Determination of mitragynine in rat plasma by LC-MS/MS: Application to pharmacokinetics

This study used for the first time LC-MS/MS for the analysis of mitragynine (MIT), a mu-opioid agonist with antinociceptive and antitussive properties, in rat plasma. Mitragynine and the internal standard (amitriptyline) were extracted from plasma with hexane-isoamyl alcohol and resolved on a Lichrospher (R) RP-SelectB column (9.80 and 12.90 min, respectively). The quantification limit was 0.2 ng/mL within a linear range of 0.2-1000 ng/mL The method was applied to quantify mitragynine in plasma samples of rats (n = 8 per sampling time) treated with a single oral dose of 20 mg/kg. The following pharmacokinetic parameters were obtained (mean): maximum plasma concentration: 424 ng/mL; time to reach maximum plasma concentration: 1.26 h; elimination half-life: 3.85 h, apparent total clearance: 6.35 L/h/kg, and apparent volume of distribution: 37.90 L/kg. (C) 2009 Elsevier B.V. All rights reserved.

Short- and long-term, salinity-induced modulation of V-ATPase activity in the posterior gills of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better understand the biochemical mechanisms underlying anisosmotic extracellular regulation in the freshwater Brachyura, we kinetically characterized the V-ATPase from the posterior gills of Dilocarcinus pagei, acclimated for 10 days to salinities up to 21%.. Specific activity was highest in fresh water (26.5 +/- 2.1 U mg(-1)), decreasing in 5 parts per thousand to 21 parts per thousand, attaining 3-fold less at 15 parts per thousand. Apparent affinities for ATP and Mg(2+) respectively increased 3.2- and 2-fold at 10 parts per thousand, suggesting expression of different isoenzymes. In a 240-h time-course study of exposure to 21%., maximum specific activity decreased 2.5- to 4-fold within 1 to 24 h while apparent affinities for ATP and Mg(2+) respectively increased by 12-fold within 24 h and 2.4-fold after 1 h, unchanged thereafter. K(I) for bafilomycin A(1) decreased 150-fold after 1 h, remaining constant up to 120 h. This is the first kinetic analysis of V-ATPase specific activity in crustacean gills during salinity acclimation. Our findings indicate active gill Cl(-) uptake by D. pagei in fresh water, and short- and long-term down-regulation of V-ATPase-driven ion uptake processes during salinity exposure, aiding in comprehension of the biochemical adaptations underpinning the establishment of the Brachyura in fresh water. (C) 2011 Elsevier Inc. All rights reserved.

Purification and Biochemical Properties of a Glucose-Stimulated beta-D-Glucosidase Produced by Humicola grisea var. thermoidea Grown on Sugarcane Bagasse

The effect of several carbon sources on the production of mycelial-bound beta-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated beta-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The beta-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50 degrees C, respectively. The purified enzyme was thermostable up to 60 min in water at 55 degrees C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, o-nitrophenyl-beta-D-galactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-beta-D-fucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude beta-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea beta-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Purification and biochemical characterization of a mycelial glucose- and xylose-stimulated beta-glucosidase from the thermophilic fungus Humicola insolens

A mycelial beta-glucosidase from the thermophilic mold Humicola insolens was purified and biochemically characterized. The enzyme showed carbohydrate content of 21% and apparent molecular mass of 94 kDa, as estimated by gel filtration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a single polypeptide band of 55 kDa, suggesting that the native enzyme was a homodimer. Mass spectrometry analysis showed amino acid sequence similarity with a P-glucosidase from Humicola grisea var. thermoidea, with about 22% coverage. Optima of temperature and pH were 60 degrees C and 6.0-6.5, respectively. The enzyme was stable up to I h at 50 degrees C and showed a half-life of approximately 44 min at 55 degrees C. The beta-glucosidase hydrolyzed cellobiose, lactose, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-galactopyranoside, o-nitrophenyl-beta-D-galactopyranoside, and salicin. Kinetic studies showed that p-nitrophenyl-beta-D-fucopyranoside and cellobiose were the best enzyme substrates. Enzyme activity was stimulated by glucose or xylose at concentrations up to 400 mM, with maximal stimulatory effect (about 2-fold) around 40 mM. The high catalytic efficiency for the natural substrate, good thermal stability, strong stimulation by glucose or xylose, and tolerance to elevated concentrations of these monosaccharides qualify this enzyme for application in the hydrolysis of cellulosic materials. (C) 2009 Elsevier Ltd. All rights reserved.

