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Biomedical subjects

A Gertler

Publications and source records attributed to A Gertler.

At least 109 records · Page 6Linked to original sources

Effect of prolactin on casein and fat synthesis and casein secretion in explants of bovine mammary tissue pretreated in athymic nude mice.

Bovine mammary gland tissue from 4- to 6-mo-old calves was grafted s.c. (dorsum) to 7- to 9-wk-old female athymic "nude" mice. The graft-bearing mice were divided into three groups and were treated with the following hormonal combinations: 1) implantation of a pellet containing estrogen and progesterone followed by injection of estrogen and progesterone, 2) injections of estrogen and progesterone, and 3) injections of estrogen, progesterone, and prolactin. Following in vivo treatment, the grafts were removed and prepared for organ culture. Explants from each treatment were subdivided into two hormonal regimens: 1) insulin and cortisol; 2) insulin, cortisol, and prolactin. An increase in in vitro fat synthesis was observed in explants cultured in the presence of prolactin. This increase was not affected by previous in vivo hormonal treatment. In vitro casein synthesis was not stimulated by the presence of prolactin in the culture medium; however, casein secretion was elevated in explants from grafts from animals that were treated in vivo with estrogen, progesterone, and prolactin. Casein secretion was further increased in explants cultured in the presence of prolactin but rapidly dropped in its absence. Explants that were not implanted in mice responded in long-term organ culture similarly to explants pretreated in vivo.

Animals↗

[Fixateur externe as a definitive treatment method of unstable thoracolumbar spinal fractures in emergency conditions--technical modification and clinical experiences].

A survey on 31 injuries of thoracolumbar spine, 27 were accompanied with neurological deficiency. Treatment was exclusively by means of percutan attached f.e. until bony consolidation. An average treatment time of 152 days took place. Mean rate of correction was about 0.24 (Beck's SI-Index). The former applied frame construction was substituted by self developed spindle fixateur system, adapted to the requirements of traumatology of the spine in emergency situations.

Adolescent↗

Inhibition of the proliferation of Nb2 cells by femtomolar concentrations of cholera toxin and partial reversal of the effect by 12-O-tetradecanoyl-phorbol-13-acetate.

One hour of exposure to cholera toxin is sufficient to elicit a significant delay in the initiation of DNA synthesis and cell division in lactogenic hormone-dependent Nb2-11C lymphoma cells. The inhibitory effect occurs already at very low concentrations of cholera toxin (5-50 fM), at which it is not accompanied by a detectable increase in intracellular cAMP, or ADP-ribosylation of the alpha subunit of Gs, the stimulatory guanine nucleotide binding protein of adenylate cyclase; IBMX, the phosphodiesterase inhibitor, acts synergistically to cholera toxin, indicating that a minute increase in cAMP may be sufficient for the inhibition. This indication is substantiated by the finding that dibutyryl cAMP also inhibits cell proliferation. Phorbol diester reverses partially the inhibitory activity of cholera toxin. It is most likely that this effect does not result from blocking the increase in cAMP, but rather from some subsequent, yet unidentified, events. The inhibitory effect of cholera toxin is not dependent on the concentration of the proliferation-stimulating lactogenic hormone and cannot be abolished or reduced by excess of the hormone. Cholera toxin also inhibits the autonomous proliferation of a lactogenic hormone-independent cell line (Nb2-SP); however, in this case the inhibition is not affected by TPA.

Animals↗

Preparation of high-titre anti-P1 sera for blood typing in man.

For the last 25 years, antisera for testing P1 in man have been obtained by i.v. injections in goats using hydatid cyst fluid from livers of pigs infected by Echinococcus granulosus. Due to the fact that this antigen has become very rare today, efforts have been made by the authors to substitute it by different antigens and to save antigen by modifying the technique of immunisation as well. By this, very high titres up to 1:4096 could be reached after specification absorption.

Animals↗

Post-mortem examination and certification of death under G.D.R. law.

Post-mortem examination and certification of death in the G.D.R. are regulated by the Order on the Medical Post-mortem Examination (O.M.P.E.) issued in 1949 and confirmed in 1978. The tasks of the doctor certifying death are described as well as the co-operation between police, district attorney, and pathologist, and legal doctor, respectively, in all cases of death 'under suspicious circumstances.' Medical as well as judicial aspects of post-mortem examination are discussed.

Autopsy↗

Effect of insulin-like growth factor I on deoxyribonucleic acid synthesis and galactopoiesis in bovine undifferentiated and lactating mammary tissue in vitro.

