In vitro safety evaluation and anticlastogenic effect of BacoMind on human lymphocytes.

OBJECTIVE: BacoMind (BM) is a standardized extract of Bacopa monnieri, which belongs to the family Scrophulariaceae and is a creeping annual plant found throughout the Indian subcontinent. It has been used by Ayurvedic medicinal practitioners in India for almost 3000 years and is classified as a medharasayana, a substance which improves memory and intellect. With the widespread traditional use as well as scientific validation of Bacopa monnieri for nootropic activity, a bioactive-rich unique phytochemical composition-BacoMind was developed from B. monnieri for use as a cognition and memory enhancing agent. The present study aimed to investigate the in vitro toxicity of this formulation of BacoMind on human lymphocytes and to rule out its possible contribution to mutagenicity. METHODS: In the present investigation the active ingredients present in BM were identified and quantified by high performance liquid chromatography (HPLC) and high performance thin-layer chromatography (HPTLC). Antioxidant and anticlastogenic properties of BM were studied in vitro with and without metabolic activation. Doses of BM were chosen on the basis of mitotic index (MI) and cytokinesis-block proliferation index (CBPI). Clastogenicity assays were performed at 31.2 microg/mL, 62.5 microg/mL, and 125 microg/mL, while the Salmonella reverse mutation assay (Ames test) was performed at doses of 61.72, 185.18, 555.55, 1666.67, and 5000.00 microg/plate. RESULTS: HPLC and HPTLC analysis of BM revealed the presence of bacoside A3, bacopaside I, bacopaside II, jujubogenin isomer of bacopasaponin C, bacosine, luteolin, apigenin, bacosine, and beta-sitosterol D glucoside. BM demonstrated significant antioxidant activity. The number of chromosomal aberrations and the frequency of micronuclei induced by BM were not statistically significant up to a dose of 62.5 microg/mL. A subsequent dose of 125 microg/mL prior to metabolic activation induced mild clastogenicity, but it was found to be biologically insignificant as this effect was not seen post metabolic activation. BM also demonstrated a dose-dependent protection against the clastogens used in this study using the above tests for clastogenicity. Maximum protection was observed in presence of metabolic activation. Moreover, BM demonstrated no mutagenic effect on the tested strains, as observed in the Ames test. CONCLUSION: BM protected human lymphocytes against various clastogens. BM also exhibited high antioxidant activity which might be responsible for the observed protective effects against the clastogens since the used clastogens are known to induce their clastogenic effects via production of oxidative radicals.

Identification of a heterodimer-specific epitope present in human chorionic gonadotrophin (hCG) using a monoclonal antibody that can distinguish between hCG and human LH.

Human chorionic gonadotrophin (hCG) is secreted during early pregnancy and is required for implantation and maintenance of the pregnancy. Active or passive immunoneutralization of hCG results in termination of pregnancy and this forms the basis of the hCG-based female contraceptive vaccine. However, the beta subunit of hCG possesses 85% sequence homology with the first 114 amino acids of the beta subunit of pituitary human LH (hLH), which is required for ovulation and maintenance of the corpus luteum function during the menstrual cycle. Immunization against hCG or its beta subunit leads to generation of antibodies that can neutralize hLH due to many shared epitopes and hence may cause abnormal menstrual cycles. Therefore, it is essential to identify epitopes that are different in the two hormones. In the present study, we report a monoclonal antibody (MAb) specific for hCG that shows no binding to the isolated subunits. Interestingly, the MAb also does not bind hLH at all. The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCG beta that is absent in hLH beta. The MAb, either as IgG or its recombinant single-chain variable region fragment, inhibited the response to hCG, but not to hLH. Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception.

Identification of the structural and functional determinants of the extracellular domain of the human follicle stimulating hormone receptor.

The extracellular domain (ECD) of the human follicle-stimulating hormone receptor (hFSHR) is believed to be the major determinant of hormone selectivity. Different discrete, discontinuous regions on the ECD of the hFSHR have been suggested to be crucial for hormone binding. However, the role of the ECD in signal transduction is not well understood. This study provides some insight into these aspects of the structure-function relationship of the ECD of hFSHR. Ten peptides were selected from the ECD on the basis of their ability to be surface oriented, synthesized by the solid-phase method using fluorenylmethyloxycarbonyl chemistry, purified and characterized. They were further studied for their ability to modulate both human follicle-stimulating hormone (hFSH)-FSHR binding and cAMP generation. Competitive inhibition studies showed that, of all the peptides studied, peptides 285-300 and 297-310 hFSHR were able to inhibit hFSH binding to FSHR. Both peptides function as weak competitive inhibitors of hFSH-FSHR binding. Peptides 285-300 hFSHR, 216-235 hFSHR, 184-195 hFSHR, 79-89 hFSHR and 15-31 hFSHR were observed to inhibit FSH-induced cAMP production. In summary, this study suggests that discrete, functional domains of the ECD have a role in hormone binding and signal transduction. Region 285-300 has been identified as a novel region crucial for both FSH binding and cAMP generation.

