carrier proteins play a critical role in the maintenance of pregnancy, studies on the immunology of these proteins have become very relevant. This paper describes our attempts to identify the sequential antigenic determinants of chicken riboflavin carrier protein (cRCP). Potential surface oriented regions of cRCP were identified by constructing acrophilicity and hydrophilicity profiles of the protein. Of the three regions identified, the pentadecapeptide 10-24 of cRCP forms the subject of the present study. The pentadecapeptide amide was synthesized by solid phase synthesis using Fmoc chemistry. Immunization of rabbits with the peptide-diphtheria toxoid conjugate elicited high titres of the antipeptide antibodies, revealing the high immunogenicity of the peptide. The antipeptide antibodies bound to both cRCP and reduced carboxymethylated RCP (RCM-RCP). Antisera to RCM-RCP showed significant binding to the peptide showing thereby that the region 10-24 of cRCP is perhaps one of the major epitopes of RCM-RCP. Thus, the studies have resulted in the identification of the region 10-24 as an antigenic determinant of cRCP.

The work reported herein describes our attempts to identify peptide immunogens of the beta-subunit of the pregnancy-specific placental hormone, human chorionic gonadotropin (hCG), capable of eliciting hormone specific and neutralizing antisera. Hydrophilicity profiles of the beta-subunits of hCG and the homologous pituitary hormone, human luteinizing hormone (hLH) were compared and two sequences which are hydrophilic but unique to hCG-beta were identified. They are 6-12 and 109-119 of hCG-beta. Results of the studies with the undecapeptide 109-119 of hCG-beta are reported in this paper. The undecapeptide amide was synthesized using the p-(acyloxy) benzhydrylamine resin and antisera to the peptide were elicited in rabbits using the peptide-diphtheria toxoid conjugate. The peptide is highly immunogenic as both the rabbits responded with high titers of antibodies to the peptide. The antipeptide antibodies bound to hCG but not to hLH showing thereby that the region 109-119 of hCG-beta is a unique determinant of hCG. However, the antibodies were found not to neutralize the biological activity of hCG.

Sperm is a highly differentiated cell streamlined for fertilization. The function is thus heavily dependent on the cytoskeletal organization. Conventional methods limit the appreciation and correlation of this intricate cytoskeletal filament network in the context of an entire sperm. Our recent successful localization of nonmuscle myosin IIA on sperm nuclear matrix-intermediate filament (NM-IF) preparations from fertile men by embedment-free electron microscopy (EF-EM), prompted us to investigate the antigenic distribution of two major cytoskeletal proteins—actin and tubulin. The NM-IF preparations were subjected to a cocktail of buffered paraformaldehyde (2%) with a low concentration of glutaraldehyde (0.05%). These proteins were localized by indirect immunogold technique using EF-EM on sperm NM-IF whole mounts. Ultrastructure analysis revealed well preserved centrioles, outer dense fibers, axonemal filaments, and submitochondrial reticulum in the sperm NM-IF. Immunoreactive actin was localized along the length of the sperm whereas β-tubulin was present in the axoneme alone. The spatial distribution of actin and tubulin in normal human sperm NM-IF reported here together with that of myosin on whole mount offers a powerful technique to understand sperm cytoskeletal supramolecular structure. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.

The capsid protein (CP) of Sesbania mosaic virus (SeMV, a T=3 plant virus) consists of a disordered N-terminal R-domain and an ordered S-domain. Removal of the R-domain results in the formation of T=1 particles. In the current study, the R-domain was replaced with unrelated polypeptides of similar lengths: the B-domain of Staphylococcus aureus SpA, and SeMV encoded polypeptides P8 and P10. The chimeric proteins contained T=3 or larger virus-like particles (VLPs) and could not be crystallized. The presence of metal ions during purification resulted in a large number of heterogeneous nucleoprotein complexes. N Delta 65-B (R domain replaced with B domain) could also be purified in a dimeric form. Its crystal structure revealed T=1 particles devoid of metal ions and the B-domain was disordered. However, the B-domain was functional in N Delta 65-B VLPs, suggesting possible biotechnological applications. These studies illustrate the importance of N-terminal residues, metal ions and robustness of the assembly process. (C) 2015 Elsevier Inc. All rights reserved.

