Atropine inhibits the degranulation of Paneth cells in ex-germ-free mice

Summary Previous studies have shown that the secretory products of Paneth cells contain antibacterial agents (lysozyme, IgA) that are affected by the bacterial milieu in the intestine. To investigate whether Paneth-cell secretion is controlled via cholinergic mechanisms, the ultrastructure of Paneth cells was studied in four animal groups: (1) germfree (GF) control mice (Jcl: ICR [GN], male, 13 weeks old), (2) GF mice injected subcutaneously with atropine sulfate (200 mg/kg body weight, dissolved in physiological saline 20 mg/ml), (3) ex-GF mice inoculated with feces from specific-pathogen-free (SPF) mice, and (4) ex-GF mice injected with atropine and inoculated with feces from SPF mice. In ex-GF mice inoculated with feces, 70–90% of the Paneth cells showed fewer secretory granules than those from GF mice (p<0.01). Approximately 30% of the Paneth cells had a large vacuole (3–10 m diameter) in the apical cytoplasm. Exocytosed electron-dense material from secretory granules was observed in a few crypt lumens. In ex-GF mice inoculated with feces and given atropine, about 90% of the Paneth cells contained numerous secretory granules, like those in GF control mice, but vacuolated Paneth cells and exocytotic figures were rare; thus the secretion of Paneth cells was blocked by atropine. It is therefore possible that the bacterial milieu in the intestine affects the secretory activity of Paneth cells via cholinergic mechanisms.     

Transient states of geosmin,pigments, carbohydrates and proteins in continuous cultures of Oscillatoria brevis induced by changes in nitrogen supply

Transitions in the growth limiting factor from light (I) to nitrogen (N) and vice versa caused changes in geosmin production, protein and carbohydrate content, and the synthesis of pigments such as chlorophyll a (Chl a), phycobiliproteins (PBPs), and -carotene of the cyanobacterium Oscillatoria brevis. Following IN transition the first 150h, the decrease in protein content was compensated for by an increase of carbohydrates, and thereby, a constant biomass level was maintained in this period. Thereafter, biimass dropped to 15% of its initial level. A decrease in geosmin and pigment content was observed during transition from IN-limited growth. However, geosmin increased relative to phytol (Chl a) and -carotene which may indicate that a lowered demand for phytol and -carotene during N-limited growth allows isoprenoid precursors to be directed to geosmin rather than to pigment synthesis. Synthesis of Chl a and -carotene at the expense of geosmin was suggested for the observed start of increase in geosmin production only at the time that Chl a and -carotene had reached their I-limited steady state. Transition from nitrogen to light limited growth caused an acceleration of metabolism shown by a rapid decrease in carbohydrate content accompanied by an increase in protein content. The growth rate of the organisms temporarily exceeded the dilution rate of the culture and the biomass level increased 6-fold. Due to the only modest changes in geosmin production (2-fold) compared to changes in biomass level (6-fold) during I-or N-limited growth, environmental factors seem to have limited effect on geosmin production.Abbreviations Chl a chlorophyll a - dry wt dry weight; - I-limited light-limited - N-limited nitrogen-limited - PBP phycobiliprotein This research was performed at the Department of Microbiology, University of Amsterdam, with finacial support provided by the Royal Norwegian Ministry of Foreign Affairs and the Royal Norwegian Council for Scientific and Industrial Research     

Nitrite reduction in Bacteroids of Rhizobium “hedysari” strain HCNT 1

Ex planta, bacteroids of the sulla-symbiont Rhizobium hedysari strain HCNT 1 terminated reduction of nitrite at nitrous oxide irrespective of the presence or absence of acetylene. Nitrate was not reduced during the experimental period, but slight nitrate reductase activity occurred if incubation with nitrate was prolonged (up to 15 h). As was observed in free-living cells, exposure of the bacteroids to the metal chelator, diethyldithiocarbamate, prevented reduction of nitrite, indicating the presence of a copper-containing nitrite reductase. Pulses of 10–75 M nitrite transiently impeded O₂ uptake in bacteroids, which resumed consumption of O₂ when the nitrite had been reduced. Exposure to >1.0 mM nitrite for 24h greatly inhibited nitrogenase activity (assayed as acetylene reduction activity) of bacteroids in planta. Exposure to the same concentrations of nitrite after 1h of incubation in the presence of acetylene almost completely stopped ongoing ethylene production in bacteroids of strain HCNT 1 extracted from nodules. Free cells of the non-nitrite-reducing R. hedysari strain CC 1335 were lacking in nitrogenase (acetylene-reduction) activity, whereas identically cultured (low-oxygen) strain HCNT 1 cells reduced both nitrite and acetylene.Abbreviations PMS phenazine methosulfate - DDC diethyldithiocarbamate     

Catabolism of dl-α-phenylhydracrylic,phenylacetic and 3- and 4-hydroxyphenylacetic acid via homogentisic acid in a Flavobacterium sp.