We have demonstrated that insulin-like growth factor I (IGF-I), at physiological concentrations, is a potent mitogen of bovine undifferentiated mammary epithelial cells cultured in collagen in serum-free medium. Its activity is independent of insulin, although at pharmacological concentrations insulin may substitute for IGF-I. The maximal [3H]thymidine incorporation stimulated by either IGF-I or insulin was only 25-40% of that in medium supplemented with 10% fetal calf serum (FCS) only. Epidermal growth factor (EGF) exhibited low mitogenic activity which was not synergistic with IGF-I in serum-free medium. IGF-I and EGF had low synergistic activity when added separately to 10% FCS-supplemented medium. Strong synergism (100% or more) was observed, however, when both factors were added simultaneously, indicating that their maximum mitogenic effect is dependent on a simultaneous presence of other factors existing in FCS. The galactopoietic effect of IGF-I was tested in organ culture of bovine lactating mammary gland. Neither fatty acid synthesis nor alpha-lactalbumin secretion was stimulated by IGF-I, even at 2000 ng/ml. These results indicate that, at least in our in vitro system, galactopoiesis is not affected by IGF-I.

Animals↗

Characterization of lactogen receptors in lactogenic hormone-dependent and independent NB2 lymphoma cell lines.

A comparative study of lactogen receptors in lactogenic hormone-dependent (Nb2-11C) and independent (Nb2-SP) rat lymphoma cell lines revealed no significant differences in their binding and structural characteristics. The affinity for human growth hormone (hGH) and ovine prolactin is identical (Kd = 1.3 X 10(-10) M). Exposure to hGH results in a rapid receptor down-regulation. This process is faster in Nb2-SP cells, however, similar receptor levels are reached within 3 h. Affinity labeling revealed 2 major binding species (Mr 75,000 and 29,000) in both cell lines. These observations suggest that the spontaneous proliferation of Nb2-SP cells probably results from an aberration in some post-receptoral event of the mitogenic pathway.

Animals↗

Partial purification and characterization of bovine mammary gland prolactin receptor.

Prolactin (PRL) receptors from the mammary gland of the lactating cow were solubilized with 3-[(3-cholamidopropyl)dimethylamonio]-1-propane sulfonate (CHAPS). Affinite chromatography on human growth hormone (hGH) coupled to Affi-Gel 10 resulted in over 500-fold purification, as compared to microsomal fractions. Scatchard analysis of the binding of hGH indicated an increase in the affinity constant of 2.5-fold after solubilization and of further 2-fold after the affinity purification. The specific binding activity of the affinity-purified fraction was 9000 fmol hGH/mg protein. Complexes of Triton X-100-solubilized receptors with [125I]hGH were analyzed by gel filtration on Sephadex G-150, in the presence of Triton X-100. A minor fraction of the complexes eluted as high molecular weight (Mr) aggregates, whereas a major fraction eluted as a 150 kDa peak. Assuming a contribution of approximately 30% to the Mr by the bound detergent and a hormone: receptor ratio of 1:1 in the complex, a Mr of 80-85 kDa can be calculated for the receptor molecule. Affinity labelling of the receptor with [125I]hGH revealed a Mr of 37 +/- 0.5 kDa (n = 7) for the binding subunit. Specific high Mr aggregates were also observed after crosslinking; however, the size of the labelled species was not affected by reducing agents. Homologous and heterologous competitive binding studies with ovine PRL (oPRL) or hGH revealed a considerably higher affinity for hGH as compared to oPRL. The competitive displacement patterns obtained with oPRL or hGH as tracers were similar, indicating that both hormones bound to the same receptor sites with different affinities. A similar difference in affinity was retained by the affinity-purified receptors.

Affinity Labels↗

Comparative study of in vitro and in vivo modulation of lactogenic and somatotropic receptors by native human growth hormone and its modified analog prepared by recombinant deoxyribonucleic acid technology.

A modified analog of human GH (hGH), prepared by recombinant DNA technology, that lacks 13 amino acids at the amino terminus (Met14hGH), was able to compete with [125I]hGH for binding to lactogenic receptors in Nb2-11C rat lymphoma cells, to somatotropic receptors in IM-9 human lymphocytes, and to both lactogenic and somatotropic receptors in the microsomal fraction of virgin female rat liver. Exposure of intact Nb2 or IM-9 cells to Met14hGH did not reduce the number of surface or intracellular receptors, as compared to the control without hormone. A parallel exposure to 500-fold lower concentrations of hGH resulted in 77-93% reduction in both surface and intracellular receptors. In contrast to [125I]hGH, [125I]Met14hGH was not taken up by the intact Nb2 lymphoma cells. Infusion of anesthetized female virgin rats for 3 h with hGH down-regulated both lactogenic and somatotropic receptors in the liver. A similar infusion with up to 200-fold higher amounts of Met14hGH did not lower the number of total receptors, indicating lack of down-regulation. Some decrease in the binding to free receptors was observed, suggesting that Met14hGH is capable of binding to liver receptors in vivo.

