Conserved water-mediated H-bonding dynamics of catalytic Asn 175 in plant thiol protease

The role of invariant water molecules in the activity of plant cysteine protease is ubiquitous in nature. On analysing the 11 different Protein DataBank (PDB) structures of plant thiol proteases, the two invariant water molecules W1 and W2 (W220 and W222 in the template 1PPN structure) were observed to form H-bonds with the O_b atom of Asn 175. Extensive energy minimization and molecular dynamics simulation studies up to 2 ns on all the PDB and solvated structures clearly revealed the involvement of the H-bonding association of the two water molecules in fixing the orientation of the asparagine residue of the catalytic triad. From this study, it is suggested that H-bonding of the water molecule at the W1 invariant site better stabilizes the Asn residue at the active site of the catalytic triad.     

Effect of anti-mosquito antibodies on the infectivity of the rodent malaria parasite Plasmodium berghei to Anopheles farauti

The effect of mouse anti-mosquito antibodies, present in the bloodmeal, on the infectivity of Plasmodium berghei Vincke to Anopheles farauti Laveran was investigated. Significantly fewer oocysts developed in mosquitoes feeding on mice immunized with sugar-fed mosquito midgut antigens than in mosquitoes feeding on control mice. Mosquitoes feeding on mice immunized with the midgut antigens derived from sugar-fed mosquitoes also showed reduced mortality and had lower infection rates than those fed on unimmunized mice. Blood-fed midgut antigen was less effective in producing these effects than sugar-fed midgut antigen.     

A review of the in vitro and in vivo neurochemical characterization of the NMDA/PCP/Glycine/Ion channel receptor macrocomplex

Conclusions Current neurochemical studies of the NMDA receptor macromolecular complex are yielding new insights into the interactions of the subunits of this complex and the associated potential clinical benefits of selective modulation of these subnits. Such studies offer the great potential for a new generation of pharmacotherapies for a wide range of CNS disorders, including stroke, a condition for which there is currently no effective pharmacological treatment. However, it is essential to understand that the first generation products in this area may not be optimal pharmacotherapies, such that haracterization of possible receptor subtypes and understanding the molecular biology of the component proteins of the receptor complex will be crucial in the design of the optimal pharmacological modulators of the NMDA receptor complex.Special issue dedicated to Dr. Erminio Costa     

Molecular biology of wound-inducible proteinase inhibitors in plants

Abstract. The techniques of molecular biology are being employed to investigate at the gene level the systemically mediated, wound-induced accumulation of two defensive proteinase inhibitor proteins in plant leaves. These techniques have added a new dimension to biochemical and physiological studies already underway to understand the mechanism of induction by wounding. The acquisition of cDNAs from the RNAs coding for the two inhibitors facilitated studies of mRNA synthesis in leaves in response to wounding, and provided probes to obtain wound-inducible proteinase inhibitor genes from tomato ( Lycopersicon esculentum ) and potato (Solarium tuberosum) genomes. Successful transformations of tobacco plants with fused genes, containing the 5' and 3' regions of the inhibitor genes with the open reading frame of the chloramphenicol acelyltransferase ( cat ) gene, have provided a wound-inducible chloramphenicol acetyltransferase (CATase) activity with which to seek cis- and transacting elements that regulate wound-inducibility to help to understand the interaction of cytoplasmic and nuclear components of the intracellular communication systems that activate the proteinase inhibitor genes in response to wounding by insect pests.     

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate

Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666].     

Biochemical and immunological characterization of the cloned crystal toxin of Bacillus thuringiensis var. israelensis

The protein components of the cloned crystal toxin of Bacillus thuringiensis var. israelensis were separated by polyacrylamide gel electrophoresis under denaturing conditions. Using an antiserum to the solubilized B. thuringiensis var. israelensis crystal protein as a probe, immunological homology between the crystal protein components of B. thuringiensis var. israelensis and those of the recombinant B. megaterium strain VB131 was tested. The results from this study indicate that the crystal inclusion of the recombinant strain contains only the 130 kilodalton protein and not the 68 or the 28 kilodalton proteins of the crystal toxin of B. thuringiensis var. israelensis and that the 130 kilodalton protein is primarily responsible for the mosquitocidal activity of this organism.     

Opiate actions on mesocortical dopamine metabolism in the rat

The actions of parenteral morphine were examined with regard to dopamine metabolism in the mesocortical dopaminergic pathways of the rat. The effects of morphine on dopamine metabolism in the prefrontal, cingulate, pyriform and entorhinal cortices were compared with the actions of morphine on the metabolism of dopamine in the striatum and olfactory tubercle. In all tissues, except the entorhinal cortex, morphine significantly elevated the dopamine metabolites dihydroxphenylacetic acid and homovanillic acid. These data, along with previous studies of various pharmacological agents, clearly indicate that the mesocortical dopaminergic projections possess unique opioid and non-opioid regulatory inputs.     

Expression of the cytochrome b-URF6-URF5 region of the mouse mitochondrial genome

The nature of RNA coded by the only light-strand (L-strand) open-reading frame unidentified reading frame 6 (URF6) was studied by using a variety of single- and double-strand DNA subclones derived from the 3.6-kilobase (kb) cytochrome b (cyt b)-URF5 coding region of the mouse mitochondrial genome. Northern blot experiments using single-strand-specific M13 clones indicate that both the heavy (H) and L strands of this genomic region are symmetrically transcribed and processed into poly(adenylic acid) [poly(A)] RNAs of comparable size. The 1.2- and 2.4-kb RNAs coded by the H strand, putative mRNAs for cyt b and URF5 reading frames, respectively, are derived from a common precursor of 3.6-kb RNA. The L-strand-coded 1.15-kb RNA, on the other hand, is derived from a short-lived precursor of 3.6-kb RNA by a multiple-step processing involving a 2.4-kb intermediate RNA. The S1 nuclease protection experiments using both the 3'- or 5'-end-labeled DNA probes and also affinity-purified 32P-labeled RNA probes indicate that the 1.15-kb RNA maps between the start of the URF6 reading frame (3' end) and a region 590-600 nucleotides to the 5' end of this reading frame. The 1.15-kb RNA thus contains the entire URF6 coding sequence and an about 590-nucleotide-long 3' untranslated region. The molar abundance of the three mRNAs in the steady-state mitochondrial RNA varies markedly. The 1.15-kb URF6 mRNA is only one-tenth the level of 1.2-kb cyt b mRNA, although it is nearly as abundant as the 2.4-kb URF5 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)