Detection of P-glycoprotein on endothelial and endocrine cells of the human pancreatic islets by C 494 monoclonal antibody

Summary P-glycoprotein, an integral membrane protein acting as an energy-dependent efflux pump, has been detected immunocytochemically in the human pancreatic islets using C 494 monoclonal antibody. Intense P-glycoprotein immunoreactivity was found in both endothelial cells of islet blood capillaries and in endocrine cells. Strong expression of P-glycoprotein has been found in the capillary blood vessels at blood-tissue barrier sites and in numerous kinds of cells with secretory/excretory function. Therefore the present findings suggest that P-glycoprotein may play a role in controlling the composition of the extracellular fluids and the intracellular milieu of endocrine islet cells and possibly in regulating their secretory activity.     

Correlative studies between the presence of thyrotropin-releasing hormone (TRH) receptors and the in vitro stimulation of growth-hormone (GH) secretion in human GH-secreting adenomas

Specific receptors for TRH were characterized on cellular membranes of 6 out of 13 somatotrophic adenomas obtained from acromegalic patients. These receptors had the same dissociation constant (Kd: 62 +/- 10 nM) as those found in human PRL-secreting adenomas, but their maximal number of binding sites (Bmax: 76 +/- 24 fmol/mg of protein) was six fold smaller. A good correlation was found between the presence of TRH receptors and the in vitro TRH-induced stimulation of GH secretion. The increase in GH release varied from 25 to 200%. It was thus concluded that these receptors are functional. However, why only some of the human somatotrophic adenomas possess TRH receptors and respond to TRH in vitro needs further investigations.     

Kinin-converting aminopeptidase from human urine. Further purification and characterization through kinetic and inhibitory studies

An aminopeptidase from human urine (HUA) able to hydrolyze L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and to convert both MLBK and LBK to BK has been further purified and characterized. The preparation now obtained showed a 3-fold higher specific activity than the previously described one and a single active protein band in 7% polyacrylamide gel electrophoresis accounting for 86% of total protein. Kinetic constants for this kinin-converting enzyme were determined using L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and LBK. The Km values for different naphthylamides were in the 10(-5) M range while that for L-Leu-p-nitroanilide was 3.6 X 10(-4) M. With LBK as substrate the aminopeptidase activity showed the highest catalytic efficiency in spite of a Km in the mM range. The enzyme was poorly inhibited by -SH and -S-S- group reagents. Some L-aminoacids, as well as mono- and diamines, indomethacin, puromycin and bestatin were equipotent competitive inhibitors of both arylamidase and aminopeptidase activities. Results obtained in this paper are compatible with our conclusion that human urine, unlike other enzyme sources, contains only one aminopeptidase, and that this enzyme displays both arylamidase and kinin-converting activities. The enzyme's action may be important in the metabolism of kinins, yielding peptides which could interact with both B-1 and B-2 kinin receptors in the kidney.     

Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.     

Role of Hydroxyl Radicals in Escherzchza Colz Killing Induced by Hydrogen Peroxide

Escherichia coli lethality by hydrogen peroxide is characterized by two modes of killing. In this paper we have found that hydroxyl radicals (OH -) generated by H₂O₂ and intracellular divalent iron are not involved in the induction of mode one lethality (i.e. cell killing produced by concentrations of H₂O₂ lower than 2.5 mM). In fact, the OH radical scavengers, thiourea, ethanol and dimethyl sulfoxide, and the iron chelator, desferrioxarnine, did not affect the survival of cells exposed to 2.5mM H₂O₂. In addition cell vulnerability to the same H₂O₂ concentration was independent on the intracellular iron content. In contrast, mode two lethality (i.e. cell killing generated by concentrations of H₂O₂ higher than 10mM) was markedly reduced by OH radical scavengers and desferrioxamine and was augmented by increasing the intracellular iron content.

It is concluded that OH. are required for mode two killing of E. coli by hydrogen peroxide.     

Action of Cystine in the Cytotoxic Response of Escherichia Coli Cells Exposed to Hydrogen Peroxide

Cystine markedly enhanced the cytotoxic response of Escherichia coli cells to concentrations of hydrogen peroxide resulting in mode one killing, but displayed little effect in mode two killed cells. The effect of cystine was concentration-dependent over a range of 5-50 μM and did not further increase at higher levels. Cystine had similar effects in other bacterial systems.

