Antidepressants and seizure-interactions at the GABA-receptor chloride-ionophore complex

Convulsive seizures are a potential side effect of antidepressant drug treatment and can be produced by all classes of antidepressants. It is also known that some convulsant and anticonvulsant drug actions are mediated by the GABA-receptor chloride-ionophore complex. Drugs acting at this complex appear to induce convulsions by inhibiting chloride conductance through the associated chloride channel. Using the method of GABA-stimulated 36Cl-uptake by rat cerebral cortical vesicles, we show that some antidepressant drugs (imipramine, amitryptyline, and mianserine) can inhibit the GABA-receptor chloride uptake, and that the degree of chloride channel inhibition by these drugs correlates with the frequency of convulsive seizures induced by them.     

Egg yolk lipoprotein,a new supplement for the growth of mammalian cells in serum-free medium

Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity.     

Fermentation of methanethiol and dimethylsulfide by a newly isolated methanogenic bacterium

From dilution series in defined mineral medium, a marine iregular coccoid methanogenic bacterium (strain MTP4) was isolated that was able to grow on methanethiol as sole source of energy. The strain also grew on dimethylsulfide, mono-, di-, and trimethylamine, methanol and acetate. On formate the organism produced methane without significant growth. Optimal growth on MT, with doubling times of about 20 h, occurred at 30°C in marine medium. The isolate required p-aminobenzoate and a further not identified vitamin. Strain MTP4 had a high tolerance to hydrogen sulfide but was very sensitive to mechanical forces or addition of detergents such as Triton X-100 or sodium dodecylsulfate. Methanethiol was fermented by strain MTP4 according to the following equation:

Cloning, Nucleotide Sequences and Differential Expression of the nifH and nifH-Like (frxC) Genes from the Filamentous Nitrogen-Fixing Cyanobacterium Plectonema boryanum

The frxC gene, found in liverwort chloroplast DNA, encodes aprotein of unknown function. The deduced amino acid sequenceof the protein shows significant homology to that of ni-trogenaseFe-protein encoded by the nifH gene. We have cloned the frxCand nifH genes from the nitrogen-fixing cyanobacterium Plectonemaboryanum, using frxC- and nifH-specific probes, and have determinedtheir nucleotide sequences. The amino acid sequence deducedfrom the frxC gene of P. boryanum exhibits 83% homology to thatof the protein encoded by the/rxCgene from liverwort, whereasit exhibits only 34% homology to that encoded by the nifH genefrom the same organism, namely, P. boryanum. Northern blot analysisshowed that the frxC gene was transcribed more actively undernitrogenase-repressed conditions than under nitrogenase-inducedconditions, suggesting that the FrxC protein has a functiondistinct from nitrogen fixation. These results, together withthe phylogenetic relationship between the nifH and frxC genes,indicate that the frxC and nifH genes are derived from a commonancestral gene but have evolved independently to encode proteinswith different functions. (Received April 27, 1991; Accepted August 12, 1991)     

Molecular cloning of the gene coding for the human T cell differentiation antigen CD7

The CD7 molecule is a differentiation antigen found on the surface of T lymphocytes and also on a very minor fraction of acute nonlymphocytic leukemia (ANLL). To study the genomic structure of the CD7 gene, two clones (SY4 and SY22) were isolated by screening a genomic library with a CD7 cDNA probe. Restriction mapping of these two phage clones showed that both overlapped each other, covering a total length of 23 kilobases (kb). Transfection of mouse L cells demonstrated that SY22 contains the gene expressing the CD7 antigen reactive with monoclonal CD7 antibody (Tp40), while SY4 does not. Subcloning of a 10.5 kb fragment from a 14.4 kb insert of SY22 contained the structural gene for the CD7 antigen. Detailed restriction mapping and partial sequence analysis revealed the CD7 gene to consist of four exons. By RNase protection assay, multiple initiation sites — 122 base pairs (bp) to — 38 bp from ATG translation initiation site were demonstrated. The promoter region had high G+C content and contained two SP1 binding sites (CCGCCC) and an AP2 binding site (CCCCAGGC), but lacked CAAT and TATA motifs.     

The production of anorexigenic substances by intestinal flora

The role of intestinal flora in the production of anorexigenic substance was investigated. Proteus mirabilis (P. mirabilis) and Escherichia coli (E. coli) were found to produce an anorexigenic substance, while Enterococcus faecalis (E. faecalis, type 1 and 2) and Staphylococcus intermedius (S. intermedius) did not. The anorexigenic substance was purified and was detected as, a single though broad band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of the final form of the purified substance was 120 units/mg carbohydrate. The substance contained no protein residue and appeared to be a lipopolysaccharide. The evidence that intestinal flora produces an anorexigenic substance leads to an interesting assumption that the intestinal flora may be responsible for regulating food intake.     

