Coronary flow-induced inotropism is modulated by binding of dextrans to the endothelial luminal surface

In isolated perfused guinea pig hearts, coronary flow causes a positive inotropic effect [positive coronary flow-induced effect (+CFIE)] that could be altered by dextrans (Dx) in the coronary perfusion solution. To test this possibility, Dx of 20, 40, 70, and 500 kDa were infused and found to modulate +CFIE; however, when Dx infusion was terminated, the effect persisted, i.e., was irreversible/nonwashable, suggesting that Dx may bind to luminal endothelial lectinic structures. This hypothesis was tested when Dx [with fluorescent traces (D*)] bound to the vessel wall was hydrolyzed by dextranase infusion and washout of D* fragments completely reverted the +CFIE, and it was found that bound D* to be displaced by free Dx required concentrations 50-100 times that used during binding. In addition, dose-response curves for Dx on +CFIE show that the higher the Dx molecular mass, the lesser the concentration required to have an effect. Because a large Dx molecule has a greater number polymeric glucose branches, it can bind to a larger number of endothelial lectinic sites, requiring a lower concentration to affect +CFIE. Our results suggest that luminal endothelial lectinic structures are part of the flow-sensing assembly.     

Polyprotein processing protease of African swine fever virus: purification and biochemical characterization

The purified recombinant African swine fever virus polyprotein processing protease cleaves the two GG-X sites in polyprotein pp62 with the same efficiency. Cleavage at the site that is first recognized in vivo is not a requisite for cleavage at the second site, suggesting the existence of mechanisms that control the ordered processing of the polyprotein during infection.     

The three-dimensional structure of the core domain of Naf Y from Azotobacter vinelandii determined at 1.8-A resolution

The Azotobacter vinelandii NafY protein (nitrogenase accessory factor Y) is able to bind either to the iron molybdenum cofactor (FeMo-co) or to apodinitrogenase and is believed to facilitate the transfer of FeMo-co into apodinitrogenase. The NafY protein has two domains: an N-terminal domain (residues Met1-Leu98) and a C-terminal domain (residues Glu99-Ser232), referred here to as the "core domain." The core domain of NafY is shown here to be capable of binding the FeMo cofactor of nitrogenase but unable to bind to apodinitrogenase in the absence of the first domain. The three-dimensional molecular structure of the core domain of NafY has been solved to 1.8-A resolution, revealing that the protein consists of a mixed five-stranded beta-sheet flanked by five alpha-helices that belongs to the ribonuclease H superfamily. As such, this represents a new fold capable of binding FeMo-co, where the only previous example was that seen in dinitrogenase.     

Phylogenetic analysis in the genus Cicer and cultivated chickpea using RAPD and ISSR markers

Seventy five accessions belonging to 14 species of the genus Cicer were analysed with PCR-based molecular markers to determine their phylogenetic relationships. Eight of the species were annuals and included the Section Monocicer which contains cultivated chickpea (Cicer arietinum L.). The remaining six species were perennials (five from Section Polycicer and one from Section Acanthocicer). More than one accession per species was analysed in most of the wild species; within C. arietinum, 26 accessions including Kabuli and Desi types, were studied. RAPD analyses using 12 primers gave 234 polymorphic fragments. Variability within species was detected. A dendrogram based on the Jaccard similarity index showed that the distribution pattern of variability between species was related to both growth habit and geographical origin. An accession of Cicer reticulatum was closer to accessions of Cicer echinospermum than to the four remaining of C. reticulatum, suggesting the possibility of gene flow between species. Cluster analysis for cultivated chickpea differentiated Kabuli and Desi types but we did not detect a clear relationship between groups and the geographical origin of the accessions. Received: 5 April 2001 / Accepted: 13 July 2001     

Effects of water stress on antioxidant enzymes of leaves and nodules of transgenic alfalfa overexpressing superoxide dismutases

