Prostaglandin-E2-induced activation of adenosine 3'-5' cyclic monophosphate-dependent protein kinases of a murine macrophage-like cell line (P388D1)

Changes in the activities of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinases in response to prostaglandin (PG)E2-induced elevation of intracellular cAMP level were investigated with a murine macrophage-like cell line, P388D1. Photoaffinity labeling with 8-azido-[32P]cAMP showed that untreated P388D1 cells possess two types of cAMP-binding proteins of m.w. 49,000 and 52,000, respectively, in the cytosol fraction in a ration of 1:8. They must represent regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinases, because affinity chromatography on a column of omega-aminohexyl-agarose of the cytosol fraction clearly separated two fractions that exhibited the enzymatic activities and cAMP-binding activities. Photoaffinity labeling of these fractions with 8-azido-[32P]cAMP confirmed the separation of two types of isoenzymes, because each cAMP-dependent protein kinase active fraction was associated with only one type of regulatory subunit. The exposure of P388D1 cells to exogenously added PGE2 (1 microM) caused about 7.5-fold increase in the intracellular cAMP level within 30 sec. The cAMP level then sharply dropped to about 100 pmol/10(7) cells, remained at this level for about 20 min, and then gradually increased to 200 pmol/10(7) (about fivefold over the control). The enzyme assay of the cytosol demonstrated that the activation of cAMP-dependent protein kinases closely follows the kinetics of the intracellular cAMP level. The activation of the enzyme was specific for PGE2 and was not triggered by 1 microM PGF2 alpha or PGD2 which have been shown to be unable to activate adenylate cyclase of P388D1 cells. The PGE2-induced increase in the intracellular cAMP level appeared to activate preferentially the type I isoenzyme, inasmuch as the enzymatic activity of this type separated by the affinity chromatography of the cytosol of PGE2-exposed cells was lower in the presence than in the absence of cAMP, whereas the type II enzyme activity remained responsive to exogenously added cAMP.     

Glutamine Synthetase of the Human Brain: Purification and Characterization

Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver.     

Solubilization and Characterization of the Nitrendipine Receptor in Rat Brain

The nitrendipine receptor associated with the voltage-dependent calcium channel in rat brain was solubilized by detergent extraction and sonication. The detergent solution used for extraction consisted of 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.25% (wt/vol) polyoxyethylene 20 cetyl ether (Brij 58), and 0.025% (wt/vol) polyoxyethylene 17 cetyl stearyl ether (Lubrol WX) in the presence of 30% (wt/vol) glycerol as a stabilizer. The molecular weight of the receptor was estimated to be 1,800K by Sephacryl S-500 gel filtration and 800K by sucrose density gradient sedimentation. The equilibrium dissociation constant of [3H]nitrendipine to the solubilized receptors was 5.6 nM, which is approximately 10 times that of the membrane-bound receptor. The binding of nitrendipine to the receptor was inhibited noncompetitively by the structurally unrelated calcium channel inhibitors verapamil and prenylamine; their concentrations for 50% inhibition were both 1.0 X 10(-7) M, and they caused maximal inhibitions of 70 and 100%, respectively.     

Detection of lipid hydroperoxides and hydrogen peroxide at picomole levels by an HPLC and isoluminol chemiluminescence assay

An isoluminol assay is utilized for the detection of hydrogen peroxide and lipid hydroperoxides in biological samples. The combination of this assay as a post-column detection for HPLC avoids interference of antioxidants and enables characterization of hydroperoxides at picomole levels. Two useful HPLC conditions for the separation of hydrogen peroxide, lipid hydroperoxides, antioxidants, and unoxidized lipids are described.     

Generation of Tn5 insertions in streptococcal conjugative transposon Tn916.

A method has been developed for the introduction of Tn5 into Escherichia coli plasmid chimeras containing Streptococcus faecalis DNA. Tn5 could be introduced via a lambda::Tn5 delivery vehicle. The system proved to be particularly efficient and facilitated insertions at numerous sites on DNA containing the 16-kilobase conjugative transposon Tn916. It was possible to introduce some of the resulting Tn916::Tn5 derivatives back into S. faecalis by using a recently developed protoplast transformation procedure. A presumed zygotic induction resulted in insertion of the Tn916 derivatives at multiple sites in the S. faecalis chromosome.     

Effect of roasting on ochratoxin A level in green coffee beans inoculated with Aspergillus ochraceus

The heat stability of ochratoxin A in green coffee beans inoculated with Aspergillus ochraceus was studied. Heat treatment (roasting) at 200 °C for 10 or 20 min reduced the levels of ochratoxin A by only 0–12% in the dried whole beans. Almost all of the ochratoxin A was infused into the coffee decoction when the roasted samples were ground and extracted with boiling water. Therefore, the reduction of ochratoxin A concentration of contaminated coffee beans by roasting under these conditions is ineffective.     

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.     

Genetic characterization of the mei-41 locus in Drosophila melanogaster

Summary The mutagen-sensitive mutant mus(1)104 ^D1 of Drosophila melanogaster maps to a position on the X chromosome very close to the meiotic mutant mei-41 ^D5 . Both mutants have been characterized as mutagen-sensitive and defective in post-replication repair. In the present report we show by complementation studies that mus(1)104 and mus(1)103 are allelic with mei-41. In addition, two reported alleles of mus(1)104 lie between the mei-41 alleles A10 and D5. The size of the mei-41 locus is estimated to be about 0.1 centimorgans (cM). Because several alleles of mei-41 have been shown to reduce recombination and increase meiotic chromosome loss and nondisjunction, mus(1)104 ^D1 females were examined for defects in meiosis. Although there was no evidence for reduced recombination on the second chromosome in homozygous mus(1)104 ^D1 females, heterozygous mus(1)104 ^D1 /mei-41 ^>D5 and mus(1)104 ^D1 /deficiency females showed reduced levels of recombination. However, there was no evidence of an increase in nondijunction in these females.We dedicate this article to the memory of Larry Sandler, who passed away suddenly on February 7, 1987