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K Ramasharma

Publications and source records attributed to K Ramasharma.

At least 19 recordsLinked to original sources
PubMed1994

Effects of alpha-inhibin-92 fragments and alpha-inhibin-92 antiserum on the control of follicle-stimulating hormone release in male rats.

Alpha-inhibin-92 (alpha-IB-92) has been characterized from human seminal plasma and found to be active in suppressing FSH release in vitro and in vivo. In order to determine if smaller fragments of this 92 amino acid peptide would still be active to suppress FSH release, we have evaluated 5 of these fragments for their effects on FSH and LH release in the present study. Five alpha-IB-92 fragments (1-34, 1-46, 35-65, 35-92 and 66-92) were synthesized and injected intravenously (iv) into castrated adult rats (2 days post operation). Only fragments alpha-IB-92-(35-65) and alpha-IB-92-(66-92) significantly lowered plasma FSH, but not LH, at doses of 10 micrograms. These fragments exerted a preferential FSH-suppressing effect, but their activities were less than that of alpha-IB-92. In view of the rapid action of these peptides and the preferential FSH suppressing effect, they could be useful clinically to suppress FSH release. To determine the possible physiologic significance of alpha-IB-92, we injected antiserum raised against alpha-IB-92 into immature male rats and evaluated its effects on FSH and LH release. Normal rabbit serum (NRS) or anti-alpha-IB-92 serum was injected iv through indwelling jugular catheters into conscious, unrestrained 18 day-old male rats. Blood samples (0.2 ml) were collected at various intervals. Intravenous injection of alpha-IB-92 antiserum (0.1 ml/rat) selectively elevated plasma levels of FSH but not LH from 2-8 h post-injection in 18 day-old male rats (P < 0.01). Since immunoneutralization of alpha-IB-92 significantly elevated FSH release in immature rats, alpha-IB-92 has a physiological inhibiting role in control of FSH but not LH release at this stage of development.

Animals↗
PubMed1989

Insulin-like growth factor-II (IGF-II): a potential autocrine/paracrine growth factor for human breast cancer acting via the IGF-I receptor.

Insulin-like growth factor-II (IGF-II) is a potent mitogen for several types of cultured cells and tissues. We have studied the interaction of IGF-II with a panel of cultured human breast cancer cell lines, examining the possibility that these cells synthesize and secrete IGF-II activity which could have autocrine/paracrine functions. Synthetic IGF-II was mitogenic in five of seven cell lines tested, including the estrogen receptor-positive lines MCF-7L, ZR75-1, and T47D and the estrogen receptor (ER)-negative lines Hs578T and MDA-231. IGF-II was slightly less potent than IGF-I in stimulating DNA synthesis in MCF-71 cells, an effect that paralleled its ability to compete for [125I]IGF-I binding in these cells. Affinity labeling studies revealed that IGF-II could also compete for binding to the 130,000 mol wt alpha-subunit of the IGF-I receptor. A monoclonal antibody to the IGF-I receptor inhibited the mitogenic effects of IGF-II in MCF-7L and MDA-231 cells, suggesting that this receptor mediates the growth effects of IGF-II in these breast cancer cells. Using a RIA and a RRA, IGF-II-like activity was detected in conditioned medium extracts processed to remove IGF-binding proteins from several breast cancer cell lines, with the highest levels found in conditioned medium from MCF-7L and T47D cell lines. IGF-II mRNA transcripts in MCF-7L and T47D cells were identified by Northern blot analysis and were confirmed by RNase protection assay. IGF-II mRNA was increased by estrogen in MCF-7L cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1988

Human follicular gonadotropin releasing peptide analogs. Evaluation of biological (in vitro) and immunological activity.

Human follicular gonadotropin releasing peptide (hF-GRP) has been shown to stimulate pituitary LH and FSH secretion in vitro. Six hF-GRP analogs have been synthesized and evaluated for gonadotropin releasing activity in a rat anterior pituitary primary cell culture system. A tyrosine analog of hF-GRP, [Tyr4]-hF-GRP, retained comparable biological activity in releasing gonadotropins. However, acetylation of hF-GRP in Ac-hF-GRP greatly reduced the in vitro activity. The shorter segments of hF-GRP, hF-GRP-(5-14), and hF-GRP-(10-14), were tested for LH and FSH releasing activity, and it was found that the decapeptide retained moderate activity while the activity of the pentapeptide was markedly lower than hF-GRP. The baboon alpha 1 antitrypsin-(27-40) peptide, b-alpha 1 AT-(27-40), is relatively less potent in releasing LH than hF-GRP. Interestingly, the baboon peptide is more potent (2.5-fold) in releasing FSH under identical conditions. The effect of hF-GRP in releasing LH and FSH was not affected by the presence of LHRH antagonists in cell culture systems. When these peptides were tested for immunological activity in a hF-GRP radioimmunoassay, it was found that hF-GRP and [Tyr4]-hF-GRP have comparable activities. The C-terminal decapeptide of hF-GRP is more active (1.5-fold) in the RIA, and the C-terminal pentapeptide had only one third of the immunoreactivity. The b-alpha 1-AT-(27-40) failed to cross-react in the RIA even at a concentration of 20 micrograms per tube.

