Cross-bridge kinetics modeled from myoplasmic [Ca2+] and LV pressure at 17 degrees C and after 37 degrees C and 17 degrees C ischemia

We modeled changes in contractile element kinetics derived from the cyclic relationship between myoplasmic [Ca(2+)], measured by indo 1 fluorescence, and left ventricular pressure (LVP). We estimated model rate constants of the Ca(2+) affinity for troponin C (TnC) on actin (A) filament (TnCA) and actin and myosin (M) cross-bridge (A x M) cycling in intact guinea pig hearts during baseline 37 degrees C perfusion and evaluated changes at 1) 20 min 17 degrees C pressure, 2) 30-min reperfusion (RP) after 30-min 37 degrees C global ischemia during 37 degrees C RP, and 3) 30-min RP after 240-min 17 degrees C global ischemia during 37 degrees C RP. At 17 degrees C perfusion versus 37 degrees C perfusion, the model predicted: A x M binding was less sensitive; A x M dissociation was slower; Ca(2+) was less likely to bind to TnCA with A x M present; and Ca(2+) and TnCA binding was less sensitive in the absence of A x M. Model results were consistent with a cold-induced fall in heart rate from 260 beats/min (37 degrees C) to 33 beats/min (17 degrees C), increased diastolic LVP, and increased phasic Ca(2+). On RP after 37 degrees C ischemia vs. 37 degrees C perfusion, the model predicted the following: A x M binding was less sensitive; A x M dissociation was slower; and Ca(2+) was less likely to bind to TnCA in the absence of A. M. Model results were consistent with reduced myofilament responsiveness to [Ca(2+)] and diastolic contracture on 37 degrees C RP. In contrast, after cold ischemia versus 37 degrees C perfusion, A x M association and dissociation rates, and Ca(2+) and TnCA association rates, returned to preischemic values, whereas the dissociation rate of Ca(2+) from A x M was ninefold faster. This cardiac muscle kinetic model predicted a better-restored relationship between Ca(2+) and cross-bridge function on RP after an eightfold longer period of 17 degrees C than 37 degrees C ischemia.     

Aqueous Leaf Extract of Jatropha gossypiifolia L. (Euphorbiaceae) Inhibits Enzymatic and Biological Actions of Bothrops jararaca Snake Venom

Snakebites are a serious public health problem due their high morbi-mortality. The main available specific treatment is the antivenom serum therapy, which has some disadvantages, such as poor neutralization of local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies is relevant. Therefore, the aim of this study was to evaluate the antiophidic properties of Jatropha gossypiifolia, a medicinal plant used in folk medicine to treat snakebites. The aqueous leaf extract of the plant was prepared by decoction and phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins. The extract was able to inhibit enzymatic and biologic activities induced by Bothrops jararaca snake venom in vitro and in vivo. The blood incoagulability was efficiently inhibited by the extract by oral route. The hemorrhagic and edematogenic local effects were also inhibited, the former by up to 56% and the latter by 100%, in animals treated with extract by oral and intraperitoneal routes, respectively. The inhibition of myotoxic action of B. jararaca reached almost 100%. According to enzymatic tests performed, it is possible to suggest that the antiophidic activity may be due an inhibitory action upon snake venom metalloproteinases (SVMPs) and/or serine proteinases (SVSPs), including fibrinogenolytic enzymes, clotting factors activators and thrombin like enzymes (SVTLEs), as well upon catalytically inactive phospholipases A₂ (Lys49 PLA₂). Anti-inflammatory activity, at least partially, could also be related to the inhibition of local effects. Additionally, protein precipitating and antioxidant activities may also be important features contributing to the activity presented. In conclusion, the results demonstrate the potential antiophidic activity of J. gossypiifolia extract, including its significant action upon local effects, suggesting that it may be used as a new source of bioactive molecules against bothropic venom.     

Expression analysis of LEDGF/p75, APOBEC3G, TRIM5alpha, and tetherin in a Senegalese cohort of HIV-1-exposed seronegative individuals

Background

HIV-1 replication depends on a delicate balance between cellular co-factors and antiviral restriction factors. Lens epithelium-derived growth factor (LEDGF/p75) benefits HIV, whereas apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and tetherin exert anti-HIV activity. Expression levels of these proteins possibly contribute to HIV-1 resistance in HIV-1-exposed populations.

Methodology/Principal Findings

We used real-time PCR and flow cytometry to study mRNA and protein levels respectively in PBMC and PBMC subsets. We observed significantly reduced LEDGF/p75 protein levels in CD4+ lymphocytes of HIV-1-exposed seronegative subjects relative to healthy controls, whereas we found no differences in APOBEC3G, TRIM5α, or tetherin expression. Untreated HIV-1-infected patients generally expressed higher mRNA and protein levels than healthy controls. Increased tetherin levels, in particular, correlated with markers of disease progression: directly with the viral load and T cell activation and inversely with the CD4 count.

Conclusions/Significance

Our data suggest that reduced LEDGF/p75 levels may play a role in resistance to HIV-1 infection, while increased tetherin levels could be a marker of advanced HIV disease. Host factors that influence HIV-1 infection and disease could be important targets for new antiviral therapies.     

