Retention of radioactive substances in the hypothalamus, anterior pituitary, and reproductive organs of male rats after 3H-melatonin administration

Radioactive concentrations were determined in serum, lung, hypothalamus, anterior pituitary, testis, and accessory sex organs of adult male rats at 2, 20, 30 and 60 min after intravenous 3H-melatonin administrations. The retention patterns of 3H-melatonin and other radioactive substance (3H-non-melatonin) in these tissues were compared. The anterior pituitary demonstrated best tissue retention with highest concentration of 3H-melatonin. The testis and prostate gland accumulated 3H-non-melatonin in an increasing manner from 2 to 60 min. The radioactive substances were also preferentially and progressively located in the nuclear fraction of the anterior pituitary, hypothalamus, testis, and prostate gland. This study leads to the following suggestions: the anterior pituitary is another target organ of melatonin; melatonin is converted into other active material which exerts its action in the testis and prostate gland; melatonin and its active derivative exert their action through the nuclei of their respective cells.     

Mechanisms responsible for autocrine regulation of the epiphysis function

The effect of injection of exogenous melatonin on formation of the nocturnal peak of the melatonin concentration and intrapineal hormonogenesis was studied on mature male rats. It was shown that³H-melatonin is selectively uptaken by pinealocytes, and the uptake intensity depends on the degree of saturation of an organism with exogenous melatonin. Exogenous melatonin exerts an inhibitory influence on the hormone self-production, at the same time apparently stimulating synthesis of epiphyseal peptides. The results can be considered an indication of the existence of an ultra-short connection between the mechanisms of production of epiphyseal indoles and peptides, which apparently plays an important role in autocrine regulation of the epiphyseal functions.     

Radioimmunoassay for melatonin and its application to fowl pineal research

A radioimmunoassay for melatonin has been developed after raising anti-melatonin antibodies in rabbit. Melatonin was extracted from serum or pineal gland of chickens (Gallus domesticus). The radioimmunoassay was performed by using 3H-melatonin as tracer. The standard curve covered the range 0.022-0.345 pmol/vial and the KD value for melatonin was estimated at 1.37 x 10(10) l/mol. The antiserum specificity has been analysed, none of the common melatonin analogues influencing this method of melatonin measurement. The intra-assay variability was 7.2% for serum samples and 8.6% for pineal extract. The inter-assay variability for this biological sample was 15.3% and 6.4% respectively.     

A modified enzymatic-isotopic microassay for serotonin (5HT) using 5HT-N-acetyltransferase partially purified from Drosophila

Serotonin-N-acetyltransferase (EC 2.3.1.5), partially purified from $\text{Drosophila}$$\text{melanogaster}$ (DNAT) was substituted for rat liver enzyme (RNAT) in a previously published radioenzymatic assay for serotonin (5HT). The purpose was to determine if this modification would increase the sensitivity and reliability of the original assay. Compared with RNAT, DNAT is 3–4 times more active; it lacks secondary 5HTP decarboxylase activity; and it is more stable. Under conditions of the modified assay, the only radioactive product formed from hypothalamic tissue extracts is ³H-melatonin; which can be measured from as little as 15 pg of serotonin substrate. Thus, substitution of DNAT for RNAT improves the original radioenzymatic assay allowing measurement of endogenous 5HT in hypothalamic nuclei from individual animals.     

Characterization of the adaptive response to trichloroethylene-mediated stresses in Ralstonia pickettii PKO1

In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression.     

Epithelial polarity and tubulogenesis in vitro

The most fundamental type of organization of cells in metazoa is that of epithelia, which comprise sheets of adherent cells that divide the organism into topologically and physiologically distinct spaces. Some epithelial cells cover the outside of the organism; these often form multiple layers, such as in skin. Other epithelial cells form monolayers that line internal organs, and yet others form tubes that infiltrate the whole organism, carrying liquids and gases containing nutrients, waste and other materials. These tubes can form elaborate networks in the lung, kidney, reproductive passages and vasculature tree, as well as the many glands branching from the digestive system such as the liver, pancreas and salivary glands. In vitro systems can be used to study tube formation and might help to define common principles underlying the formation of diverse types of tubular organ.     