Animals↗

Inhibition of casein and fat synthesis and alpha-lactalbumin secretion by progesterone in explants from bovine lactating mammary glands.

The effect of progesterone on casein and fat synthesis, on alpha-lactalbumin secretion and on accumulation of casein mRNA was studied in explants from bovine lactating mammary glands, cultured in the presence of insulin, cortisol or prolactin. All four biological activities were inhibited down to or below the level obtained in medium without prolactin. However, inhibition was only achieved by using concentrations of progesterone exceeding by 1000-fold those required for similar effects in tissue from non-lactating animals. A similar inhibitory effect was also found using high levels of other steroids without 11 beta-hydroxyl, such as deoxycorticosterone and dehydroepiandrosterone, and to some extent with oestradiol-17 beta, but not with cortisol or corticosterone. The inhibition by progesterone did not result from a decrease in explant viability, as determined by glucose uptake.

Animals↗

A comparative study of lactogenic hormone binding sites in the adrenal gland, ovary and kidney of the lactating cow.

The specific binding of 125I-oPRL to microsomal fractions from the adrenal gland, ovary and kidney of the lactating cow was significantly lower than binding of iodinated hGH. In addition, the ability of oPRL to compete with iodinated hGH as compared to hGH, was 50-fold lower in the adrenal gland 35-fold lower in the ovary and 18-fold lower in the kidney. These results are similar to those obtained in the mammary gland and liver, whereas the ability of oPRL to compete with iodinated hGH was 90-fold lower, as compared to hGH. In the kidney the difference between the binding of iodinated hGH and iodinated oPRL was smaller. Results obtained with a solubilized kidney microsomal fraction also show a slightly higher affinity for oPRL than in other tissues. Thus the phenomena of differential affinities of oPRL and hGH to lactogenic hormone binding sites, characterizes most lactogenic hormone target tissues in the lactating cow. The distribution of these sites in different parts of the tissues was also studied. In the adrenal gland, the binding capacity in the cortex was 8-fold higher than in the medulla. In the ovary most of the binding sites were found in the corpus luteum, while in the kidney the binding capacity was higher in the cortex as compared to the medulla.

Adrenal Glands↗

Down-regulation of lactogenic hormone receptors in Nb2 lymphoma cells by cholera toxin.

Exposure of lactogen-dependent (Nb2-11C) and lactogen independent (Nb2-SP) lymphoma cells to cholera toxin (0.05-50 pM) resulted within 18-28 h in a 50% decrease in the binding capacity of the intact cells to iodinated human growth hormone, and 40% decrease in cell-homogenates. Scatchard analysis revealed that the reduction in binding resulted from loss of cell-surface receptors accompanied by degradation of intracellular receptors. No alterations in receptor binding affinity were observed. One to 3 h of exposure to the toxin was sufficient to reduce the binding to the level obtained after continuous incubation with the toxin for 28 h. Addition of dibutyryl cAMP (0.1mM) to the medium resulted in similar down-regulation of lactogenic receptors.

Animals↗

Short-term regulation of prolactin receptors in the liver, mammary gland and kidney of pregnant and lactating rats infused with ovine prolactin or human growth hormone.