Developmental and hormonal regulation of type II DNA topoisomerase in rat testis.

Type II DNA topoisomerase (topo II) is required for diverse biological functions including DNA replication, maintenance of genome stability, chromosome segregation and chromosome condensation. While the identity of topo II in rodent testis has been established, the regulation of topo II expression during the development of the postnatal testis and gametogenesis is unclear. Here, we report that rat testis topo II is developmentally and hormonally regulated. Topo IIalpha mRNA levels peaked prior to the onset of puberty, declined sharply thereafter and stabilized in adult testis. In contrast, the topo II enzyme content was lower in prepubertal testis but increased after the onset of puberty. Topo II was expressed in a cell-specific manner within germ cells, being detected only in pachytene spermatocytes. While testosterone markedly increased topo IIalpha mRNA levels in prepubertal testis, continued treatment failed to enhance topo IIalpha mRNA above postpubertal control levels. The extent of topo II activity remained steady regardless of the testosterone-induced increase in topo IIalpha mRNA levels. Inhibition of testosterone function in postpubertal animals by ethanedimethane sulphonate (EDS) and flutamide resulted in a significant decrease in topo IIalpha gene expression and topo II activity. The administration of exogenous testosterone (T) to EDS- and flutamide-treated rats restored topo IIalpha mRNA levels and topo II activity similar to the levels seen in the testis of age-matched control animals. Histochemical analyses of testes indicated that the effect of T on spermatogenesis was separable from its effect on topo IIalpha expression. Our results reveal that testosterone acts as a positive regulator of topo IIalpha gene expression and is required for the maintenance of topo IIalpha expression during the development of the postnatal testis and spermatogenesis.

Biological activity of single chain chorionic gonadotropin, hCGalphabeta, is decreased upon deletion of five carboxyl terminal amino acids of the alpha subunit without affecting its receptor binding.

The strategy of translationally fusing the two subunits of human chorionic gonadotropin (hCG) has been used to produce recombinant single chain hCG in which the C-terminus of the alpha subunit is fused to the N-terminus beta without any linker using Pichia pastoris expression system. The Pichia-expressed hCGalphabeta (phCGalphabeta) attained an overall conformation similar to that of hCG, and could bind to the receptor and elicit biological response, suggesting that receptor binding and signal transduction can take place even with a molecule having blocked the C-terminus of the alpha subunit. The carboxyl terminal of the alpha subunit has been shown to be involved in hormone binding and signal transduction of all the heterodimeric glycoprotein hormones. However, deletion of five amino acids from the C-terminus of the alpha subunit in the single chain hCG did not alter the overall conformation of the fusion molecule and its receptor binding ability, but led to a significant reduction in its ability to elicit biological response. These data show that these five amino acids at the C-terminus of the alpha subunit in the single chain hCG are not absolutely essential for attaining a conformation required for receptor binding, but are essential for obtaining a full biological response.

Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris.

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hCG. The level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermentor. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hCG and is suitable for structure-function studies of the hormone.

Identification of an attenuating region in the bovine follicle-stimulating hormone beta subunit mRNA that decreases its expression in E. coli.

Follicle-stimulating hormone (FSH) is one of the key regulators of gonadal function in mammals. Recombinant DNA expression of this hormone has proved to be a difficult task as expression levels are invariably low, irrespective of the expression system employed. In the present study, we have attempted to identify reasons for this low expression using bacterial expression vectors, and we report here the identification of a theoretically predicted hairpin structure in the mRNA corresponding to the N-terminal portion of the mature coding portion of bFSHbeta cDNA that is responsible for attenuating its expression in E. coli. When full-length FSHbeta was expressed using the bacterial expression vector, a very low expression was obtained. However, when fragments of FSHbeta with N-terminal deletions (amino acids 24-110 and 13-110) were expressed using the same expression strategy, a 30- to 40-fold higher expression was observed. This low expression of FSHbeta could be attributed to a hairpin structure present in the first 12 codons of mature FSHbeta mRNA. Disruption of this structure without changing the amino acid sequence resulted in a higher level of expression of FSHbeta. The predicted hairpin structure, though away from the transcriptional and translational start site, was able to downregulate the expression of FSHbeta probably by impeding the movement of ribosomes.