Identification of methyl coenzyme M as an intermediate in methanogenesis from acetate in Methanosarcina spp

The transfer of the methyl group of acetate to coenzyme M (2-mercaptoethanesulfonic acid; HS-CoM) during the metabolism of acetate to methane was investigated in cultures of Methanosarcina strain TM-1. The organism metabolized CD3COO- to 83% CD3H and 17% CD2H2 and produced no CDH3 or CH4. The isotopic composition of coenzyme M in cells grown on CD3COO- was analyzed with a novel gas chromatography-mass spectrometry technique. The cells contained CD3-D-CoM and CD2H-S-CoM) in a proportion similar to that of CD3H to CD2H2. These results, in conjunction with a report (J.K. Nelson and J.G. Ferry, J. Bacteriol. 160:526-532, 1984) that extracts of acetate-grown strain TM-1 contain high levels of CH3-S-CoM methylreductase, indicate that CH3-S-CoM is an intermediate in the metabolism of acetate to methane in this organism.     

Relationship between lysostaphin endopeptidase production and cell wall composition in Staphylococcus staphylolyticus.

Mutants of Staphylococcus staphylolyticus incapable of producing an extracellular staphylolytic glycylglycine endopeptidase were isolated and found to have cells in the population susceptible to lysis by this enzyme, as did the wild-type organism under conditions in which the endopeptidase was not produced. These results suggest that cultures of this organism normally contain a heterogeneous population of cells with regard to cell wall composition and susceptibility to the enzyme. Production of the endopeptidase appears to act as a selective pressure which removes the susceptible cells in the population as the enzyme appears in the medium. A comparison of the peptidoglycan of the wild-type organism grown under conditions in which the endopeptidase was produced with that of this organism grown under nonproducing conditions and with those of endopeptidase-less mutants showed that in the presence of the endopeptidase the cell population had peptidoglycan with shorter peptide cross bridges and a greater percentage of serine in these cross bridges than was found in cells grown in the absence of the enzyme. The inability of the endopeptidase to hydrolyze glycylserine and serylglycine peptide bonds suggests that at least part of the resistance this organism has to the endopeptidase is due to relative amounts of serine found in the peptide cross bridges of some cells in the population.     

Malignant transformation of NIH-3T3 and CV-1 cells by a helical mycoplasma,Spiroplasma mirum,strain SMCA

Summary A helical mycoplasma,Spiroplasma mirum strain SMCA, produced malignant transformation in mouse NIH 3T3 cells and monkey kidney CV-1 cells. The transformed cells exhibited morphological changes consistent with the transformed phenotype, grew in soft agar and produced tumors in athymic and BALB/c mice. Transmission electron microscopy revealed structures morphologically similar to mycoplasmas present in the cytoplasm of transformed but not untransformed 3T cells. The time of inoculation ofS. mirum SMCA to 3T3 cells and the passage level of 3T3 cells affected transformation. Editor's statement This paper describes the possible role of a mycoplasma organism, which induces cataracts and brain pathology similar to Creutzfeld Jacob and other degenerative diseases, in malignant transformation of mammalian cells. In addition to this surprising and novel finding is the observation that the mycoplasma resides in an intracellular position. These findings may have important implications for understanding malignant transformation and the nature of the diseases produced by this organism.     

The fate of Bacillus anthracis in unpasteurized and pasteurized milk

Growth of Listeria monocytogenes at pH 5·0 did not increase growth of the organism at pH 7·0 after exposure to low pH (3·0, 3·5), compared with cells initially grown at pH 7·0. However, growth at pH 5·0 significantly increased the survival of cells at low pH as determined by plate counts compared with cells grown at neutral pH. Thus, pH adaptation not only occurs in enteric bacteria but also in this Gram-positive organism. Alterations in the cytoplasmic membrane could be responsible.     

Adherence and receptor relationships of Candida albicans.

