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Biomedical subjects

I Parikh

Publications and source records attributed to I Parikh.

At least 19 recordsLinked to original sources
PubMed1992

Amino acid sequence determination of ancrod, the thrombin-like alpha-fibrinogenase from the venom of Akistrodon rhodostoma.

The thrombin-like serine protease and antithrombotic agent, Ancrod, was rapidly purified from the crude venom of Akistrodon rhodostoma by agmatine-Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N-Terminal sequencing and analysis of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N-linked glycosylation sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N-glycanase-treated peptides. Monoclonal antibodies raised against the denatured protein and HF-deglycosylated protein recognized Ancrod on Western blots. Sequence comparison to other thrombin-like serine proteases and reptilian fibrinogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.

Amino Acid Sequence↗
PubMed1991

Isolation and complete amino acid sequence of two fibrinolytic proteinases from the toxic Saturnid caterpillar Lonomia achelous.

The major toxic and fibrinolytic activity of the saliva and hemolymph of the larval form of Lonomia achelous was purified to homogeneity by a combination of metal chelate and affinity chromatography. Two apparent isozymes, Achelase I (213 amino acids, pIcalc = 10.55) and Achelase II (214 amino acids, pIcalc = 8.51), were sequenced by automated Edman degradation, and their C-termini confirmed by Fourier-transform mass spectrometry. The calculated molecular weights (22,473 and 22,727) correspond well to Mr estimates of 24,000 by SDS-PAGE. No carbohydrate was detected during sequencing. The enzymes degraded all three chains of fibrin, alpha greater than beta much greater than gamma, yielding a fragmentation pattern indistinguishable from that produced by trypsin. Chromogenic peptides S-2222 (Factor Xa and trypsin), S-2251 (plasmin), S-2302 (kallikrein) and S-2444 (urokinase) were substrates while S-2288 (broad range of serine proteinases including thrombin) was not hydrolyzed. Among a range of inhibitors Hg+2, aminophenylmercuriacetate, leupeptin, antipain and E-64 but not N-ethylmaleimide or iodoacetate abolished the activity of the purified isozymes against S-2444. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and aprotinin were less effective. The presence of the classic catalytic triad (histidine-41, aspartate-86 and serine-189) suggests that Achelases I and II may be serine proteinases, but with a potentially free cysteine-185 which could react with thiol proteinase-directed reagents.

Amino Acid Sequence↗
PubMed1991

Estrogens reduce bone loss in the ovariectomized, lactating rat model.

Estrogen treatment of ovariectomized, lactating rats improved retention of bone mineral mass by 15-25% compared to ovariectomized, lactating rats receiving vehicle only. On the second day postpartum all lactating rats were ovariectomized and were placed along with age-matched non-mated controls on a whole-wheat flour-based diet with 0.1% calcium and 0.4% phosphorus. On day 6 postpartum estrogen treatment was begun with either implantation of a slow-release 17 beta-estradiol pellet or with the first of daily subcutaneous injections in sesame oil (vehicle). Increasing doses of estrogen resulted in decreased food consumption and decreased litter weight gain, both well-known effects of estrogens. Ovariectomized, lactating rats implanted with a slow-release pellet containing 0.35 mg 17 beta-estradiol had mean serum estradiol levels of 113.5 pg/ml. At the end of 21 days of lactation, femurs of dams with placebo pellets showed loss of 54% of bone ash weight compared with the non-mated controls versus only 42% loss by rats receiving estradiol treatment. Rats were also injected with estradiol benzoate in a sesame oil vehicle at 3 dose levels of 1.6, 5, or 16 micrograms/day. Only the 5 and 16 micrograms/day doses significantly improved retention of bone mineral mass during lactation (+17% and +18%, respectively, vs vehicle-injected, lactating rats). Estrone administered by subcutaneous injection also improved retention of bone during lactation; however, injection of 50 micrograms/day of estrone was required to produce an equivalent bone retention compared to 5 micrograms/day of estradiol. Thus, treatment of ovariectomized, lactating rats with estrogens results in a significant reduction of the loss of bone mineral mass associated with lactation.

Animals↗
PubMed1990

Insulinlike growth factor I receptors in rabbit gastrointestinal tract. Characterization and autoradiographic localization.

