Characterization of ribonucleoproteins and ribosomes isolated from lymphocytic choriomeningitis virus.

Disruption of purified lymphocytic choriomeningitis (LCM) virus with Nonidet P-40 in 0.5 M KCl followed by sucrose gradient centrifugation in 0.3 M KCl led to the isolation of two viral nucleoproteins (RNPs) as well as 40S and 60S ribosomal subunits. The largest viral RNP sedimented heterogenously at 123S to 148S and was associated with 23S and 31S viral RNA. The other viral RNP sedimented at 83S and was associated with 23S viral RNA. The buoyant density in CsCl was determined to be 1.32 g/cm3 for the viral RNP. Densities of 1.52 and 1.60 g/cm3 were determined for the 40S and 60S subunits, similar to those of the BHK-21 cells subunits dissociated by 0.5 M KCl. The viral RNPs were partly sensitive to RNase.     

Determination of recognition-sequences for DNA-binding proteins by a polymerase chain reaction assisted binding site selection method (BSS) using nitrocellulose immobilized DNA binding protein.

We have developed a simple procedure for rapid determination of a DNA sequence recognized by a DNA binding protein based on immobilization of the protein on nitrocellulose filters. The procedure consists of the following steps: A recombinant protein with a functional DNA binding domain is expressed in E. coli. The protein is purified to homogeneity, immobilized on nitrocellulose paper, and exposed to a pool of double stranded oligonucleotides carrying in the central part a 20 bp random sequence, which is flanked by conserved sequences with restriction endonuclease recognition sites for analytical and subcloning purposes and sequences complementary to polymerase chain reaction primers. Oligonucleotides retained by the DNA-binding protein are liberated by increasing the ionic strength and used in a new binding process after amplification by the polymerase chain reaction technique. Finally the amplified product is cloned for determination of the DNA sequence selected by the DNA-binding protein. Murine Zn-finger and basic helix-loop-helix DNA binding proteins were used to demonstrate the efficiency of the method. We show that the yield of oligonucleotides binding to the protein was increased by several consecutive rounds of filter binding and amplification, and that the protein extracted a specific sequence from the pool of random oligonucleotides.     

Correlation of the conformation of a modified ribonuclease octapeptide, homologous to peptide T, with its ability to induce CD4-dependent monocyte chemotaxis

Peptide T, from the human immunodeficiency virus (HIV), whose sequence is Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, has been shown to inhibit attachment of this virus to T cells and neural cells bearing the CD4 receptor. This peptide shares extensive homology with the 19–26 segment of ribonuclease A (RNase A), whose sequence is Ala-Ala-Ser-Ser-Ser-Asn-Tyr-Cys. Based on comparison of the structures of peptides occurring in proteins of known structure that are homologous to peptide T,viz, RNase A and endothiapepsin and on conformational energy calculations, we predicted that peptide T adopts a structure much like that for residues 19–26 in RNase A. A critical feature is a bend involving residues Thr 4-Asn 7 in peptide T corresponding to Ser 22-Tyr 25 in the RNase A peptide. Our proposed structure for peptide T has recently been confirmed by Cotelleet al. (Biochem. Biophys. Res. Commun. 171, 596–602). We now show directly that the RNase A peptide, with Met replacing Cys 26 to prevent disulfide exchange reactions, strongly induces monocyte-chemotaxis that is blocked by anti-CD4 monoclonal antibody. Both peptide T and RNase A fail to induce chemotaxis, however, in neutrophils which do not express surface CD4 receptors. These results suggest that both peptides interact with the CD4 receptor in inducing monocyte chemotaxis. We have also prepared cyclo-RNase A peptide with Met 26. Using molecular dynamics and conformational energy calculations, we find that the cyclic peptide cannot form a bend structure involving Ser 22-Tyr 25 that is superimposable on the RNase A bend. Indeed, we find that this peptide is inactive in inducing monocyte chemotaxis despite the fact that its amino acid sequence is identical to that of the open chain form. This result suggests that a correlation between the -bend structure of the RNase A peptide and peptide T and their abilities to bind to the CD4 receptor.     

Lactobacillus succession in the piglet digestive tract demonstrated by plasmid profiling.

Plasmid profiling was used to distinguish strains of lactobacilli inhabiting the digestive tract of piglets and the feces of sows. Fifteen plasmid profile types were detected among 328 isolates of lactobacilli. Plasmid profiling of lactobacilli permitted the following conclusions to be made: the maternal feces were a major source of lactobacilli colonizing the piglet digestive tract; the lactobacillus population of the gastric region of the piglet digestive tract was composed of lactobacillus strains different from those present in the rectal population; and a lactobacillus succession was observed in the digestive tract of piglets drawn from a single litter, and one plasmid profile type became dominant in the gastric region of these animals.     

Generation of daunomycin radicals on the outer side of the erythrocyte membrane

The generation of the daunomycin semiquinone was studied in intact red blood cells under CO atmosphere by ESR spectroscopy. The undialyzed hemolysates and the spin broadening agent chromium oxalate quenched the ESR signal, suggesting external location of the ESR-detectable radicals and their slow diffusion inside. A constant outward flow of O2- was detected by monitoring the approach to the steady state of the ESR signal of Cu,Zn superoxide dismutase externally added to red blood cells plus daunomycin in air. This suggests a reductase on the outer side of the erythrocyte membrane as the source of daunomycin radicals.     