Biochemical properties of an extracellular beta-D-fructofuranosidase II produced by Aspergillus phoenicis under Solid-Sate Fermentation using soy bran as substrate

The filamentous fungus A. phoenicis produced high levels of beta-D-fructofuranosidase (FFase) when grown for 72 hrs under Solid-State Fermentation (SSF), using soy bran moistened with tap water (1:0.5 w/v) as substrate/carbon source. Two isoforms (I and II) were obtained, and FFase II was purified 18-fold to apparent homogeneity with 14% recovery. The native molecular mass of the glycoprotein (12% of carbohydrate content) was 158.5 kDa with two subunits of 85 kDa estimated by SDS-PAGE. Optima of temperature and pH were 55 degrees C and 4.5. The enzyme was stable for more than 1 hr at 50 degrees C and was also stable in a pH range from 7.0 to 8.0. FFase II retained 80% of activity after storage at 4 degrees C by 200 hrs. Dichroism analysis showed the presence of random and beta-sheet structure. A. phoenicis FFase II was activated by Mn(2+), Mg(2+) and Co(2+), and inhibited by Cu(2+), Hg(2+) and EDTA. The enzyme hydrolyzed sucrose, inulin and raffinose. K(d) and V(max) values were 18 mM and 189 U/mg protein using sucrose as substrate.

STATIC IMAGES WITH DIFFERENT INDUCED INTENSITIES OF HUMAN BODY MOVEMENTS AFFECT SUBJECTIVE TIME

Modulation of subjective time was examined using static images eliciting perceptions of different intensities of body movement. Undergraduate students were exposed to photographs of dancer sculptures in different dance positions for 36 sec. and asked to estimate the exposure duration. Lower movement intensities were related to shorter estimated durations. Mean durations for images of unmoving dancers were underestimated and for dancers taking a ballet step were overestimated. Temporal estimations were also related to the order of presentation of the stimuli, which suggested that subjective time estimations were influenced by the experimental context. Subjective time is related not only to the visual perception of moving images, but also of elicited perceptions of movement in static images, suggesting an embodiment effect on subjective time estimation.

The unconditioned fear produced by morphine withdrawal is regulated by mu- and kappa-opioid receptors in the midbrain tectum

We have recently shown that morphine withdrawal sensitizes the neural substrates of fear in the midbrain tectum structures-the dorsal periaqueductal gray (dPAG) and inferior colliculus (IC). In the present study, we investigated the role of mu- and kappa-opioid receptors in the mediation of these effects. Periadolescent rats chronically treated with morphine (10 mg/kg; s.c.) twice daily for 10 days were implanted with an electrode glued to a guide-cannula into the dPAG or the IC. Forty-eight hours after the interruption of this treatment, the effects of intra-dPAG or intra-IC microinjections of [D-Ala(2) N-Me-Phe(4) Gly(5)-ol]-enkephalin (DAMGO; 0.6 and 1 nmol/0.2 mu l) - a selective mu-receptor agonist - or nor-binaltorphimine (BNI; 2.5 and 5 mu g/0.2 mu l) - a selective K-receptor antagonist with tardive action - on the freezing and escape thresholds determined by electrical stimulation of the dPAG and the IC were examined. For both structures, morphine withdrawal produced pro-aversive effects. DAMGO and BNI had antiaversive effects when injected into the dPAG and IC of non-dependent rats. In morphine-withdrawn rats, only BNI continued to promote antiaversive effects in both structures. Whereas DAMGO lost its antiaversive efficacy when injected into the dPAG, only its highest dose promoted antiaversive effects in the IC of morphine-withdrawn rats, suggesting the development of an apparent tolerance. Thus, the enhanced reactivity of the midbrain tectum in morphine-withdrawn periadolescent rats may be due, at least partially, to an impairment of the inhibitory influence of mechanisms mediated by mu-receptors on the neural substrates of fear in this region. (C) 2009 Elsevier B.V. All rights reserved.

Structural and Biochemical Correlates of Na(+), K(+)-ATPase Driven Ion Uptake Across the Posterior Gill Epithelium of the True Freshwater Crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+), K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (approximate to 14 mu m(2) membrane per mu m(3) cytoplasm), deep invaginations that house the Na(+), K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 mu m(2) mu m(-2)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 mu m(2) mu m(-2)), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+), K(+)-ATPase specific activity resembles marine crabs but is approximate to 5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two alpha-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4)(+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water. J. Exp. Zool. 313A:508-523, 2010. (C) 2010 Wiley-Liss, Inc.

Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K (m) and V (max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 A mu mol/min/mg, respectively. PG was found to have temperature optimum at 65 A degrees C and was totally stable at 45 A degrees C for 90 min. Half-life at 55 A degrees C was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).

