Generalized transduction in Rhizobium meliloti.

Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3. Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids. Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances. Bacteriophage N3 was capable of infecting several commonly used strains of R. meliloti.     

Effects of ethanol on the Escherichia coli plasma membrane.

The effects of ethanol on the fluidity of Escherichia coli plasma membranes were examined by using a variety of fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene, perylene, and a set of n-(9-anthroyloxy) fatty acids. The anthroyloxy fatty acid probes were used to examine the fluidity gradient across the width of the plasma membrane and artificial membranes prepared from lipid extracts of plasma membranes. Ethanol caused a small decrease in the polarization of probes primarily located near the membrane surface. In comparison, hexanol decreased the polarization of probes located more deeply in the membrane. Temperature had a large effect on probes located at all depths. The effects of ethanol on E. coli membranes from cells grown with or without ethanol were also examined. Plasma membranes isolated from cells grown in the presence of ethanol were more rigid than those from control cells. In contrast to plasma membranes, artificial membranes prepared from lipid extracts of ethanol-grown cells were more fluid than those from control cells. These differences are explained by analyses of membrane composition. Membranes from cells grown in the presence of ethanol are more rigid than those from control cells due to a decrease in the lipid-to-protein ratio. This change more than compensates for the fluidizing effect of ethanol and the ethanol-induced increase in membrane C18:1 fatty acid which occurs during growth. Our results suggest that the regulation of the lipid-to-protein ratio of the plasma membrane may be an important adaptive response of E. coli to growth in the presence of ethanol.     

Role of IIIGlc of the phosphoenolpyruvate-glucose phosphotransferase system in inducer exclusion in Escherichia coli.

The phosphoenolpyruvate-D-glucose phosphotransferase system of Enterobacteriaceae is thought to regulate the synthesis and activity of a number of catabolite uptake systems, including those for maltose, lactose, and glycerol, via the phosphorylation state of one of its components, IIIGlc. We have investigated the proposal by Kornberg and co-workers (FEBS Lett. 117(Suppl.):K28-K36, 1980) that not IIIGlc, but an unknown protein, the product of the iex gene, is responsible for the exclusion of the above-mentioned compounds from the cell. The iex mutant HK738 of Escherichia coli contains normal amounts of IIIGlc as measured by specific antibodies, in contrast to crr mutants that lack IIIGlc. The IIIGlc of the iex strain functions normally in glucose and methyl alpha-glucoside transport, and the specific activity in in vitro phosphorylation is approximately 60% of that of the parent. The IIIGlc activity of the iex strain is, however, heat labile, in contrast to the parental IIIGlc, suggesting that the mutant contains an altered IIIGlc. This is supported by the observation that IIIGlc from the iex strain cannot bind to the lactose carrier. Thus it cannot inhibit the carrier, and this explains why the uptake of non-phosphotransferase system compounds in an iex strain is resistant to phosphotransferase system sugars. The introduction of a plasmid containing a wild-type crr+ allele into the iex strain restores the iex phenotype to that of the iex+ parent. The IIIGlc produced from the plasmid in the iex strain is heat stable and binds normally to the lactose carrier. These results lead to the conclusion that the iex mutation is most likely allelic with crr and results in an altered, temperature-sensitive IIIGlc that is still able to function D-glucose and methyl alpha-glucoside uptake and phosphorylation and in the activation of adenylate cyclase, but is unable to bind to and inhibit the lactose carrier.     

Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome

A membrane-associated b-type cytochrome (a proposed component in the neutrophil microbicidal superoxide generating system) has been partially purified from nonactivated beef granulocytes to a specific heme content of 20 nmol of heme/mg of protein, a value about 10-fold higher than those previously reported. The hemoprotein was solubilized at low temperature (4 degrees C) from mixed granule (30,000 X g) cell fractions using Triton X-114 detergent. Warming the extract to 25 degrees C allowed separation into detergent and aqueous phases; cytochrome b558 partitioned exclusively into the detergent phase, allowing separation from other visible-absorbing species (e.g. myeloperoxidase) and indicated an intrinsic membrane localization (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). The partitioned cytochrome was chromatographed on hydroxylapatite and a hydrophobic affinity matrix, allowing a 185-fold (heme content) purification from the granule extract. The cytochrome preparation revealed three equal-staining protein bands by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis; apparent molecular weights were 14,000, 12,000, and 11,000. The question of heterogeneity of the preparation versus subunit structure is not resolved at present. The hemoprotein binds carbon monoxide, consistent with a proposed role as a terminal oxidase, and has an unusually negative oxidation-reduction potential (-225 mV) similar to that observed in granulocyte membranes. The preparation is devoid of NAD(P)H-diaphorase and cytochrome c reductase activities.     