The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP). Molecular cDNA cloning of most of the mammalian OGP has been accomplished. The nucleotide and deduced amino acid sequences show a remarkable homology across species and also to chitinase protein. Even though OGP has been shown to interact with gametes and the early embryo, the protein’s direct function has not yet been established. A prerequisite for such studies is the availability of well-characterized protein in bulk. We used recombinant DNA technology to obtain OGP (rOGP). An authentic partial cDNA clone encoding bonnet monkey (Macaca radiata) OGP (accession number AF132 215) was recloned into expression vector pET20b. Overexpression of the protein could be demonstrated after induction with isopropylthio-b-galactopyranoside. Recombinant protein was purified by gel filtration of Escherichia coli lysate through Sephadex G75. The protein migrated with a molecular weight of ;14 kDa on SDS-PAGE. The molecular weight as assessed by matrix-assisted laser adsorption time-of-flight was 14 439 daltons. With Western blot procedures the protein could be immunostained with antibodies to human OGP, baboon OGP, and antipeptide antibodies generated against a well-conserved region of mammalian OGP. The monospecificity of rabbit antibodies generated against rOGP was established by its ability to immunostain human OGP (100‐110 kDa) isolated from hydrosalpinx by Western blot analysis, and the antibody immunostained epithelial cells that secrete OGP in human fallopian tubes. OGP binding sites on the head and tail region of monkey sperm could be demonstrated by using antibody against rOGP.

Purpose:Our purpose was to determine whether lymphocytes synthesize proteins during pregnancy, to observe whether one of the proteins synthesized has early pregnancy factor (EPF)–like activity and to isolate and purify this molecule from culture supernatants obtained from stimulated lymphocytes of pregnant women. Methods:Lymphocyte proliferation assay and35S-methionine labeling were done to study de novo synthesis of proteins followed by autoradiography to confirm synthesis of proteins. The rosette inhibition assay was used for detection of the EPF-like molecule. Gel filtration on Sephadex G-100 and RPHPLC were used for purification of the EPF-like molecule. Results:The rate of incorporation of35S-methionine was significantly higher in the lymphocytes of pregnant women compared to those of the control, and autoradiography confirmed the synthesis of proteins during pregnancy. There is a total protein enhancement trend observed during the first trimester that declines toward term. The EPF-like molecule is observed to be synthesized during all the trimesters of pregnancy. This molecule, when purified, showed a single homogeneous biologically active peak. Conclusions:The results indicated that there is an enhancement of existing protein or synthesis of new proteins during pregnancy. The EPF-like molecule is one of the many proteins synthesized and secreted by lymphocytes during pregnancy that, when purified, is biologically active.

The antigenecity of tryptic fragments of reduced and carboxymethylated chicken riboflavin carrier protein were studied. The tryptic sites of the native riboflavin carrier protein bound to riboflavin were inaccessible. The molecular weight and the elution profile on high performance liquid chromatography (TSK 545 DEAE) were unaltered at an enzyme to substrate ratio of 1:31. However, carboxymethylated riboflavin carrier protein could be cleaved into 3 or 4 fragments at an enzyme to substrate ratio of 1:250 or 1:125. Chromatographic separation of the tryptic fragments on high pressure liquid chromatography (TSK 545 DEAE) revealed the presence of two fragments with different elution profiles but similar molecular weight 26 ±2 kDa. Only one fragment (associated with peak 2) had the ability to displace chicken riboflavin carrier protein in an homologous chicken riboflavin carrier protein radioimmunoassay. Thus, carboxymethylated ribotlavin carrier protein which does not compete with chicken riboflavin carrier protein in the radioimmunoassay, on mild trypsinization generates a fragment which interacts with chicken riboflavin carrier protein in radioimmunoassay.