Expression of biologically active beta subunit of bovine follicle-stimulating hormone in the methylotrophic yeast Pichia pastoris.

Follicle-stimulating hormone (FSH), a pituitary gonadotropin, is a heterodimer composed of an alpha subunit, which is common to all the glycoprotein hormones, noncovalently associated with the hormone-specific beta subunit. The objective of the present study is to develop a recombinant DNA expression system for the beta subunit of FSH that can be applied to study structure-function relationships while producing large quantities of the hormone subunit for immuno-contraceptive, clinical, and veterinary purposes. We report here the expression of biologically active bovine FSH beta (bFSH beta) in the methylotrophic yeast Pichia pastoris. The Pichia-expressed FSH beta (pFSH beta) was secreted into the culture medium and was found to be immunologically very similar to pituitary-derived ovine FSH beta. Replacement of cognate signal peptide with the yeast alpha mating factor signal peptide increased the level of expression from 230 ng/ml (cognate signal peptide) to 4 micrograms/ml (alpha mating factor signal peptide) of the culture supernatant. pFSH beta His.tag (pFSH beta with six histidine residues at the C terminus) was purified to apparent homogeneity using one-step nickel affinity chromatography. The molecular weight of purified pFSH beta His.tag was approximately 22,000, which was slightly higher than that of the pituitary-derived ovine FSH beta. pFSH beta His.tag could assemble with the alpha subunit to yield a heterodimer capable of binding to the FSH receptors and also elicit biological response. These data show that pFSH beta His.tag is properly folded and biologically active.

Demonstration of complimentarity between monoclonal antibodies (MAbs) to human chorionic gonadotropin (hCG) and polyclonal antibodies to luteinizing hormone/hCG receptor (LH-R) and their use in better understanding hormone-receptor interaction.

We have earlier reported that polyclonal antisera raised in rabbits to a luteinizing hormone/chorionic gonadotropin receptor (LH-R) purified from sheep luteal tissue has antibodies exhibiting hormone agonistic and antagonistic activities. Western blot analysis showed this antibody (LHR-anti IgG) to be highly specific to sheep luteal LH receptor (LH-R) (Jeyakumar and Moudgal, 1991). Using this, along with a battery of mouse monoclonal antibodies (MAbs) to hCG, an attempt has been made to better understand the interaction of LH/hCG with its receptor. Of the eight hCG MAbs screened, three (B14/B7, B52/18 and A7/G4) were specific to the beta-subunit; while a second set of three (G10/F7, H9/E9 and B52/21) were specific to the alpha-subunit. Two additional MAbs (B52/28 and F9/G8) did not recognize individual subunits, but bound like the rest intact hCG. Both 125I hCG and 125I anti LHR-IgG bound specifically to ovine luteal membrane LH-R. Assuming that a certain degree of similarity should exist between hCG and LHR-anti IgG, different hCG MAbs were tested for their ability to block the binding of either 125I hCG or 125I LHR-anti IgG to sheep luteal LH-R. It appears that hCG and LH-R share a minimum of four sites that are complementary to each other and these are recognized by the hCG MAbs B14/B7, G10/F7, A7/G4, and H9/E9. Whereas two of the MAbs B14/B7 and G10/F7 blocked the binding of both 125I labeled hCG and LHR-anti IgG to the receptor, MAbs A7/G4 and H9/E9 only inhibited the binding of 125I LHR-anti IgG to the LH-R. Although individually B14/B7 and G10/F7 blocked the binding of 125I LHR-anti IgG to LH-R to a maximum extent of 43%, together they inhibited binding by as much as 80%. The ability of B14/B7 to inhibit binding of 125I LHR-anti IgG to the receptor was also significantly increased by the addition of A7/G4. Finally, by demonstrating direct binding of the immobilized hCG MAbs B14/B7, G10/F7, A7/G4, and H9/E9 to LHR-anti IgG, we have been able to establish that the receptor binding sites of hCG and LHR-anti IgG are complementary and that a set of four sites are recognizable by the hCG MAbs. From the degree of interaction, it appears that two sites recognized by MAbs B14/B7 and G10/F7 (representing a site each in the beta- and alpha-subunit of hCG) have a prominent role in the interaction of hCG with its receptor. Thus, this study has provided us with an opportunity to investigate the interaction of LH/hCG with its receptor by an indirect approach of monitoring the binding of their respective antibodies with each other.

