Characterization of thyrotropin-releasing hormone in the central nervous system of African lungfish

Central administration of thyrotropin-releasing hormone (TRH) produces potent effects on various physiological parameters, such as arousal, respiration, and cardiovascular function, in several species. As part of an investigation into the evolution of this tripeptide as a central modulator of these parameters, we examined its distribution in the central nervous system of the African lungfish (Protopterus). Lungfish brains were dissected into three regions: telencephalon, diencephalon, and medulla. Each region was assayed for TRH by radioimmunoassay and for norepinephrine, dopamine, and serotonin by HPLC/electrochemical methods. TRH immunoreactivity (IR-TRH) was present in all regions of lungfish brain examined. The telencephalon contained the highest concentrations of TRH, the diencephalon also contained a high concentration of TRH, and the medulla contained a markedly lower concentration. Similar concentration gradients (telencephalon greater than diencephalon greater than medulla) were observed for norepinephrine, dopamine, and serotonin. The identity of IR-TRH as authentic TRH was confirmed by elution profiles on HPLC. The results of this investigation demonstrated that TRH and the monoamine neurotransmitters are present in high concentrations in various regions of lungfish brain. The lungfish may represent a promising model for further studies of the interactions of TRH with these neurotransmitter systems.     

Covalent binding of the endogenous estrogen 16 alpha-hydroxyestrone to estradiol receptor in human breast cancer cells: characterization and intranuclear localization

The interactions of 16 alpha-hydroxyestrone (16 alpha-OHE1), a metabolite of estradiol (E2), with estrogen receptors (ERs) were compared in this study to the classic E2-receptor mechanism in human breast cancer cells MCF-7 in culture. When MCF-7 cells were incubated with radioinert 16 alpha-OHE1 or its 3H-labeled form for 4 weeks, the estrogen bound extensively and irreversibly in a time-dependent fashion to nuclear protein species that correspond to the ER. Here we show that the interactions of 16 alpha-OHE1 with the ER are different from those of E2 with the receptor. Dissociation of tritiated E2-ER or 16 alpha-OHE1-ER complexes, salt extraction, DNase and proteinase K digestion, and ethanol treatment demonstrated that the binding of 16 alpha-OHE1 to the ER corresponds to two different forms: a classical noncovalent interaction similar to that of E2, and a covalent adduct formation between the metabolite and the ER. These complexes localized preferentially in nuclear matrix components as revealed by cell fractionation and probing with a monoclonal anti-ER antibody. [3H]16 alpha-OHE1-ER complexes analyzed by polyacrylamide gel electrophoresis demonstrated a radiolabeled band at approximately 66 kDa that was absent when the exposure of cells was done in the presence of E2 in competition and that was also absent in [3H]E2 incubations. The present results when considered together with our previous findings of elevated activities of estrogen 16 alpha-hydroxylase, the enzyme responsible for the formation of 16 alpha-OHE1, in breast cancer patients and in women at enhanced risk for the disease, suggest that covalent modification of the ER may be one mechanism of malignant transformation in estrogen target tissues.     

Choleragen (cholera toxin): a bacterial lectin.

Choleragen (cholera toxin) agglutinated erythrocytes and liposomes containing the toxin receptor, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (ganglioside GM1). Cells that had been exposed to GM1 were agglutinated, but agglutination was not observed when cells had been exposed to other gangliosides (GM2, GM3, GD1a, GD1b). Choleragen-dependent agglutination of liposomes was slightly less specific, because liposomes containing either GM1 or GD1b, but neither GM2, GD1a, nor GM3 were agglutinated. The oligosaccharide isolated from GM1 inhibited both the agglutination of cells and liposomes containing GM1 and the binding of choleragen to liposomes containing GM1. Galactose and sialic acid were less effective inhibitors of liposomal agglutination and did not inhibit cellular agglutination or binding of choleragen to liposomes. Liposomal agglutination was dependent on choleragen concentration and occurred with the B but not the A protomer of choleragen. These results suggest that choleragen, through its binding to the oligosaccharide portion of a glycolipid, exhibits lectinlike activity, which results in agglutination of liposomes and erythrocytes.     

Endogenous digitalis-like activity in mammalian brain.

A fraction of brain has been prepared that contains a substance mimicking actions of the digitalis glycosides. It both blocks the binding of [3H]ouabain to Na+,K+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and inhibits the uptake of 86Rb+ into human erythrocytes. Partial isolation was achieved by Sephadex G-10 fractionation and subsequent desalting. This activity is potentially capable of regulating ionic fluxes and concentration gradients across the cell membrane.     

