Unlike thrombin, protein C and activated protein C do not affect vascular tone

Because plasma levels of protein C (PC) or activated protein C (APC) are altered in certain diseases associated with vascular dysfunction, and APC has therapeutic potential in preventing microvascular coagulation in severe sepsis, potential vascular effects of PC and APC were compared to those of the vasoactive peptide, thrombin. Thrombin was a more potent relaxant agonist than contractile agonist in aorta. Unlike thrombin, cumulatively administered APC (10(-9)-10(-7) M) did not exert vascular effects in rat or rabbit aorta. Noncumulative challenge of PC (10(-7) M) and APC (8 x 10(-8) M) also did not contract rat or rabbit aortae, either with or without endothelium. Likewise, the same concentrations of PC and APC also did not relax norepinephrine-induced (10(-7) M) vascular tone in either rat or rabbit aortae. Thus, in contrast to thrombin, PC and APC failed to modulate vascular tone, suggesting that the therapeutic use of APC is unlikely to be accompanied by any direct effects on vascular motility.     

Structural and interactional homology of clinically potential trypsin inhibitors: molecular modelling of cucurbitaceae family peptides using the X-ray structure of MCTI-II

Several trypsin inhibitor peptides (with 28-32 amino acid residues) belonging to the Cucurbitaceae (LA-1, LA-2, MCTI-I, CMTI-I, CMTI-III, CMTI-IV), characterized by a distinctive tertiary fold with three conserved disulphide bonds and with mostly arginine at their active centre, were modelled using the high-resolution X-ray structure of a homologous inhibitor, MCTI-II, isolated from bitter gourd. All the inhibitors were modelled in both their native and complexed state with the trypsin molecule, keeping the active site the same as was observed in the trypsin-MCTI-II complex, by homology modelling using the InsightII program. The minimized energy profile supported the binding constants (binding behaviour) of the inhibitor-trypsin complexes in the solution state. A difference accessible surface area (DASA) study of the trypsin with and without inhibitors revealed the subsites of trypsin where the inhibitors bind. It revealed that the role of mutation of these peptides through evolution is to modulate their inhibitory function depending on the biological need rather than changing the overall structural folding characteristics which are highly conserved. The minor changes of amino acids in the non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complex formation.     

Neovascularisation offers a new perspective to glutamine related therapy

Angiogenesis or the generation of new blood vessel, is an important factor in the growth of a solid tumor. Hence, it becomes a necessary parameter of any kind of therapeutic study. Glutamine is an essential nutrient of tumor tissue and glutamine related therapy involves clearance of circulatory glutamine by glutaminase. Therefore, using different murine solid tumor models, the present study was undertaken to find out whether the S-180 cell glutaminase has any effect on angiogenesis of solid tumor, or not. Result indicates that the purified S-180 cell glutaminase reduces tumor volume and restrict the generation of neo blood vessels. Therefore, it can be concluded that this enzyme may be an effective device against the cancer metastasis.     

Tau phosphorylation at serine 396 and serine 404 by human recombinant tau protein kinase II inhibits tau's ability to promote microtubule assembly

In Alzheimer's disease, hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies and fails to promote microtubule assembly. Dysregulation of the brain-specific tau protein kinase II is reported to play an important role in the pathogenesis of Alzheimer's disease (Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S., Dikkes, P., and Tsai, L.-H. (1999) Nature 402, 615-622). We report here that in vitro phosphorylation of human tau by human recombinant tau protein kinase II severely inhibits the ability of tau to promote microtubule assembly as monitored by tubulin polymerization. The ultrastructure of tau-mediated polymerized tubulin was visualized by electron microscopy and compared with phosphorylated tau. Consistent with the observed slower kinetics of tubulin polymerization, phosphorylated tau is compromised in its ability to generate microtubules. Moreover, we show that phosphorylation of microtubule-associated tau results in tau's dissociation from the microtubules and tubulin depolymerization. Mutational studies with human tau indicate that phosphorylation by tau protein kinase II at serine 396 and serine 404 is primarily responsible for the functional loss of tau-mediated tubulin polymerization. These in vitro results suggest a possible role for tau protein kinase II-mediated tau phosphorylation in initiating the destabilization of microtubules.     

