A novel function of the cAMP-CRP complex in Escherichia coli: cAMP-CRP functions as an adaptor for the CytR repressor in the deo operon

Unlike classical bacterial repressors, the CytR repressor of Escherichia coli cannot independently regulate gene expression. Here we show that CytR binding to the deoP2 promoter relies on interaction with the master gene regulatory protein, CRP, and, furthermore, that cAMP-CRP and CytR bind co-operatively to deoP2. Using mutant promoters we show that tandem, properly spaced DNA-bound cAMP-CRP complexes are required for this co-operative binding. These data suggest that CytR forms a bridge between tandem cAMP-CRP complexes, and that cAMP-CRP functions as an adaptor for CytR. The implications of this new version of negative control in E. coli on bacterial gene expression and on combinatorial gene regulation in higher organisms are discussed.     

Transfer of Lactic Acid Bacterial Strains from the Feed to the Sow,the Environment,and the Piglets

The spread of lactic acid bacterial strains to the environment and to newborn piglets was investigated after feeding of such strains to sows. Rifampicin resistant bacterial strains were fed to sows, 1010 c.f.u. per day, during the period from 1 week before expected farrowing until 1 week after farrowing. Fecal samples from the sows and samples of litter were collected for bacteriological examination together with swabs from the pens, the skin of the sows, and from the rectum of the piglets. The test strains were only excreted in relatively low amounts in the feces of the sows, approximately 103 −106 c.f.u. per gram. They were not able to displace the normal lactic acid bacterial flora in the sows nor were they transmitted to the intestinal tract of the piglets to any significant extent. After the last administration the test strains disappeared from both feces, skin, and environment, indicating that no permanent colonization had taken place, although considerable differences in duration of persistence were noticed between test strains.     

Immunocytochemical identification of the human α2-macrogiobulin receptor in monocytes and fibroblasts: Monoclonal antibodies define the receptor as a monocyte differentiation antigen

The α₂-macroglobulin receptor was recently purified from rat liver and human placenta. Three different monoclonal antibodies have now been raised against the human receptor and expression of the 440-kDa receptor protein is demonstrated in human placenta, fibroblasts, liver, and monocytes by immunoblot analysis. Flow cytometric studies showed that anti-α₂-macroglobulin receptor monoclonal antibodies bind to 90–100% of the blood monocyte population and not to other blood cells. This defines the α₂-macroglobulin receptor as a monocyte differentiation antigen, different from any of the classified leucocyte cluster determinants. Electron microscopic gold immunocytochemistry revealed the subcellular distribution of the receptor in human cultured monocytes and fibroblasts. In these cells, 18–33% of the gold particles were found on the outside of the plasma membrane, and in fibroblasts, especially, in coated invaginations. The intracellular receptors were mainly distributed in vesicles and tubular structures.     

A 21S enzyme complex from HeLa cells that functions in simian virus 40 DNA replication in vitro

A sedimentable complex of enzymes for DNA synthesis was partially purified from the combined low-salt nuclear extract-postmicrosomal supernatant solution of HeLa cell homogenates by poly(ethylene glycol) precipitation in the presence of 2 M KCl, discontinuous gradient centrifugation, Q-Sepharose chromatography, and velocity gradient centrifugation. In addition to the previously described 640-kDa multiprotein DNA polymerase alpha-primase complex [Vishwanatha et al. (1986) J. Biol. Chem. 261, 6619-6628], the enzyme complex also has associated topoisomerase I, DNA-dependent ATPase, RNase H, DNA ligase, a simian virus 40 origin recognition, dA/dT sequence binding protein [Malkas & Baril (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 70-74], and proliferating cell nuclear antigen. Essentially all of the T antigen dependent simian virus 40 in vitro replication activity in the combined nuclear extract-postmicrosomal supernatant solution resides with the sedimentable complex of enzymes for DNA synthesis. Sedimentation analysis on a 10-35% glycerol gradient in the presence of 0.5 M KCl indicates that the enzyme complex is 21S. The associated enzymes for DNA synthesis and in vitro simian virus 40 replication activity cofractionate throughout the purification of the 21S complex. The DNA polymerase and in vitro simian virus 40 replication activities are both inhibited by monoclonal antibody (SJK 132-20) to human DNA polymerase alpha and by 5-10 microM butylphenyl-dGTP, indicating that the association of DNA polymerase alpha with the 21S enzyme complex is essential for the initiation of SV40 DNA replication in vitro.     

Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium, phospholipids, and tissue factor

Previous studies indicated that factor VIIa, in complex with tissue factor, readily activates either factor X or factor IX in the presence of calcium ions. In order to assess the relative physiological importance of the activation of factor IX versus the activation of factor X by recombinant factor VIIa, we have obtained steady-state kinetic parameters for the factor VIIa catalyzed activation of factor IX and factor X under a variety of cofactor conditions that include calcium alone, calcium and phospholipids, calcium, phospholipids, and tissue factor apoprotein, and calcium and cell-surface tissue factor. Calcium alone stimulated the activation of factors IX and X by factor VIIa maximally at 1 and 2.5 mM, respectively. In the presence of 25 microM phospholipids, maximal rates of factor IX and factor X activation were achieved at 2.5-5 mM calcium. With calcium alone, or with phospholipid and calcium, the initial rates of factor IX activation by factor VIIa were significantly higher than that observed for factor X. Kinetic studies revealed that the Km for the factor VIIa catalyzed activation of factor IX was essentially constant in the presence of 5 mM calcium and 1-500 microM phospholipid, whereas the Km for factor X activation varied with phospholipid concentration, reaching a minimum at 7-20 microM phospholipid. At all concentrations of added phospholipid, the kcat/Km ratio for the activation of factor IX by factor VIIa appeared to be considerably greater than that observed for the activation of factor X.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of carrot somatic embryo development in suspension culture

Somatic embryos in liquid culture can serve as a mass cloning system in a plant propagation program. A quantitative formulation of embryo development obtained from cell suspension cultures is used to develop a segregated kinetic model. The model is based on standard classification schemes as previously developed by plant physiologists. Dependent variables include carbohydrate concentrations (sucrose, fructose, and glucose) and biomass apportioned among the inoculum (free single cells, cell clusters), normal developmental stages, and aberrant cell and embryo types. Good agreement between the model and experimental results is indicated and allows for a rigorous approach to media optimization and reactor scaleup for embryo formation.     

Viability of a marine microbial biofilm

The formation of a microbial biofilm on glass surfaces arranged in lamellar piles parallel with circulating sea water (3 cm·sec^–1) was studied. The increase in dry weight, protein content, nucleotide content (ATP, ADP), and diatoms was followed over a period of 62 days. Dry weight and protein were estimates of the total biofilm development, whereas the nucleotide measurements revealed the viability of the biofilm and reflected the dynamics in the community structure.     

Formation of (4R)- and (4S)-4-hydroxyochratoxin A from ochratoxin A by liver microsomes from various species.

Two metabolic products were formed from ochratoxin A by human, pig, and rat liver microsomal fractions in the presence of reduced nicotinamide adenine dinucleotide phosphate. They were isolated from the incubation mixture in the presence of pig liver microsomes by extraction, thin-layer chromatography, and high-pressure liquid chromatography Their structures are suggested to be (4R)- and (4S)-4-hydroxyochratoxin A on the basis of mass and nuclear magnetic resonance spectroscopy. Km and the maximum velocity for the formation of the two metabolites by human, pig, and rat microsomes were determined. Their formation was inhibited by carbon monoxide and metyrapone. The results indicate that the microsomal hydroxylation system is a cytochrome P-450 and that different species are involved in the formation of the two epimeric forms of 4-hydroxyochratoxin A.     

Analysis of the genome of an endogenous, ecotropic retrovirus of the AKR strain of mice: micromethod for detailed characterization of high-molecular-weight RNA.

A detailed characterization of the genome of an endogenous, ecotropic type C virus, the Akv virus, is presented. Approximately 100 RNase T1-resistant oligonucleotides characteristic of the Akv genome were identified by two-dimensional gel electrophoresis, and the complete nucleotide sequence is presented for 75 of these oligonucleotides. A correspondence between the sequence of some of these oligonucleotides and the amino acid sequence of some virus-coded gag gene proteins is reported. For this study we developed methods suitable for the analysis of high-molecular-weight RNA species in nanogram quantities. The in vitro labeling procedures used here led to uniform labeling of the unique oligonucleotides.     

Electron microscopy of lanthanum in plasma

Summary Aqueous solutions of lanthanum nitrate may be used as electron microscopic tracers in vivo to study vascular permeability in the experimental animal. However, with this technique the size of the tracer particles is not known. To gain information about the tracer size, we injected lanthanum nitrate into the blood circulation of living rabbits. The plasma obtained from such animals 30 min later, was studied with the electron microscope. The plasma contained an electron-dense material, readily visible in the electron microscope. A precipitate obtained after centrifugation of the whole blood to separate the cells, also contained the tracer. Lanthanum was found in large amounts in the fibrin clot obtained after treating the plasma with thrombin. The tracer was not detected in the serum (i.e. thrombin-treated plasma). The study indicates that ionic lanthanum injected into the blood circulation of living rabbits, is to a great extent bound to fibrinogen, and that the smallest possible size of the tracer is that of the fibrinogen,molecule (m. w. 330,000). Larger particles are present as well.