The oviduct is known to secrete mucins (MUC1 and MUC9) under the influence of ovarian steroids. The secreted form of MUC1 binds gametes in the oviduct, whereas the cellular form seen in breast cancers has been implicated in cell adhesion and morphogenesis. The secreted MUC9 or oviduct-specific glycoprotein (OGP), in addition to being a mucin, belongs to family 18 glycosylhydrolases and is known to bind gametes and embryos in the oviduct. Studies in our laboratory have identified non-muscle myosin IIA (involved in cell shape, polarity, and morphogenesis) as the protein partner to OGP in gametes. In view of the crucial role of the cortical cytoskeleton in the selective internalization of tight junctions (TJs) /adherent junctions (AJs) or apical junctional complex (AJC) in simple epithelial cells during tissue remodeling, the present study has been undertaken to evaluate the existence of a cellular form of OGP in oviductal tissue, which itself undergoes cyclic tissue remodeling. In silico analysis of the deduced amino-acid sequence of OGP has revealed the presence of several conserved motifs; these imply that OGP is a component of multi-protein complexes such as TJs. Corroborative immunoelectron-microscopic analysis in peri-ovulatory oviduct epithelia in the bonnet monkey has revealed the presence of OGP at the TJ. Co-localization studies of OGP and cadherin demonstrate that, whereas OGP is localized at the tonofilaments of the TJs, cadherin is localized at the intercellular space of the AJ. The possible role of OGP in oviductal tissue remodeling is discussed in light of the present findings and those reported in the literature.

The mammalian estrogen induced oviductal glycoprotein (OGP) has been known to associate with capacitated sperm, oocytes and developing embryos. This study aimed to identify the putative binding partner of OGP on gametes using N-terminal peptide of bonnet monkey (Macaca radiata) OGP, Nmon, as bait. A protein(s) of molecular size approximately 54 kDa was detected by far-western blot analysis of detergent solubilized human sperm proteins. MALDI-TOF mass spectra analysis of approximately 54 kDa tryptic peptides gave a significant hit to non-muscle myosin heavy chain. Biochemical characterization of approximately 54 kDa was done with antibodies specific to non-muscle myosin IIA, MYH9. The approximately 54 kDa protein, possible breakdown product of MYH9, immunoreacted with MYH9 antibody in western blot analysis. OGP binding to approximately 54 kDa could also be demonstrated in far-western blot analysis of detergent solubilized human sperm proteins and nuclear matrix intermediate filament (NM-IF) preparations from human sperm and mouse oocytes. Far-western blot analysis of MYH9 enriched by immunoprecipitation identified the native approximately 220 kDa protein as OGP-binding partner. The identical and characteristic immunogold localization pattern of Nmon and MYH9 on sperm NM-IF preparation substantiated these findings. The results suggest that OGP binds to both gametes through its interaction with MYH9 through the non-glycosylated N-terminal conserved region of OGP, spanning the residues 11-137.

Mammalian oviduct is the physiological site for sperm capacitation, gamete fertilization and early embryonic development. The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP) in response to estrogen. The protein has been shown to interact with gametes and early embryo. Several key functions have been postulated particularly its role in pre-implantation events which would have far reaching implications in assisted reproductive technology and in the development of non-hormonal contraceptive vaccine. The intention of this article is to discuss the current status of the protein and analyze how far the postulated function of OGP has been borne out by the available data.