Mortality among Homeless Men

The mortality in a series of 6,032 homeless men in Stockholm was investigated. The observation period was up to 3 years. The observed number of deaths amounted to 327 compared to the expected 86·7, corresponding to an excess mortality ratio of about 4. Mortality was raised for all the causes of death studied. Most striking, however, was excess mortality from accidents and diseases of the digestive and respiratory systems. Accidental deaths were remarkably numerous, indicating that these are largely correlated to the social consequences of abuse. The same applies to deaths from pulmonary tuberculosis. Excess mortality from suicide was lower than that found in some earlier studies. This discrepancy is discussed. In comparison with the general population the high mortality from diseases such as cirrhosis of the liver and pancreatitis shows that many medically serious cases of alcoholism are included in the “Homeless Men” group.     

Medical records contaminated with dried blood: A quality issue.

A routine chart review over 23 months in a 256-bed community hospital revealed 246 medical records contaminated with apparent blood. Sixty percent of the records were nursing and anesthesiology records. Analysis of systematically selected records confirmed blood as the visible contaminant in 27% of the cases (8/30). Total quality improvement methodology reduced the incidents by 75%. Actions included policy development, in-service education, and changes in work practices. Although bloodborne pathogen transmission is statistically improbable, we should improve work practices to eliminate blood contamination of charts.     

Cross reactivity of enterohemorrhagic Escherichia coli O157:H7-specific sera with non-O157 serotypes

Escherichia coli O157:H7 is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a Type III secretion system, a mechanism shared by other enterohemorrhagic E. coli (EHEC). We recently reported that vaccination of cattle with Type III-secreted proteins (TTSPs) resulted in decreased shedding of the organism following both experimental infection as well as under conditions of natural exposure. In order to extend this to non-O157 EHEC serotypes, we examined the serological cross reactivity of TTSPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Western blotting experiments with polyclonal antisera directed against serotype O157:H7 TTSPs suggested that there was significant cross reactivity, although there was limited cross reactivity when two Tir- and EspA-specific monoclonal antibodies were used. Groups of cattle were then vaccinated with TTSPs produced from each of the above serotypes and the magnitude and specificity of the responses were measured. All animals responded well with antibodies to TTSPs of the homologous serotype. However, limited cross reactivity was observed against the others. No cross reactivity was observed against Tir and EspA of serotype O157:H7. These results suggest that vaccination of cattle with TTSPs as a means of reducing the risk of EHEC transmission to humans will induce protection that is serotype specific.     

Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin.

Chemically transformed mouse fibroblasts did not raise their cyclic AMP level in response to Escherichia coli heat-labile enterotoxin. These fibroblasts did, however, incorporate exogenous mono-, di-, and trisialogangliosides. After the uptake of monosialoganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM1), the cells responded to E. coli heat-labile enterotoxin. The di- and trisialogangliosides were considerably less effective. GM1, the putative cholera toxin (choleragen) receptor, has been implicated previously as the receptor for E. coli heat-labile enterotoxin based on the ability of the free ganglioside to inhibit the effects of toxin. This investigation establishes that the ganglioside, when incorporated into fibroblasts, serves a functional role in mediating the responsiveness to the toxin.     

Models of congenital heart disease in fetal lambs

Intracardiac flow patterns were chronically altered by partially obstructing left ventricular (LV) inflow or outflow in midgestational fetal lambs. Physiological measurements of the fetal circulation were made serially through indwelling catheters and the use of radioactive microspheres. With LV inflow obstruction, mean LV output (LVO) decreased to 30% of control (P less than 0.01). Within seven days, the LV/right ventricular (RV) weight ratio decreased to 70% of control (P less than 0.01), and the mean LV/RV chamber volume decreased to less than one-half of control (P less than 0.001), simulating an early form of the hypoplastic left heart syndrome. With LV outflow obstruction, mean LVO decreased to 64% of control (P less than 0.05). Mean LV/RV wall thickness doubled (P less than 0.0001) and mean LF/RV chamber volume decreased to less than one-half of control (P less than 0.0001). Within four to ten days after increasing LV afterload, a large increase in LV mass occurred, which was demonstrated by morphometric analysis to be due to hyperplasia of ventricular myocytes. LV chamber volume decreased somewhat, simulating moderately severe congenital aortic stenosis. Over the long term (30--36 days), the mean LV/RV weight ratio decreased and the LV chamber was nearly obliterated, simulating very severe congenital aortic stenosis. The results suggest that by varying preload and afterload in both ventricles of the fetus, various forms of congenital heart disease may be simulated.