In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells. In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant. The enhancing effect of cystine on oxidative injury of E. coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide. Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant. The effect of the disulfides was not concentration-dependent over a range of 200-800 μM and was statistically significant only for cystamine.

Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide. This effect may involve a thiol-disulfide exchange reaction at the cell membrane level.     

Role for autocrine TGF-β1 in regulating differentiation of a human leukemic cell line toward osteoclast-like cells

Increasing evidence suggests that transforming growth factor-β (TGF-β) is involved in bone formation during remodeling. Using a recently cloned human leukemic cell line (FLG 29.1 cells) we demonstrate that these cells synthesize and secrete TGF-β1 and that exogenous or autocrine TGF-β1 can induce the same features of osteoclastic-like cells, exerting its effects through the binding to TGF-β specific receptors. Scatchard analysis of ¹²⁵I-labeled TGF-β1 to FLG 29.1 cells revealed the presence of a single high affinity binding site with a Kd value of ～25 pM and a binding capacity of ～900 sites/cell. Affinity labeling experiments showed that FLG 29.1 cells express type I and type II TGF-β receptors. Stimulation of FLG 29.1 cells with low TGF-β1 doses reduced cell proliferation and increased cell adhesion and tartrate resistant acid phosphatase (TRAcP) activity. Pretreatment of FLG 29.1 cells with TGF-β1 caused a significant and dose-dependent response to calcitonin. Northern blot of total mRNA and analysis of the conditioned media (CM) showed that TGF-β1 was synthesized by FLG 29.1 cells. TPA treatment, which induces partial differentiation of these cells, markedly increased TGF-β1 mRNA expression and growth factor release. The majority of TGF-β1 secreted by TPA-treated cells was in its latent form. However, anti-TGF-β antibodies inhibited TGF-β1 and TPA-induced growth inhibition, calcitonin responsiveness, and TRAcP activity, suggesting that the TPA effect is mediated in part by autocrine TGF-β1 and indicating that the cells can activate and respond to the TGF-β that they secrete. These findings support a potential autocrine role for TGF-β1 in osteoclast differentiation. © 1994 Wiley-Liss, Inc.     

Homozygotes for the autosomal dominant neoplasia syndrome (MEN1).

Families in which both parents are heterozygotes for the same autosomal dominant neoplasia syndrome are extremely unusual. Recently, we had the unique opportunity to evaluate three symptomatic siblings from the union between two unrelated individuals affected by multiple endocrine neoplasia type 1 (MEN1). When the three siblings and their parents and relatives were genotyped for 12 markers tightly linked to the MEN1 locus, at 11q13, two of the siblings were found to be homozygotes, and one a heterozygote, for MEN1. With regard to the MEN1 syndrome, no phenotypic differences were observed between the two homozygotes and the heterozygotes. However, the two homozygotes showed unexplained infertility, which was not the case for any of the heterozygotes. Thus, MEN1 appears to be a disease with complete dominance, and the presence of two MEN1 alleles with mutations of the type that occur constitutionally may be insufficient for tumor development.     

Survival of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria in soil

The survival of mesophilic Aeromonas spp. in soil in the presence or absence of indigenous microflora was evaluated in a laboratory study. Two cytotoxic ( Aer. hydrophila and Aer. caviae ) and one invasive ( Aer. sobria ) clinical isolate strains were selected for this study. After contamination of sterile or unsterilized soil with the three strains of Aeromonas , the number of living cells was determined over at least 5 months. For all strains the survival curves were characterized by an initial re-growth followed by a slow inactivation of bacteria, with significant differences due to the presence of indigenous microflora. The times necessary to achieve a 95% reduction of the initial population were > 140, 113 and 62 d in sterilized soil respectively for Aer. caviae, Aer. hydrophila and Aer. sobria , while the corresponding times in unsterilized soil were 42, 38 and 11 d. All strains preserved the virulence factors for the entire period of the study. These results suggest that the soil may be an important reservoir for Aeromonas spp. and, thus, may play an important role in the epidemiology of Aeromonas -associated human infections.