The cell surface of Candida albicans is composed of a variety of polysaccharides such as glucan, chitin, and mannan. The first two components primarily provide structure, while the mannan, often covalently linked to protein, constitutes the major antigen of the organism. Mannoproteins also have enzymatic activity (acid protease) and ligand-receptor functions. The complement receptors of C. albicans appear to be mannoproteins that are required for the adherence of the organism to endothelial cells. This is certainly true of the CR3-like protein of C. albicans. Proof that the CR3 is the Candida receptor for endothelial cells is derived from two observations. First, mutants lacking CR3 activity are less adherent in vitro and, in fact, less virulent. Second, the ligand recognized by the CR3 receptor (C3bi) as well as anti-CR3 antibodies blocks adherence of the organism to endothelial cells. The CR2 of C. albicans appears to promote the adherence of the organism to plastic substrates. Unlike the CR2 of mammalian cells, the Candida CR2 recognizes ligands containing the RGD sequence of amino acids in addition to the C3d ligand, which does not contain the RGD sequence. There is uncertainty as to whether the Candida CR2 and CR3 are, in fact, different proteins. A mannoprotein has also been described as the adhesin for epithelial cells. In this case, the receptor has a lectinlike activity and recognizes fucose- or glucosamine-containing glycoproteins of epithelial cells, depending on the strain of C. albicans. The oligosaccharide component of the receptor is probably not involved in ligand recognition and may serve to stabilize the receptor. However, the oligosaccharide factor 6 epitope of mannan may also provide adhesin activity in the recognition of epithelial cells. Mannoproteins can be extracted from cells by a number of reagents. Zymolyase, for instance, tends to remove structural mannoproteins, which contain relatively little protein and are linked to glucan. Reagents such as dithiothreitol, on the other hand, tend to extract mannoproteins containing higher amounts of protein that appear to have receptor function. The mannoproteins of C. albicans are dynamically expressed and may be growth phase and growth form specific.     

Characterization of the Adaptive Response to Trichloroethylene-Mediated Stresses in Ralstonia pickettii PKO1

In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression.     

Regulation of Dictyostelium morphogenesis by RapGAP3

Rap1 is a key regulator of cell adhesion and cell motility in Dictyostelium. Here, we identify a Rap1-specific GAP protein (RapGAP3) and provide evidence that Rap1 signaling regulates cell-cell adhesion and cell migration within the multicellular organism. RapGAP3 mediates the deactivation of Rap1 at the late mound stage of development and plays an important role in regulating cell sorting during apical tip formation, when the anterior-posterior axis of the organism is formed, by controlling cell-cell adhesion and cell migration. The loss of RapGAP3 results in a severely altered morphogenesis of the multicellular organism at the late mound stage. Direct measurement of cell motility within the mound shows that rapGAP3⁻ cells have a reduced speed of movement and, compared to wild-type cells, have a reduced motility towards the apex. rapGAP3⁻ cells exhibit some increased EDTA/EGTA sensitive cell-cell adhesion at the late mound stage. RapGAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation, which is dependent on F-actin polymerization. We suggest that the altered morphogenesis and the cell-sorting defect of rapGAP3⁻ cells may result in reduced directional movement of the mutant cells to the apex of the mound.     

Human cytokines interleukin (IL)-3 and IL-6 affect the growth and insulin binding of the unicellular organismTetrahymena

Interleukin (IL)-3 and IL-6 significantly increase the growth rate of the unicellular organism,Tetrahymena. The effect elicited by IL-3 is long lasting as it was also detectable after 20 generations. Effect of IL-6 was detectable as long as the substance was present in the cell culture. Pretreatment with IL-3 did not enhance the proliferative response to subsequent IL-3 treatment, but the second exposure to IL-3 considerably depressed the active proliferation ofTetrahymenacells. However, a positive ‘priming effect’ elicited by IL-6 resulted in an increased growth rate following repeated IL-6 stimulation. Insulin binding to the plasma membrane ofTetrahymenawas increased by IL-6 but not by IL-3 after 24 hours, and this enhancement appeared even after one hour incubation. If the cells were pretreated with insulin, IL-6 did not influence insulin binding, while an inhibition by IL-3 was observed. These results direct attention to the similarities of actions induced by IL-3 and IL-6 at different levels of phylogeny probably due to the presence of cytokine receptor-like structures on this unicellular organism.     

Hemagglutinating activity of Mycoplasma salivarium and its attachment to sheep red blood cells

Mycoplasma salivarium (ATCC 23064) and 10 other strains isolated from human saliva agglutinated red blood cells of rabbits and human types A and O weakly, and those of sheep (SRBC) and human type B strongly. Glycoproteins on the surface of the organism cells and N-acetylneuraminic acid residues and some sugars on SRBC were suggested to be involved in agglutination of SRBC. Protein A-like activity was detected in the organism cells. The organism cells were also shown to attach to SRBC in PPLO broth (Difco) supplemented with 10% horse serum, and bivalent metal ions were suggested to be involved in the attachment. The organism cells attaching to SRBC activated complement through the alternative pathway and lyzed the SRBC.     

