Chromosomal transformation in the cyanobacterium Agmenellum quadruplicatum.

Chromosomal transformation of Agmenellum quadruplicatum PR-6 (= Synechococcus sp. strain 7002) was characterized for phenotypic expression, for exposure time to DNA, and for dependence on DNA concentration with regard to Rifr donor DNA. Exponentially growing cells of PR-6 were competent for chromosomal transformation. Competence decreased in cells in the stationary phase of growth or in cells deprived of a nitrogen source. Dark incubation of cells before exposure to donor DNA also decreased competence. Homologous Rifr and Strr DNA and heterologous Escherichia coli W3110 DNA were used in DNA-DNA competition studies, which clearly showed that DNA binding by PR-6 was nonspecific. DNA binding and uptake by PR-6 exhibited single-hit kinetics. Single-stranded DNA failed to transform competent cells of PR-6, and DNA eclipse was not observed, suggesting that double-stranded DNA was the substrate for the binding and uptake reactions during the transformation of PR-6. A significant improvement in transformation frequency was achieved by increasing the nitrate content of the culture medium and by lowering the temperature at which cells were exposed to donor DNA from 39 degrees C (the optimal temperature for growth) to 30 degrees C.     

Class F Thy-1-negative murine lymphoma cells are deficient in ether lipid biosynthesis

The glycosyl phosphatidylinositol (PI) membrane anchors of several proteins contain 1-alkyl-2-acyl-glycerophosphoinositol. Although this PI analog has never been found free in cells, the presence of "alkyl-PI" as a component of some membrane anchors suggests its existence. The resistance of ether linkages to cleavage by mild alkali treatment was used to detect possible alkyl chains in the [3H]inositol-labeled phospholipids of several murine lymphoma cell lines which normally express the glycosyl PI-anchored protein Thy-1. One lipid, which arose from alkaline hydrolysis of PI and had mobility on thin layer chromatography similar to lyso-PI, was detected in all wild-type cell lines. Analysis of the base-stable inositol lipids of several lymphoma lines that are deficient in Thy-1 surface expression because of defective biosynthesis of the glycosyl PI membrane anchor revealed that the putative alkyl-PI was missing in the class F mutant. The levels of both the ethanolamine- and choline-containing plasmalogens were also decreased 10-fold in these cells, suggesting a general defect in the production of ether lipids. The activity of the peroxisomal form of dihydroxyacetonephosphate acyltransferase, which catalyzes the first step of ether lipid biosynthesis, was found to be 10-fold decreased relative to the wild-type level. Unlike previously described Chinese hamster ovary cell mutants deficient in ether lipids (Zoeller, R. A., and Raetz, C. R. H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5170-5174), the class F Thy-1- cells contain intact functional peroxisomes. Attempts to restore the putative alkyl-PI to the class F mutants by alkylglycerol supplementation were unsuccessful, despite concomitant restoration of the much larger plasmenylethanolamine pool, suggesting that there are some differences in the biosynthesis of this PI analog and plasmalogens that are presently not understood. Although the deficiencies in ether lipids and surface expression of Thy-1 in the class F mutants could also be due to separate mutations, our findings raise the possibility that alkyl-PI exists in animal cells and may be an obligate precursor for the biosynthesis of the glycosyl-PI membrane anchor of Thy-1.     

NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine

1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebroside sulfatase activator deficiency induced metachromatic leukodystrophy

Two siblings of consanguineous parents had presented with a variety of findings indicative of juvenile metachromatic leukodystrophy (MLD). However, instead of the expected profound deficiency of arylsulfatase A (ARS A), their enzyme levels were about half-normal, and enzyme from fibroblasts had properties identical with the properties of enzyme from normal fibroblasts. Nevertheless, the hydrolysis of cerebroside sulfate by growing fibroblasts was markedly attenuated. Supplementation of the fibroblasts with cerebroside sulfatase activator normalized the response in the loading test. These results imply that the fibroblasts, and by extension the patients, are deficient in activator. Although the defective catabolism of cerebroside sulfate and the clinical manifestations in these patients mimic MLD, the molecular basis is distinct from the classical forms of the disorder.     

