Biomedical subjects

J C Calvo

Publications and source records attributed to J C Calvo.

At least 19 recordsLinked to original sources

PubMed1995

Biosynthesis of bone sialoprotein by a human osteoclast-like cell line (FLG 29.1).

Biosynthesis of bone sialoprotein (BSP) by a human osteoclastic cell line (FLG 29.1) during its differentiation induced by phorbol 12-myristate 13-acetate (TPA) was studied using metabolic radiolabeling experiments. The FLG 29.1 cells were metabolically radiolabeled with [3H]glucosamine and [35S]sulfate, and the labeled glycoproteins were analyzed by anion exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation experiments. One of the major glycoproteins synthesized by the TPA-treated FLG 29.1 cells was sulfated, had an identical electrophoretic mobility to purified BSP, and could be immunoprecipitated with a specific antibody against human BSP (LF 6). Thus, this glycoprotein was tentatively identified as the BSP. Furthermore, mRNA for BSP was also detected in TPA-treated FLG 29.1 cells by RNA-polymerase chain reaction. Most BSP synthesized by FLG 29.1 cells remained cell-associated, and this is in contrast with those synthesized by osteoblasts, where the protein is rapidly released into the extracellular matrix. Immunocytochemistry using an anti-BSP antibody showed a prominent paranuclear (suggestive of Golgi apparatus) localization of BSP in the TPA-treated FLG 29.1 cells after permeabilization, while untreated cells were not significantly immunostained. Localization of BSP at the plasma membrane was also demonstrated in the TPA-treated FLG 29.1 cells by the fluorescence-activated cell sorting analysis. Since TPA has been demonstrated to induce expression of various osteoclastic characteristics in FLG 29.1 cells, induction of BSP expression by TPA suggests that the protein may play a role during the differentiation process of osteoclasts or in functions of differentiated osteoclasts.

Amino Acid Sequence↗

PubMed1995

Glucagon-like peptide-1(7-36)amide induces the release of aspartic acid and glutamine by the ventromedial hypothalamus of the conscious rat.

Glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) and its own receptor have been found in the hypothalamus and brain stem of the rat. In an attempt to gain further insight into the role of this peptide in brain functioning we investigated the effects of GLP-1 (7-36)amide on the release of excitatory amino acid neurotransmitters by the ventromedial hypothalamus using an experimental microdialysis approach. GLP-1(7-36)amide produced an immediate increase in the extracellular concentrations of aspartic acid and glutamine, p < 0.01 and p < 0.05, respectively. By contrast, extracellular concentrations of glutamic acid, alanine, threonine, and tyrosine were unaffected. The results of this study show a stimulatory effect of GLP-1(7-36)amide on the release of aspartic acid and glutamine by the ventromedial hypothalamus of the rat.

Animals↗

PubMed1995

Structural characterization by affinity cross-linking of glucagon-like peptide-1(7-36)amide receptor in rat brain.

Specific binding of glucagon-like peptide (GLP)-1(7-36)amide was detected in several rat brain areas, with the highest values being found in hypothalamic nuclei and the nucleus of the solitary tract. In hypothalamus and brainstem homogenate binding of 125I-GLP-1(7-36)amide was time, temperature, and protein content dependent and was inhibited by unlabeled proglucagon-derived peptides. The rank order of potency was GLP-1(7-36)amide >> GLP-1(1-36)amide > GLP-1(1-37) approximately equal to GLP-2 > glucagon. Scatchard analysis of the steady-state binding data was consistent with the presence of both high- and low-affinity binding sites in hypothalamus and brainstem. Brain 125I-GLP-1(7-36)amide-binding protein complexes were covalently cross-linked using disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single radiolabeled band of M(r) 56,000 identified in both hypothalamus and brainstem homogenates was unaffected by reducing agents. An excess of unlabeled GLP-1(7-36)amide abolished the band labeling, whereas glucagon had no effect. Other unlabeled GLPs inhibited M(r) 56,000 complex labeling with the following order of potency: GLP-1(1-36)amide > GLP-1(1-37) > GLP-2. The binding of 125I-GLP-1(7-36)amide and the intensity of the cross-linked band were similarly inhibited in a dose-response manner by increasing concentrations of unlabeled GLP-1(7-36)amide. Covalent M(r) 56,000 125I-GLP-1(7-36)amide-binding protein complexes solubilized by Triton X-100 were adsorbed onto wheat germ agglutinin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

DATALOAD: a user-friendly program to prepare binding data for the "LIGAND" software.