Activation of phosphorylase kinase through autophosphorylation by membrane component phospholipids

Phosphatidic acid (PtdOH) has been shown not only to stimulate autophosphorylation and autoactivation of phosphorylase kinase of rabbit skeletal muscle but also to decrease the apparent Ka for Ca2+ on autophosphorylation sharply [Negami et al. (1985) Biochem. Biophys. Res. Commun. 131, 712-719]. In this study we investigated the interaction between PtdOH and other phospholipids on autophosphorylation and autoactivation of this enzyme. Acidic phospholipids, such as phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns) and PtdOH, stimulated this reaction about 2-4-fold, and the approximate Ka values of this reaction were 10 micrograms/ml, 6.3 micrograms/ml and 30 micrograms/ml respectively. The molar ratio of PtdIns and PtdSer with maximal effect on autophosphorylation was about 1:1. Under these conditions PtdOH stimulated the initial velocity of autophosphorylation about 5.2-fold. When fully autophosphorylated, about 12-13 mol phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of mixed acidic phospholipids (PtdOH:PtdIns:PtdSer = 2:1:1), which was about twice as much as values observed without effectors. In the presence of mixed acidic phospholipids there was a concomitant enhancement of kinase activity, about 30-40-fold at pH 6.8 and 2.5-3-fold at pH 8.2. Mixed acidic phospholipids sharply decreased an apparent Ka for Ca2+ from 4 X 10(-5) M to 8 X 10(-7) M. With mixed acidic phospholipids as effectors this autophosphorylation occurred through an intramolecular mechanism. Based on these results, autophosphorylation and autoactivation of phosphorylase kinase in the presence of acidic phospholipids may account for an important regulatory mechanism of glycogenolysis in muscle contraction.     

Protease-activated protein kinase in rat liver plasma membrane

Upon limited proteolysis with trypsin, a cAMP and Ca2+-independent protein kinase was produced from rat liver plasma membrane. This enzyme showed a multifunctional capacity and phosphorylated calf thymus histone and rat liver ribosomal proteins. The molecular weight was estimated to be 5.0 X 10(4). When plasma membrane was treated with a buffer containing Triton X-100, a proenzyme with a molecular weight of 8.4 X 10(4) was extracted. By tryptic digestion, the proenzyme was converted to an active protein kinase which was similar to the enzyme obtained by the direct digestion of membrane. However, this proenzyme phosphorylated H1 histone in the presence of Ca2+ and phospholipid without proteolytic digestion. These results indicate the existence of a protease-activated protein kinase in rat liver plasma membrane and the proenzyme seems to be same as protein kinase C.     

Calcium- and calmodulin-dependent phosphorylase kinase activity in porcine uterine smooth muscle

Porcine uterine smooth muscle phosphorylase kinase has been partially purified. The enzyme was activated about 1.5-2.0-fold by exogenous calmodulin. Half maximal stimulation was observed at about 100 nM calmodulin. The activation was dependent on calcium and was maximum at pH 7.5 in the range of pH from 6 to 9. This activation was completely abolished by 100 microM trifluoperazine. The result suggested that unlike slow and cardiac muscles, phosphorylase kinase of uterine smooth muscle showed similar response to calmodulin with that of fast muscle. The physiological role of the calcium and calmodulin-dependent activation of myometrium phosphorylase kinase is briefly discussed.     

Bombesin evoked acetylcholine release from the guinea pig antrum

Bombesin induced contraction and acetylcholine (ACh) release of the longitudinal muscle strip of the guinea pig antrum were examined using the standard organ bath technique and the superfusion system. Bombesin increased frequency and tonus of rhythmic contraction in a dose dependent manner (10(-10)M - 10(-7)M). The effects of bombesin on frequency of contraction were not affected by atropine, propranolol, phentolamine, hexamethonium and tetrodotoxin. The effects on tonus, on the other hand, were significantly reduced by atropine, and the dose response curve to bombesin was shifted to the right. There was a remarkable increase of 3H-ACh release by the superfusion of bombesin (10(-8)M), which was almost completely abolished in Ca-free medium, but not affected by hexamethonium and tetrodotoxin. These results suggest that mechanism of bombesin effects on frequency is different from that on tonus; frequency response to bombesin is not dependent on autonomic nervous system but due to a direct effect on smooth muscle cells, whereas tonic response to the peptide is partly mediated by ACh release via a mechanism independent of sodium spike.