Amino Acid Sequence↗
PubMed1987

Characteristics of binding of human seminal alpha-inhibin-92 to human pituitary membranes.

We investigated the binding of 125I-labeled alpha-inhibin-92 (a 92-residue peptide) to human pituitary membrane preparations. Unlabeled alpha-inhibin-92 competed effectively with the labeled peptide for binding to the membranes. Binding was also inhibited by both alpha-inhibin-52 and alpha-inhibin-31, but less effectively. Scatchard analysis of the alpha-inhibin-92 binding data indicated the presence of high-affinity binding sites (1.35 nM/mg of membrane protein) with an apparent Kd of 0.37 nM. When 125I-labeled alpha-inhibin-92 was covalently crosslinked to the pituitary membrane preparation with disuccinimidyl suberate and the solubilized labeled receptor complex was analyzed by NaDodSO4/PAGE under either reducing or nonreducing conditions, a single radioactive band at an apparent molecular weight of 90,000 +/- 5000 was observed. These data suggest that human pituitary has specific binding sites for alpha-inhibins.

Activin Receptors↗
PubMed1987

Gonadotropin-releasing peptide from human follicular fluid: isolation, characterization, and chemical synthesis.

A gonadotropin-releasing peptide has been isolated from human follicular fluid. Its amino acid composition and sequence are completely different from the hypothalamic lutropin-releasing hormone. It is designated human follicular gonadotropin-releasing peptide and abbreviated as hF-GRP. The primary structure of this peptide (H-Thr-Asp-Thr-Ser-His-His-Asp-Gln-Asp-His-Pro-Thr-Phe-Asn-OH) has been confirmed by chemical synthesis. In the mouse pituitary incubation assay, the ED50 value for follitropin or lutropin release is estimated to be 1.2-1.6 nM.

Body Fluids↗
PubMed1987

Synthetic peptide with inhibin-like activity preferentially inhibits follitropin secretion in comparison with lutropin-releasing hormone antagonists.

Biological activity of a synthetic peptide with inhibin-like activity under in vitro and in vivo conditions was compared with three highly potent synthetic lutropin-releasing hormone antagonists. Unlike the synthetic lutropin-releasing hormone antagonists, which effectively inhibited both lutropin and follitropin secretion from the pituitary, the inhibin-like peptide showed a preferential effect by inhibiting follitropin release both in vitro and in vivo. Thus, small peptides such as inhibin-like peptide with a sequence unrelated to lutropin-releasing hormone may provide a basis for design of selective inhibitors of gonadotropin release.

Amino Acid Sequence↗
PubMed1987

Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells.

Human granulosa cells cultured with calf serum actively proliferated for 18-20 generation and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-II into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-II secretion from these cells. When synthetic lutropin-releasing hormone and alpha-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-II into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin.

Cells, Cultured↗
PubMed1987

Inhibin.

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Amino Acid Sequence↗
PubMed1986

Identification of insulin-like growth factor-II in human seminal and follicular fluids.

Antisera raised in rabbits against synthetic insulin-like growth factor-II (IGF-II) were used to develop a specific radioimmunoassay (RIA) for IGF-II. Affinity purified antibodies showed 6% cross-reactivity with IGF-I but failed to recognize insulin even at 10 micrograms/tube. Utilizing this RIA system, immunoreactive IGF-II was identified in the pooled samples of human follicular fluid and seminal plasma. The acid-ethanol precipitates of human seminal and follicular fluids were chromatographed on Sephadex G-50 column and the IGF-II immunoreactive fractions were subjected to reversed-phase high performance liquid chromatography. It was found that immunoactive IGF-II was eluted in the same location as that of synthetic IGF-II. The data indicate for the first time that human seminal plasma and follicular fluid contain significant amounts of IGF-II.

Body Fluids↗
PubMed1986

Human seminal alpha-inhibins: detection in human pituitary, hypothalamus, and serum by immunoreactivity.

An antiserum generated in rabbits against human seminal alpha-inhibin-52 has been used to develop a sensitive radioimmunoassay for the detection of alpha-inhibins. The alpha-inhibin-52 antiserum reacts with alpha-inhibin-92 and alpha-inhibin-31 with equal avidity. These peptides were found to be present in human pituitary, hypothalamus, and serum. In exclusion chromatography on Sephadex G-100, the immunoreactive material eluted in a large molecular size region. Immunoblot analysis of column-derived fractions of these extracts revealed the presence of alpha-inhibin-92. The mean concentrations of immunoreactive alpha-inhibin were found to be 7.2 ng/ml in normal adult male serum, 70.3 ng/g (wet weight) of pituitary, and 12.9 ng/g (wet weight) of hypothalamus. This communication reports on the evidence for the existence of gonadal peptides in the brain.

Humans↗
PubMed1986

Chemical synthesis of alpha-inhibin-92 by the thiocarboxyl segment coupling method.