This paper presents a program for the preparation of binding data to be used with the LIGAND software. This program will analyze saturation and competition/displacement binding data, and prepare all the necessary files that provide the input values for the final processing by the LIGAND package. Features include choices of different units for specific activity of the labeled tracer, and screen editing of the data, and results are provided as an average of replicates or as individual points. The program creates a graphic file to observe the raw data as a B/T vs T graph, prior to the analysis with the LIGAND program. The program saves all the information in a session file. Calculation of the bound/free values for the raw data is also provided.

Computer Graphics↗

PubMed1993

Molecular cloning of the rat adipocyte hormone-sensitive cyclic GMP-inhibited cyclic nucleotide phosphodiesterase.

Two distinct but related cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE) cDNAs were cloned from rat adipose tissue cDNA libraries. The open reading frame (3324 base pairs) of RcGIP1 encodes 1108 amino acids, including a hydrophobic membrane-associated domain in the NH2-terminal portion and, in the COOH-terminal portion, a putative catalytic domain conserved among all mammalian PDEs which is preceded by a putative regulatory domain that contains three consensus cAMP-dependent protein kinase phosphorylation sites and followed by a hydrophilic COOH-terminal domain. The carboxyl-terminal portion including the conserved domain was expressed as a glutathione S-transferase fusion protein and exhibited cAMP PDE activity which was inhibited by cilostamide, a specific cGI PDE inhibitor. RcGIP1 cDNA hybridizes strongly with RNA from isolated adipocytes, and its mRNA increases dramatically during differentiation of 3T3-L1 adipocytes. The deduced sequence of the second partial cDNA clone (RcGIP2 clone 53B) is highly homologous to the corresponding region of human cardiac cGI PDE cDNA. RcGIP2 cDNA hybridized strongly with rat cardiac tissue RNA and weakly if at all with RNA from rat adipocytes or 3T3-L1 fibroblasts or adipocytes. We suggest that RcGIP1 represents the hormone-sensitive, membrane-associated rat adipocyte cGI PDE and RcGIP2, a cGI PDE from vascular elements in rat adipose tissue.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1993

Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures.

The viscoelastic properties of culture medium obtained from confluent 3T3-L1 preadipocytes, after differentiation with isobutyl-methylxanthine and dexamethasone, were studied with a rotational Couette viscometer. In close association with adipocyte differentiation, the culture medium showed gel-like properties, in concert with an increase in viscosity. This behavior vanishes after digestion by Streptomyces hyaluronidase or chondroitinase ABC, but not after application of collagenase, pronase, trypsin, DNase, or neuraminidase, or by treatment with EDTA or mercaptoethanol, indicating that the primary substance responsible for this behavior is hyaluronic acid. The material revealed a non-Newtonian behavior with an irreversible disruption of the network by shear force at high speeds. The viscosity of the medium, containing about 1 microgram/ml of hyaluronic acid, was calculated to be similar to that of a solution containing 1.7 mg high molecular weight hyaluronic acid per milliliter of stock culture medium. The comparison of rheological properties between the culture medium and solutions of hyaluronic acid indicated the possibility of a highly organized network in the culture medium that is more complicated than a simple interaction between homologous hyaluronic acid molecules. The non-Newtonian behavior depends on the hyaluronic acid concentration in the medium as well as on the length of exposure of the 3T3-L1 cells to the isobutyl-methylxanthine/dexamethasone mixture. The results point toward the possibility of interaction between hyaluronic acid and binding proteins.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Seasonal variations in testicular LH, FSH, and PRL receptors; in vitro testosterone production; and serum testosterone concentration in adult male vizcacha (Lagostomus maximus maximus).

Seasonal changes in the reproductive activity of the adult male vizcacha (Lagostomus maximus maximus), a South American rodent, were investigated. Monthly, for 1 year, the animals were captured during the night near their burrows in the vicinity of San Luis, Argentina. The animals were killed, and samples of blood and the testes were collected. The testosterone serum levels were determined and the testicular tissue was used for biochemical and structural studies. Significant changes associated with seasonal cycles were found. In July-August (winter in South America), a short hibernal period of sexual quiescence, decline in testicular volume, and a significant decrease in serum testosterone were observed. The in vitro testosterone production and testicular LH, FSH, and PRL receptors revealed a significant decrease during this period. During September-November (spring season), recovery was observed, with a significant increase in gonadal activity during April-May (autumn season). During this season, the gonadal volume was increased, the serum testosterone and the in vitro stimulation of testosterone production by hCG (as well as the basal levels) also revealed a significant increase. Correspondingly, the concentration of testicular LH, FSH, and PRL receptors was elevated. These results indicate an increase in parameters related to testicular function. A gradual reduction in the testicular activity was observed in June-July (early winter). Our findings indicate that the male adult vizcacha, under natural conditions, exhibits characteristics of an annual reproductive cycle.

