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Biomedical subjects

J Ramachandran

Publications and source records attributed to J Ramachandran.

At least 73 records · Page 4Linked to original sources
PubMed1984

Preparation of a specifically tritiated locust adipokinetic hormone analog with full biological potency.

A synthetic peptide related to locus adipokinetic hormone ( AKH ) and shrimp red pigment concentrating hormone ( RPCH ) containing a tyrosine residue in place of phenylalanine was iodinated and the 3,5- diiodotyrosyl derivative was isolated by reverse phase HPLC. Catalytic dehalogenation of the diiodo derivative in the presence of tritium yielded the tritiated AKH analog which was isolated by gel filtration on Sephadex LH-20 and reverse phase HPLC. The tritiated peptide was formed to be identical to AKH in its ability to stimulate lipid release into the hemolymph of locusts in vivo where the diiodotryrosyl derivative was inactive. The specific radioactivity of the tritiated peptide was 57.2 Ci/mmol, or 99% of the theoretical value.

Animals↗
PubMed1984

Preparation and characterization of photoreactive derivatives of GnRH. Persistent activation of function by photoaffinity labeling.

A photoreactive derivative of the highly potent gonadotropin releasing hormone (GnRH) agonist, D-Lys6-GnRH(1-9)-ethylamide, was prepared by selective modification of the epsilon-amino group with 2-nitro-4-azidophenyl sulfenyl chloride (2,4-NAPS C1). The modified peptide [D-Lys(NAPS)]6-GnRH-(1-9)-ethylamide was found to be a full agonist of LH release from rat pituitary cells with a relative potency 23 compared to GnRH. Covalent attachment of the photoreactive analog to rat pituitary cells resulted in prolonged activation of LH secretion which could not be inhibited by a potent GnRH antagonist. Persistent stimulation of pituitary gonadotrophs caused by covalently bound hormone led to desensitization of the LH releasing mechanism.

Affinity Labels↗
PubMed1984

Radioimmunocytochemistry using a tritiated goat anti-rabbit second antibody.

Affinity-purified goat anti-rabbit immunoglobulin G (GAR) was conjugated with (3H)-propionyl succinimidate and used to localize substance P (SP), enkephalin (ENK), and serotonin immunoreactive sites in the spinal dorsal horn and medulla of the rat and cat. Autoradiographic localization was demonstrated on paraffin, frozen, Vibratome, and 2 micron plastic sections. The latter were obtained from radiolabeled Vibratome sections that were embedded in epoxy resin. The distribution of SP, ENK, and serotonin demonstrated by radioimmunocytochemistry was comparable to that observed on semiadjacent sections using peroxidase-antiperoxidase (PAP) immunocytochemistry. The autoradiograms, however, were generated using primary antibody concentrations up to five times more dilute than concentrations used for the PAP procedure. Indirect radioimmunocytochemistry using a (3H) anti-immunoglobulin G second antibody can be used to localize a variety of monoclonal and polyclonal antisera. It is quantifiable at the light microscopic level and can be potentially used with peroxidase histochemistry to double label immunoreactive structures at the ultrastructural level.

Animals↗
PubMed1984

A rapid sensitive assay for the quantitation of elastin.

A rapid, sensitive and direct assay was developed for the quantitation of elastin using reverse phase high performance liquid chromatography. The method is based on the separation of the alkaline hydrolysis-resistant dipeptide valyl-proline, a dipeptide found in high concentrations in elastin. The methodology is applicable to unlabelled elastin samples in the range of 10-100 micrograms utilizing fluorescamine detection and to hot alkali insoluble as well as soluble forms of elastin. Increased sensitivity was achieved by the use of in vitro radiolabelling of elastin with [3H]proline. This was useful for following synthesis of elastin in cultured cells. Since this technique is not dependent on detection of the elastin-specific crosslink groups, desmosine and isodesmosine, precursor forms of elastin can be measured as well as other forms of elastin deficient in cross-linking. The same chromatographic procedure however, is able to detect these desmosine and isodesmosine crosslinks. Therefore, in cell cultures under the proper radiolabelling conditions, biosynthesis and cross-linking of elastin can be followed simultaneously in a single chromatographic procedure.