Specific immunoneutralization of FSH leads to apoptotic cell death of the pachytene spermatocytes and spermatogonial cells in the rat.

Although requirement for follicle stimulating hormone (FSH) in the initiation of spermatogenesis is well documented, its role in adult spermatogenesis is still debated. In the present communication, we have investigated the effect of specific immunoneutralization of FSH on apoptotic cell death in the testicular germ cells both in immature and adult rats. The germ cells of control animals showed predominantly high molecular weight DNA while the antiserum (a/s) treated group showed DNA fragmentation characteristic of apoptosis. The pattern could be detected within 24 hours of a/s treatment, and became more pronounced after 48 hours. The germ cells were purified from FSH a/s treated rats by centrifugal elutriation and vulnerability of each cell type to undergo apoptosis on FSH neutralization was investigated. The pachytene spermatocytes were found to be most sensitive to absence of FSH, even in the adult animals suggesting the involvement of FSH in spermatogenesis. The in situ analysis of DNA strand breakage following FSH a/s treatment showed fragmentation of the DNA of the pachytene spermatocytes confirming this observation. The in situ analysis also showed that the spermatogonia undergo apoptosis in addition to the pachytene spermatocytes. These data clearly demonstrate the role of FSH in the adult rat spermatogenesis.

Selective immunoneutralization of luteinizing hormone results in the apoptotic cell death of pachytene spermatocytes and spermatids in the rat testis.

The selective withdrawal of pituitary gonadotropins through specific antibodies is known to cause disruption of spermatogenesis. The cellular mechanism responsible for the degenerative changes under isolated effect of luteinizing hormone (LH) deprivation is not clear. Using antibodies specific to LH we have investigated the effect of immunoneutralization of LH on apoptotic cell death in the testicular cells of the immature and the adult rats. Specific neutralization of LH resulted in apoptotic cell death of germ cells, both in the immature and the adult rats. The germ cells from control animals showed predominantly high molecular weight DNA, while the antiserum treated group showed DNA cleavage into low molecular weight DNA ladder characteristic of apoptosis. This pattern could be observed within 24 h of a/s administration and the effect could be reversed by testosterone. The germ cells were purified by centrifugal elutriation and the vulnerability of germ cell types to undergo apoptosis under LH deprivation was investigated. The round spermatids and the pachytene spermatocytes were found to be the most sensitive germ cells to lack of LH and underwent apoptosis. Interestingly, spermatogonial cells were found to be the least sensitive germ cells to the lack of LH in terms of apoptotic cell death. Results show that LH, in addition to being involved in the germ cell differentiation, is also involved in cell survival and prevent degeneration of germ cells during spermatogenesis. Apoptotic DNA fragmentation may serve as a useful marker for the study of hormonal regulation of spermatogenesis and the specific neutralization of gonadotropic hormones can be a reliable model for the study of the molecular mechanism of apoptosis.

Long-term contraceptive efficacy of vaccine of ovine follicle-stimulating hormone in male bonnet monkeys (Macaca radiata).

A group of ten healthy fertile adult male bonnet monkeys were actively immunized using procedures acceptable for human use with pure follicle-stimulating hormone (oFSH) isolated from sheep pituitaries. The vaccine elicited an immunogenic response in all ten monkeys; the antibody-binding capacity, determined by Scatchard analysis, varied from 3 to 18 micrograms oFSH ml-1, the binding affinity ranging from 0.13 to 2.0 x 10(10) mol-1. A substantial population of antibodies against oFSH crossreacted with 125I-labelled human (h) FSH, used here as a representative ligand of primate FSH. The bioneutralization activity of the antisera assessed by a specific bioassay in vitro, when the antibody titre was high, was 6.9 +/- 0.18 micrograms hFSH ml-1. Immunization for 4.7-5.7 years did not affect the health and libido of the animals. Concentration of testosterone in serum remained normal throughout the study, but, within 150 days of immunization, there was a marked decrease (75-100%) in the number of spermatozoa in seminal ejaculates. Oligospermic status interspersed with azoospermia was maintained by periodic boosting. The fertility of these animals was monitored between 6 months and 2 years after primary immunization. All the ten animals proved infertile in repeated mating experiments with females of proven fertility. After stopping booster injections, nine of ten animals regained fertility, but the time taken for this depended upon the rate of decline of antibody titres. Re-boosting these monkeys with 100 micrograms oFSH after confirming that recovery had occurred revealed prompt increases in antibody titres followed once again by onset of oligo-azoospermia and infertility, underscoring the specificity of immunization effect. The immunized monkeys, apart from being acutely oligospermic, ejaculated spermatozoa that were markedly deficient in key acrosomal enzymes, such as acrosin and hyaluronidase, and motility as well as in their ability to penetrate a gel in vitro, suggesting that the infertility observed was due to gross reductions in the numbers of spermatozoa that could effectively interact with the oocyte and cause successful fertilization.