Short-term regulation of prolactin (PRL) receptors was studied in ketamine-anaesthetized 18-day pregnant or 7-day lactating female rats, by infusing them with various doses of oPRL or human growth hormone (hGH) for 0-3 h and measuring the binding of [125I]oPRL of [125I]hGH to the microsomal fractions prepared from the liver, mammary gland and kidneys of animals sacrificed at various states of infusion. Our main findings are: In pregnant rats, only 30% of liver receptors are unoccupied and infusion with 25 micrograms/h for 3 h of either oPRL of hGH decreased both free and total receptors by 22-30% while infusion with 250 micrograms/ml caused an additional decrease only in the free receptors. In the mammary gland and the kidney of pregnant rats, all receptors seem to be unoccupied; infusion with 25 micrograms/ml had none or a slight elevating effect on the number of both free and total receptors in the mammary gland but caused a significant 3-fold increase in the kidney; infusion with 250 micrograms/ml, however, resulted in a slight decrease in the mammary gland and a significant decrease in the kidney in both total and free receptors. In the liver of the lactating rats, there was no significant difference between the number of free and total receptors, but in mammary gland, specific binding to the total receptor was higher than to the free ones indicating partial occupancy; infusion with 25 micrograms/ml caused a significant decrease in free and total liver receptors without a remarkable change in the mammary gland and some decrease (by infusion with hGH only) in the kidney. In all cases, the changes in the specific binding resulted from the increase or decrease in receptor number and not from the change in receptor-hormone affinity. In almost all cases, infusion with oPRL or hGH yielded similar results. Infusion with both hormones did not affect the level of the endogenous rat prolactin. In conclusion, our results indicate the short-term regulation of PRL receptors by exogenous hormones is a complicated process which is affected by the level of the infused hormone, physiological state of the animal and may yield, simultaneously, different or even opposite changes in receptor number in various organs.

Animals↗

Human growth hormone-stimulated mitogenesis of Nb2 node lymphoma cells is not mediated by an immediate acceleration of phosphoinositide metabolism.

Lactogenic hormones stimulate the mitogenesis of Nb2-11C rat lymphoma cells and this stimulation is enhanced by 12-O-tetradecanoyl phorbol ester (TPA). The effect of human growth hormone (hGH) and TPA on phosphoinositide metabolism in Nb2-11C cells was tested by measuring the incorporation of [3H]myo-inositol or 32P or measuring the breakdown of polyphosphoinositides in cells prelabeled with [3H]myo-inositol, 32P or [3H]arachidonic acid. We found that none of these processes is immediately stimulated by either hGH and/or TPA. Our results indicate that the overall phosphoinositide turnover is slow or confined to small fractions only. On the other hand, the phosphorylation-dephosphorylation cycle of polyphosphoinositides is quite fast and remarkably exceeds the rate of incorporation of [3H]myo-inositol. Our present results indicate, therefore, that the mitogenic effect of hGH and its enhancement by TPA is not mediated by an immediate increase in phosphoinositide metabolism.

Animals↗

A model for in vitro proliferation of undifferentiated bovine mammary epithelial cells.

A method for culturing bovine undifferentiated mammary epithelial cells embedded in collagen gels is described. Cell growth was quantitated by incorporation of tritiated thymidine. Maximal rate of incorporation was achieved in media supplemented with sera, however considerable growth was observed in serum-free medium supplemented with insulin, transferrin and selenium. The growth promoting effect of insulin was maximally expressed only at high nonphysiological concentrations.

Animals↗

Partial amino acid sequence of porcine elastase II. Active site and the activation peptide regions.

The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (Mr = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP-Ser195. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.

Amino Acid Sequence↗

Inhibition of lactogenic activities of ovine prolactin and human growth hormone (hGH) by a novel form of a modified recombinant hGH.

A recombinant analog of human GH (hGH) lacking 13 amino acids at the amino-terminus (Met14hGH) inhibited the hGH- or ovine PRL (oPRL)-stimulated proliferation of Nb2 lymphoma cells and bovine PRL-stimulated fat synthesis and alpha-lactalbumin secretion in explants from bovine lactating mammary gland. The inhibition was competitive in nature, and in Nb2 cells could be abolished by an excess of hGH or oPRL. Inhibition of oPRL-stimulated proliferation of Nb2 cells by Met14hGH could also be specifically abolished by anti-hGH monoclonal antibodies. Met14hGH had no growth-stimulating activity in Nb2 cells and was not cytotoxic. It also did not affect glucose uptake by the mammary gland explants. Met14hGH competed with [125I]hGH for binding to intact Nb2 cells, IM-9 lymphocytes, solubilized microsomal fraction from lactating bovine mammary gland, and microsomal fraction from the liver of female virgin rats, but its affinity for those receptors was 2 orders of magnitude lower than the affinity of hGH. Since Met14hGH used in most experiments contained about 25% impurities and degradation products, a small amount of it was further purified by immunoaffinity chromatography. Two purified fractions, one consisting of a single 20K protein and the other accompanied by a small amount of 25K protein, were obtained. Both fractions exhibited increased inhibition of hGH- or oPRL-stimulated proliferation of Nb2 cells, thus indicating that the inhibitory activity results from the intact Met14hGH molecule. To the best of our knowledge, this is the first report describing the inhibition of lactogenic hormone activities by a modified hGH.

Animals↗