Animals↗
PubMed1984

Photoreactive derivative of Kunitz's soybean trypsin inhibitor. Preparation by selective modification of a tryptophan residue and formation of a covalent complex of the modified inhibitor with trypsin.

The photoreactive arylsulfenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (SBTI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-SBTI indicated that only one of the two tryptophans (93 or 117) present in SBTI was modified. CNBr cleavage of 2,4-NAPS-SBTI resulted in two fragments 1-114 and 115-181. Amino acid analysis of the two separated fragments showed that only tryptophan 93 underwent modification. 2,4-NAPS-SBTI fully retained its inhibitory activity against trypsin. The photoaffinity labeling of trypsin with 2,4-NAPS-Cl was performed on tritiated trypsin prepared by reacting bovine trypsin with [3H]-succinimidyl propionate. The covalent attachment of 2,4-NAPS-SBTI to the tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride.

Affinity Labels↗
PubMed1983

Studies of corticotropin receptors on rat adipocytes.

Synthetic [125I]-Tyr23, Phe2, Nle4-adrenocorticotropin (ACTH)-(1-38) [( 125I]-ACTH analog) with full biological potency and near theoretical specific radioactivity (1800 +/- 75 Ci/mmol) was used to investigate ACTH receptors on isolated rat adipocytes derived from 42-day-old rats. Binding to adipocytes was studied in the presence of 1% bovine serum albumin (BSA) as well as 4% BSA. The interaction of the [125I]-ACTH analog with adipocytes was highly specific, rapid, saturable, and reversible. Scatchard analysis of the binding data obtained in medium containing 1% BSA revealed a single class of binding sites with an apparent KD = 170 +/- 11.9 pM. Competition experiments with unlabeled ACTH also yielded a comparable value for the apparent KD (143 +/- 16.5 pM). The number of receptors per adipocyte was quite low (521-841/cell). The stimulation of lipolysis by ACTH was closely correlated with the binding, the apparent Km being 145-177 pM. At a concentration of 4% BSA in the incubation medium, the binding curve was shifted significantly to the right (apparent KD = 446 +/- 77 pM) and the binding capacity was also significantly enhanced (1663 +/- 208/cell) without any change in the apparent Km for glycerol release (187 +/- 7.1 pM).

Adipose Tissue↗
PubMed1983

Determination of locust vitellogenin by radioimmunoassay with [3H]Propionyl-vitellogenin.

A simple procedure for radiolabeling of locust vitellogenin is described. This procedure involves coupling of [3H]propionyl succinimidate to purified vitellogenin with high yield and specific activity. Using this radiolabeled analog, a specific and sensitive radioimmunoassay was developed for determining locust vitellogenin content, with a lower detection limit of 1 ng. [3H]Propionyl-vitellogenin binds completely to rabbit anti-vitellogenin (locust) and can be completely competed out by locust vitellogenin. The structural similarity of locust vitellogenin with that of locust egg vitellin, male locust lipophorin (a diglyceride-carrying lipoprotein), Xenopus laevis vitellogenin, and chicken egg yolk lipovitellin was examined with this RIA procedure. Comparable binding competition was obtained with locust vitellin only. Male locust lipophorin, Xenopus vitellogenin, and chicken lipovitellin did not inhibit vitellogenin binding at concentrations 1000-fold greater than that of locust vitellogenin. The use of this RIA in determination of vitellogenin synthesis in vivo and in vitro, using isolated fat body preparations, is described.

Animals↗
PubMed1983

Cardiovascular responses to alpha-melanocyte stimulating hormone during the perinatal period in sheep.

