Complementarity of regulation for the two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile

Chromatophores from Rhodopseudomonas capsulata cells grown semiaerobically in the dark oxidize NADH, succinate, and dichlorophenolindophenol. In the presence of N₃^? these activities are inhibited, but light induces oxidation of dichlorophenolindophenol with O₂ as a terminal electron acceptor. Cyanide also inhibits electron transport but much higher concentrations are required to inhibit the photooxidation than the dark oxidation. The photooxidation was studied in a mutant strain of Rhodopseudomonas capsulata (YIV) which cannot grow anaerobically in the light, but similarly to the wild type, grows in the presence of oxygen. Chromatophores from YIV mutant catalyze photophosphorylation and dark oxidation activities with the same properties as those of the wild type. However, the rate of photooxidation in the mutant is only one-third that of the wild type. Based on the differential inhibitor sensitivity and on the mutation it is suggested that the photooxidase is different from the two respiratory oxidases and that this photooxidation activity might be essential for growth of the cells under anaerobic conditions in the light.     

Properties of the selenium-containing moiety of nicotinic acid hydroxylase from Clostridium barkeri

The nicotinic acid hydroxylase from Clostridium barkeri is a selenoenzyme, as evidenced by the copurification of selenium with enzyme activity. This conclusion is supported by data showing a 23-fold increase in nicotinic acid hydroxylase activity when C. barkeri was cultured in media supplemented with selenium. A labile, selenium-containing compound was released from the native protein by treatment with either chaotropic agents and heat or by heating alone. A stable selenium compound was formed when the enzyme was alkylated prior to denaturation. This compound had the same chromatographic properties as dialykyl selenide in a number of systems. The formation of dialkyl selenide upon alkylation is not consistent with the selenium moiety being selenocysteine. Thus, nicotinic acid hydroxylase represents a new type of selenoenzyme.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Calcium control of glycogen synthase activities in mouse diaphragms, rat adipocytes and rat hepatocytes

The following article provides evidence that cellular calcium controls the activity of glycogen synthase in all three major glycogen storage tissues; muscle, fat, and liver. Depletion of cellular calcium resulted in a moderate increase of glycogen synthase %I activities in intact mouse diaphragms, in isolated rat adipocytes, and in rat hepatocytes. The increase in %I activity of glycogen synthase was more pronounced when the uridine di-phosphoglucose concentration in the glycogen synthase assay was lowered from 4.4 mM to 0.2 mM. Calcium depletion resulted in an approximately two-fold decrease in the Ka values for glucose-6-phosphate in all three tissues. The activities of glycogen synthase also correlated well with the content of cell-associated calcium in rat hepatocytes. The glucose-6-phosphate independent activities of glycogen synthase in extracts of calcium-replete and calcium-depleted tissue approached the same value following the exposure to crude phosphoprotein phosphatase. The activities of glycogen phosphorylase decreased in calcium-depleted tissues and cells. Insulin stimulated the activity of glycogen synthase in muscle and fat in the absence of added sugar and in the absence of extracellular calcium. It is concluded that glycogen synthase is under the control of calcium in the three main glycogen storage tissues. The actions of calcium are probably mediated through the actions of calcium-sensitive protein kinase(s).     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Trichinella spiralis: Comparison with an Arctic isolate

The muscle phase of Trichinella spiralis and of Trichinella sp. isolated in the Arctic was compared in experimental and wild animals. Reproductive capacity indices (RCI) of the Trichinella sp. isolate were significantly lower in laboratory rodents but were similar to T. spiralis in wild rodents. Sprague-Dawley rats were the most refractory to the Trichinella sp. isolate of all laboratory rodents. Outbred strains of mice were more susceptible to both T. spiralis and the Trichinella sp. isolate than inbred strains of mice. T. spiralis muscle larvae survived longer in mice and the survival of both T. spiralis and the Trichinella sp. isolate larvae was higher in female mice. While single pair interbreeding experiments showed reproductive isolation between T. spiralis and the Trichinella sp. isolate, multiple pair and transplant breeding experiments showed reproductive compatibility. Male and female infective larvae of T. spiralis and the Trichinella sp. isolate differed morphometrically, but a convergence in size of worms was observed after prolonged passages of the parasites in mice. Passaging history of the isolate and host species was found to have a significant effect on Trichinella morphology. It is proposed that the Trichinella sp. isolate is a physiological variant of T. spiralis and not a distinct species.     

Sequential polymerization of flagellin A and flagellin B into Caulobacter flagella

The mode of polymerization of two species of flagellins, flagellin A and flagellin B, in polar flagella of Caulobacter crescentus was examined. By immunological staining we found that 1 to 1.2 μm of the portion of the flagellar filament proximal to the cell was composed of flagellin B, whereas about 5 μm of the distal portion was composed of flagellin A. This result, together with the previous observation that a flagellin B-less mutant cannot form normal flagella but instead forms stubs in spite of their high level of flagellin A synthesis, indicates that flagellin B is very important for the formation of complete flagella and/or for the initiation of filament formation from the hook.     

Reverse transformation of Chinese hamster ovary cells by methyl xanthines : Structure-function relationships

Using a number of drugs that increase cellular cAMP levels, alterations in the amount of cell surface fibronectin and other transformation parameters were studied in Chinese hamster ovary (CHO) cells. The drugs include db-cAMP, different methylxanthines (theophylline, aminophylline, methyl isobutyl xanthine (MIX), caffeine and theobromine), papaverine and cholera toxin. Methylxanthines that have a methyl group at the seventh position lack reverse transforming potential; those that lack a methyl group at the seventh position induced reverse transformation in CHO cells, causing an increase in surface fibronectin, cell substratum adhesive strength and anchorage dependence for growth. Further, as methyl xanthines are substituted in other positions different from the seventh position, the more efficient they become in restoring normal phenotypic properties; the later agents also induced low saturation density via a cytostatic state causing accumulation of cells in the S and G2 phases of the cycle in contrast to the G1 arrest of normal cells at low saturation density. db-cAMP and cholera toxin induced cell elongation but like caffeine and theobromine, did not induce surface fibronectin. The non-methylxanthine phosphodiesterase inhibitor papaverine induced neither cell elongation nor surface fibronectin but produced a cytostatic effect similar to aminophylline and MIX. These studies suggest that the reverse transformation properties fall into two groups: (a) Differentiation-related properties including cell morphology, parallel alignment and surface matrix fibronectin, etc.; (b) cell cycle-related properties-low saturation density, cell arrest at G1 phase and anchorage-dependent growth. Phosphodiesterase inhibitors reversibly eliminate indefinite division potential of CHO cells by inducing a cytostatic situation and not by inducing a G1-specific arrest.     

Developmental delays in exploration and locomotor activity in male rats exposed to low level lead

Delays in the development of exploratory and locomotor behavior in neonatal male rats (up to 21 days of age) are shown to accrue as a consequence of low level lead exposure. Cross fostering experiments indicate that these delays are primarily due to prenatal exposure. These Pb induced behavioral modifications appear to be associated with the delays in synaptogenesis and biochemical development of the cerebral cortex reported previously (4, 18). A new behavioral bioassay for detecting delays in brain development is described.