Characteristics of a lipolytic and fatty acid-requiring Butyrivibrio sp. isolated from the ovine rumen.

A naturally occurring fatty acid-requiring Butyrivibrio sp. (strain S2), isolated from the ovine rumen, deacylates plant galactolipids, phospholipids and sulpholipids to obtain sufficient fatty acid for growth. Growth in vitro was promoted by adding to the growth medium a single straight-chain saturated fatty acid (C13 to C18) or vaccenic acid. Palmitoleic and oleic acids also supported growth but gave lengthy lag phases probably due to their toxicity. Linolenic and linoleic acids supported good growth but they were completely hydrogenated to trans-11-octadecenoic acid which was incorporated into the bacterial complex lipids. No chain elongation, chain shortening or desaturation of the added fatty acids occurred and all were substantially incorporated into bacterial lipids of the plasmalogen type, partially as a new type of hydrophobic grouping derived from two molecules of fatty acid. The absence of fatty acid unsaturation poses the question of the maintenance of membrane fluidity within this bacterium.     

THE AFFINITY OF THE FUCOSE-BINDING LECTIN FROM LOTUS TETRAGONOLOBUS FOR GLYCOPEPTIDES AND OLIGOSACCHARIDES ACCUMULATING IN FUCOSIDOSIS

Abstract— The affinity of the fucose-binding lectin from Lotus tetragonolobus for fuco-oligosaccharides accumulating in the brain and other tissues of a patient with fucosidosis was studied by two methods: by inhibition of the co-precipitation of the lectin with porcine stomach mucin and by one-step affinity chromatography on a column of the lectin bound to Sepharose-4B. Both methods indicated that the lectin had greater affinity for the disaccharide Fuc(α, 1-6)GlcNAc than for either the main fucosidosis storage material in brain, a fuco-dekasaccharide, or the heterogeneous fuco-glycopeptide fractions obtained from normal human and rat brain glycoproteins. Our results suggest that the fucose residue linked α(1-6) to the N -acetylglucosamine residue involved in the N -glycosidic linkage to asparagine is not available to the lectin in the intact N -glycosidic chains of normal brain glycopeptide fractions and that the lectin has poor affinity for the Fuc(α, 1-3)Glc N Ac linkage in rat brain glycoproteins.     

Phosphatidylinositol-degrading enzymes in liver lysosomes.

The major pathway by which liver lysosomal enzymes degrade phosphatidylinositol is through an EDTA-insensitive formation of phosphorylinositol. This is in distinct contrast with the Ca2+-dependent production of phosphorylinositol from phosphatidylinositol, which is located in the cytosol. Lysosomal enzymes can also totally deacylate phosphatidylinositol, producing glycerophosphorylinositol.     

The effects of temperature, pH and growth rate on secondary metabolism in Streptomyces thermoviolaceus grown in a chemostat.

Streptomyces thermoviolaceus was grown in a chemostat under conditions of glutamate limitation. The effects of growth rate on production of the antibiotic granaticin, extracellular protein and protease activity as components of secondary metabolism were studied at 37, 45 and 50 degrees C. The amount of each secondary metabolite synthesized was highly dependent on growth rate and temperature. Granaticin yields were highest at growth rates of 0.1 to 0.15 h-1 at 37 degrees C, 0.175 h-1 at 45 degrees C and 0.045 h-1 at 50 degrees C. Protease activity of culture supernatants responded to low nutrient concentration and/or low growth rate. Measurements of extracellular protein revealed complex changes in amount which were dependent on growth rate and temperature. At 45 degrees C and a growth rate of 0.15 h-1, biomass yield was highest between pH 5.5 to 6.5 whereas granaticin synthesis was low at pH 5.5 and rose to highest values at between pH 6.5 and 7.5.     

Intracellular pH regulates basolateral K+ and Cl- conductances in colonic epithelial cells by modulating Ca2+ activation.

The role of intracellular pH as a modulator of basolateral K+ and Cl- conductances in epithelial cells was studied using digitonin-permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K+ and Cl- conductances were activated by stepwise increases in intracellular free Ca2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca2+, but changing extracellular pH had no effect. Maximal K+ and Cl- currents activated by Ca2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K1/2 for Ca2+ activation. In the absence of Ca2+, changes in intracellular pH did not have a significant effect on the basolateral K+ and Cl- conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control."     

Nuclear factor I is specifically targeted to discrete subnuclear sites in adenovirus type 2-infected cells.

During the S phase of the eukaryotic cell cycle and in virus-infected cells, DNA replication takes place at discrete sites in the nucleus, although it is not clear how the proteins involved in the replicative process are directed to these sites. Nuclear factor I is a cellular, sequence-specific DNA-binding protein utilized by adenovirus type 2 to facilitate the assembly of a nucleoprotein complex at the viral origin of DNA replication. Immunofluorescence experiments reveal that in uninfected cells, nuclear factor I is distributed evenly throughout the nucleus. However, after a cell is infected with adenovirus type 2, the distribution of nuclear factor I is dramatically altered, being colocalized with the viral DNA-binding protein in a limited number of subnuclear sites which bromodeoxyuridine pulse-labeling experiments have identified as sites of viral DNA replication. Experiments with adenovirus type 4, which does not require nuclear factor I for viral DNA replication, indicate that although the adenovirus type 4 DNA-binding protein is localized to discrete nuclear sites, this does not result in the redistribution of nuclear factor I. Localization of nuclear factor I to discrete subnuclear sites is therefore likely to represent a specific targeting event that reflects the requirement for nuclear factor I in adenovirus type 2 DNA replication.     

Some growth and metabolic characteristics of monensin-sensitive and monensin-resistant strains of Prevotella (Bacteroides) ruminicola.

New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by stepwise exposure to increasing concentrations of monensin. The resulting resistant strains (23MR2 and GA33MR) could initiate growth in concentrations of monensin which were 4 to 40 times greater than those which inhibited the parental strains. Resistant strains also showed enhanced resistance to nigericin and combinations of monensin and nigericin but retained sensitivity to lasalocid. Glucose utilization in cultures of the monensin-sensitive strains (23 and GA33) and one monensin-resistant strain (23MR2) was retarded but not completely inhibited when logarithmic cultures were challenged with monensin (10 mg/liter). Monensin challenge of cultures of the two monensin-sensitive strains (23 and GA33) was characterized by 78 and 51% decreases in protein yield (milligrams of protein per mole of glucose utilized), respectively. Protein yields in cultures of resistant strain 23MR2 were decreased by only 21% following monensin challenge. Cell yields and rates of glucose utilization by resistant strains GA33MR were not decreased by challenge with 10 mg of monensin per liter. Resistant strains produced greater relative proportions of propionate and less acetate than the corresponding sensitive strains. The relative amounts of succinate produced were greater in cultures of strains 23, GA33, and 23MR2 following monensin challenge. However, only minor changes in end product formation were associate with monensin challenge of resistant strain GA33MR. These results suggest that monensin has significant effects on both the growth characteristics and metabolic activities of these predominant, gram-negative ruminal bacteria.     

Effects of Hydrostatic Pressure on Growth of Hyperthermophilic Archaebacteria from the Juan de Fuca Ridge

Two new strains (AL1 and AL2) of hyperthermophilic, sulfur-reducing, heterotrophic archaebacteria from high-temperature (350°C) vents on the Juan de Fuca Ridge were highly barotolerant at their optimal growth temperatures (90 and 100°C, respectively). A trend towards barophily at pressures greater than those encountered in situ at the sea floor was demonstrated for the more extremely thermophilic strain (AL2), implying an ability to thrive in (unexplored) habitats well below accessible vent formations.     

Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.