To determine the effects of alpha-melanocyte stimulating hormone (alpha MSH) on the circulation of undisturbed fetal sheep and young lambs and to explore possible mechanisms of actions, we examined the responses of ascending aortic blood flow, heart rate, stroke volume, and arterial blood pressure to a single intravenous injection of alpha MSH, ACTH, cortisol and to alpha MSH after beta-adrenergic blockade. We also measured cardiac output and organ blood flows before and after alpha MSH injection by the radionuclide labeled microsphere technique. alpha MSH increased ascending aortic blood flow from 249 +/- 23 to 327 +/- 30 ml X min-1 X kg-1 (P less than 0.001), heart rate from 111 +/- 10 to 126 +/- 11 min-1, (P less than 0.001) and stroke volume from 2.32 +/- 0.21 to 2.75 +/- 0.20 ml X kg-1, (P less than 0.001) in 10 young lambs 15-30-day-old. alpha MSH decreased mean systemic arterial blood pressure from 42.6 +/- 1.7 to 39.3 +/- 1.6 mmHg (P less than 0.001) in fetal sheep and min-1, (P less than 0.001) and stroke volume from 2.32 +/- 0.21 to 2.75 +/- 0.20 ml X kg-1, (P less than 0.001) in 10 young lambs 15-30-days-old. alpha MSH decreased mean systemic arterial blood pressure from 42.6 +/- 1.7 to 39.3 +/- 1.6 mmHg (P less than 0.001) in fetal sheep and from 77.5 +/- 4.5 to 72.7 +/- 4.3 mmHg (P less than 0.001) in young lambs. In six additional young lambs (20-35-day-old) cardiac output, measured by microspheres, increased with alpha MSH from 216 +/- 20 to 261 +/- 19 ml X min-1 X kg-1 (P less than 0.001). alpha MSH also increased blood flow to the myocardium from 104 +/- 22 to 151 +/- 26 ml X min-1 X 100 g-1 (P less than 0.001), to the adrenals from 195 +/- 38 to 243 +/- 41 ml X min-1 X 100 g-1, (P less than 0.05), and to the lungs from 157 +/- 31 to 468 +/- 81 ml X min-1 X 100 g-1 (P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1983

Studies on corticotropin-induced desensitization of normal rat adrenocortical cells.

The effects of prior exposure of normal rat adrenocortical cells to ACTH on the responsiveness of the cells to subsequent stimulation with the hormone have been studied. ACTH induces a time- and concentration-dependent refractoriness of both cAMP formation and steroidogenesis. Desensitization of either response was observed only upon activation of the response. Thus, both ACTH and 8-Br-cAMP caused desensitization of the steroidogenic response. The ACTH-induced desensitization of steroidogenesis, however, was completely prevented by blocking the steroidogenic action of ACTH with aminoglutethimide during exposure of cells to the hormone. Aminoglutethimide had no effect on ACTH-induced desensitization of the cAMP response. Studies with analogs of the hormone also confirmed that induction of desensitization of the steroidogenic response is independent of the desensitization of the cAMP response. Binding studies showed that the insignificant decrease in ACTH receptors could not account for the large changes in the responsiveness induced by prior exposure of cells to ACTH. Desensitization of the steroidogenic response appears to result from a defect in the rate-limiting first step of the steroidogenic pathway, namely conversion of cholesterol to pregnenolone.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1982

Photoaffinity labeling of the canine renal receptor for parathyroid hormone.

Studies were undertaken to identify and characterize components of the parathyroid hormone receptor. An analog of the bovine parathyroid hormone(1-34) sequence was derivatized at the 23-tryptophan position with the photoreactive reagent 2-nitro-5-azidophenylsulfenyl chloride. 2-Nitro-5-azidophenylsulfenyl-bovine parathyroid hormone (NAPS-bPTH) analog retained full biological activity with respect to receptor binding and activation of adenylate cyclase in canine renal cortical plasma membranes. 125I-bPTH(1-34) analog was also derivatized without loss of receptor-binding activity. When 125I-NAPS-bPTH(1-34) analog was incubated with canine renal plasma membranes and then photolyzed, at least two 125I-labeled membrane components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The incorporation of 125I radioactivity into one of these components (Mr congruent to 60,000) was inhibited when incubation and photolysis were performed in the presence of excess, unlabeled bPTH(1-34). Furthermore, photolysis of membranes in the presence of NAPS-bPTH(1-34) analog led to activation of adenylate cyclase which persisted following washing to remove noncovalently bound peptide, suggesting a functional, covalent hormone-receptor complex had been formed. We conclude that the M r congruent to 60,000 membrane component may be a part of the renal receptor for parathyroid hormone.

Adenylyl Cyclases↗
PubMed1982

Synthesis of human corticotropinyl-thiolglycine and its specific conjugation to bovine serum albumin.