Studies on Oxidative Fermentation

It has been found that Gluconobacter liquefaciens metabolized 5-ketogluconic acid. In order to clarify metabolic pathways of this compound, the oxidation products by resting cells of this organism were investigated. Rubiginol, rubiginic, comenic, 2,5-diketogluconic, glycolic and tartronic acids were detected or identified in the reaction fluid. On the basis of these results and the data obtained by means of manometric experiments, the oxidation pathways of 5–ketogluconic acid were discussed.

Oxidation pathways of 5-ketogluconie acid by resting cells of Gluconobacter liquefaciens were further investigated. Arsenite inhibited the oxidation of this compound. The amount of carbonyl compounds in the oxidation products of 5–ketogluconic acid was increased by addition of 10^-3 m arsenite. Pyruvic and α-ketoglutaric acids were identified among these carbonyl compounds. Members of the tricarboxylic acid cycle were oxidized actively by resting cells or cell-free extracts of this organism. These results suggested the presence of the tricarboxylic acid cycle in the terminal oxidatjon of 5-ketogluconic acid by this organism.     

Function of the p53 gene: choice between life and death

Gene p53 is a central component of a system that eliminates pathologically damaged cells from an organism. Multiple signal pathways monitor the state of a cell and when damage or a fault is found that could cause heritable changes, p53 protein is activated to either coordinate the repair process or induce cell suicide. Thus, the p53 gene acts as a supreme judge that decides the fate of cells and guarantees their social behavior. Loss of the p53 gene results in uncontrolled accumulation of genetic damage causing failure of control by the organism, malignant cell growth, and death of the organism.     

Some biochemical effects of 4-deoxy-4-fluoro-D-glucose on Escherichia coli.

The uptake of 4-deoxy-4-fluoro-D-glucose (4FG), without subsequent catabolism, by resting cells of Escherichia coli (ATCC 11775) is 0.06 mg/mg dry weight. In frozen-thawed cells of this organism, 4FG is a substrate for the phosphoenolpyruvate phosphotransferase system with a rate of phosphorylation twice that found for the isomeric 3-deoxy-3-fluoro-D-glucose. 4FG is not a carbon source for growth of this organism and it inhibits the extent of growth of cells in the presence of glucose. The inhibition of growth of E. coli K12 on lactose by 4FG is also observed and this is considered to be consistent with the fact that 4FG is an uncompetitive inhibitor of beta-galactosidase (EC 3.2.1.23) activity and that 4FG or 4-deoxy-4-fluoro-D-glucose-6 phosphate repress beta-galactosidase synthesis. These results support the view that catabolite repression may be produced by compounds which are not necessarily metabolised further than hexose-6-phosphates.     

Methanol production by Mycobacterium smegmatis.

Mycobacterium smegmatis cells produce [3H]methanol when incubated with [methyl-3H]methionine. The methanol is derived from S-adenosylmethionine rather than methyltetrahydrofolate. M. smegmatis cells carboxymethylate several proteins, and some of the methanol probably results from their demethylation, but most of the methanol may come from an unidentified component with a high gel mobility. Although methanol in the medium reached 19 microM, it was not incorporated into the methylated mannose polysaccharide, a lipid carrier in this organism.     

Biofilm and planktonic Enterococcus faecalis elicit different responses from host phagocytes in vitro

Enterococcus faecalis is a commensal organism of the gastrointestinal tract but can also cause serious opportunistic infections. In addition to high levels of antibiotic resistance, the ability to form biofilms on abiotic surfaces and on in-dwelling devices within the host complicates treatment strategies and successful outcomes of antibiotic therapy. Despite rapid advances made in recent years in understanding the genomics and virulence of this organism, much remains to be learned regarding the host response to enterococcal infections. In this study, we investigated the interaction of RAW264.7 macrophages and JAWS II dendritic cells with biofilm and planktonic E.?faecalis, in vitro. Specifically, we compared phagocytosis, intracellular survival, secretion of proinflammatory cytokines, and the activation and maturation of phagocytes. Our results revealed that both macrophages and dendritic cells phagocytize biofilm mode cells at levels equal to or better than their planktonic counterparts. Internalized biofilm bacteria showed relatively greater survival at 24?h in macrophages than in dendritic cells and led to slightly higher expression of phagocyte activation markers. Macrophages infected with biofilm cells also secreted lower levels of proinflammatory cytokines studied. Overall, these results suggest that biofilm E.?faecalis may be better adapted to overcome host defenses in vivo.