Insulinlike growth factor I is a potent mitogen with insulinlike metabolic effects. Insulinlike growth factor I is synthesized in the liver, intestine, and other organs. Insulinlike growth factor I receptors are widely distributed and structurally similar to insulin receptors. Frozen sections of rabbit gastrointestinal tract were incubated in buffer containing 40 pmol/L [125I]insulinlike growth factor I. Binding was saturable, temperature- and time-dependent, and reversible. Saturation binding experiments showed a single class of high-affinity receptors (Kd = 0.9 nmol/L, Bmax = 0.36 pmol/mg protein). The IC50s for insulinlike growth factor I and insulinlike growth factor II were 3 nmol/L and 90 nmol/L, respectively; whereas insulin at 1-3 mumol/L displaced 50% of specific binding. Autoradiography of insulinlike growth factor I binding demonstrated significant differences in receptor density in gastrointestinal smooth muscle, epithelium of the esophagus, stomach, small intestine, and colon. These results indicate that a single class of specific, high-affinity insulinlike growth factor I receptors were distributed in muscular and mucosal layers of the entire rabbit gastrointestinal tract. Insulinlike growth factor I is likely to be an important local mediator of intestinal growth and metabolism.

Animals↗
PubMed1990

The ovariectomized, lactating rat as an experimental model for osteopenia: calcium metabolism and bone changes.

The ovariectomized, lactating rat (Sprague-Dawley) is proposed as an experimental model for the rapid development of osteopenia which may be used to test the effectiveness of bone-retentive drugs potentially useful in treating osteoporotic women. Rats were ovariectomized (OVX) on day 2 postpartum and were kept on a low-calcium diet (0.1%). Measurements of serum total calcium, ionic calcium, albumin and parathyroid hormone were conducted between days 4 and 21 of lactation. Serum total and ionic calcium and albumin were significantly lower and serum parathyroid hormone was significantly higher in all lactating rats at 16 days postpartum compared to nonlactating controls. Mean bone mass of the femurs of OVX lactating rats measured at day 21 was approximately 50% of that of non-lactating intact controls. The enhanced duodenal calcium absorption (in everted gut sacs) associated with lactation was not affected by OVX and neither was the average litter weight gain between 2 and 14 days of lactation. In conclusion, lactation coupled with a low-calcium diet resulted in marked osteopenia, depressed serum calcium (both total and ionic) and significantly elevated serum parathyroid hormone concentration. The rapid and extensive bone loss of this model makes it appropriate for the study of therapeutic agents designed to retain bone mass.

Animals↗
PubMed1987

Chemical identification of a tumor-derived angiogenic factor.

Neoplasms produce substances that induce blood vessel formation (angiogenesis). Fractions from ethanol extracts of the Walker 256 carcinoma were isolated by silica column chromatography and C18 reversed-phase high-performance liquid chromatography. Two of the isolated fractions induced neovascularization when tested in the rabbit corneal micropocket assay. One of the fractions was identified as nicotinamide by desorption-electron impact mass spectrometry, nuclear magnetic resonance spectroscopy, and gas chromatography-mass spectrometry. The second active fraction contained nicotinamide as part of a more complex, as yet unidentified, molecular arrangement. Microgram quantities of commercial nicotinamide induced neovascularization in the corneal micropocket assay and in the chick chorioallantoic membrane assay.

Angiogenesis Inducing Agents↗
PubMed1987

Immunocytochemical demonstration of estrogen receptors by monoclonal antibodies in human breast cancer: correlation with estrogen receptor assay by dextran-coated charcoal method.

Immunocytochemical demonstration of estrogen receptors in 115 human breast cancer specimens was performed using mouse monoclonal antibodies against estrogen receptor and avidin-biotin as the displaying system. The antibody indicated a highly heterogeneous endowment of neoplastic cells with estrogen receptor at both nuclear and cytoplasmic levels. The percentage of labeled cells within each tumor specimen was recorded to compare this immunocytochemical assay with the biochemical assay of estrogen receptors by the dextran-coated charcoal method. A significant correlation was observed between these two assays. The present results show that estrogen receptors can be confidently demonstrated at the single cell level, thus providing additional information to quantitative biochemical assays. Their prognostic and therapeutic predictive powers may be usefully integrated, particularly in view of the heterogeneous distribution of receptors among cancer cells.

Antibodies, Monoclonal↗
PubMed1987

Estrogenic effect of phenol red in MCF-7 cells is achieved through activation of estrogen receptor by interacting with a site distinct from the steroid binding site.

MCF-7 cells serially subcultured in media containing phenol red show poor stimulation of progesterone receptor (PR) synthesis in response to estradiol compared to cells grown in phenol red-free media. Phenol red, when added to cytosol, did not compete with [3H]estradiol for estrogen binding sites in concentrations ranging from 2 microM-1 mM. However 25 microM of the dye was sufficient to increase nuclear translocation of estrogen receptor (ER) in the intact cell. Phenol red activates cytoplasmic ER as indicated by DNA-cellulose binding studies. When cells grown in phenol red-free medium were exposed to phenol red for 48 h, PR levels increased in a dose dependent manner. From these data, it may be concluded that phenol red causes estrogenic effect in MCF-7 cells through activation of cytoplasmic receptor by interacting at a site distinct from the steroid binding site.

