Development of mutants of the mosquitocidal bacterium Bacillus thuringiensis subspecies morrisoni (PG-14) toxic to lepidopterous or dipterous insects

The parasporal body of the mosquitocidal isolate (PG-14) of Bacillus thuringiensis subsp. morrisoni (BTM) contains five major proteins with molecular masses of, respectively, 27.3, 65, 128, 135, and 144 kDa. Proteins corresponding in mass to the first four of these also occur in the mosquitocidal strain, B. thuringiensis subsp. israelensis (BTI), and it is thought therefore that the mosquitocidal activity of both strains is due to these four proteins. In other studies it has been shown that each of these proteins exhibits from moderate to high toxicity to mosquitoes, though the specific toxicity of the 144 kDa protein in PG-14 to mosquitoes remains unknown. In the present study, two parasporal body mutants (M146 and M242) of PG-14 were developed growing the wild-type strain at 42 degrees C. The parasporal body of M146 contained less of the 65-kDa protein and was less toxic (LC50 = 108 ng/ml) to mosquitoes than the wild-type strain (LC50 = 8.3 ng/ml). The parasporal body of M242 consisted of a bipyramidal crystal composed of a 144-kDa protein that was not toxic to the mosquito, Aedes aegypti, but exhibited substantial toxicity (LC50 = 2.5 micrograms/ml) to the lepidopteran. Trichoplusia ni. Because the parasporal bodies of BTI and BTM PG-14 are similar in mosquitocidal toxicity on a weight basis, the latter results suggest the 144-kDa protein, though not mosquitocidal alone, can contribute to mosquitocidal, activity when in the presence of other mosquitocidal proteins.     

2''-Deoxycoformycin Inhibition of Adenosine Deaminase in Rat Brain: In Vivo and In Vitro Analysis of Specificity, Potency, and Enzyme Recovery

2'-Deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase (ADA), is increasingly used as a tool to investigate adenosine metabolism and neuromodulation. To advance further the usefulness of DCF for studies of purines in the CNS, we determined the inhibitory potency of this compound against ADA and adenylate deaminase (AMPDA) in brain, the rate of ADA recovery in various brain regions after single or repeated intraperitoneal DCF administrations, and the effect of DCF on several neurotransmitter synthetic enzymes. In vitro, the Ki values for inhibition of ADA and AMPDA were found to be 23 pM and 233 microM, respectively. In vivo, DCF inhibited ADA with ED50 values ranging from 155 to 280 micrograms/kg at 2 h posttreatment, and 98% inhibition was achieved with 1 mg/kg. AMPDA activity was not affected by doses up to 5.0 mg/kg. In contrast to the greater than 95% inhibition of ADA seen 1 day after DCF at 5 mg/kg, the effectiveness of a second similar DCF treatment on the activity that had recovered by 14 days was dramatically reduced. Eight days after DCF treatment with doses of 5-50 mg/kg, the degree of ADA activity recovery in 10 brain regions examined was similar; it averaged 35% of control values at the low dose but showed some heterogeneity, ranging from 15 to 54% of control values, at the higher doses. Forty days after treatment with a single dose of 5 mg/kg, ADA activity recovered by 68-78% of control values in brain regions with normally high levels of activity and by 44-59% of control values in other regions. The activities of choline acetyltransferase, glutamic acid decarboxylase, and histidine decarboxylase (an enzyme colocalized with ADA in hypothalamic neurons) were unaffected by DCF treatment, a result suggesting the lack of a generalized neurotoxic effect. The very low doses of DCF required for ADA inhibition in vivo are consistent with the high potency of this drug against ADA in vitro, and any physiological effects observed at low doses might therefore be ascribed to inhibition of ADA.     

