Chemotaxis in Methanospirillum hungatei

Methanospirillum hungatei gave a positive chemotactic response to acetate.     

Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA.

The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative.     

Nucleotide sequence of the Methylobacterium extorquens AM1 moxF and moxJ genes involved in methanol oxidation

The nucleotide sequence has been determined for two genes involved in methanol oxidation in the facultative methylotroph, Methylobacterium extorquens AM1. The two genes are moxF, encoding the 66-kDa subunit of the methanol dehydrogenase and moxJ, located immediately downstream from moxF, which encodes a 30-kDa protein with unknown function. This information completes the sequence of the 5.86-kb XhoI-SalI fragment containing the moxFJGI region in M. extorquens AM1, and the structure of this gene cluster is presented. Evidence is presented that moxJ is also present in Paracoccus denitrificans. The aa sequence of MoxJ has provided little information concerning its function, but it does appear to contain a signal sequence suggesting a periplasmic location.     

Amylase gene expression in intraspecific and interspecific somatic transformants of Drosophila.

The Amylase locus in Drosophila melanogaster normally contains two copies of the structural gene for alpha-amylase, a centromere-proximal copy, Amy-p, and a distal copy, Amy-d. Products of the two genes may display discrete electrophoretic mobilities, but many strains known to carry the Amy duplication are characterized by a single amylase electromorph, e.g., Oregon-R, which produces the mobility variant AMY-1. A transient expression assay was used in somatic transformation experiments to test the functional status of the Amy genes from an Oregon-R strain. Plasmid constructs containing either the proximal or distal copy were tested in amylase-null hosts. Both genes produced a functional AMY-1 isozyme. Constructs were tested against an AMY-3 reference activity produced by a coinjected plasmid that contains the Amy-d3 allele from a Canton-S strain. With reference to the internal control, the Amy-p and Amy-d genes from Oregon-R expressed different relative activity levels for AMY-1 in transient assays. The transient expression assay was successfully used to test the functional status of Amy-homologous sequences from strains of other species of Drosophila characterized by a single amylase elctromorph, namely, Drosophila pseudoobscura ST and Drosophila miranda S 204. The amylase-null strain of D. melanogaster provided the hosts for these interspecific somatic transformation experiments.     

3H]prostaglandin uptake in vivo by rabbit uterine tissues and blastocysts

Day-6 pregnant rabbits were anesthetized and subjected to a mid-ventral laparotomy. [3H] Prostaglandin F2alpha) (PGF2alpha) [3H]PGE2, [14C]Urea or [14C]Sucrose were instilled into the uterine lumen via the uterotubal junction. The amounts instilled/uterine horn were respectively 3.7 +/- 0.3, 3.5 +/- 0.3, 5.7 +/- 1.3 and 2.7 +/- 1.6 muCi in 20mul of buffer. Animals were killed at 1, 2, 9, 19 or 21 h after radioactive instillation, and the amounts of radioactivity in blastocysts, uterine tissue, peritoneal cavity washings and urine evaluated by liquid scintillation spectrometry. A gradient of radioactivity was observed from the uterotubal junction to the cervical end of the uterus. Large amounts of [3H]PG were found in the injected horn and associated blastocysts with a considerable crossover to the non-injected horn, but little in the associated blastocysts. Much of the blastocysts associated- [3H]PG remained unmetabolized. Large amounts of metabolized [3 H] were found in urine. [14C]Urea was taken up by uterine tissue in the injected horn, but there was little cross over to the non-injected horn. Urea was also found in urine. Much of the [14C]Sucrose remained in the injected horn, and little was recovered from the urine. It was found that at 9 h, but not at 19 h, after [3 H]PG instillation, the PG was localized at the site of the blastocysts in the injected but not in the contralateral horn. Significantly more [3H]PGF2alpha than [3H]PGE2 was localized in this situation. [14C]Urea was not localized at the site of the blastocysts in urea injected horns. (ABSTRACT TRUNCATED AT 250 WORDS)     

Neutralization of respiratory syncytial virus by individual and mixtures of F and G protein monoclonal antibodies.

We identified several types of neutralization effected by F and G protein monoclonal antibodies (MAbs) reacted individually or as mixtures against respiratory syncytial virus (RSV). Neutralizing activity was identified by a microneutralization test in which virus replication was determined by enzyme immunoassay. Complete neutralization was seen only with MAbs against the F protein. Strain-specific neutralization, complete neutralization against some strains of RSV, and no neutralization against other strains were seen with an additional MAb against the F protein. Partial neutralization, virus replication significantly reduced but still present, and no neutralization were seen with MAbs against both the F and G proteins. Enhanced neutralization, enhanced efficacy of neutralization, or increased neutralizing titer with a mixture of two MAbs over that for the individual MAbs was seen with all MAbs against the F protein and all but three MAbs against the G protein. Most (10 of 13) of the MAbs that exhibited neutralizing activity reacted with some but not all strains of RSV in an enzyme immunoassay. The epitopes corresponding to these 10 MAbs probably contribute to the strain-specific component of the neutralizing antibody response to RSV. Our results suggest that interpretation of RSV neutralization with MAbs is complex and that studies of such neutralization should include mixtures of MAbs and multiple RSV strains.     

Fasciola hepatica: development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.     

Selective binding of Dolichos biflorus agglutinin to L3T4-, Lyt-2- thymocytes. Expression of terminal alpha-linked N-acetyl-D-galactosamine residues defines a subpopulation of fetal and adult murine thymocytes

Using histochemical and immunocytochemical techniques, a lectin with nominal specificity for alpha-linked N-acetyl-D-galactosamine, Dolichos biflorus agglutinin (DBA), was found to preferentially label thymocytes with an L3T4-, Lyt-2- phenotype from fetal/newborn and adult mice. Through days 14 to 16 of gestation, virtually all thymocytes bound DBA, followed by a dramatic reduction of DBA labeling during the last 4 days of gestation, reaching adult levels of about 2 to 4% of total thymocytes. At later stages of fetal development, the DBA+ cells were confined to the subcapsular area of the thymus. This apparent loss of DBA+ cells was caused by an expansion of the thymocyte population not labeled with this lectin. Affinity purification of thymocyte cell surface components with insolubilized DBA indicated that virtually all of the lectin binding to fetal thymocytes was mediated by a 120-kDa glycoprotein. In addition to thymocytes, DBA also labeled about 5% of bone marrow cells from both normal or nude mice and a small population of spleen cells as well. These results suggest that this lectin may be useful to positively select for LT34-, Lyt-2- thymocytes, and, possibly, other immature populations within the T cell lineage.