Phosphorylation ofEscherichia coli NADP+-specific glutamate dehydrogenase

Glutamate dehydrogenase fromEscherichia coli is phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein, when exposed to acid conditions, releases the phosphate; this implies that the phosphorylation site is not on a serine, tyrosine, or threonine residue(s). Treatment of glutamate dehydrogenase with diethyl pyrocarbonate, a highly specific histidine-modifying reagent, blocks incorporation of³²P-phosphate from [-³²P]ATP into the enzyme, suggestive that the phosphorylation site is a histidine residue(s). The phosphorylated glutamate dehydrogenase was identified on the basis of its comigration with highly purified glutamate dehydrogenase, isolated fromE. coli, on denaturing, nondenaturing, and isoelectric focusing polyacrylamide gels and by sequence analysis.     

Phorbol ester receptors in cerebral cortex of cats with GM1 gangliosidosis

The pathogenesis of neuronal dysfunction in the gangliosidoses is poorly understood. Studies of the feline gangliosidoses and in vitro experiments implicate ganglioside inhibition of protein kinase C (PKC) in the pathogenesis of these neurological diseases. Therefore, in the present study, the binding of [³H]phorbol-12, 13 dibutyrate was measured to determine the levels of PKC in cerebral cortex of cats with GM₁ gangliosidosis (mutant) and age matched normal siblings. This binding of ([³H]PDB) to cerebral cortex homogenates in both normal and mutant cats was highly specific. The specificity of receptors was ascertained also from displacement studies using nonradioactive phorbol ester analogues to displace [³H]PDB bound to its receptors. In both mutant and normal cat brain, phorbol 12, 13-dibutyrate (PDB), 4--phorbol 12,13-didecanoate (-PDA) and 4--phorbol 12,13-dibenzoate (-PDBz) were highly potent (approximately to same degree) and effective in displacing [³H]PDB. On the other hand, 4- phorbol 12,13-diacetate (-PDA) was a weak displacer and 4--phorbol did not displace the bound [³H]PDB in either normal or mutant brain. Scatchard analysis of the binding data indicated a homogenous single class of binding sites in normal and mutant brain (Normal: K_d=1.42×10^–7 M, B_max=8.40 pmoles/mg protein. Mutant: K_d=1.60×10^–7 M, B_max=10.00 pmoles/mg protein). Sphingosine inhibited the binding to approximately the same extent in normal and mutant cortex. These studies demonstrate the presence of highly specific, homogenous, single type phorbol ester receptors in cerebral cortex of cats with GM₁ gangliosidosis which are qualitatively and quantitatively similar to normal cat brain.     

Duplication of HRAS1, INS, and IGF2 is not a common event in Beckwith-Wiedemann syndrome

A few cases of Beckwith-Wiedemann syndrome (BWS) have in common a duplication of 11p15. Among the genes located in 11p15, c-Ha-ras 1 (HRAS1), insulin (INS), and insulin-like growth factor II (IGF2) may account for the clinical features and the increased risk for malignancy. Using eight 11p15 markers including HRAS1, INS and IGF2 we have studied eight sporadic and hereditary cases of BWS whether or not associated with a nephroblastoma. By gene dosage determination and family studies, we have shown the following: the eight patients examined had an apparent diploid representation of all of the eight markers studied, thus indicating that a microduplication of these markers or of the region characterized by these markers is not a common event in BWS; in a family with three affected sibs the genes for HRAS1 and INS/IGF2 did not cosegregate with BWS and therefore may not participate in the pathogenic processes here observed.     

A Bacillus thuringiensis subsp. israelensis gene encoding a 125-kilodalton larvicidal polypeptide is associated with inverted repeat sequences.

