Tryptophan environments in glutathione transferase of human placenta from temperature-dependent phosphorescence studies

An investigation of the tryptophan emission properties of glutathione transferase from human placenta was conducted in order to characterize the environments of the two aromatic residues. The low-temperature phosphorescence spectra and temperature dependence of the phosphorescence quantum yield of the tryptophan residues revealed a difference in the chemical nature and dynamical structure of the surrounding protein matrix. Thus, one tryptophan residue seems to be deeply embedded within the polypeptide in a rigid weakly polar environment, characteristic of a beta-type secondary structure. The other is located in a more polar site, probably near the surface, in a rather flexible region of the macromolecule. At high temperature, the heterogeneity in the triplet lifetime of the internal residue attests to the presence of multiple conformers which are not in rapid equilibrium in the phosphorescence time scale. The anisotropy of the phosphorescence emission of glutathione transferase indicates that no energy transfer occurs between the two residues, and measurement of the rotational correlation time yields an hydrodynamic volume which is in good agreement with the molecular weight reported in the literature for the dimer.     

Influence of lactate on isoproterenol-induced lipolysis and beta-adrenoceptors distribution in human fat cells

The influence of lactate on human adipocytes lipolysis and the possible relationship between lactate-induced metabolic effects and beta-adrenoceptor binding sites were investigated. beta-sites were identified in membranes with (125I)-cyanopindolol and in intact cells with (125I)-cyanopindolol and (3H)-CGP 12177. Lactate reduced isoproterenol-induced lipolysis in a dose-response fashion and such inhibition became significant only at 16 mmol/l lactate. Exposure of human fat cells to 16 mmol/l lactate significantly reduced beta-adrenoceptors density on crude membranes. When the binding assay was performed on intact cells using (125I)-cyanopindolol at 37 degrees C, the radioligand identified the same number of receptors, regardless of the presence of lactate in the preincubation medium. When (3H)-CGP 12177 was used, it bound to about 35% less receptors in lactate pre-treated cells than in control. Seemingly, at 37 degrees C, because of its lipophilicity, (125I)-cyanopindolol can cross the plasma membrane and bind to intracellular sites whereas, (3H)-CGP 1277, due to its hydrophilicity, identifies surface receptors only. Thus, the present in vitro study provides evidence that high levels of lactate, similar to the concentrations usually achieved in overt lactic acidosis, are able per se to inhibit human lipolysis and to redistribute beta-adrenoceptors from cell surface to a domain not accessible to hydrophilic ligands.     

Transformation of NIH 3T3 cells by overexpression of the normal coding sequence of the rat neu gene.

While the normal human erbB-2 gene is potently transforming when overexpressed in NIH 3T3 cells, its rat homolog, the neu gene, seems to acquire transforming properties only upon alteration of its coding sequence. In this study, we compared the effects of different levels of expression of normal erbB-2 and neu in NIH 3T3 cells. Our results revealed that the normal rat neu gene acts as a potent oncogene when sufficiently overexpressed in NIH 3T3 cells.     

Synthesis of rhodanese in Hep 3B cells

Summary The biosynthesis of rhodanese was studied in human hepatoma cell lines by immunoblotting and pulselabeling experiments using polyclonal antibodies raised against the bovine liver enzyme. Rhodanese, partially purified from human liver, showed an apparent molecular weight of 33,000 daltons, coincident with that of rhodanese from Hep 3B cells. After pulse labeling of Hep 3B cells both at 37°C and 25°C, rhodanese in the cytosol fraction exhibited the same molecular weight as the enzyme isolated from the particulate fraction containing mitochondria. Moreover, newly synthesized rhodanese from total Hep 3B RNA translation products showed the same electrophoretic mobility as rhodanese from Hep 3B cells. These results suggest that rhodanese, unlike most mitochondrial proteins, is not synthesized as a higher molecular weight precursor.     

Thermoregulatory adjustments during continuous heat exposure

Body temperature regulation was studied in 6 male subjects during an acclimation procedure involving uninterrupted heat exposure for 5 successive days and nights in a hot dry environment (ambient temperature = 35 degrees C, dew-point temperature = 7 degrees C; air velocity = 0.2 m.s-1). Data were obtained at rest and during exercise (relative mechanical workload = 35% VO2max). At rest, hourly measurements were made of oesophageal and 4 local skin temperatures, to allow the calculation of mean skin temperature, and of body motility and heart rate. During the working periods these measurements were made at 5 min intervals. Hourly whole-body weight loss was measured at rest on a sensitive platform scale while in the working condition just before starting and immediately after completing the bicycle exercise. The results show that, in both exercise and at rest, the successive heat exposures increased the sweat gland output during the first 3 days. Afterwards, sweat rate decreased without any corresponding change in body temperature. For the fixed workload, the sweat rate decline was associated with a decrease in circulatory strain. Adjustments in both sweating and circulatory mechanisms occur in the first 3 days of continuous heat exposure. The overall sweat rate decline could involve a redistribution of the regional sweating rates which enhances the sweat gland activities of skin areas with maximal evaporative efficiencies.     