Animals↗

PubMed1992

Interleukin 6 reduces lipoprotein lipase activity in adipose tissue of mice in vivo and in 3T3-L1 adipocytes: a possible role for interleukin 6 in cancer cachexia.

To investigate whether interleukin 6 (IL-6) might be a potential mediator of the depleted fat reserves observed in malignancy-associated cachexia, we measured lipoprotein lipase (LPL) activity in adipose tissue of mice after administration of IL-6 or tumor necrosis factor and in cultured adipocytes after addition of these cytokines. Injection of IL-6 i.p. reduced adipose tissue LPL activity by 53% within 4.5 to 5.5 h. Injection of tumor necrosis factor elevated serum IL-6 levels and reduced adipose tissue LPL activity by 70%. Both human and murine IL-6 reduced heparin-releasable LPL activity in 3T3-L1 adipocytes in a dose-dependent manner; half-maximal inhibition of LPL activity was achieved with 5000 hybridoma growth factor units/ml. Thus, IL-6 reduces adipose LPL activity and may contribute to the loss of body fat stores associated with some cases of cancer cachexia. Since tumor necrosis factor increases circulating IL-6, some of its effects may be mediated or potentiated by IL-6.

3T3 Cells↗

PubMed1992

Mouse mammary epithelium produces a soluble heat-sensitive macromolecule that inhibits differentiation of 3T3-L1 preadipocytes.

Coculture of normal mouse mammary gland (NMMG) epithelial cells with 3T3-L1 preadipocytes resulted in inhibition of triglyceride accumulation. This inhibition was also observed when the NMMG cells were grown in inserts and placed within a 100-mm dish containing confluent 3T3-L1 cells. As the number of NMMG-containing inserts was increased, there was a progressive decline in triglyceride content of the 3T3-L1 cells. Conditioned medium from NMMG cells also resulted in a dose-dependent inhibition of adipocyte formation, and when concentrated 10-fold by passage through a filter with a cutoff of 30 kDa, all of the inhibitory activity was recovered. Heating the concentrated conditioned medium at 98 degrees C for 30 min resulted in complete loss of activity. Of several peptides tested, transforming growth factor-beta, platelet-derived growth factor, tumor necrosis factor, interleukin 6, and basic fibroblast growth factor showed inhibitory activity, whereas epidermal growth factor, insulin-like growth factor I, and transforming growth factor-alpha did not.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans.

The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with [35S]sulfate and [3H] glucosamine for 24 h and then extracted and analyzed. There was a 1.68 +/- 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of approximately 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of approximately 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 +/- 0.2-fold in media and 3.2 +/- 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Developmental changes in testicular interstitial cell populations, LH receptors and in the response to hCG in the rat.

High doses of hCG were administered to immature rats of different ages and the animals killed 48 h later. Serum testosterone increased 2 to 4-fold over control values 48 h after hCG. In-vitro androgen production showed different patterns according to age. Animals younger than 35 days, when treated with hCG, retained the ability to respond to in-vitro gonadotrophic stimulation. This ability was lost in testes from rats aged 45 days. The number of free LH-receptors 48 h after hCG diminished with increasing age to become non-detectable at 35 and 45 days. In control animals the proportion of differentiated Leydig cells in relation to their mesenchymal precursors increased progressively with age to reach highest values at 45 days. hCG administration induced a shift of the cellular composition of the interstitium toward the more mature cell types. hCG has a predominantly trophic action on mesenchymal precursors in young rats, promoting their differentiation. These effects are minimal in the differentiated Leydig cells in older animals. It is proposed that the observed biochemical responses are the result of the balance between the increase in LH receptors and steroidogenic enzymes in the developing new generation of young Leydig cells and the down-regulation of receptors and enzymatic lesions in fully differentiated Leydig cells.

Aging↗

PubMed1988

Prolactin differentially affects bacterial toxin-induced ADP-ribosylation of Nb2 lymphoma cell membrane proteins.

Exposure of lactogen-dependent Nb2 lymphoma cells to prolactin for up to 72 hr caused time- and dose-dependent changes in the ability of specific 38 kDa and 41.5 kDa membrane proteins from these cells to be subsequently ADP-ribosylated by pertussis and cholera toxins, respectively. Whereas the sensitivity of the 41.5 kDa substrate to cholera toxin was already reduced after 1 hour, that of the 38 kDa substrate for cholera toxin was increased for up to 72 hours. These findings suggest that membrane G-proteins may mediate the effects of prolactin binding to its receptor, leading to the proliferation of Nb2 lymphoma cells.

Adenosine Diphosphate Ribose↗

PubMed1987

DNA topoisomerase I from Leydig cells is modulated by luteinizing hormone and cyclic adenosine monophosphate.