Two simple and rapid methods to detect monoclonal antibodies with identical epitope specificities in a large population of monoclonal antibodies.

Two novel and simple methods have been developed for detecting monoclonal antibodies (MAbs) with identical epitope specificities from a large population of MAbs against human chorionic gonadotropin (hCG). The first method was based on the observation that following radioiodination many molecules of an antigen are altered in such a manner that they cannot be recognized by MAbs. The proportion of radiolabelled antigen (Bmax) able to bind to a MAb was characteristic of that MAb and MAbs having identical specificities showed identical Bmax values. Using this principle it was possible to identify MAbs having identical epitope specificities within a large population of MAbs against hCG. In the second method one test MAb was immobilized on a plastic surface through an immunochemical bridge. This MAb was then incubated with 125I-hCG previously complexed with a second MAb. Such a complex could bind to the solid phase MAb only if the two MAbs were not identical. The results obtained with both methods were concordant. With such methods it is possible to identify MAbs with identical epitope specificities immediately after the initial screening of the fusion. These methods do not require subcloning, ascites production, or the purification and iodination of large number of MAbs.

Conformation of the alpha-subunit of glycoprotein hormones: a study using polyclonal and monoclonal antibodies.

The conformation of the common alpha-subunit of human glycoprotein hormones, luteinizing hormone (hLH), follicle-stimulating hormone (hFSH), thyroid-stimulating hormone (hTSH) and chorionic gonadotropin (hCG) was probed using a highly specific polyclonal antiserum against the alpha-subunit of hCG and several monoclonal antibodies (MAbs) produced against hCG which recognized the alpha-subunit in free and combined form. The alpha-subunit was found to be conformationally altered (compared to its conformation in the isolated state) when it was in combination with various beta-subunits as indicated by shifts in the displacement curves of binding of [125I]hCG alpha to the polyclonal antiserum. The extent of the change was dependent on the beta-subunit present with minimum change being observed with hLH beta, intermediate with hCG beta and maximum change with hFSH and TSH beta-subunits. However, the affinity constants of this antiserum for all four hormones were nearly similar. Further, it was also found that binding of any one of the glycoprotein hormones to this antibody could be completely inhibited by any other hormone suggesting that the conformation of the alpha-subunit in all the four hormones is probably very similar. This was further investigated using five hCG MAbs capable of recognizing the alpha-subunit, but with different epitope specificities. All these MAbs could recognize all the four hormones suggesting the presence of the epitopes in these proteins. These epitopes were conformation specific since the MAbs did not bind reduced and carboxymethylated alpha-subunit. Displacement analysis using [125I]hCG as the tracer showed that two epitopes have nearly the same conformation in all the four hormones, while two were partially modified depending on the beta-subunit present. Based on these results, it is concluded that the alpha-subunit of glycoprotein hormones has nearly the same conformation, though subtle differences do exist.

Regulation of protein synthesis in the first trimester human placenta by 17 beta-oestradiol and progesterone.

Addition of 17 beta-oestradiol or progesterone to first trimester human placental explants in vitro resulted in the stimulation of protein synthesis, as seen by autofluorographic analysis of placental tissue and medium proteins. An increase in the incorporation of [35S]methionine into trichloroacetic acid-precipitable proteins was seen, following the addition of 17 beta-oestradiol. Use of aromatase inhibitor to block the synthesis of 17 beta-oestradiol inhibited the protein synthesis and while addition of cyclohexamide blocked both basal- and 17 beta-oestradiol-induced protein synthesis, actinomycin-D blocked only 17 beta-oestradiol induced protein synthesis. Double labelling of placental proteins in the presence and absence of 17 beta-oestradiol also indicated that there is a significant stimulation of protein synthesis by 17 beta-oestradiol. Based on these results it is suggested that oestradiol has a role in regulation of placental protein synthesis.