Eukaryotic DNA topoisomerase I reaction is topology dependent.

The effects of supercoiling on the topoisomerization reaction by eukaryotic DNA topoisomerases I have been analyzed. The systems used were: DNA topoisomerase I from wheat germ, chicken erythrocyte and calf thymus on a 2.3 kb DNA fragment which encompasses the immunoglobulin kappa-light chain (L kappa) promoter of the mouse plasmacytoma MPC11; S. cerevisiae DNA topoisomerase I on a 2.2 kb DNA fragment from the same organism which encompasses the regulatory and the coding region of the ADH II gene; wheat germ DNA topoisomerase I on the plasmid pUC18. It was found in every system that lack of torsional stress prevents topoisomerization of the substrate. A simple regulatory model of DNA topoisomerase I function, based on topological considerations, is presented.     

Secreted proteins induced by epidermal growth factor and transforming growth factor beta in EL2 rat fibroblasts. Role in the mitogenic response

Most growth active hormones and peptides are mitogenic only in the presence of other growth factors [e.g., Platelet Derived Growth Factor (PDGF) and Epidermal Growth Factor (EGF) in "competence-progression" fibroblast model]. We have previously described that EGF alone is able to induce the signals which appear necessary for the mitogenic stimulation of EL2 rat embryo fibroblast line. Recently, we have demonstrated that Transforming Growth Factor beta (TGF beta) slightly stimulates the mitogenic response in EL2 cells. Here, we show that in EGF-treated EL2 cells the induction of at least four inducible-secreted proteins (ISPs, range from 29,000 to 68,000 Mr) is accompanied by a marked increase in DNA synthesis. In contrast, TGF beta or different concentrations of EGF induce a slow increase of the ISPs proportional to slow induction in DNA synthesis. Our results suggest that the mitogenic response in EL2 cell line may be connected with the qualitative and quantitative induction of these secreted proteins.     

Long-delay learning in rats with parabrachial pontine lesions

Two experiments were conducted to test the hypothesis that ratswith lesions in the parabrachial nucleus of the pons (PbN) canacquire conditioned taste aversions only if the conditionedand unconditioned stimuli are presented close together in time.In experiment 1, rats with lesions in the medial PbN, lateralPbN or outside of the PbN (lesioned control), and unoperatedcontrol rats were trained to avoid Na-saccharin in a one-bottletest. In this procedure, a 5-min delay was imposed between the15-min Na-saccharin presentation and an injection of LiCl (0.3M, 1 % body weight, i.p.). Results showed that, after threeNa-saccharin-LiCl pairings, all rats, except the medial PbNgroup, acquired a strong aversion to Na-saccharin. In experiment2, the same rats were presented with LiCl (0.12 M) in their15-min daily access to fluid on 3 alternate days. Although ratsin the medial PbN group drank more LiCl on day 1 than rats inthe other groups, they significantly reduced their LiCl consumptionon day 2 and did not differ from other groups by day 3. Resultsare discussed in terms of possible behavioral and physiologicalmechanisms that might account for these phenomena.     

DNA flow cytometry of endoscopically examined colorectal adenomas and adenocarcinomas

DNA ploidy of 64 colorectal adenomas and 49 adenocarcinomas, examined endoscopically, was studied by flow cytometry. We found DNA aneuploidy in none of the 105 normal mucosa samples (0%), in 20 adenomas (31%), and in 36 adenocarcinomas (74%). DNA ploidy of adenomas correlated with size (P = 0.02) and degree of dysplasia (P less than 0.01) but not with histologic type. Adenomas had a 45% incidence of DNA aneuploid stem lines in the DNA index range of 0.80-1.20, compared with 8% in the case of adenocarcinomas. The distribution of the DNA index values of adenocarcinomas was approximately normal, with a mean value 1.63 +/- 0.28. The mean DNA index for the three cases of "carcinoma in adenoma" with invasion of the stalk of the adenoma was 1.52 +/- 0.18. These results, using DNA flow cytometry, provide evidence for the progression of colorectal adenoma to adenocarcinoma. The classification of adenomas according to DNA ploidy may be information of considerable practical value to the clinician in predicting risk of further adenomas and/or risk of cancer.