Glutathione synthetase in tobacco suspension cultures: catalytic properties and localization

Glutathione synthetase activity (EC 6.3.2.3) was analysed in ammonium sulfate precipitates of extracts l'rom photohetevotrophically grown cells of Nicotiana tabactm L. cv. Samsun by determination of glutathione as its monobromobimane derivative. Maximal enzyme activity was obtained at pH 8.0–9.0 in Tris-HCl and CHES as buffer systems. The enzyme showed an absolute requirement for Mg2+ and was slightly stimulated by K+. When Mg2+ was replaced by Mn2+ less synthetase activity was observed, and above 30 m M Mn2+ no activity was found. The enzyme was specific for glycine (KM = 0.308 m M ). No product formation was observed with ß -alanine and γ y-aminobutyrate using substrate conccntrations of 10 m M . The apparent KM values for γ -glutamylcysteine and γ -glutamyl- l -α-aminobutyrate were, respectively, 0.022 and 0.033 m M . By chloroplast Isolation ca 24% of the total glutathione synthetase activity of the cells could be shown to be localized in the chloroplasts, the rest being attributed to the cytoplasm of the tobacco cells.     

The respiratory response to heat shock in Neurospora crassa

A sharp decrease in oxygen uptake occurred in Neurospora crassa cells that were transferred from 30 degrees C to 45 degrees C, and the respiration that resumed later at 45 degrees C was cyanide-insensitive. Energization of mitochondria, measured in vivo with fluorescence microscopy and a carbocyanine dye, also declined sharply in cells at 45 degrees C. Electron microscopy showed no changes in mitochondrial complexity; however, the cytoplasm of heat-shocked cells was deficient in glycogen granules.     

Biliary lipid secretion in patients with heterozygous familial hypercholesterolemia and combined hyperlipidemia. Influence of bezafibrate and fenofibrate

Serum and biliary lipid metabolism were examined in 13 patients with different types of hyperlipoproteinemia before and after 4 weeks of treatment with either bezafibrate or fenofibrate. In patients with heterozygous familial hypercholesterolemia (FH), bezafibrate (n = 5) and fenofibrate (n = 7) produced a similar significant reduction of total cholesterol, LDL-cholesterol, and triglycerides by 21, 23, and 32%, respectively. In patients with familial combined hyperlipidemia (CHL), only triglycerides decreased markedly. Biliary lipid secretion rates in patients with heterozygous FH were not different from those of young male volunteers, indicating that a reduction of hepatic LDL receptors did not affect hepatic elimination of cholesterol or bile acids. Biliary cholesterol secretion increased significantly from 57 to 75 mg/hr during bezafibrate therapy (n = 8) and from 62 to 71 mg/hr during fenofibrate therapy (n = 9). No consistent change in bile acid or phospholipid secretion was observed. The elevated output of biliary cholesterol increased cholesterol saturation significantly from 147 to 185% and from 152 to 173% during administration of bezafibrate and fenofibrate, respectively. The present study indicates that treatment with bezafibrate or fenofibrate is effective in lowering LDL cholesterol in patients with heterozygous FH, but both drugs increase cholesterol saturation of bile, which might enhance the risk of cholesterol gallstone formation.     

Processing of the asparagine-linked oligosaccharides of secreted and intracellular forms of the vesicular stomatitis virus G protein: in vivo evidence of Golgi apparatus compartmentalization

The structures of the asparagine-linked oligosaccharides of several variant forms of the vesicular stomatitis virus glycoprotein transiently expressed from cloned cDNAs have been determined. Glycopeptides isolated from forms of the G protein that reach the cell surface or that are secreted into the medium are virtually identical; they contain complex-type oligosaccharides whose nonreducing ends terminate in galactose and sialic acid residues. In contrast, forms of the G protein that remain intracellular possess oligosaccharides at intermediate stages in the processing pathway. One deletion mutant, delta 1473, codes for a protein that remains in the rough endoplasmic reticulum (Rose, J. K., and J. E. Bergmann, 1982, Cell, 30:753-762) and contains only high mannose-type oligosaccharides. Another mutant, delta 1554, codes for a glycoprotein that contains oligosaccharides of primarily two classes. One class is of the high mannose type and is similar to those found on the protein coded for by delta 1473. However, the major class contains biantennary and more highly branched complex-type oligosaccharides that terminate in N-acetylglucosamine rather than galactose or sialic acid residues. These data suggest that the protein coded for by delta 1554 migrates to the Golgi apparatus, but does not enter the more distal compartment(s) of the organelle which contains galactosyl- and sialyltransferases.     

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The lectin neuraminidase inhibition test: a new method for the detection of antibodies to neuraminidase

Two methods for the detection of neuraminidase antibodies were compared. The lectin neuraminidase inhibition test (LNI-test) gave results comparable with those provided by the conventional neuraminidase inhibition test (NI-test). Reproducibility and repeatability were better with the LNI-test which used smaller amounts of materials, was less time consuming than the NI-test and was more sensitive.     

Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein

We have altered the structure of the COOH-terminus of the vesicular stomatitis virus (VSV) glycoprotein (G) by introducing deletions into a cDNA clone encoding G protein. We examined the effects of these deletions on intracellular transport of G protein after expression of the deleted genes in eucaryotic cells under control of the SV40 late promoter. To prevent readthrough of translation into vector sequences, we introduced synthetic DNA linkers containing translation stop codons at the site of the deletion. G proteins that lacked the cytoplasmic domain and most of the transmembrane domain were secreted slowly from the cells. Deletion mutants affecting the structure of the cytoplasmic domain fell into two classes. The first class completely arrested transport of the protein to the cell surface at a stage prior to acquisition of complex oligosaccharides. The second class showed severely reduced rates of complex sugar addition although the proteins were eventually transported to the cell surface. Indirect immunofluorescence microscopy suggested that mutant proteins in both classes may accumulate in the rough endoplasmic reticulum.     

Diploids in Microsporum gypseum

The paper studies diploids in dermatophyteMicrosporum gypseum. They were isolated as the more rapidly growing sectors from heterokaryons on minimal medium. They are characterized by their wild morphology, conidiation and growth rate, and they are prototrophic. In their genome they contain all the markers present in both mutant components.