Hydrosoluble formazan XTT: its application to natural products drug discovery for Leishmania

A colorimetric method for measuring the viability of Leishmania promastigotes is described that is based on the reduction of the tetrazolium salt, XTT, to a water-soluble formazan. Values obtained by the XTT method correlated well with parasite number (r=0.965) and with methods that rely upon the reduction of MTT or MTS (r=0.96 and 0.97, respectively). The IC(50) values obtained by XTT method with amphotericin-B, miltefosine and ketoconazole were similar to those previously reported by other methods. The XTT method proved to be a reliable and convenient method for the screening of methanolic extracts from 1059 plants and was used for the bioassay-guided fractionation of the alkaloid aegeline from Sarcorhachis naranjoana.     

Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris

Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.     

Seed lectin from pisum arvense: isolation, biochemical characterization and amino acid sequence

A glucose/mannose lectin was purified by affinity chromatography from Pisum arvense seeds (PAL) and the 50 kDa molecular mass in solution determined by size exclusion chromatography. SDS-PAGE and electrospray ionization mass spectrometry showed two distinct polypeptide chains: alpha (Mr. 5591 Da) and beta (19986 Da). The lectin was extensively characterized in terms of its biochemical and biological aspects. The amino acid sequence was established by Edman degradation of overlapping peptides. PAL in solution behaves as a dimer and has its monomeric structure formed by two distinct polypeptide chains named alpha (Mr. 5591 Da) and beta (19986 Da) by Electrospray ionization (ESI) mass spectrometry. PAL possesses identical amino acid sequences to that of pea seed lectin but undoubtedly does not exhibit sequence heterogeneity. It is discussed that P. arvense should be considered as a synonym of P. sativum. Furthermore, like pea lectin, PAL discriminates biantennary fucosylated glycan, determined by surface plasmon resonance.     

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.     

Synaptic behaviour of some structural and numerical chromosome anomalies in female and male rats (Rattus norvegicus)

The processes of synapsis and synaptic adjustment have been detected in some structural and numerical anomalies in two female rat foetuses and in one male rat in the course of a study on X-ray genotoxicity. The synaptic characteristics and adjustment of one pericentric inversion and a deletion have been analysed by electron microscopy in synaptonemal complex spreads from two female foetuses, and the synaptic behaviour of a trisomy has been studied in a testicular biopsy from an adult male. In a large proportion (from 50% to 90%) of the analysed cells, the abnormal meiotic configuration could not be detected either because the anomaly was present in mosaic from trisomy or because synaptic adjustment had already taken place (inversion) or as result of a combination of two of the above (deletion).     

Elevated interstitial adenosine concentrations do not activate the muscle reflex

The purpose of the present study was to examine the effects of adenosine perfusion of the isolated triceps surae muscle group in the decerebrate cat on interstitial adenosine concentrations as well as heart rate and blood pressure responses. In six male cats (6.0 +/- 0.21 kg), the triceps surae muscle group of both legs was perfused with an artificial blood solution containing no additives (control) and then with blood containing 20 mM or 100 microM adenosine for 10 min. An intact muscle reflex was confirmed by bolus injections of 50 mM phosphate and/or saturated KCl administered into the triceps surae muscle via the cannulated popliteal artery before and after adenosine blood perfusion. Microdialysis of the triceps surae muscle group during muscle perfusion revealed that interstitial adenosine was elevated (P < 0.05) from 0.9 +/- 0.3 microM during control blood perfusion to 2,421 +/- 547 microM during 20 mM adenosine perfusion. In addition, interstitial adenosine levels were increased (P < 0.05) from 1.1 +/- 0.3 microM during control blood perfusion to 4.1 +/- 1.2 microM during perfusion with 100 microM adenosine. Despite the large increases in interstitial adenosine levels, perfusion of the triceps surae muscle group with the two blood adenosine solutions resulted in no significant increases in heart rate or blood pressure. These data strongly suggest that elevated interstitial adenosine concentrations do not play a role in activating the muscle reflex and confirm our previous in vivo human findings (J Appl Physiol 83: 1045-1053, 1997).     

