Ca2+/Calmodulin Distinguishes Between Guanyl-5''-yl-Imidodiphosphate-and Opiate-Mediated Inhibition of Rat Striatal Adenylate Cyclase

The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p-mediated inhibition accurately reflected hormone-mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p-mediated inhibition was dependent on the presence of Ca2+/CaM at 24 degrees C; the inhibition was independent of Ca2+/CaM at 18 degrees C; and inhibition could not be detected in the presence, or absence, of Ca2+/CaM at 30 degrees C. In contrast, naloxone-reversible, morphine-induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+ dose-response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p- and morphine-induced inhibition. Because Gpp(NH)p-mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18 degrees C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24 degrees C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p- and morphine-induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p-mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone-mediated inhibition is Ca2+/CaM independent under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Short-term metabolic fate of [13N]ammonia in rat liver in vivo

The short-term metabolic fate of [13N]ammonia in the livers of adult male, anesthetized rats was determined. Following a bolus injection of tracer quantities of [13N]ammonia into the portal vein, the single pass extraction was approximately 93%, in good agreement with the portal-hepatic vein difference of approximately 90%. High performance liquid chromatographic analysis of deproteinized liver samples indicated that labeled nitrogen is exchanged rapidly among components of: mitochondrial aspartate aminotransferase and glutamate dehydrogenase reactions and cytoplasmic aspartate aminotransferase and alanine aminotransferase reactions (t1/2 for the exchange of label toward equilibrium is on the order of seconds). Comparison of specific activities of glutamate and ammonia suggests that at 5 s most labeled glutamate was mitochondrial, whereas at 60 s approximately 93% was cytosolic; this change is presumably brought about by the combined action of the mitochondrial and cytosolic aspartate aminotransferases and the aspartate carrier of the malate-aspartate shuttle. Specific activity measurements of glutamate, alanine, and aspartate are in accord with the proposal by Williamson et al. (Williamson, D.H., Lopes-Vieira, O., and Walker, B. (1967) Biochem. J. 104, 497-502) that the components of the aspartate aminotransferase reaction are in thermodynamic equilibrium, whereas the components of the alanine aminotransferase reaction are in equilibrium but compartmented in the rat liver. Despite considerable label in citrulline at early time points, no radioactivity (less than or equal to 0.25% of the total) was detected in carbamyl phosphate, suggesting very efficient conversion to citrulline with little free carbamyl phosphate accumulating in the mitochondria. Our data also show that some portal vein-derived ammonia is metabolized to glutamine in the rat liver, but the amount is small (approximately 7% of that metabolized to urea) in part because liver glutamine synthetase is located in a small population of perivenous cells "downstream" from the urea cycle-containing periportal cells. Finally, no tracer evidence could be found for the participation of the purine nucleotide cycle in ammonia production from aspartate. The present work continues to emphasize the usefulness of [13N]ammonia for short-term metabolic studies under truly tracer conditions, particularly when turnover times are on the order of seconds.     

Characterization of three group A klebicin plasmids: localization of their E colicin immunity genes

We have investigated the immunity to E colicins conferred by three group A klebicin plasmids. pP5a, which encodes klebicin A1-P5, like pClo-DF13, confers immunity to colicin E6 on Escherichia coli K12, whilst pP5b and pP3, which encode klebicins A2-P5 and A3-P3 respectively, both confer immunity to colicin E3. We have determined the restriction endonuclease and functional maps of the three group A klebicin plasmids. By sub-cloning and transposon mutagenesis we have investigated the relationship between the klebicin immunity and the E colicin immunity conferred by these plasmids. The colicin E6 and the klebicin A1 immunity are encoded by a single gene present on pP5a. The colicin E3 and the klebicin A2 immunity are encoded by a single gene present on pP5b. The colicin E3 and the klebicin A3 immunity are encoded by separate genes present on pP3. Recombinant pML8412, which is derived from the ColE6-CT14 plasmid and encodes colicin E6 immunity, confers klebicin A1-P5 immunity upon Klebsiella pneumoniae UNF5023. Recombinant pKC23, which is derived from the ColE3-CA38 plasmid and confers colicin E3 immunity, confers immunity to klebicin A2-P5, but not to klebicin A3-P3.     

2-Phenylethylamine catabolism by Escherichia coli K12

Escherichia coli K12 grows on 2-phenylethylamine as sole carbon and energy source by converting it, via phenylacetaldehyde, to phenylacetic acid. Phenylacetaldehyde was formed by the action of an inducible amine oxidase and catalase activity was increased sixfold, presumably to ensure removal of the H2O2 that was expected to be a product of the amine oxidation. The phenylacetaldehyde was oxidized to phenylacetic acid by an inducible NAD+-dependent dehydrogenase. Mutants defective in phenylacetaldehyde dehydrogenase cannot grow on 2-phenylethylamine as carbon and energy source but can still use it as a nitrogen source.     