Differential induction of peroxisomal oxidation of palmitic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid in rat liver

The effect of feeding rats 20% partially hydrogenated marine oil (PHMO), 20% soybean oil, or clofibrate on the conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid to cholic acid was studied in light mitochondrial (L) fractions prepared from liver. 20% PHMO gave a doubling both of the specific and of the total activity of the cholic acid formation compared to those found in the L-fraction from animals given standard pellets. 20% soybean oil induced the specific and the total activity to a lesser extent, 1.4- and 1.2-fold, respectively. The specific and total activity of the peroxisomal beta-oxidation of palmitic acid were induced 2.4- and 2.7-fold, respectively, by PHMO feeding. Soybean oil gave a smaller increase, 2-fold, in both specific and total activity. Clofibrate, a known peroxisomal proliferator, induced the specific and total activity of the peroxisomal fatty acid beta-oxidation 5.2- and 5.7-fold, respectively, whereas the specific activity of the cholic acid formation remained unchanged compared to standard pellet feeding. The same pattern was found in the postnuclear supernatants (E-fractions), excluding the possibility that different treatments caused different distributions of organelles between the fractions. This differential induction of two similar peroxisomal reaction sequences suggests that at least two mechanisms for peroxisomal induction exist.     

Effect of tunicamycin on the synthesis of herpes simplex virus type 1 glycoproteins and their expression on the cell surface.

Herpes simplex virus specifies five glycoproteins which have been found on the surface of both the intact, infected cells and the virion envelope. In the presence of the drug tunicamycin, glycosylation of the herpes simplex virus type 1 glycoproteins is inhibited. We present in this report evidence that the immunologically specificity of the glycoproteins designated gA, gB, and gD resides mainly in the underglycosylated "core" proteins, as demonstrated by the immunoblotting technique. We showed also that tunicamycin prevented exposure of the viral glycoproteins on the cell surface, as the individual glycoproteins lost their ability to participate as targets for the specific antibodies applied in the antibody-dependent, cell-mediated cytotoxicity test. Immunocytolysis was reduced between 73 and 97%, depending on the specificity of the antibodies used. The intracellular processing of the herpes simplex virus type 1-specific glycoprotein designated gC differed from the processing of gA, gB, and GD, as evidenced by the identification of an underglycosylated but immunochemically modified form of gC on the surface of infected cells grown in the presence of tunicamycin.     

Sensitivity of early mouse embryos to [H]thymidine

Effects of intranuclear radiation on the developmental capacity of early mouse embryos were studied by exposing embryos to [³H]thymidine and counting the number of embryos forming blastocysts, trophoblast outgrowths, inner cell masses (ICMs), and two-layer ICMs (differentiated into primary endoderm and ectoderm). When embryos were cultured from the 2-cell stage for 8 days in the continuous presence of [³H]thymidine, concentrations as low as 0.1 nCi/ml reduced the number of embryos forming two-layer ICMs. At 1 nCi/ml, the number of both ICMs and two-layer ICMs was reduced, and at 10 nCi/ml the number of embryos developing to all three post-blastocyst endpoints was reduced. Blastocyst formation was not affected even at the highest concentration tested (100 nCi/ml). When embryos were cultured from the 2-cell stage for 3 days in the presence of [³H]thymidine and then cultured further in unlabelled medium, the effects were similar to those of 8-day exposure. When embryos were exposed to [³H]thymidine for 24 h at various developmental stages, effects were less severe than when they were exposed continuously for 3 or 8 days, and the sensitivity of embryos differed between stages; the lowest concentration that interfered with development was 10 nCi/ml, and exposure at the morula stage was most detrimental to the subsequent development of embryos, particularly that of ICMs. The 24-h exposure of immunosurgically isolated ICMs to [³H]thymidine revealed that the high sensitivity of the ICM to [³H]thymidine persists through the late blastocyst stage and declines progressively thereafter. Autoradiography indicated that the change in radiosensitivity of embryos or ICMs is generally related to their ability to incorporate [³H]thymidine into the DNA.     

The Influence of Sow Colostrum Trypsin Inhibitor on the Immunoglobulin Absorption in Newborn Piglets

The influence of sow colostrum trypsin inhibitor (SCTI) on the immunoglobulin absorption from the gut of 16 newborn colostrum-deprived piglets was investigated in a paired feeding experiment. Three times at 1 h intervals the piglets were fed an experimental diet consisting of sow milk, purified swine serum immunoglobulins containing agglutinins against Bordetella bronchiseptica, and purified SGTI (diet I) or saline (diet II). The serum concentrations of IgG, IgM, IgA, and antibodies for B. bronchiseptica were measured by single radial immunodiffusion and by a tube agglutination procedure and used to evaluate the immunoglobulin absorption. Four and 6 h after the first experimental meal, blood samples from the piglets given SGTI in their diet had a generally higher level of IgG, IgA and aggutinins against B. bronchiseptica than blood samples from the piglets d no SGTI. No real differences were found in the IgM levels. Although the piglets fed no SGTI all showed a considerable immunoglobulin absorption, the SCTI was found to have a statistically significant positive influence on the IgG and IgA absorption.