Purification and some catalytic properties of phosphoglucomutase from maize leaves

Phosphoglucomutase (EC 2.7.5.1, PGM) was purified to homogeneity from maize (Zea mays L.) leaves. The enzyme had specific activity 11. 7 U/mg protein and molecular mass (determined by gel-chromatography) of 133 +/- 4 kD. The molecular mass of PGM subunits determined by SDS-electrophoresis was 66 +/- 3 kD. The enzyme had Km for glucose-1-phosphate and glucose-1,6-diphosphate of 20.0 +/- 0.9 and 16.0 +/- 0.8 &mgr;M, respectively. Concentrations of glucose-1-phosphate and glucose-1,6-diphosphate above 3 and 0.4 mM, respectively, cause substrate inhibition. The enzyme activity was maximal at pH 8.0 and temperature 35 degreesC. Magnesium ions activate the enzyme and manganese ions inhibit it. 3-Phosphoglycerate is an uncompetitive inhibitor of the enzyme (Ki = 1.22 +/- 0.05 mM). Fructose-6-phosphate, 6-phosphogluconate, and ADP activate PGM, whereas ATP, UTP, and AMP inhibit the enzyme. Citrate was also a potent inhibitor, inhibitory effects of isocitrate and cis-aconitate being less pronounced.     

Plastid enzymes of terpenoid biosynthesis. Purification and characterization of gamma-tocopherol methyltransferase from Capsicum chromoplasts

gamma-Tocopherol methyltransferase was solubilized and purified from Capsicum chromoplast membranes by a combination of standard fractionation techniques. The purified enzyme was electrophoretically homogeneous, and its molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 33,000. In the absence of detergent, the enzyme formed high molecular weight aggregates. Several properties of the enzyme have been determined. The Km values were 2.5 and 13.7 microM for S-adenosylmethionine and gamma-tocopherol, respectively. The enzyme was able to transfer the methyl group S-adenosylmethionine to N-4-azido-2-nitrophenyl-beta-alanyl-gamma-tocopherol. The rate of transfer was less efficient compared to gamma-tocopherol. In the presence of ultraviolet light, this analog inhibited the gamma-tocopherol methyltransferase activity.     

Ischemia-reperfusion injury changes the dynamics of Ca2+-contraction coupling due to inotropic drugs in isolated hearts.

Positive inotropic drugs may attenuate or exacerbate the deleterious effects of ischemia and reperfusion (IR) injury on excitation-contraction coupling in hearts. We 1) quantified the phase-space relationship between simultaneously measured myoplasmic Ca2+ concentration ([Ca2+]) and isovolumetric left ventricular pressure (LVP) using indexes of loop area, orientation, and position; and 2) quantified cooperativity by linearly modeling the phase-space relationship between [Ca2+] and rate of LVP development in intact hearts during administration of positive inotropic drugs before and after global IR injury. Unpaced, isolated guinea pig hearts were perfused at a constant pressure with Krebs-Ringer solution (37 degrees C, 1.25 mM CaCl2). [Ca2+] was measured ratiometrically by indo 1 fluorescence by using a fiber-optic probe placed at the left ventricular free wall. LVP was measured by using a saline-filled latex balloon and transducer. Drugs were infused for 2 min, 30 min before, and for 2 min, 30 min after 30-min global ischemia. IR injury worsened Ca2+-contraction coupling, as seen from decreased orientation and repositioning of the loop rightward and downward and reduced cooperativity of contraction and relaxation with or without drugs. Dobutamine (4 microM) worsened, whereas dopamine (8 microM) improved Ca2+-contraction coupling before and after IR injury. Dobutamine and dopamine improved cooperativity of contraction and relaxation after IR injury, whereas only dopamine increased cooperativity of relaxation before IR injury. Digoxin (1 microM) improved Ca2+-contraction coupling and cooperativity of contraction after but not before ischemia. Levosimendan (1 microM) did not alter Ca2+-contraction coupling or cooperativity, despite producing concomitant increases in contractility, relaxation, and Ca2+ flux before and after ischemia. Dynamic indexes based on LVP-[Ca2+] diagrams (area, shape, position) can be used to identify and measure alterations in Ca2+-contraction coupling during administration of positive inotropic drugs in isolated hearts before and after IR injury.     

Saccharomyces cerevisiae and Candida tropicalis as starter cultures for the alcoholic fermentation of tchapalo, a traditional sorghum beer

Starter cultures of Candida tropicalis and Saccharomyces cerevisiae isolated from tchapalo were tested in pure culture and co-culture of four ratios [2:1, 25:4, 1:4, 2:3 (cells/cells)] for their ability to ferment sorghum wort to produce tchapalo. All the starters showed means growth rate between 0.043 and 0.101 h^?1. Only C. tropicalis in pure culture showed growth rate lower than that of S. cerevisiae in single culture. During fermentation, according to total soluble solids depletion, yeast starters could be grouped in four different profiles. But in the beer produced, total soluble solids contents were statistically identical. The lowest values were obtained with co-culture C. tropicalis + S. cerevisiae in the ratios of 2:1 and 2:3. Starter cultures with large ratio of C. tropicalis produced a higher organic acids and 2-butanone than S. cerevisiae in pure culture. However, co-culture C. tropicalis + S. cerevisiae (2:1) was the alone starter which produced higher ethanol than S. cerevisiae in pure culture. The beers produced with C. tropicalis + S. cerevisiae (25:4), C. tropicalis + S. cerevisiae (1:4) and C. tropicalis were widely different from those produced with the others starter cultures.