Molecular structures of human argininosuccinate synthetase pseudogenes. Evolutionary and mechanistic implications

In the human genome there is one expressed gene for argininosuccinate synthetase and 14 pseudogenes. A cDNA coding for human argininosuccinate synthetase was used to screen a human genomic library. Twenty-five unique genomic clones were isolated and extensively characterized. At least seven clones represented processed argininosuccinate synthetase pseudogenes that lost the introns in the expressed gene. Restriction mapping demonstrated that these processed pseudogenes were located in distinct regions of the human genome. Complete nucleotide sequences of two processed pseudogenes, psi AS-1 and psi AS-3, and a partial sequence of psi AS-7 were determined. Both psi AS-1 and psi AS-3 had an adenine-rich region at their 3' end and were flanked by distinct imperfect direct repeats. A comparison of these pseudogene sequences to that of the cDNA demonstrated that psi AS-1 and psi AS-3 were 93% homologous to the cDNA, whereas psi AS-7 was 89% homologous to the cDNA. Therefore, it is estimated that psi AS-1 and psi AS-3 were created 10-11 million years ago, whereas psi AS-7 arose approximately 21 million years ago. We have estimated the evolutionary rate for the expressed argininosuccinate synthetase gene based on the sequences of psi AS-1 and psi AS-3. These data indicate that the expressed argininosuccinate synthetase gene is evolving at a rate similar to that of the beta-globin gene and much faster than the alpha-tubulin gene. Furthermore, a comparison of the sequences of psi AS-1 and psi AS-3 suggests the possibility that these pseudogenes arose from a common intermediate.     

Photoaffinity labeling of mammalian alpha 1-adrenergic receptors. Identification of the ligand binding subunit with a high affinity radioiodinated probe

We have synthesized and characterized a novel high affinity radioiodinated alpha 1-adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido - 3 - [125I]iodophenyl) pentanoyl] - 1 - piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (KD = 130 pM) in a reversible and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an alpha 1-adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical alpha 1-adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at Mr = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the Mr = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the alpha 1-adrenergic receptor.     

Neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii

The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59 mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosaccharides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal(beta 1-4)Gal-Cer, Gal(beta 1-4)Glc-Cer, and Man(beta 1-4)Glc-Cer; ceramide trisaccharide, Man(alpha 1-3)Man(beta 1-4)Glc-Cer; ceramide tetrasaccharides, Man(alpha 1-3)[Xyl(beta 1-2)]Man(beta 1-4)Glc-Cer, GlcNAc(beta 1-2)Man(alpha 1-3)Man(beta 1-4)Glc-Cer, Man(alpha 1-3)[Gal(beta 1-2)]Man(beta 1-4)Glc-Cer, and Man(alpha 1-2?)Man(alpha 1-3)Man(beta 1-4)Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C18-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.     

A comparative study of the enzymatic hydrolysis of acid-pretreated white pine and mixed hardwood

Removal of hemicellulose by acid pretreatment in a flow reactor followed by enzymatic hydrolysis of the neutralized slurry has resulted in glucose yields as high as 95% for mixed hardwood. For white pine, however, the maximum glucose yield is 65%. Although pine has a higher extractives content, removal of the extractives prior to enzymatic hydrolysis does not increases the glucose yield. Pore size measurements reveal that the increase in pore volume, in the size range of the cellulase molecule, following pretreatment for pine is only about one-half the value obtained with mixed hardwood. This suggests that pore volume is an important determinant of substrate-enzyme reactivity.     

Multistage continuous culture to examine secondary metabolite formation in plant cells: Phenolics from Nicotiana tabacum

The feasibility of operating a multistage continuous culture of plant cells was demonstrated for Nicotiana tabacum. Cells in the second stage of a two-stage chemostat were morphologically distinct from cells in the first stage or cells in a single-stage unit with a holding time equal to the combined holding times in the two-stage system. Cells in the second stage produced much higher levels of phenolics per unit weight of cells than cells in either the first-stage or single-stage unit. The steady-state was reproduced. When a glucose side stream was fed to the second stage, an increase in apparent cell division was observed with a simultaneous decrease in phenolics productivity. When the toxic precursor phenylalanine was pulsed into the reactor, the quantity of biomass decreased temporarily while phenolic productivity increased. These experiments demonstrate that multistage continuous culture may be useful in increasing secondary metabolite formation in cells and in exploring mechanisms controlling secondary metabolite formation.     

Mechanisms of serotonin-induced lymphocyte proliferation inhibition

When human peripheral blood lymphocytes were stimulated with phytohemagglutinin in the presence of serotonin, inhibition of [3H]thymidine incorporation occurred, the most marked inhibition occurring at high (10(-3)M) serotonin concentrations. This effect could not be reversed by the addition of Interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analysis showed that virtually all of the cells remained in the G0 phase (unactivated) at 24 hr while some of the cells entered the G1a and G1b phases of the cell cycle by 42 hr. The cellular production of IL-2 was not affected by serotonin, as supernatants of treated cultures contained essentially the same IL-2 titers as did control cultures. Serotonin seemed to primarily affect cell activation and had little or no effect on proliferating cells. This was further confirmed by the lack of effects of serotonin on a variety of established proliferating lymphocyte, macrophage, and fibroblast cell lines. By contrast, dose-dependent inhibition of IL-2-dependent CTLL cells occurred. Serotonin was not toxic even at 10(-3) M concentrations. A marked decrease in IL-2 receptors and a change in their distribution on responder cells was seen when treated cultures were examined with the anti-Tac monoclonal antibody. At 24 hr this effect was contrastingly not seen for the OKT-8 marker, although a slight decrease in OKT-4-positive cells was seen. Serotonin thus produced an inhibition of lectin-stimulated lymphocyte proliferation via a mechanism independent of IL-2 production, and caused a decrease in the expression and distribution of IL-2 receptors on the surface of responder cells.