Regulation of Ryanodine Receptor Calcium Release Channels by Diadenosine Polyphosphates

Abstract: [³H]Ryanodine binding to, as well as functions of, ryanodine receptor intracellular Ca²⁺ release channel complexes are modulated by several adenosine-based compounds. In this study, we determined the effects of endogenous compounds termed diadenosine polyphosphates (Ap_nAs; n = 2–6 phosphate groups) on [³H]ryanodine binding to membranes prepared from rat brain and skeletal and cardiac muscle. Under low ionic strength buffer conditions, [³H]ryanodine binding to brain membranes was significantly increased by 171% with 333 µMP¹,P⁵-di(adenosine-5′) pentaphosphate (Ap₅A) and by 209% with the same concentration of the metabolism-resistant ATP analogue βγ-methyleneadenosine 5′-triphosphate (AMP-PCP) compared with control values for [³H]ryanodine binding of 9.6 ± 1.8 fmol/mg of protein. Dose-related increases in [³H]ryanodine binding were observed for all five Ap_nAs tested [P¹,P²-di(adenosine-5′) pyrophosphate (Ap₂A), P¹,P³-di(adenosine-5′) triphosphate (Ap₃A), P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A), Ap₅A, and P¹,P⁶-di(adenosine-5′) hexaphosphate (Ap₆A)] as well as AMP-PCP; oxidized salts of Ap_nAs stimulated [³H]ryanodine binding to a greater degree than did nonoxidized Ap_nAs. The apparent rank order for the capacity of these agents to increase [³H]-ryanodine binding was oxidized Ap₄A = oxidized Ap₅A > oxidized Ap₃A > Ap₆A > AMP-PCP > Ap₅A > Ap₂A. Addition of the approximate EC₅₀ dose of oxidized Ap₄A (37 µM) increased the affinity (K_D) of ryanodine receptors from 34 ± 7 to 12 ± 2 nM; the apparent binding site density (B_max) was not significantly different from control values of 107 ± 33 fmol/mg of protein. Increases in [³H]-ryanodine binding by either oxidized Ap₄A or nonoxidized Ap₅A were not further enhanced by coincubation with AMP-PCP, which suggests a similar site of action for the Ap_nAs and AMP-PCP. [³H]Ryanodine binding to skeletal and cardiac muscle membranes was enhanced by addition of oxidized Ap₄A, Ap₅A, and AMP-PCP. Oxidized Ap₄A increased the specific binding by ninefold in skeletal muscle and by threefold in cardiac muscle. These results suggest that Ap_nAs, at physiologically relevant concentrations, may serve as endogenous modulators of ryanodine receptor-gated Ca²⁺ release channels.     

Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury

To examine whether basic fibroblast growth factor (bFGF) administered to the heart by perfusion can improve cardiac resistance to injury we employed an isolated rat heart model of ischemia-reperfusion injury and determined the extent of functional recovery in bFGF-treated and control hearts. Global ischemia was simulated by interruption of flow for 60 min. Recovery of developed force of contraction (DF), recorded after reestablishment of flow for 30 min, reached 63.8±1.5% and 96.5±3.5% of preischemic levels in control and bFGF-treated hearts (10 g/heart), respectively, indicating that bFGF induced significantly improved recovery of mechanical function. Recoveries of the rates of contraction or relaxation were also significantly improved in bFGF-treated hearts. Extent of myocardial injury, assessed by determination of phosphocreatine kinase in the effluent, was reduced as a result of bFGF treatment. As a first step towards understanding the mechanism and direct cellular target(s) of bFGF-induced cardioprotection, we investigated its fate after perfusion. Perfusion of 10 g bFGF/heart resulted in a 4-fold increase in bFGF associated with the heart compared to control levels, as estimated by biochemical fractionation and immunoblotting. Immunofluorescent staining of the bFGF-perfused hearts revealed intense anti-bFGF staining in association with blood vessels as well as the periphery of cardiomyocytes, suggesting that the latter may be a target for direct bFGF action. In conclusion, our findings of bFGF-induced increases in cardiac resistance to, and improved functional recovery from, ischemia-reperfusion injury indicate that bFGF may have clinical applications in the treatment of ischemic heart disease.     

Lisinopril quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of Lisinopril in human plasma using Enalaprilat as internal standard. The analyte and internal standard were extracted from the plasma samples by solid-phase extraction using Waters HLB Oasis SPE cartridges and chromatographed on a C8 analytical column. The mobile phase consisted of acetonitrile/water (60:40, v/v) + 20 mM acetic acid + 4.3 mM of triethylamine. The method had a chromatographic total run-time of 6.5 min and was linear within the range 2.00-200 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM). The precision (CV%) and accuracy, calculated from limit of quantification (LOQ) samples (n = 8), were 8.9 and 98.9%, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of Lisinopril 20mg.     