A degradation pathway for dl--phenylhydracrylic, phenylacetic, 3- and 4-hydroxyphenylacetic acid by a Flavobacterium is presented. Experiments with washed cells and enzyme studies revealed that dl--phenylhydracrylic acid in an initial reaction was oxidatively decarboxylated to phenylacetaldehyde. Whole cells oxidized both stereoisomers of phenylhydracrylic acid at different rates. The product phenylacetaldehyde in turn was oxidized to phenylacetic acid. No hydroxylation of phenylacetic acid was detected in cell extracts, but on the basis of experiments with washed cells it is assumed that phenylacetic acid is mainly metabolized via 3-hydroxyphenylacetic acid. This latter product was subsequently hydroxylated yielding the ring-cleavage substrate homogentisate. 4-Hydroxyphenylacetic acid was also degraded via homogentisate. Ringcleavage of homogentisate gave maleylacetoacetate which was further degraded through a glutathione-dependent pathway. Homoprotocatechuate was not an intermediate in the metabolism of dl-phenylhydracrylic acid, phenylacetic, 3- and 4-hydroxyphenylacetic acid metabolism, but it could be hydroxylated aspecifically to 2,4,5-trihydroxyphenylacetic acid by the action of the 3-hydroxyphenylacetic acid-6-hydroxylase.Abbreviations HPLC high-performance liquid chromatography - PHA phenylhydracrylic acid - PA phenylacetic acid - HPA hyxdroxyphenylacetic acid - PMS phenazine methosulphate - PMA phenylmalonic acid - GSH glutathione     

Structural studies on cyanobacterial photosystem I

The cyanobacterial photosystem, I complex from Synechococcus sp. PCC6301 contains polypeptides of apparent M_r of 70,000, 18,000, 17,700, 16,000 and 10,000. Procedures were developed for the purification of the M_r 17,700 and 10,000 polypeptides. Amino acid analyses showed the absence of cystine and cysteine from these polypeptides. Amino-terminal sequences of 98 residues for the M_r 17,700 polypeptide and of 42 residues for the M_r 10,000 polypeptide were determined. Studies of pigment distribution within the photosystem I complex indicated that the binding of chlorophyll a and -carotene is in part dependent on the presence of these polypeptides.Abbreviations PSI photosystem I - P700 reaction center of PSI - SDS sodium dodecylsulfate - TBS tris-buffered saline - TTBS TBS containing Tween-20     

Purification and characterisation of an inducible β-galactosidase from Corynebacterium murisepticum

The -galactosidase (EC 3.2.1.32) of Corynebacterium murisepticum (inducible by lactose and galactose) was purified by successive column chromatography on Sephadex G-200, DEAE-Sephadex A-50 and DEAE-cellulose (DE52). The enzyme was found to be a dimer of identical subunits of molecular mass 100,000 daltons. The K _m values of the enzyme for the substrates lactose and o-nitrophenyl--d-galactopyranoside (ONPG) are 16.7 mM and 4.4 mM, respectively, indicating, its low affinity for the substrates. The Ouchterlony immunodiffusion method exhibited immunological homogeneity of the enzyme preparation. The catalytic site of the enzyme does not take part in antigen-antibody reaction.     

A re-examination of the Na+-independent binding of [3H]β-alanine to rat brain stem-spinal cord

The Na⁺-independent binding of [³H]-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described -alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na⁺-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na⁺-independent binding, using frozen/thawed/washed synaptosomal-mitochodrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200–415 g/ml incubation medium. Binding was completely inhibited by glycine, alanine, -aminobutyric acid, -aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl--alanine, brucine and gelsemine. It was insensitive to taurine, -aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (K _D 20 M), and heat sensitive.     

Serum copper,zinc levels,and copper

Serum copper and zinc levels were determined in 20 healthy women and in 100 women with gynecological tumors. Malignant and benign tumor cases were separated according to their postoperative, histopathological examinations. The stages of malignant and benign tumors were also established histologically. Seventy benign and 30 malignant genital tumors (carcinoma of cervix in situ, cervix, ovary endometrium, and vulva) of the patients were differentiated histopathologically. The serum Cu/Zn ratios of patients were increased significantly from the control group (0.32±0.35) to the benign group (1.22±0.63) and from the benign group to the malignant group (2.24±1.03). Nine of 30 malignant cases were determined as false negative (30%) and 15 of 70 benign cases were determined as false positive (14.2%) according to the serum Cu/Zn ratios of patients. Serum copper levels of 30 malignant and 10 benign tumor cases showed linear correlation with serum ceruloplasmin values.     

Transformation with SV40 virus prevents retinoic acid inhibition of plasma membrane NADH diferric transferrin reductase in rat liver cells

Retinoic acid inhibits the reduction of diferric transferrin through the transplasma membrane electron transport system on fetal rat liver cells infected with a temperature-sensitive SV40 virus when the cells are in the nontransformed state cultured at 40°C. When the cells are in the transformed state (grown at the permissive 33°C temperature), retinoic acid does not inhibit the diferric transferrin reduction. Inhibition of activity of nontransformed cells is specific for retinoic acid with only slight inhibition by retinol and retinyl acetate at higher concentrations. Isolated rat liver plasma membrane NADH diferric transferrin reductase is also inhibited by retinoic acid. The effect of transformation with SV40 virus to decrease susceptibility to retinoic acid inhibition stands in contrast to much greater adriamycin inhibition of diferric transferrin reduction in the transformed cells than in nontransformed cells.     

Proteolytic specificity of chicken cathepsin L on bovineβ-casein

The proteolytic specificity of chicken cathepsin L was studied using bovine -casein as substrate. The peptide mixtures obtained after various times of hydrolysis were separated by RP-HPLC and ten peptides were identified. Chicken cathepsin L accepts proline residues in all positions except P ₁ . Looking at the amino acid residues on the amino side of the scissile bond we found three times the Tyr-Pro pair at P ₁ –P ₂ positions and that the S ₁ subsite can interact with modified amino acids such as phosphoserine.Abbreviations RP-HPLC reverse phase high performance liquid chromatography - NMec N-methyl coumarylamide - TEA triethylamine - TFA trifluoroacetic acid