Acute Toxic Leukoencephalopathy: Potential for Reversibility Clinically and on MRI With Diffusion-Weighted and FLAIR Imaging

OBJECTIVE. Toxic leukoencephalopathy may present acutely or subacutely with symmetrically reduced diffusion in the periventricular and supraventricular white matter, hereafter referred to as periventricular white matter. This entity may reverse both on imaging and clinically. However, a gathering together of the heterogeneous causes of this disorder as seen on MRI with diffusion-weighted imaging (DWI) and an analysis of their likelihood to reverse has not yet been performed. Our goals were to gather causes of acute or subacute toxic leukoencephalopathy that can present with reduced diffusion of periventricular white matter in order to promote recognition of this entity, to evaluate whether DWI with apparent diffusion coefficient (ADC) values can predict the extent of chronic FLAIR abnormality ( imaging reversibility), and to evaluate whether DWI can predict the clinical outcome ( clinical reversibility). MATERIALS AND METHODS. Two neuroradiologists retrospectively reviewed the MRI examinations of 39 patients with acute symptoms and reduced diffusion of periventricular white matter. The reviewers then scored the extent of abnormality on DWI and FLAIR. ADC ratios of affected white matter versus the unaffected periventricular white matter were obtained. Each patient`s clinical records were reviewed to determine the cause and clinical outcome. Histology findings were available in three patients. Correlations were calculated between the initial MRI markers and both the clinical course and the follow-up extent on FLAIR using Spearman`s correlation coefficient. RESULTS. Of the initial 39 patients, seven were excluded because of a nontoxic cause (hypoxic-ischemic encephalopathy [HIE] or congenital genetic disorders) or because of technical errors. In the remaining 32 patients, no correlation was noted between any of the initial MRI markers (percentage of ADC reduction, DWI extent, or FLAIR extent) with the clinical outcome. Three patients had histologic correlation. However, moderate correlation was seen between the extent of abnormality on initial FLAIR and the extent on follow-up FLAIR (r = 0.441, p = 0.047). Of the 13 patients who underwent repeat MRI at 21 days or longer, the reduced diffusion resolved in all but one. Significant differences were noted between ADC values in affected white matter versus unaffected periventricular white matter on initial (p < 0.0001) but not on follow-up MRI (p = 0.13), and in affected white matter on initial versus follow-up (p = 0.0014) in those individuals who underwent repeat imaging on the same magnet (n = 9), confirming resolution of the DWI abnormalities. CONCLUSION. Acute toxic leukoencephalopathy with reduced diffusion may be clinically reversible and radiologically reversible on DWI, and may also be reversible, but to a lesser degree, on FLAIR MRI. None of the imaging markers measured in this study appears to correlate with clinical outcome, which underscores the necessity for prompt recognition of this entity. Alerting the clinician to this potentially reversible syndrome can facilitate treatment and removal of the offending agent in the early stages.

CR3 (CD11b/CD18) activation of nasal neutrophils: a measure of upper airway endotoxin exposure

Inhaled endotoxin (lipopolysaccharide, LPS) initiates an inflammatory response and leads to the expression of CR3 (CD11b/CD18) receptors on polymorphonuclear leukocytes (PMNs). We determined if PMN activation in nasal lavage fluid (NLF) is a possible biomarker of occupational endotoxin exposure. Seven subjects exposed to endotoxin provided NLF samples that were split into three aliquots (negative control - 1 M nicotinamide; sham; positive control - 11 eta g of exogenous LPS) and PMN activation was measured using a chemiluminometer. Differences in mean PMN activation were apparent, negative control: 548 +/- 15.65 RLU 100 mu l(-1); sham: 11469 +/- 2582 RLU 100 mu l(-1); positive control: 42026 +/- 16659 RLU 100 mu l (n = 7; p < 0.05). This technique shows promise as a diagnostic method for measuring upper airway LPS exposure.

Novel mutations of the BSCL2 and AGPAT2 genes in 10 families with Berardinelli-Seip congenital generalized lipodystrophy syndrome