Mechanisms regulating endothelial cell barrier function

Endothelium forms a physical barrier that separates blood from tissue. Communication between blood and tissue occurs through the delivery of molecules and circulating substances across the endothelial barrier by directed transport either through or between cells. Inflammation promotes macromolecular transport by decreasing cell-cell and cell-matrix adhesion and increasing centripetally directed tension, resulting in the formation of intercellular gaps. Inflammation may also increase the selected transport of macromolecules through cells. Significant progress has been made in understanding the molecular and cellular mechanisms that account for constitutive endothelial cell barrier function and also the mechanisms activated during inflammation that reduce barrier function. Current concepts of mechanisms regulating endothelial cell barrier function were presented in a symposium at the 2000 Experimental Biology Conference and are reviewed here.     

The SSU rDNA coding region of a filose amoeba contains a group I intron lacking the universally conserved G at the 3'-terminus

We sequenced small subunit ribosomal DNA (rDNA) PCR-fragments of sizes 2.3 kb and 2.9 kb isolated from a culture of the red alga, Porphyra spiralis var. spiralis. Phylogenetic analysis of the 2.3-kb fragment showed that it encoded the sequence of a contaminant filose amoeba. The Nuclearia-like amoeba (named strain N-Por) was identified with scanning electron microscopy. Its rDNA sequence was positioned with strong bootstrap support within a diverse protist assemblage that includes filose amoebae, chlorarachniophytes, cercomonads, and Plasmodiophora brassicae. The rDNA of N-Por contained a group I intron at the conserved 943 position that remarkably, had a U at the 3'-terminus rather than the universally conserved G.     

Genomic diversity among Vibrio cholerae O139 strains isolated in Bangladesh and India between 1992 and 1998

In order to assess the extent of genomic diversity among Vibrio cholerae O139 strains, restriction fragment length polymorphisms in two genetic loci, rrn and ctx, were studied. Analysis of 144 strains isolated from different regions of Bangladesh and India between 1992 and 1998 revealed the presence of at least six distinct ribotypes (B-I through B-VI) of which three were new ribotypes, and one of these was represented by a nontoxigenic O139 strain. Strains of ribotypes B-I through B-V shared 11 different CTX genotypes (A through K). Antimicrobial resistance patterns of the strains varied independently of their ribotypes and CTX genotypes. Results of this study suggest that V. cholerae O139 is undergoing rapid genetic changes leading to the origination of new variants, and temporal changes in antimicrobial resistance patterns may be contributing to the selection of different variants.     

Widespread occurrence of spliceosomal introns in the rDNA genes of ascomycetes

Spliceosomal (pre-mRNA) introns have previously been found in eukaryotic protein-coding genes, in the small nuclear RNAs of some fungi, and in the small- and large-subunit ribosomal DNA genes of a limited number of ascomycetes. How the majority of these introns originate remains an open question because few proven cases of recent and pervasive intron origin have been documented. We report here the widespread occurrence of spliceosomal introns (69 introns at 27 different sites) in the small- and large-subunit nuclear-encoded rDNA of lichen-forming and free-living members of the Ascomycota. Our analyses suggest that these spliceosomal introns are of relatively recent origin, i.e., within the Euascomycetes, and have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. The spliceosome itself, and not an external agent (e.g., transposable elements, group II introns), may have given rise to these introns. A nonrandom sequence pattern was found at sites flanking the rRNA spliceosomal introns. This pattern (AG-intron-G) closely resembles the proto-splice site (MAG-intron-R) postulated for intron insertions in pre-mRNA genes. The clustered positions of spliceosomal introns on secondary structures suggest that particular rRNA regions are preferred sites for insertion through reverse-splicing.     

Behavioural alterations in rats induced by single prenatal exposure of haloperidol

Haloperidol (50 mg/kg, i.p.) treatment was given once to two different groups of pregnant Charles Foster rats on gestational day 9 and 14, these being respectively the critical periods of neural morphogenesis and rapid neural cell proliferation in this species. Pregnant control rats were similarly treated with equal volume of vehicle. The pups born were subjected to open-field exploratory behaviour and elevated plus-maze behaviour tests of anxiety and learned helplessness test of depression at 9 weeks of age. The results indicate that prenatal haloperidol treatment on gestational day 14 induces a significant increase in open-field ambulation and faecal droppings whereas haloperidol treatment on gestational day 9 caused significantly decreased rearing and unaltered ambulation in rat offsprings. Rat offsprings treated with haloperidol on gestational day 9 and 14 also displayed significant anxiogenic behaviour pattern on elevated plus-maze. Significantly increased number of escape failures were observed in learned helplessness tests indicating presence of depression in haloperidol treated rat offsprings. These behavioural alterations were found to be more marked in rat offsprings treated with haloperidol on gestational day 14. The results suggest that prenatal single exposure of high dose of haloperidol during critical period of neural cell proliferation leaves a lasting imprint on offsprings resulting in abnormal emotional state.