Human corticotropin containing a thiolglycine residue at the carboxyl terminal (I) was synthesized by the solid-phase method. The citraconyl derivative of peptide I was coupled to either a model tetrapeptide or bovine serum albumin by reaction with silver nitrate/N-hydroxysuccinimide in water. The extent of reaction with bovine serum albumin was determined by radioimmunoassay, and the peptide-protein conjugate was shown to possess 12% of the steroidogenic activity of porcine adrenocorticotropin in isolated rat adrenal cells. Peptide I and its conjugate with the model tetrapeptide were fully active in the same system.

Adrenal Cortex↗
PubMed1982

Mechanism of induction of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in rat adrenocortical cells by corticotropin.

The regulation of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) was studied in primary cultures of rat adrenocortical cells. In the absence of ACTH, this enzymic activity was found to decay with a half-life of 3.1 days, which was similar to the half-life of the enzyme activity induced by ACTH in vitro (3.5 days). The increase in 3 beta-HSD activity was highly specific for ACTH and dibutyryl cAMP; the activity was not increased by other hormones known to affect adrenocortical growth or function. The induction of 3 beta-HSD activity by ACTH or dibutyryl cAMP required a lag period of approximately 4 h and was dependent on RNA and protein syntheses. The increase in 3 beta-HSD activity observed after ACTH treatment was not a result of ACTH-induced inhibition of degradation of the enzyme, nor was it due to the synthesis of a soluble intermediate which could directly activate the enzyme. ACTH stimulated the incorporation of [35S]methionine into a protein associated with 3 beta-HSD activity detected on polyacrylamide gels after electrophoresis of Triton X-100 extracts of adrenocortical cells. The induction of this protein by ACTH was inhibited by actinomycin D. A protein band of a partially purified preparation of rat adrenal 3 beta-HSD was found to comigrate with the ACTH-induced protein on sodium dodecyl sulfate-polyacrylamide gel. These results suggest that ACTH caused the de novo synthesis of 3 beta-HSD by a mechanism dependent on RNA synthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1981

Persistent activation of steroidogenesis in adrenocortical cells by photoaffinity labeling of corticotropin receptors.

Photolysis of rat adrenocortical cells in the presence of the photoreactive derivative [(2-nitro-5-azidophenylsulfenyl)Trp9]-adrenocorticotropic hormone (2,5-NAPS-ACTH) at 24 degrees C resulted in persistent activation of corticosterone production. The basal rate of steroidogenesis became maximal when photolysis was performed at 24 degrees C but remained the same as that of control cells when irradiation was performed at 0 degrees C. No increase in basal rate was observed with dark controls or cells photolyzed with [(2,4-dinitrophenylsulfenyl)Trp9]ACTH, a photoresistant analog of the hormone. Prephotolyzed 2,5-NAPS-ACTH failed to induce persistent activation. Both ACTH and 2,4-(dinitrophenylsulfenyl)Trp9-ACTH blocked the photo-induced activation of steroidogenesis elicited by 2,5-NAPS-ACTH. Under photolysis conditions which caused the basal rate of steroidogenesis to become maximal, a 3-fold increase in the basal rate of cAMP formation was observed.

Adrenal Cortex↗
PubMed1981

Preparation of photoreactive derivatives of glutathione and [9-(2-mercaptotryptophan)]corticotropin by selective modification of the sulfhydryl group.

The photoreactive arylsulfenyl chlorides 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) and 2-nitro-5-azidophenylsulfenyl chloride (2,5-NAPS-Cl) have been used for the selective modification of thiol groups in glutathione and [Trp(SH)9]corticotropin (ACTH). Both reagents reacted rapidly with both types of thiol groups to form unsymmetrical disulfides. The photoreactive derivatives of glutathione and [Trp(SH)9]ACTH were stable to neutral and acidic conditions but were readily cleaved above pH 9 and by beta-mercaptoethanol. Photolysis of the NAPS derivatives of [Trp(SH)9]-ACTH at neutral pH resulted in the formation of covalently liked polymers and dimers which yielded monomer upon treatment with beta-mercaptoethanol. Analysis of the amino acid composition of acid hydrolysates of photolysed monomeric and dimeric products indicated a decrease in proline, valine, tyrosine, and phenylalanine.

Adrenocorticotropic Hormone↗