Recombinant interferon-alpha, -beta, and -gamma enhance the proliferative response of human B cells

Recombinant interferons (IFN-alpha, -beta, and -gamma) were examined for their effects on B cell activation. Relatively small IgM+ B cells from human blood samples were isolated by fluorescence-activated cell sorting and were used as target cells. Although the interferons themselves were nonmitogenic, each enhanced the proliferative response induced by a mitogenic anti-mu monoclonal antibody, with IFN-beta usually showing the greatest enhancement and IFN-gamma the least. Pretreatment with the interferons primed resting B cells to undergo enhanced DNA synthesis in response to the anti-mu antibody DA4. Conversely, anti-mu pretreatment, followed by IFN treatment, did not induce B cells to enter the S phase. Time-course analysis revealed that IFN could augment the anti-mu response even when added as late as the final 24 hr of a 3-day culture interval. Combinations of IFN-gamma plus IFN-alpha or -beta were synergistic in the anti-mu response, whereas the IFN-alpha plus IFN-beta combination was not. The data suggest that interferons produced by both lymphocytes (IFN-gamma) and nonlymphoid inflammatory cells (IFN-alpha and -beta) can enhance B cell growth via different mechanisms.     

Characterization of B lymphocyte lineage progenitor cells from mice with severe combined immune deficiency disease (SCID) made possible by long term culture

A single gene mutation results in near absence of B and T lymphocytes and their immediate progenitors in mice with severe combined immunodeficiency disease (SCID). However, long term culture conditions allowed rapid outgrowth of lymphocytes from SCID bone marrow suspensions, and this permitted their detailed analysis. The cells were judged to be committed to the B lymphocyte lineage on the basis of expression of the BP-1 antigen, as well as by the density and pattern of expression of other markers. Cultured SCID lymphocytes were indistinguishable from control BALB/c cells in terms of morphology, typing for 13 cell surface markers, and changes in cell surface antigen expression with time in culture. In contrast to cultures of normal cells, which always included IgM synthesizing cells, SCID lymphocytes rarely expressed mu heavy chains. Southern blot analysis demonstrated that at least the first Ig gene rearrangement step had occurred in most of the cultured cells. The patterns of JH gene rearrangements suggested that relatively limited population diversity existed in individual cultures of SCID and normal BALB/c marrow. In addition, there was evidence that abnormal Ig heavy chain gene rearrangements had taken place in lymphocytes from approximately 25% of the SCID cultures. These cells were distinguished by the absence of detectable JH gene segments. kappa light chain genes appeared to be unrearranged in SCID cultured lymphocytes. We conclude that the lymphopoietic microenvironments of SCID mice are probably normal, and the animals have infrequent progenitors of B cells. Aberrant or nonproductive IgH gene rearrangements may account for the absence of pre-B and B cells in SCID mice. This study demonstrates the usefulness of long term culture methodology for isolating rare subsets of non-transformed lymphoid cells from normal and genetically defective hemopoietic tissues.     

Identification of Lotus Rhizobia by Direct DNA Hybridization of Crushed Root Nodules

Hybridization of crushed Lotus pedunculatus root nodules with ³²P-labeled total genomic DNA probes was used to identify Rhizobium loti and Bradyrhizobium sp. (Lotus rhizobia). Probes always hybridized with homologous target DNA and frequently with DNAs of other strains from the same genus. Intergeneric hybridization did not occur. Results were comparable to those from colony hybridization.     

Toxicity of enzymically-oxidized low-density lipoprotein

Intravenous injection of cholesterol oxidase into hyperlipidemic rabbits in which aortic atheromatous lesions have been induced by dietary means is lethal within hours, whereas injection of the same enzyme into normal rabbits has no visible adverse effect. The lethal effect of the enzyme is explicable by the finding that injection of cholesterol-oxidase treated low-density lipoprotein kills normal rabbits, in contrast to untreated low-density lipoprotein which does not. Enzymically oxidized low-density lipoprotein was also found to be cytotoxic for two human cell lines and for cultured bovine aortic endothelial cells. We suggest that in vivo enzymic conversion of low-density lipoprotein cholesterol to low-density lipoprotein cholestenone may possibly play a role in the initiation of atheromatous lesions in humans.     

Purification and characterization of Rhodobacter sphaeroides acyl carrier protein

Acyl carrier protein (ACP) has been purified from the facultative phototrophic bacterium Rhodobacter sphaeroides. The ACP preparation was greater than 95% homogeneous as determined by native and disodium dodecyl sulfate (Na2DodSO4)-polyacrylamide gel electrophoreses and N-terminal amino acid analysis. Amino acid compositional analysis revealed that the protein contains approximately 75 amino acids, has a calculated minimum molecular weight of 8700, and lacks the amino acids tyrosine and tryptophan. The presence of the characteristic 4'-phosphopantetheine prosthetic group was indicated by the occurrence of equimolar quantities of beta-alanine and taurine in amino acid hydrolysates and was confirmed by independent chemical analysis. The protein displayed a pI of 3.8 and had a calculated partial specific volume of 0.732 mL/g. The primary structure of the protein has been determined for the first 46 amino acid residues from the N terminus of the molecule, and the region of the molecule encompassing the amino acids from residues 31 to 44 was found to have 100% homology with the identical residues in Escherichia coli ACP. In contrast to E. coli ACP, R. sphaeroides ACP migrated according to its molecular weight during Na2DodSO4 gel electrophoresis, was resistant to pH-induced denaturation, and comigrated with the cis-vaccenoyl-ACP derivative during native gel electrophoresis. It is proposed that the basis for these properties is the enhanced hydrophobic character of the protein.