A developmental change in dolichyl phosphate mannose synthase activity in pig brain

The initial rate of dolichyl phosphate mannose biosynthesis was measured in white-matter membranes from pig brain at various ages from before birth throughout the period of most rapid brain development. Dolichyl phosphate mannose synthase activity increased from prenatal values to a maximum in 3 week-old animals, and gradually decreased to adult values after 8 weeks of age. The nature of the developmental change was investigated by enzymic and biochemical comparisons of the membrane preparations from the most active age (3 weeks) and adult controls. The specific activity of dolichyl phosphate mannose synthase in preparations from actively myelinating animals was approx. 3-fold higher than adults when mannolipid formation was assayed with saturating concentrations of GDP-[¹⁴C]mannose and utilizing only endogenous acceptor lipid. No major variations were found in the apparent K_m values for GDP-mannose or exogenous dolichyl monophosphate. However, the ratio of dolichyl phosphate mannose synthase activity for myelinating animals/adult animals decreased significantly when large amounts of exogenous dolichyl monophosphate were added to the incubation mixtures. Dolichyl phosphate mannose synthase activity was also compared in white-matter membranes depleted of endogenous dolichyl monophosphate by enzymic mannosylation or treatment with butanol. When these preparations were assayed with identical amounts of exogenous dolichyl monophosphate, the dolichyl monophosphate-depleted membranes from actively myelinating animals contained only 20–30% more dolichyl phosphate mannose synthase activity. Overall, these studies strongly suggest that the developmental change in dolichyl phosphate mannose synthase activity is due primarily to the presence of a relatively lower amount of endogenous dolichyl monophosphate being accessible to the mannosyltransferase in the white-matter membranes from adult animals.     

Kinetic studies of the binding of inhibitors to thermolysin

The K_I values for inhibition of thermolysin activity by N-β-phenylpropionyl-aliphatic amino acids (Gly, Ala, Val, Leu, Ile) are correlated by π, the hydrophobic substituent parameter for the amino acid side chain (log K_I = ?0.73π ?1.80, correlation coefficient = 0.990). By contrast, the K_I values for the corresponding benzyloxycarbonyl amino acids are poorly correlated by π, but show a good correlation with the steric parameter E_s(log K_I = 0.880E_s ? 3.086, correlation coefficient = 0.985). Binding of β-phenylpropionyl-l-alanine is associated with an acidic residue of pK 7.3 and a basic residue of pK 8.0 in the E · I complex, and appears to raise the pK of Glu-143 by 2 units. Binding of benzyloxycarbonyl-Ala and -Phe is associated with an acidic residue of pK 8.0 and two basic residues, both with pK 8.3. Three similar pK values are observed with benzyloxycarbonyl-Phe. These results are interpreted in terms of different modes of binding of β-phenylpropionyl and benzyloxycarbonyl inhibitors.     

Newly biosynthesized cytochrome P-450 associated with the golgi apparatus from livers of rats induced with phenobarbital.

1. Cytochrome P-450 has been detected in preparations of golgi apparatus from the livers of phenobarbital-induced rats. 2. Newly biosynthesized cytochrome P-450 is also present in preparations of golgi apparatus. By using three different techniques to fractionate the golgi into vesicle contents and membrane components it was found that newly biosynthesized cytochrome P-450 is associated solely with the membrane fraction. 3. By investigating the susceptibility of cytochrome P-450, present in the golgi apparatus, to the action of trypsin it was found that the majority was oriented on the cytosolic face of the membrane.     

Low hydration phase properties of phospholipid mixtures

An experimental investigation of the low hydration phase properties of phospholipid mixtures is described. ²H (D₂O) NMR, X-ray diffraction and differential scanning calorimetry have been used to elucidate the phase properties of mixtures of the mixed chain phospholipids palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylethanolamine (POPE). At 10% hydration pure POPE exhibited a H_II phase above 330 K, a fluid lamellar phase below 315 K, and a minimally hydrated crystalline phase below 300 K. For the 1:1 mixture, the samples exhibited only gel or fluid phases between 270 K and 360 K for hydrations in the range 15% to 30%. Below 15% hydration the mixture exhibited two fluid phases with different repeat spacings, as predicted previously.     

Effect of Hydrogenase and Mixed Sulfate-Reducing Bacterial Populations on the Corrosion of Steel

The importance of hydrogenase activity to corrosion of steel was assessed by using mixed populations of sulfate-reducing bacteria isolated from corroded and noncorroded oil pipelines. Biofilms which developed on the steel studs contained detectable numbers of sulfate-reducing bacteria (10⁴ increasing to 10⁷/0.5 cm²). However, the biofilm with active hydrogenase activity (i.e., corrosion pipeline organisms), as measured by a semiquantitative commercial kit, was associated with a significantly higher corrosion rate (7.79 mm/year) relative to noncorrosive biofilm (0.48 mm/year) with 10⁵ sulfate-reducing bacteria per 0.5 cm² but no measurable hydrogenase activity. The importance of hydrogenase and the microbial sulfate-reducing bacterial population making up the biofilm are discussed relative to biocorrosion.     