The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP). Molecular cDNA cloning of most of the mammalian OGP has been accomplished. The nucleotide and deduced amino acid sequences show a remarkable homology across species and also to chitinase protein. Even though OGP has been shown to interact with gametes and the early embryo, the protein's direct function has not yet been established. A prerequisite for such studies is the availability of well-characterized protein in bulk. We used recombinant DNA technology to obtain OGP (rOGP). An authentic partial cDNA clone encoding bonnet monkey (Macaca radiata) OGP (accession number AF132 215) was recloned into expression vector pET20b. Overexpression of the protein could be demonstrated after induction with isopropylthio-beta-galactopyranoside. Recombinant protein was purified by gel filtration of Escherichia coli lysate through Sephadex G75. The protein migrated with a molecular weight of approximately 14 kDa on SDS-PAGE. The molecular weight as assessed by matrix-assisted laser adsorption time-of-flight was 14 439 daltons. With Western blot procedures the protein could be immunostained with antibodies to human OGP, baboon OGP, and antipeptide antibodies generated against a well-conserved region of mammalian OGP. The monospecificity of rabbit antibodies generated against rOGP was established by its ability to immunostain human OGP (100-110 kDa) isolated from hydrosalpinx by Western blot analysis, and the antibody immunostained epithelial cells that secrete OGP in human fallopian tubes. OGP binding sites on the head and tail region of monkey sperm could be demonstrated by using antibody against rOGP.

The next generation of contraceptives will be based on the identification of novel molecules essential for reproductive processes and will rely on the refinement of older as well as newer technologies. Functional analysis of naturally occurring reproductive genetic disorders and creation of mice null for specific genes would greatly assist in the choice of genetic targets for contraceptive development. Structure-based design of drugs as exemplified by the preparation of an orally active non-peptide gonadotropin releasing hormone (GnRH) would revolutionize drug formulation and delivery for a peptide analogue. This review examines some of the molecular targets that may change contraceptive choices in the future.

Several monoclonal antibodies (MAbs) specific to chicken riboflavin carrier protein (cRCP) were developed and characterized. Of the several MAbs analyzed, four were directed against nonoverlapping epitopes as demonstrated by MAb inhibition assay. Many of these epitopes appeared to be in close proximity and only three were situated at distinct part of the molecule as revealed by sandwich assay. A combination of chemical modification, peptide cleavage by chemical and enzymatic methods, was used to analyze the possible antigenic structure recognized by these MAbs. An assembled epitope spanning the region 22-87 forms the antigenic site recognized by 4999.1; while MAb 5555.3 interacted with the C-terminal peptide 203-219.

Riboflavin carrier protein is an essential protein required for the growth and development of the embryo and hence for the maintenance of pregnancy. Our efforts to delineate the antigenic determinants of chicken riboflavin carrier protein (cRCP) resulted in the identification of a bioneutralization epitope in the region 10-24 of cRCP. The present work compares the properties of the antibodies raised against the peptide epitope by classical and multiple antigen peptide (MAP) system approaches. The extent of cross-reaction of the antibodies to the MAP construct with the parent protein was found to be significantly less as compared to the antibodies raised against the peptide-diphtheria toxoid conjugate. Furthermore, the bioneutralizing ability of the antisera to the MAP construct was also found to be very poor. The results suggest that there are serious limitations in the ability of antibodies raised against MAP constructs to cross-react with the native proteins.

Progesterone has been implicated to play a critical role in mediating LH induction of ovulation and possibly luteinization. The present study was undertaken to determine the effects of various agonists (LH, FSH, forskolin, and GnRH) known to stimulate ovulation on their abilities to induce progesterone receptor (PR) mRNA and protein in rat preovulatory follicles and in cultured rat granulosa cells exhibiting a preovulatory phenotype. In cultured granulosa cells, PR mRNA was induced by LH in a dose- and time-dependent manner. Transcripts of approximately 11.0, 7.2, 6.8, 6.2, 3.4, and 3.1 kilobases in size were induced by ovulatory (500 ng/ml), but not low (50 ng/ml), concentrations of LH and FSH as well as by forskolin (10 microM), GnRH (1 microM), and phorbol 12-myristate 13-acetate (200 nM). Two forms (A and B) of PR protein were also induced in an agonist- and time-dependent manner, with the shorter form A (mol wt, 83,000-85,000) appearing in greater abundance than the longer form B (mol wt, 115,000). Indirect immunofluorescent analyses verified nuclear localization of the induced receptor. Forskolin and progesterone, but not progesterone alone, were able to activate a glucocorticoid (progesterone) response element-E1b-chloramphenicol acetyltransferase reporter construct (12-fold) after transfection into cultured granulosa cells. The antiprogestins RU486 and ZK98299 did not inhibit induction of PR mRNA and protein, but effectively blocked agonist activation of glucocorticoid response element2-E1b-chloramphenicol acetyltransferase, as well as LH stimulation of luteinization in vitro. These results provide direct evidence that agonists stimulating diverse intracellular pathways can induce PR in granulosa cells, that progesterone plays a functional role in the luteinization process triggered by the LH surge, and that the effects are mediated at least in part by induction of PR.