Induction of sister chromatid exchanges by ergot compounds in Chinese hamster ovary cells in vitro.

Five ergot alkaloids, d-lysergic acid derivatives, were tested for the induction of sister chromatid exchange (SCE) frequencies in cultured Chinese hamster ovary cells. The concentrations of the test substances ranged between 10(-5) and 10(-8) M. Ergotamine, ergonovine, and methylergonovine induced a significantly high number of SCEs at all the concentrations used, while with ergocristine this occurred only at the highest concentration. alpha-Ergocryptine failed to induce SCEs at any concentration. It is therefore surmised that ergotamine, ergonovine, and methylergonovine are effective inducers, ergocristine is a weak inducer, and alpha-ergocryptine is a noninducer in Chinese hamster ovary cells in vitro.

Testicular peptides and their endocrine and paracrine functions.

A number of testicular peptides appear to have endocrine and/or paracrine functions. Most, if not all, of these peptides are secreted by the Sertoli cells into the tubular fluid. Although their physiological role has not been fully clarified, these secretory products are believed to play an important role in the local regulation of testicular functions, particularly the spermatogenic process. Presence of high affinity, specific binding sites for transferrin and androgen binding protein, both secreted by the Sertoli cells, has been demonstrated on the round spermatids and/or pachytene spermatocytes. Since within the seminiferous tubules, only the Sertoli cells respond directly to follicle stimulating hormone and testosterone, they could mediate the hormonal effects on spermatogenesis by providing certain products which may be essential during specific stages of germ cell differentiation.

Studies on characterization of mosquito cell lines.

Results of studies on characterization of mosquito cell lines are described; these include chromosome analysis, image cytometric, and flow cytometric estimation of DNA, tumorogenicity and angiogenicity, concanavalin-A-induced agglutination, and proton spin-lattice relaxation time (T1). The established mosquito cell lines are not diploid lines, although their stem-lines are diploid. Feulgen cytometry and flow cytometry reveal the inherent heterogeneity in DNA contents and support the observations on chromosome frequency distribution. Two cell lines exhibit angiogenic property and cell infiltration in chorio-allantoic membrane of chick embryos; however, none of the cell lines tested by inoculating hamsters, mice, or adult mosquitoes exhibit tumorogenicity. The agglutinability patterns of these cells in response to concanavalin-A treatment reveal the coexistence of agglutinable and unagglutinable cells. The mosquito cells differ from mammalian and chick cells in respect of their proton spin-lattice relaxation time.

Glucagon-stimulable adenylyl cyclase in rat liver. Effects of chronic uremia and intermittent glucagon administration.

The effects of chronic uremia and glucagon administration on glucagon-stimulable adenylyl cyclase in rat liver were assessed by determinations of adenylyl cyclase activities, specific iodoglucagon binding, and the activity of the stimulatory regulatory component of adenylyl cyclase. Glucagon-stimulated adenylyl cyclase was reduced in uremia to 75-80% of control levels (P less than 0.05), in the presence or absence of saturating levels of guanosine triphosphate (GTP) and 5'-guanylylimidodiphosphate [GMP-P(NH)P]. Although these changes were accompanied by a concomitant 20% reduction in sodium fluoride-stimulated activity, basal, GTP-, GMP-P(NH)P-, and manganese-dependent adenylyl cyclase activities were unchanged. Using [125I-Tyr10]monoiodoglucagon as a receptor probe, the number of high affinity glucagon-binding sites was reduced 28% (P less than 0.01) in uremic as compared with control liver membranes. However, the affinity of these binding sites was unaltered. The S49 cyc- -reconstituting activity with respect to both GMP-P(NH)P- and isoproterenol plus GTP-stimulable adenylyl cyclase was unaltered in membranes from uremic as compared with control rats. Intermittent glucagon (80-100 micrograms) injections administered at 8-h intervals to normal rats reproduced all of the above described effects of chronic experimental uremia on the adenylyl cyclase system. It is concluded that changes in the hormone-stimulable adenylyl cyclase complex in uremia and with glucagon treatment result primarily from a decrease in the number of hormone-specific receptor sites in hepatic plasma membranes. Since the changes in liver adenylyl cyclase are qualitatively and quantitatively the same in glucagon-treated and uremic rats, it is suggested that these may be the result of the hyperglucagonemia of uremia. Further, the data reveal an unexpected dissociation between guanine nucleotide and sodium fluoride stimulation of adenylyl cyclase. Possible causes for this dissociation based on the known subunit composition of cyclase coupling proteins are discussed.