Rat corticotropin-releasing hormone gene: sequence and tissue-specific expression

The rat corticotropin releasing hormone (CRH) gene has been isolated and characterized by DNA sequence analysis. The gene exhibits a structural organization similar to that of the human CRH gene. The nucleotide sequence encoding the entire rat CRH precursor is located on the second exon, while exon I encodes the 5'-untranslated region of the mRNA. Analysis of the nucleotide sequence homology between the human and rat CRH genes reveals several highly conserved regions including the CRH peptide-encoding sequence and the 5'-flanking sequence. RNA blot analysis demonstrates that CRH mRNA can be observed in numerous regions of the rat brain as well as the spinal cord, adrenal gland, pituitary, and testis.     

Identification of Lotus Rhizobia by Direct DNA Hybridization of Crushed Root Nodules

Hybridization of crushed Lotus pedunculatus root nodules with ³²P-labeled total genomic DNA probes was used to identify Rhizobium loti and Bradyrhizobium sp. (Lotus rhizobia). Probes always hybridized with homologous target DNA and frequently with DNAs of other strains from the same genus. Intergeneric hybridization did not occur. Results were comparable to those from colony hybridization.     

Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ

ras and myc oncogenes were able to induce distinct phenotypic alterations, resembling different types of premalignant lesions, when introduced into approximately 0.1% of the cells used to reconstitute the mouse prostate gland. While ras induced dysplasia in combination with angiogenesis, myc induced a hyperplasia of the otherwise normally developed organ. ras and myc together induced primarily carcinomas. However, tumor progression was also associated with additional genetic alterations involving gene amplification. Our data indicate that specific types of benign premalignant lesions may reflect the activation of different single oncogenes, and that the consecutive activation of multiple oncogenes could be a causal event in the step-like progression of tumorigenesis.     

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Concepts of coevolution

The word coevolution has become a standard part of the lexicon of evolutionary biology. More than 1000 papers during the past decade have used coevolution in the title or abstract. Hundreds more have used the word in passing in the body of the paper. A half dozen books now include coevolution in the title. As usage of coevolution has increased, so have the views on the processes and mechanisms of coevolutionary change. The problem now is to understand the ecological and genetic conditions that favor different modes and outcomes of coevolution.     

Microbial growth and accumulation in industrial metal-working fluids.

The dynamics of microbial growth in metal-working fluids (MWF) and the effect of the addition of biocides were studied in large fluid systems, in this case, one central tank which holds 150 m3. In this system, populations of Pseudomonas pseudoalcaligenes (greater than 10(8) CFU/ml) were sustained for a year, although large quantities of biocides were added. Quantitation of 3-OH lauric acid, a marker for many Pseudomonas spp., by gas chromatography indicated that the bacterial biomass exceeded the viable counts by approximately 15 times. Fungi were grown on several occasions, the dominating genera being Fusarium and Candida. Soon after the old MWF was removed and the tank was provided with fresh MWF, which consisted of an emulsion of mineral oil in water, there was a massive growth of P. pseudoalcaligenes that reached levels of greater than 10(8) bacteria per ml. Initially, only low concentrations of other species were found for some weeks. After this period, different enterobacteria and other gram-negative rods often appeared at high concentrations (10(7) and 10(8) bacteria per ml, respectively). Bacteria identified as P. pseudoalcaligenes showed great variation with respect to colony morphology and a certain heterogeneity with respect to biochemical characteristics. Certain bacterial species grew as microcolonies on metal strips immersed in the circulating MWF, but P. pseudoalcaligenes was not recovered from this habitat. The total bacterial count in the air surrounding the machines in the metal-working shop showed an inverse relation to increasing distance from the machine. The concentration of bacteria in the air varied because of the number of machines in use, temperature, and humidity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The temporal relationship between phospholipase activation, diradylglycerol formation and superoxide production in the human neutrophil.

Fluctuations in the amounts of choline, inositol 1,4,5-trisphosphate (IP3) and diradylglycerol have been used to monitor phospholipase activation in the human neutrophil. Stimulation of human neutrophils by formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) resulted in a rapid activation of both phosphatidylinositol 4,5-bisphosphate breakdown by phospholipase C and phosphatidylcholine breakdown by phospholipase D. Diradylglycerol accumulation occurred more slowly than that of either choline or IP3 and was inhibited by 30 mM-butanol, suggesting that the bulk was derived from the phospholipase D pathway via phosphatidate phosphohydrolase. Consistent with this is the observation that choline and diradylglycerol are produced in similar amounts. 1,2-Diacylglycerol (DAG) and 1-O-alkyl-2-acyl-sn-glycerol species accumulated with different time courses, indicating that one or more steps in the phospholipase D pathway was selective for the diacyl species. Superoxide production by fMet-Leu-Phe-stimulated neutrophils paralleled DAG accumulation over the first 5 min, but thereafter this production stopped, despite the fact that DAG remained elevated. We conclude that DAG derived from the phospholipase D pathway is only one of the second messengers important in controlling this functional response.     

Efficient site directed in vitro mutagenesis using ampicillin selection.

A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin. In tests of the vector, highly efficient (60-90%) mutagenesis was obtained.     

Lactic acid bacteria: model systems for in vivo studies of sugar transport and metabolism in gram-positive organisms

Lactic acid bacteria provide a model system for the in vivo study of mechanisms pertaining to the regulation of sugar transport and metabolism by microorganisms. Recent studies with resting and growing cells of the homofermentative Streptococci and Lactobacilli have yielded evidence for hitherto unsuspected regulatory mechanisms in this group of industrial and medically important bacteria. These regulatory mechanisms mediate the exclusion and expulsion of sugars, the preferential transport of sugar from sugar mixtures, resistance to non-metabolizable sugar analogs and participate in the establishment of energy-dissipating futile cycles. Transport experiments conducted with novel sugar analogs, data from enzymatic analyses and 31P-NMR spectroscopy studies with wild type and mutant strains of Streptococci, have provided new insight into the fine- and coarse-controls responsible for the modulation of activity of the sugar transport: glycolysis cycle. The purpose of this review is to summarize our current knowledge of these regulatory mechanisms and to suggest avenues for future investigation. Although specifically addressed to the lactic acid bacteria, it seems likely that some of the mechanisms described will be found in other Gram-positive species.