A gene encoding a 125-kilodalton (kDa) mosquitocidal delta-endotoxin was cloned from the 72-MDa resident plasmid of Bacillus thuringiensis subsp. israelensis. This gene is similar in its 3' region to the gene encoding the 135-kDa protein previously cloned (C. Bourgouin, A. Klier, and G. Rapoport, Mol. Gen. Genet. 205:390-397, 1986). Escherichia coli recombinant clones harboring the 125-kDa gene were toxic to larvae of the three mosquito species Aedes aegypti, Anopheles stephensi, and Culex pipiens. In addition, the B. thuringiensis subsp. israelensis DNA fragment carrying the 125-kDa protein gene contains two sets of inverted repeat sequences, identified either by the S1 nuclease method or by electron microscopic observation. The structural organization of inverted repeat sequences and of the 125-kDa gene was analyzed and suggests that this B. thuringiensis subsp. israelensis delta-endotoxin gene is located within a transposable element.     

Enhancement of Butanol Formation by Clostridium acetobutylicum in the Presence of Decanol-Oleyl Alcohol Mixed Extractants

Extractive fermentation has been proposed to enhance the productivity of fermentations that are end product inhibited. Unfortunately, good extractants for butanol, such as decanol, are toxic to Clostridium acetobutylicum. The use of mixed extractants, namely, mixtures of toxic and nontoxic coextractants, was proposed to circumvent this toxicity. Decanol appeared to inhibit butanol formation by C. acetobutylicum when present in a mixed extractant that also contained oleyl alcohol. However, maintenance of the pH at 4.5 alleviated the inhibition of butanol production and the consumption of butyrate during solventogenesis. A mixed extractant that contained 20% decanol in oleyl alcohol enhanced butanol formation by 72% under pH-controlled conditions. The production of acetone and acetoin was also increased, even though these two products were not extractable. The enhancement of butanol formation was not limited by the toxicity of decanol. Supplementation of glucose and butyrate in the extractive fermentation yielded a 47% increase in butanol. The enhancement of butanol formation appeared to be dependent on the presence of dissolved decanol in the broth but was not observed unless an organic phase was present to extract butanol. A mechanism for the effects of decanol on product formation is proposed.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity

The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis. The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine. Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree. Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol. Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol. The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM). The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM). Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis. Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity. Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.     

Stereocontrolled routes to cis-hydroxyamino sugars, part VII: synthesis of daunosamine and ristosamine

The nitrogen of an allylic amine can serve as the fulcrum for stereocontrolled delivery of oxygen to an adjacent trigonal site, and cis-hydroxyamino sugars can thus be prepared. Methods for achieving the complementary procedure, namely, control of the delivery of nitrogen to an adjacent site by an allylic oxygen, are described. For example, treatment of methyl 2,3,6-trideoxy-- -erythro-hex-2-enopyranoside with trichloroacetonitrile gave an imidate ester which reacted with iodonium dicollidine perchlorate to give 2-trichloromethyl-(methyl 2,3,4,6-tetradeoxy-2-iodo-- -altropyranoside)-[3,4-d]-2-oxazoline. Exhaustive reductive dehalogenation of this product followed by hydrolysis led to methyl N-acetyl-- -ristosaminide. An analogous series of reactions was used to prepare the corresponding daunosaminide.     

Converting Sendai virus into a specific fusogen whose cell target can be selected

Covalent intermolecular hybrids of Fab anti-hemagglutinin-neuraminidase (HN) monoclonal antibody and avidin were prepared and characterized. These conjugates were used to block and redirect the fusion activity of Sendai virus (SV). After incubation of SV with Fab anti-HN: avidin conjugate on ice for 1-2 h, the SV fused only those P815 or BW5147 cells which were labeled with biotin-modified anti-cell surface immunoglobulin. The levels of cell-cell fusion obtained were at least as high as those achieved with unmodified SV and unlabeled P815 or BW5147 cells. These results demonstrate that it is possible to block the normal agglutinating activity of the HN molecules of SV and to introduce a new cell recognition feature without negating the fusogenic potential of the virus. Such an approach may be useful in harnessing the fusion activity of SV to a targeted delivery system for microinjection of macromolecules into selected cell populations.