Age-related motor and catecholomine alterations in mice on levodopa supplemented diet

Old mice reared on regular diet show reduced motor activity, decreased basal adenylate cyclase, and increased MAO activities compared to adults. Brain DDC and COMT activities, DA, NE levels and DA-stimulated adenylate cyclase remained unchanged. By contrast, mice fed levodopa for life did not develop decreased motor activity with aging, lived about 50% longer, had slightly elevated whole brain DA and NE levels and failed to develop the expected rise in MAO activity with aging. Levodopa did not alter the number of dopaminergic and muscarinic cholinergic receptors or the adenylate cyclase activity in the striatum during aging. On levodopa, hepatic and renal DA, dopa, and HVA increased but the latter two returned to basal levels by mid life. In liver, DDC was unchanged but MAO tended to be higher in levodopa-fed mice. Thus, motor impairment is an age-related phenomenon in mice associated with selective alterations in brain dopaminergic systems, which may be prevented by dietary levodopa. Extracerebral tissues, through possibly adaptive metabolic mechanisms, play a significant role in regulating brain catecholamines during chronic administration of large doses of levodopa.     

Piroxicam, a structurally novel anti-inflammatory compound. Mode of prostaglandin synthesis inhibition

Piroxicam is a potent inhibitor of prostaglandin biosynthesis. Experiments utilizing cell culture and microsomes derived from various sources have demonstrated that piroxicam is a selective inhibitor of the cyclooxygenase step of arachidonic acid metabolism. Little blocking activity is observed at the phospholipase, thromboxane or prostacyclin synthetase, and arachidonic acid lipoxygenase steps.     

The effect of 4-isothiocyanatobenzene sulfonic acid on the oxygen affinity of human hemoglobin

Human hemoglobin reacts with 4-Isothiocyanatobenzene sulfonic acid at the four amino groups of the N-terminal valines. The modified protein shows a decreased oxygen affinity over a wide pH range, a reduced alkaline Bohr effect, decreased co-operativity, and a reduced effect of inositol hexasulfate on the oxygen affinity.     

Vacuum-ultraviolet circular dichroism of chondroitins and their complexes with poly(L-arginine)

The vacuum-ultraviolet circular dichroism (VUCD) of chondroitin and chontroitin-6-sulfate has been measured to 160 nm for films and to 170 nm for D₂O solutions. The pD-dependent dichroic behavior of these glycosaminoglycans in D₂O is similar above 200 nm and is in agreement with previous studies. Near 190 nm, the CD band sign is also dependent on pD. VUCD spectra were recorded for films and solutions of poly(L -arginine). In trifluoroethanol the polypeptide is α-helical, while in D₂O it exists as a random coil. The well-characterized coil–helix transition of poly(L -arginine) during complexation with chondroitin-6-sulfate was observed by VUCD, including the previously inaccessible entire π → π* band. By construction of difference spectra it was also possible to monitor the VUCD of the polysaccharide component during complexation.     

New method for large-scale growth; and concentration of the Epstein-Barr viruses.

Efficacious systems are described for the large-scale growth in tissue culture and concentration of infectious (P3HR-1) and transforming (B95-8) Epstein-Barr virus. Also recorded here are our updated procedures for growing stock cultures and protocols to harvest fluids containing biologically active virus which is infectious or transforming. Various methods of concentrating biologically active Epstein-Barr virus have been evaluated. Cellular debris can be removed efficiently and rapidly from culture harvest fluids by clarification through a JCF-Z continuous-flow rotor. Efficient and reliable virus concentration was achieved by molecular filtration with Millipore Pellicon cassettes, using flow rates to 10 liters/h to produce fivefold concentrates followed by pelletization in a fixed-angle rotor. Data from recent production lots showed an average infectivity titer for P3HR-1 virus of 10(4.5) early antigen units per ml (100-fold concentrate) and 10(5.7) transforming units per ml (200-fold concentrate) for B95-9 virus lots.