Zein adsorption to hydrophilic and hydrophobic surfaces investigated by surface plasmon resonance

Zein, the prolamine of corn, has been investigated for its potential as an industrial biopolymer. In previous research, zein was plasticized with oleic acid and formed into sheets/films. Physical properties of films were affected by film structure and controlled in turn by zein-oleic acid interactions. The nature of such interactions is not well understood. Thus, protein-fatty acid interactions were investigated in this work by the use of surface plasmon resonance (SPR). Zein adsorption from 75% aqueous 2-propanol solutions, 0.05% to 0.5% w/v, onto hydrophilic and hydrophobic self-assembled monolayers (SAMs) formed by 11-mercaptoundecanoic acid and 1-octanethiol, respectively, was monitored by high time resolution SPR. Initial adsorption rate and ultimate surface coverage increased with bulk protein concentration for both surfaces. The initial slope of plotted adsorption isotherms was higher on 11-mercaptoundecanoic acid than on 1-octanethiol, indicating higher zein affinity for hydrophilic SAMs. Also, maximum adsorption values were higher for zein on hydrophilic than on hydrophobic SAMs. Flushing off loosely bound zein in the SPR cell allowed estimation of apparent monolayer values. Differences in monolayer values for hydrophobic and hydrophilic surfaces were explained in terms of zein adsorption footprint.     

Bone Marrow Involvement in a Patient with Paracoccidioidomycosis: A Rare Presentation of Juvenile Form

Paracoccidioides brasiliensis rarely shows bone marrow involvement and its response to treatment with itraconazole in children needs further assessment. We describe here a child with a juvenile disseminated form of paracoccidioidomycosis, which showed reticuloendothelial system involvement and the presence of Paracoccidioides brasiliensis in the bone marrow. The patient showed an effective and rapid response to itraconazole therapy.     

Effect of whole body vibration training on lower limb performance in selected high-level ballet students

The aim of this study was to examine the effects of 8 weeks of whole body vibration (WBV) training on vertical jump ability (CMJ) and knee-extensor performance at selected external loads (50, 70, and 100 kg; leg-press exercise) in elite ballerinas. Twenty-two (age, 21.25 +/- 1.5 years) full-time ballerinas were assigned randomly to the experimental (E, n = 11) and control (C, n = 11) groups. The experimental group was submitted to WBV training 3 times per week before ballet practice. During the training period, the E and C groups undertook the same amount of ballet practice. Posttraining CMJ performance significantly increased in E group (6.3 +/- 3.8%, p < 0.001). Furthermore, E group showed significant (p < 0.05-0.001) posttraining average leg-press power and velocity improvements at all the external loads considered. Consequently, the force-velocity and power-velocity relationship shifted to the right after WBV training in the E group. The results of the present study show that WBV training is an effective short-term training methodology for inducing improvements in knee-extensor explosiveness in elite ballerinas.     

Influences of experimental factors on spinal stretch reflex latency and amplitude in the human triceps surae.

The spinal stretch reflex (SSR) is commonly assessed via electromyographic (EMG) analysis of joint perturbations inducing changes in muscle length. Previous literature indicates that when large experimental changes in magnitude of agonist background EMG, perturbation velocity, and perturbation amplitude are employed, SSR latency and amplitude are significantly altered. The purpose of this investigation was to evaluate the relative dependence of SSR latency and amplitude on inherent variability in these experimental variables. Soleus SSR latency and amplitude were assessed in 40 healthy subjects following dorsiflexion perturbation under an active state ( approximately 14% MVC). Experimental variables displayed limited variability (means +/- SD): soleus background EMG (13.47 +/- 7.08% MVC), perturbation velocity (96.1 +/- 30 degrees /s), and perturbation amplitude (4 +/- 1 degrees ). SSR latency was not significantly related to soleus background EMG (r = 0.189), perturbation velocity (r = 0.213), or perturbation amplitude (r = 0.202). Similarly, SSR amplitude was not significantly related to soleus background EMG (r = 0.306), perturbation velocity (r = 0.053), or perturbation amplitude (r = 0.056). Variability in experimental variables was much smaller than what has been reported in the literature to significantly impact SSR characteristics. These results suggest that SSR latency and amplitude are independent of agonist background EMG, perturbation velocity, and perturbation amplitude when experimental variability is relatively limited.