The action of luteinizing hormone on topoisomerase I activity from rat Leydig cells was studied. Stimulation of the enzyme was observed after long-term (24 and 48 h) gonadotrophin treatment in in vivo experiments. No change could be detected for shorter times than 12 h using two different experimental approaches. Topoisomerase I was stimulated by cAMP in a whole cell extract in a phosphorylation-dependent manner. These results suggest that topoisomerase I could be a target for nuclear events induced by peptide hormone action.

Animals↗

PubMed1987

Possible interaction of the adrenal-gonadal systems on brain catecholamines of adult male rats.

Studies from this laboratory showed that gonadectomy (GDX) alters biogenic amines concentrations in diencephalon during the first 40 days. While the GDX females maintain the differences at day 60, the differences are eliminated in males at that time. In the present work, we have studied in three cerebral regions the adrenal involvement in the mechanism responsible for this normalization of catecholamine concentration in long-term castrated adult male rats. A hypersecretion of adrenal steroids seems to compensate for the lack of gonadal effect when the orchidectomized rats reach adulthood only for diencephalic dopamine.

Adrenal Glands↗

PubMed1987

Acute hCG administration induces seminiferous tubule damage in the adult rat.

In order to study the early (or short time) effects of an hCG stimulation on the seminiferous tubules of the adult rat, a single dose of 100, 200 or 400 IU hCG was administered to eighty to ninety day old rats of the Sherman strain. The histological analysis revealed a tubular damage already noticeable six hours after the injection. This precocious lesion becomes more pronounced two to five days later, consisting of degeneration and hypocellularity of the germinal epithelium, margination of the chromatin in round spermatids and formation of multinuclear giant cells. This damage involves big areas of the testis, particularly of peripheral location. The analysis performed three months after the acute stimulation still showed tubular regressive changes, thus indicating an incomplete reversion of the damage. Changes in the hormonal environment of hCG treated rats were found. Serum testosterone significantly increased from 6 to 72 hours following a 200 IU hCG injection. A severe diminution of serum FSH levels and a significant increase of the serum estradiol were observed. Intratesticular administration of estradiol benzoate was able to reproduce the tubular damage in some animals, while restoration of FSH circulating levels by simultaneous administration of hCG and purified hFSH did not prevent the changes induced by hCG alone. These results suggest that the high intratesticular level of estradiol and not the lowering of serum FSH might be the mechanism responsible for the described testicular injury.

Animals↗

PubMed1986

Photoperiod, follicle-stimulating hormone receptors, and testicular function in vizcacha (Lagostomus maximus maximus).

In the present work we investigated the presence of testosterone in serum and follicle-stimulating hormone (FSH) receptors in testes of the vizcacha (Lagostomus maximus maximus), a South American rodent. We also investigated the effect of constant light on both parameters. The control group consisted of vizcachas caught in their natural habitat and maintained under continuous darkness; the experimental group consisted of animals maintained under constant light (1076 lx) for 8 days. The results revealed a significant decrease in serum testosterone and FSH receptors when the animals were maintained under constant light, as compared to the control group. Androstenedione was elevated in the serum obtained from the experimental group. It is postulated that the pineal gland may regulate testosterone secretion through FSH receptors and through an enzymatic blockade in the steroidogenic pathway; this supposition, however, remains to be proved.

Androstenedione↗

PubMed1985

Effects of radioiodination on hormone binding ability.

This paper presents the effects of 125I labelling on human chorionic gonadotropin binding ability. The results obtained showed a marked difference in the half-life of the specific radioactivity of the radiolabelled hormone comparing the one obtained by "self-displacement" analysis and that calculated considering the half-life of the radioactive isotope (60 days for 125I). The decrease in the hormone half-life (t 1/2 = 3.10 days) was demonstrated by binding experiments performed at various days after hormone labelling. The affinity constant (Ka) and the number of binding sites obtained after Scatchard analysis using rat testes as receptor source, remained constant when the "self-displacement" specific radioactivity was used while they varied significantly if the 60 days half-life was considered for the data processing. The importance of the labelled hormone degradation as a possible cause of the decrease in the binding ability is also described.

Animals↗

PubMed1984

Hypothalamic-hypophyseal-gonadal axis in the streptozotocin-induced diabetic male rat.

In the present work it was observed that the diabetic state alters the hypophyseal response to castration, without the expected increase in the LH serum levels, as found in the control rats. On the other hand, the stimulation of the hypophysis with LHRH resulted in a lower response in the case of the diabetic animal. The results presented herein are in good agreement with the finding of a reduction in the number of androgen binding sites and also with a diminished activity of the 5 alpha-reductase in the hypophysis from diabetic animals. The present results indicate an alteration in the hypophyseal gonadotropin production as well as in the overall process of hypophyseal response in experimental diabetes.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