Binding Sites↗
PubMed1987

Are estrogen receptors cytoplasmic or nuclear? Some immunocytochemical and biochemical studies.

The subcellular localization of estradiol receptor (ER) has been examined using various experimental approaches. Immunocytochemical studies using the monoclonal antibody JS 34/32, raised against calf uterine cytosolic ER, yielded only equivocal results. In general, cells and tissues pretreated with estradiol showed positive immunostaining in the nuclei whereas those not exposed to the steroid did not show any staining. Nuclear translocation of ER was examined in intact MCF-7 cells using compounds which are known to influence receptor activation. When MCF-7 cells were exposed to molybdate (20 mM), nuclear translocation was completely inhibited while dithiothreitol (20 mM), dibutyryl cAMP (1 microM) and dibutyryl cGMP (1 microM) increased the translocation 2-3-fold. Phenol red, at the range of concentrations generally used in tissue culture media, also increased translocation. The physiological validity of such translocation was examined using cellular progesterone receptor (PR) synthesis as a specific parameter. When MCF-7 cells were grown in media containing phenol red for 48 h, the PR synthesis increased significantly. We further examined whether cytoskeletal proteins are involved in the translocation of ER. Colchicine, an inhibitor of microtubule assembly, inhibited translocation of ER in MCF-7 cells at 1-10 microM. PR synthesis was also inhibited by colchicine in a dose-dependent manner. It may be concluded from these and other published data that ER may not be located at all times in a single subcellular compartment but may rather exist in a dynamic equilibrium between the plasma membrane, cytoplasm and nucleus.

Antibodies, Monoclonal↗
PubMed1987

Peroxisome proliferator-binding protein: identification and partial characterization of nafenopin-, clofibric acid-, and ciprofibrate-binding proteins from rat liver.

Peroxisome proliferators (PP) induce a highly predictable pleiotropic response in rat and mouse liver that is characterized by hepatomegaly, increase in peroxisome number in hepatocytes, and induction of certain peroxisomal enzymes. The PP-binding protein (PPbP) was purified from rat liver cytosol by a two-step procedure involving affinity chromatography and ion-exchange chromatography. Three PP, nafenopin and its structural analogs clofibric acid and ciprofibrate, were used as affinity ligands and eluting agents. This procedure yields a major protein with an apparent Mr of 70,000 on NaDodSO4/PAGE in the presence of reducing agent and Mr 140,000 (Mr 140,000-160,000) on gel filtration and polyacrylamide gradient gel electrophoresis under nondenaturing conditions, indicating that the active protein is a dimer. This protein has an acidic pI of 4.2 under nondenaturing conditions, which rises to 5.6 under denaturing conditions. The isolation of the same Mr 70,000 protein with three different, but structurally related, agents as affinity ligands and the immunological identity of the isolated proteins constitute strong evidence that this protein is the PPbP capable of recognizing PP that are structurally related to clofibrate. The PPbP probably plays an important role in the regulation of PP-induced pleiotropic response.

Animals↗
PubMed1986

Immunohistochemical localization of estrogen receptor in rat brain, pituitary and uterus with monoclonal antibodies.

Localization of estrogen receptor (ER) in rat brain, pituitary and uterus is shown by the avidin-biotin complex technique using a monoclonal antibody, JS34/32. Immunostaining is observed in nuclei of certain neurons in the preoptic-septal region, hypothalamus and amygdala, and in cells of the anterior pituitary and uterus after estradiol stimulation. Staining is specific since preadsorbed JS34/32 antibody with purified cytoplasmic ER as well as a control monoclonal antibody do not show positive immunoreaction. In the brain, neither cytoplasmic nor nuclear staining is seen in the absence of estradiol stimulation, nor with the progesterone and dihydrotestosterone treatments. The distribution of ER-containing neurons in specific areas of the brain overlaps with the distribution of estrogen target neurons demonstrated by autoradiography. The results demonstrate the usefulness of the monoclonal antibody for the detection of ER in target tissues.

Animals↗
PubMed1976

Immobilized glucuronosyltransferase for the synthesis of conjugates.

Partially purified rabbit liver UDPglucuronosyltransferase is immobilized on agarose by the cyanogen bromide activation method. Both soluble and matrix-bound enzyme preparations display very similar Km and pH optimum. The storage stability of the immobilized enzyme at 4 degrees is 5-10 times improved over the soluble preparations. The agarose-bound UDPglucuronosyltransferase is successfully used in the synthesis of p-nitrophenyl glucuronide in an overall yield of 50-70%. The matrix-bound enzyme is reusable over an extended period of time and offers an easy and convenient synthetic tool for various drug glucuronides.

Glucuronates↗