The antioxidant composition and relative water stress tolerance of nodulated alfalfa plants ( Medicago sativa L. ×  Sinorhizobium meliloti 102F78) of the elite genotype N4 and three derived transgenic lines have been studied in detail. These transgenic lines overproduced, respectively, Mn-containing superoxide dismutase (SOD) in the mitochondria of leaves and nodules, MnSOD in the chloroplasts, and FeSOD in the chloroplasts. In general for all lines, water stress caused moderate decreases in MnSOD and FeSOD activities in both leaves and nodules, but had distinct tissue-dependent effects on the activities of the peroxide-scavenging enzymes. During water stress, with a few exceptions, ascorbate peroxidase and catalase activities increased moderately in leaves but decreased in nodules. At mild water stress, transgenic lines showed, on average, 20% higher photosynthetic activity than the parental line, which suggests a superior tolerance of transgenic plants under these conditions. However, the untransformed and the transgenic plants performed similarly during moderate and severe water stress and recovery with respect to important markers of metabolic activity and of oxidative stress in leaves and nodules. We conclude that the base genotype used for transformation and the background SOD isozymic composition may have a profound effect on the relative tolerance of the transgenic lines to abiotic stress.     

Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells

Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERT flanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Cre-expressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, after hTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence.     

Purification,cofactor analysis,and site-directed mutagenesis of Synechococcus ferredoxin-nitrate reductase

The narB gene of the cyanobacterium Synechococcus sp. strain PCC 7942 encodes an assimilatory nitrate reductase that uses photosynthetically reduced ferredoxin as the physiological electron donor. This gene was expressed in Escherichia coli and electrophoretically pure preparations of the enzyme were obtained using affinity chromatography with either reduced-ferredoxin or NarB antibodies. The electronic absorption spectrum of the oxidized enzyme showed a shoulder at around 320 nm and a broad absorption band between 350 and 500 nm. These features are indicative of the presence of an iron-sulfur centre(s) and accordingly metal analysis showed ca. 3 atoms of Fe per molecule of protein that could represent a [3Fe-4S] cluster. Further analysis indicated the presence of 1 atom of Mo and 2 molecules of ribonucleotide-conjugated molybdopterin per molecule of protein. This, together with the requirement of a mobA gene for production of an active enzyme, strongly suggests the presence of Mo in the form of the bis-MGD (bis-molybdopterin guanine dinucleotide) cofactor in Synechococcusnitrate reductase. A model for the coordination of the Mo atom to the enzyme is proposed. Four conserved Cys residues were replaced by site-directed mutagenesis. The effects of these changes on the enzyme activity and electronic absorption spectra support the participation of those residues in iron-sulfur cluster coordination. This revised version was published online in June 2006 with corrections to the Cover Date.     

β1 Integrin triggering affects leukemic cell line sensitivity to natural killer cells

The role of beta1 (CD29) integrins in natural killer (NK) cell-target cell conjugation and cytotoxicity has not been clearly established. Ligation of beta1 integrins in NK cells can modulate the lytic capacity in both a positive and a negative manner; however, the contribution of the beta1 integrins present on target cells remains to be evaluated. Here, we analyzed the effect of beta1 integrins expressed by potential tumor target cells on conjugation and cytotoxicity. Using normalized flow cytometry binding assays, we demonstrated that the pretreatment of MOLT-4, K562, U-937 and HL-60 human leukemia target cell lines with selected anti-beta1 monoclonal antibodies (mAb) increased conjugation to human NK cell line NKL as well as to purified NK cells. Only mAb recognizing residues 207-218 of the beta1 subunit and functionally involved in the induction of homotypic adhesion (functional epitope A1) increased conjugation of all the target cells. Moreover, mAb to adhesion molecules different from beta1 but also inducers of homotypic adhesion of the target cells, i.e. CD43 and CD50 (ICAM-3), failed to increase conjugation to NKL cells. Cytotoxicity assays demonstrated that lysis of NK-sensitive target cells (MOLT-4) also increased after pretreatment with anti-beta1 epitope A1 mAb. Importantly, pretreatment of NK-resistant target cells (U-937 and HL-60) with anti-beta1 mAb was not able to outweigh the cytotoxic inhibitory mechanisms controlled by HLA class I molecules. However, simultaneous masking of HLA class I molecules with mAb and pretreatment with anti-beta1 mAb rendered NK-resistant cells susceptible to lysis, as predicted by the missing self hypothesis. Triggering of tumor target cells through beta1 integrins may thus play a role in conjugation to NK cells as well as in co-stimulation of cell-mediated cytotoxicity.