Two principal physiological events occur within preovulatory follicles as a consequence of the LH/FSH surge: ovulation and luteinization. The ovulation process culminates in the extrusion of the oocyte-cumulus complex and is dependent on a specific sequence of biochemical events. These biochemical changes include the rapid but transient increase in a novel, distinct isoform of prostaglandin endoperoxide synthase (PGS-2) (1–8), tissue plasminogen activator (tPA) (9, 10), and progesterone receptor (PR) (11–13). In contrast, luteinization is a process by which follicular granulosa and theca cells become nonmitotic and establish a specific, stable luteal cell phenotype. The biochemical changes that occur in response to the LH surge and are associated with luteinization include the marked and sustained induction of cholesterol side-chain cleavage cytochrome P450 (P450scc), as well as the dramatic and rapid decreases in mRNA for the LH receptor (LH-R), the regulatory subunit β of type II cyclic AMP-dependent protein kinase (RIIβ), aromatase, and 17α-hydroxylase (P45017α) (Fig. 10.1) (14). Once luteinization has occurred, genes regulated by FSH/LH/cAMP in the follicle are no longer regulated by these agonists in the corpus luteum. This apparent transition from cAMP-dependent regulation to cAMP-independent regulation of these genes has prompted us to ask why surge (as opposed to basal) concentrations of LH are required to initiate these two diverse processes, what might be unique about the genes induced by the LH surge, and what intracellular signaling pathways might be involved.

Luteinizing hormone is known to stimulate the enzyme ornithine decarboxylase in the ovary. Highly purified human follicle stimulating hormone that is devoid of significant biologically active luteinizing hormone can also induce ornithine decarboxylase activity in intact immature rats with a time course of induction similar to that reported for luteinizing hormone. A maximum of 8–10-fold stimulation above controls was observed 4 h following intravenous administration of human follicle stimulating hormone. This stimulation followed a strict dose response relationship. Ovine luteinizing hormone and human chorionic gonadotropin always induced more ovarian ornithine decarboxylase activity than that achieved by maximally effective doses of follicle stimulating hormone. This could not be attributed solely to the ability of specific cell population to respond to the respective gonadotropins. Although granulosa cells contained little receptor for luteinizing hormone/human chorionic gonadotropin and the residual tissue contained little receptor for follicle stimulating hormone, each tissue responded to these gonadotropins in a manner suggestive of the mediation by one or more diffusable factors. A relationship between gonadotropin induced 3’5’-cyclic adenosine monophosphate (cyclic adenosine monophosphate) concentration and ornithine decarboxylase activity suggests that the mediation of gonadotropin stimulated ovarian ornithine decarboxylase is not solely through cyclic adenosine monophosphate, indicating the presence of other factors in the induction of gonadotropin increased ornithine decarboxylase activity.

The importance of riboflavin-carrier protein (RCP) in the maintenance of pregnancy in mice has been studied. Selective passive immunoneutralization of the maternal RCP resulted in fetal death and resorption. Six hours after chicken RCP antiserum treatment, the following observations were made: there was profuse vaginal bleeding in all the animals, a 60% reduction in embryonic ornithine decarboxylase (ODC) activity, a 70% reduction in the maternal progesterone levels, and a 50% reduction in the 14C-riboflavin uptake by the embryo. The above observations are indicative of fetal distress and resorption. By 24 h after treatment, there was 100% resorption of fetuses and the mouse progesterone levels dropped to 20% of untreated or normal rabbit serum (NRS)-treated values. Cytological studies of the fetal liver revealed the classical signs of cellular degeneration in hepatocytes as well as hematopoietic cells. The effect was apparent as early as 1 h after antiserum administration. The erythroid aplasia supports the biochemical evidence that fetal demise is due to preferential riboflavin deficiency of the fetus.