The amino acid residue peptide, alpha-inhibin-92 (alpha-IB-92), has been synthesized by the thiocarboxyl segment strategy. Three segments were synthesized by the solid phase method, purified, and characterized: [GlyS34]-alpha-IB-92-(1-34) (I), CF3CO-[GlyS65]-alpha-IB-92-(35-65) (II), and Msc-alpha-IB-92-(66-92) (III). All were reacted with citraconic anhydride followed by removal of the Msc group in III to give Ia, IIa, and IIIa, respectively. Peptide IIIa was coupled to IIa by the silver nitrate/N-hydroxysuccinimide procedure and, after removal of uncoupled segments and the trifluoroacetyl group, Ia was coupled followed again by removal of uncoupled segments. Final deblocking to remove citraconyl groups was accomplished under exceptionally mild conditions in aqueous acetic acid. The synthetic product was identical to natural alpha-IB-92 in amino acid analysis, HPLC, gel electrophoresis, and tryptic mapping. The synthetic peptide was indistinguishable from natural alpha-IB-92 in a radioimmunoassay and in an in vitro mouse pituitary assay for measuring suppression of FSH release in the presence of LHRH.

Amino Acid Sequence↗
PubMed1985

Human seminal alpha inhibins: isolation, characterization, and structure.

Two additional peptides with inhibin-like activity have been isolated from human seminal plasma. One consists of 52 amino acids and the other, 92 amino acids. They are designated alpha-inhibin-52 and alpha-inhibin-92. Sequence analyses show that the NH2-terminal 31 amino acids of alpha-inhibin-52 are identical to the structure of the inhibin-like peptide previously reported [ILP-(1-31), now designated alpha-inhibin-31], and the COOH-terminal 52 amino acids of alpha-inhibin-92 are identical to the structure of alpha-inhibin-52. The amino acid sequence of alpha-inhibin-92 is: (sequence in text) Bioassay data in mouse pituitaries in vitro show that alpha-inhibin-52 is 3.4 times more active and alpha-inhibin-92 is greater than 40 times more active than alpha-inhibin-31 in suppressing follitropin-release. Radioimmunoassay data indicate that alpha-inhibin-52 and alpha-inhibin-92 have only 60% immunoreactivity.

Amino Acid Sequence↗
PubMed1984

Partial amino acid sequence of a human seminal plasma peptide with inhibin-like activity.

An extract of human seminal plasma was found to have inhibin-like activity. The activity. The active factor was purified to homogeneity by ion exchange chromatography, molecular sieving and high performance liquid chromatography. The purified material has a mass of approximately 5 kDa and is very basic. Amino acid analysis showed the presence of approximately 35 residues while the sequencing data allowed the determination of the N-terminal 31 amino acids. There is a possibility of an additional 2-4 residues at the C-terminus, which could not be determined.

Amino Acid Sequence↗
PubMed1984

Synthesis and biological activity of human inhibin-like peptide-(1-31).

A peptide containing 31 amino acids with inhibin-like activity has been synthesized by the solid-phase method. The synthetic product was shown to be homogeneous in high-performance liquid chromatography and paper electrophoresis. In the mouse pituitary incubation system, the follitropin-suppressing activity of the synthetic peptide was comparable to that of the natural product. It was also active in suppressing lutropin-releasing hormone-induced follitropin release in vivo in immature male rats.

Animals↗
PubMed1983

Effect of porcine follicular fluid preparations on gonadotrophin secretion by the mouse pituitary gland in vitro.

The acute effects of pooled porcine follicular fluid (PFF), before and after various methods of processing to eliminate steroids, were studied on the luteinizing hormone releasing hormone (LH-RH)-induced release of FSH and LH by whole pituitary glands, from 34-day-old mice, incubated in vitro for 3-4 h. Charcoal treatment of PFF eliminated the steroids and reduced the inhibitory potency on gonadotrophin secretion. On the other hand, dialysis or ultrafiltration (mol. wt greater than 10,000) did not reduce the inhibitory activity on gonadotrophin secretion. Of the three steroids tested, only oestradiol at a concentration of 10(-10) mol/l inhibited FSH and LH secretion in vitro. This inhibitory effect was counteracted by the inclusion of the oestrogen antagonist tamoxifen in the incubation medium. The presence of tamoxifen did not decrease the suppression of FSH and LH induced by PFF, suggesting that the inhibition observed under the conditions of incubation was not due to oestrogen. Preincubation of mouse pituitary tissue for 1 h with PFF reduced the subsequent release of bioactive FSH and LH induced by LH-RH. The inhibitory effect of PFF was rapid and sustained. The continuous presence of PFF throughout the incubation period was not necessary for manifestations of the inhibitory effects on gonadotrophin release. The suppression of gonadotrophin secretion was related to the dose of PFF with the curve showing a biphasic pattern. The degree of FSH suppression was uniformly greater than that of LH, showing the preferential nature of the inhibitory effect of PFF. At high doses of PFF, the degree of FSH suppression was decreased significantly. This effect on LH release was less pronounced. The inhibition caused by PFF in the in-vitro incubation procedure was not due to destruction of LH-RH or the released gonadotrophins.

Animals↗