Rat pineal gland phosducin: cDNA isolation, nucleotide sequence, and chromosomal assignment in the mouse

The pineal gland contains a soluble phosphoprotein, phosducin, which is homologous to that of retinal photoreceptors. Phosducin has been shown to bind the beta, gamma subunits of the retinal G-protein transducin. Retinal phosducin has been cloned and now we report a rat pineal cDNA encoding phosducin. A 1217-nucleotide cDNA was isolated from a rat pineal library by DNA-DNA hybridization with a polymerase chain reaction-amplified cDNA of bovine retina mRNA for phosducin. Northern blot analysis demonstrates that the mRNA for phosducin is approximately 1.3 kb in both rat pineal and rat retina. The translated mRNA from rat pineal encodes a protein with 246 amino acids, compared to the 245 amino acids of bovine retina phosducin. The predicted molecular weight of rat pineal phosducin is 28,201. Immunoblot analysis with affinity-purified antibodies against bovine retina phosducin identify a single immunoreactive protein of approximately 33 kDa in both rat retina and rat pineal. The amino acid sequence of rat pineal phosducin is homologous to that of bovine retina phosducin, revealing 89% identity and another 5.7% similarity. Both rat pineal and bovine retina phosducins are acidic proteins with pIs of 4.3 and 4.5, respectively. The translated protein lacks hydrophobic domains that would suggest an integral membrane protein. Rat pineal phosducin has a single consensus phosphorylation domain for protein kinase A that is nearly identical to that of retinal phosducin, which is phosphorylated by protein kinase A in situ. Rat phosducin also contains three potential phosphorylation domains for protein kinase C and nine for casein kinase II as well as a predicted site for N-glycosylation. The cDNA encoding phosducin was used to localize the gene within a linkage group to a large segment of mouse chromosome 1 in a conserved region with the long arm of human chromosome 1 with a panel of DNA samples from an interspecific cross. In keeping with a proposed role of retinal phosducin in down-regulation of the photo-transduction cascade, a modulatory role in signal transduction is proposed for pineal phosducin.     

Three viruses found in the braconid parasitoid Microplitis croceipes and their implications in biological control programs

Productivity and longevity decreased in a laboratory colony of the parasitoid wasp Microplitis croceipes (Cresson) (Hymenoptera: Braconidae). Using light microscopy, it was determined that the colony was free of microsporidia. However, samples of the colony examined for pathogens by electron microscopy revealed three types of viruses: a nonpathogenic polydnavirus which is produced by all female wasps; a nonoccluded baculovirus which is pathogenic to late-stage pupae and adults; and a picorna-like virus which is present in larvae, pupae, and adults. The nonoccluded baculovirus was eliminated from the laboratory colony of M. croceipes by selection of progeny from wasps which had oviposited within 2 to 3 days after emergence from the cocoons and which had lived for at least 14 days post-emergence. Upon death, the wasps were examined by negative stain electron microscopy and only progeny from baculovirus-free wasps were retained. Parasitoid colonies should be systematically examined for pathogenic viruses that may reduce their productivity and efficacy as biological control agents. In addition, exotic parasitoids and predators should be evaluated for viruses and other pathogens while in quarantine.     

The effect of pH on the production of cellulases in Aspergillus terreus

Summary The optimal pH for the production of extracellular cellulolytic enzymes in the wild strain of Aspergillus terreus was shown to be pH 5.0. After 160 h of cultivation, carboxymethyl cellulase reached 9.0 IU/ml, filterpaper, cellulase 0.5 IU/ml and -glucosidase 0.9 IU/ml. The rate of synthesis of CM- and FP-cellulases decreased after 90 h of cultivation but -glucosidase was produced linearly for 160 h. Some of the enzymes produced were released into the medium during the fungal growth while others remained bound. The binding of enzymes to cells and residual crystalline cellulose was strongly affected by the pH of the medium. FP-cellulase and particularly -glucosidase were bound more effectively, at lower pHs. Cold shock treatment of the cell suspension increased the activities of FP- and CM-cellulases but -glucosidase activity was not affected.     

Versatile Method for Evaluating a Cell Culture by Various Morphological Techniques

Cellulose acetate is a versatile material for evaluating cells grown under identical conditions by various morphological techniques. This inexpensive material is transparent, easily cut to size and shape, nontoxic to cell cultures, and resistant to most chemicals used in histochemistry and in scanning and transmission electron microscopy. Samples may be obtained during and after the culture process. Cellulose acetate slides can be mounted directly over glass slides for direct observation and are easily peeled off plastic blocks for electron microscopy, leaving the cells behind. Relative disadvantages include its autofluorescence and a tendency to soften in strong acids or pure solutions of organic solvents such as xylene and propylene oxide.     

MORPHOLOGY AND DISTRIBUTION OF NUCLEI DURING DEVELOPMENT IN CYMOPOLIA BARBATA (CHLOROPHYCOPHYTA,DASYCLADALES)1

The morphology and distribution of a variety of types of nucleus in the apex, in young and mature gametangia, and in older regions of the Cymopolia cell were studied by light and electron microscopy. Spherical and convoluted nuclei 2–7μm in diameter were observed in apical regions of the vegetative siphon. Nuclei 4 μm in diameter were present in the young primary laterals and in developing gametangia. A single characteristic nucleus migrates from the siphon into the primary lateral of the second basipetal whorl. It is further transported into one of several possible secondary laterals and determines the development of gametangia which become multinucleate in the fourth or fifth whorls. Nuclei are characterized by size, shape, nucleolar morphology, nucleoplasmic inclusions and the ultrastructure of the perinuclear cytoplasm. Although no “primary nucleus” characteristic of the uninucleate genera, Acetabularia and Batophora was observed, some nuclei of Cymopolia have features in common with secondary nuclei of these genera.