The effect of seminal plasma inhibin on ornithine decarboxylase activity (ODC) in the rat prostate was studied. A single bolus injection of seminal plasma inhibin caused a 3- to 4-fold increase in ODC activity within 2 h whereas testicular ODC was unchanged. The ODC response to seminal plasma inhibin was neutralized by specific antibodies generated against the inhibin preparation. Another peptide, thyroid releasing hormone has no effect on prostatic ODC activity, but it blocked the increase in enzyme activity induced by treatment with seminal plasma inhibin.

Day 12 pregnant mice administered antibodies to cRCP exhibited riboflavin deprival, which resulted in progressive alteration in the fetal hepatic cell structure, which eventually led to fetal wastage and termination of pregnancy. These changes were evident as early as 1 h following antiserum treatment. Three hours following treatment, other degenerative changes such as disorganization of glycogen particles and endoplasmic reticulum and degranulation of mitochondria were observed. Nuclear pycnosis, and increased myelin figures, accumulation of lysosomes, all indicative of autolytic changes, occurred 6 h following treatment.

The role of gonadotropins and estrogen on the regulation of ovarian ornithine decarboxylase was studied during follicular differentiation/maturation. In intact immature rats follicular differentiation/maturation was initiated with sequential administration of estrogen and follicle stimulating hormone. Ornithine decarboxylase activity in response to human chorionic gonadotropin was markedly enhanced (2-fold) in rats with preovulatory antral follicles when compared to rats with non-ovulatory follicles. This increase could be attributed to the alteration in the turnover of the enzyme. Following follicle maturation the half life of the human chorionic gonadotropin stimulated ornithine decarboxylase was increased from 18 to 62 min. This increase in half life was associated with differentition of follicles. In the estrogen treated group (which does not induce follicular differentiation), the half life of the enzyme remained unaltered. The regulation of ornithine decarboxylase through the formation of protein inhibitor antizyme induced by diamino hexane, was unaltered during follicular differentiation.

The properties of antibodies generated in rabbits against native riboflavin carrier protein (cRCP), riboflavin carrier protein that had been denatured/renatured by SDS treatment (SDS-RCP) or disulphide-bond-reduced then S-carboxymethylated (Carb-RCP) were studied. SDS-RCP could displace native RCP in radioimmunoassay (r.i.a.), whereas Carb-RCP could not. By using antibodies raised in five different rabbits against native cRCP, 125I-labelled Carb-RCP could bind between 0 and 30% of the native antibodies. Antibodies raised against native RCP appear to be largely directed towards specific conformational determinants of RCP. Carb-RCP displaced native RCP in an r.i.a. using antibodies raised against SDS-RCP. SDS denaturation presumably unmasks cryptic epitopes in native RCP. Carb-RCP was a weak immunogen and elicited, presumably, antibodies to sequential epitope/epitopes. When injected into pregnant mice the antibodies caused neutralization of RCP, leading to termination of pregnancy, indicating highly conserved sequential epitopes in chicken and rodent RCP. Antibodies raised against Carb-RCP or native RCP reacted with CNBr fragments of native RCP, further confirming the presence of sequence-specific antibodies elicited by Carb-RCP.

The presence of protein(s) which is immunologically and functionally similar to the well characterised avian riboflavin carrier protein has been demonstrated in female common marmosets during pregnancy. The protein(s) that interacts with [ 14 C]riboflavin has an immunological homology to chicken riboflavin carrier protein. The termination of pregnancy in four out of six marmosets following administration of antiserum to chicken riboflavin carrier protein supports the hypthesis that vitamin carrier protein ensures an uninterrupted supply of the vitamin to the growing embryo and that immunoneutralization of maternal riboflavin carrier protein leads to fetal death and termination of pregnancy.