Gender affects sympathetic and hemodynamic response to postural stress

We tested the hypothesis that differences in sympathetic reflex responses to head-up tilt (HUT) between males (n = 9) and females (n = 8) were associated with decrements in postural vasomotor responses in women. Muscle sympathetic nerve activity (MSNA; microneurography), heart rate, stroke volume (SV; Doppler), and blood pressure (Finapres) were measured during a progressive HUT protocol (5 min at each of supine, 20 degrees, 40 degrees, and 60 degrees ). MSNA and hemodynamic responses were also measured during the cold pressor test (CPT) to examine nonbaroreflex neurovascular control. SV was normalized to body surface area (SV(i)) to calculate the index of cardiac output (Q(i)), and total peripheral resistance (TPR). During HUT, heart rate increased more in females versus males (P < 0.001) and SV(i) and Q(i) decreased similarly in both groups. Mean arterial pressure (MAP) increased to a lesser extent in females versus males in the HUT (P < 0.01) but increases in TPR during HUT were similar. MSNA burst frequency was lower in females versus males in supine (P < 0.03) but increased similarly during HUT. Average amplitude/burst increased in 60 degrees HUT for males but not females. Both males and females demonstrated an increase in MAP as well as MSNA burst frequency, mean burst amplitude, and total MSNA during the CPT. However, compared with females, males demonstrated a greater neural response (DeltaTotal MSNA) due to a larger increase in mean burst amplitude (P < 0.05). Therefore, these data point to gender-specific autonomic responses to cardiovascular stress. The different MSNA response to postural stress between genders may contribute importantly to decrements in blood pressure control during HUT in females.     

Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.     

Effects of dynamic exercise on mean blood velocity and muscle interstitial metabolite responses in humans

We examined the effects of dynamic one-legged knee extension exercise on mean blood velocity (MBV) and muscle interstitial metabolite concentrations in healthy young subjects (n = 7). Femoral MBV (Doppler), mean arterial pressure (MAP) and muscle interstitial metabolite (adenosine, lactate, phosphate, K(+), pH, and H(+); by microdialysis) concentrations were measured during 5 min of exercise at 30 and 60% of maximal work capacity (W(max)). MAP increased (P < 0.05) to a similar extent during the two exercise bouts, whereas the increase in MBV was greater (P < 0.05) during exercise at 60% (77.00 +/- 6.77 cm/s) compared with 30% W(max) (43.71 +/- 3.71 cm/s). The increase in interstitial adenosine from rest to exercise was greater (P < 0.05) during the 60% (0.80 +/- 0.10 microM) compared with the 30% W(max) bout (0.57 +/- 0.10 microM). During exercise at 60% W(max), interstitial K(+) rose at a greater rate than during exercise at 30% W(max) (P < 0.05). However, pH increased (H(+) decreased) at similar rates for the two exercise intensities. During exercise, interstitial lactate and phosphate increased (P < 0.05) with no difference observed between the two intensities. After 5 min of recovery, MBV decreased to baseline levels after exercise at 30% W(max) (4.12 +/- 1.10 cm/s), whereas MBV remained above baseline levels after exercise at 60% W(max) (Delta19.46 +/- 2.61 cm/s; P < 0.05). MAP and interstitial adenosine, K(+), pH, and H(+) returned toward baseline levels. However, interstitial lactate and phosphate continued to increase during the recovery period. Thus an increase in exercise intensity resulted in concomitant changes in MBV and muscle interstitial adenosine and K(+), whereas similar changes were not observed for MAP or muscle interstitial pH, lactate, or phosphate. These data suggest that K(+) and/or adenosine may play an active role in the regulation of skeletal muscle blood flow during exercise.