P>Context Congenital generalized lipodystrophy, or Berardinelli-Seip syndrome, is a rare autosomal recessive disease caused by mutations in either the BSCL2 or AGPAT2 genes. This syndrome is characterized by an almost complete loss of adipose tissue usually diagnosed at birth or early infancy resulting in apparent muscle hypertrophy. Common clinical features are acanthosis nigricans, hepatomegaly with or without splenomegaly and high stature. Acromegaloid features, cardiomyopathy and mental retardation can also be present. Design We investigated 11 kindreds from different geographical areas of Brazil (northeast and southeast). All coding regions as well as flanking intronic regions of both genes were examined. Polymerase chain reaction (PCR) amplifications were performed using primers described previously and PCR products were sequenced directly. Results Four AGPAT2 and two BSCL2 families harboured the same set of mutations. BSCL2 gene mutations were found in the homozygous form in four kindreds (c.412C > T c.464T > C, c.518-519insA, IVS5-2A > G), and in two kindreds compound mutations were found (c.1363C > T, c.424A > G). In the other four families, one mutation of the AGPAT2 gene was found (IVS3-1G > C and c.299G > A). Conclusions We have demonstrated four novel mutations of the BSCL2 and AGPAT2 genes responsible for Berardinelli-Seip syndrome and Brunzell syndrome (AGPAT2-related syndrome).

Low bone mass in juvenile onset sclerosis systemic: the possible role for 25-hydroxyvitamin D insufficiency

Juvenile onset systemic sclerosis (JoSSc) is a rare disease, and there are no studies focusing in bone mineral density and biochemical bone parameters. Ten consecutive patients with JoSSc and 10 controls gender, age, menarche age, and physical activity matched were selected. Clinical data were obtained at the medical visit and chart review. Laboratorial analysis included autoantibodies, 25-hydroxyvitamin D (25OHD), intact parathyroid hormone, calcium, phosphorus, alkaline phosphatase and albumin sera levels. Bone mineral density was analyzed by dual-energy X-ray absorptiometry, and bone mineral apparent density (BMAD) was calculated. A lower BMAD in femoral neck (0.294 +/- A 0.060 vs. 0.395 +/- A 0.048 g/cm(3), P = 0.001) and total femur (0.134 +/- A 0.021 vs. 0.171 +/- A 0.022 g/cm(3), P = 0.002) was observed in JoSSc compared to controls. Likewise, a trend to lower BMAD in lumbar spine (0.117 +/- A 0.013 vs. 0.119 +/- A 0.012 g/cm(3), P = 0.06) was also found in these patients. Serum levels of 25OHD were significantly lower in JoSSc compared to controls (18.1 +/- A 6.4 vs. 25.1 +/- A 6.6 ng/mL, P = 0.04), and all patients had vitamin D insufficiency (< 20 ng/mL) compared to 40% of controls (P = 0.01). All other biochemical parameters were within normal range and alike in both groups. BMAD in femoral neck and total femur was correlated with 25OHD levels in JoSSc (r = 0.82, P = 0.004; r = 0.707, P = 0.02; respectively). We have identified a remarkable high prevalence of 25OHD insufficiency in JoSSc. Its correlation with hip BMAD suggests a causal effect and reinforces the need to incorporate this hormone evaluation in this disease management.

Adult rats are more sensitive to the vascular effects induced by hyperhomocysteinemia than young rats

We aimed to investigate the vascular effects of hyperhomocysteinemia (HHcy) on carotid arteries from young and adult rats. With this purpose young and adult rats received a solution of DL-homocysteine-thiolactone (1 g/kg body weight/day) in the drinking water for 7, 14 and 28 days. Increase on plasma homocysteine occurred in young and adult rats treated with DL-homocysteine-thiolactone in all periods. Vascular reactivity experiments using standard muscle bath procedures showed that HHcy enhanced the contractile response of endothelium-intact, carotid rings to phenylephrine in both young and adult rats. However, in young rats, the increased phenylephrine-induced contraction was observed after hyperhomocysteinemia for 14 and 28 days, whereas in adult rats this response was already apparent after 7 day treatment. HHcy impaired acetylcholine-induced relaxation in arteries from adult but not young rats. The contraction induced by phenylephrine in carotid arteries in the presence of Y-27632 was reversed to control values in arteries from young but not adult rats with hyperhomocysteinemia. HHcy did not alter the contraction induced by CaCl(2) in carotid arteries from young rats, but enhanced CaCl(2)-induced contraction in the arteries from adult rats. HHcy increased the basal levels of superoxide anion in arteries from both groups. Finally, HHcy decreased the basal levels of nitrite in arteries from adult but not young rats. The major new finding of the present work is that arteries from young rats are more resistant to vascular changes evoked by HHcy than arteries from adult rats. Also, we verified that the enhanced vascular response to phenylephrine observed in carotid arteries of DL-homocysteine thiolactone-treated rats is mediated by different mechanisms in young and adult rats. (C) 2010 Published by Elsevier Inc.

Rehabilitation of executive dysfunction: A controlled trial of an attention and problem solving treatment group

In this study, the effectiveness of a group-based attention and problem solving (APS) treatment approach to executive impairments in patients with frontal lobe lesions was investigated. Thirty participants with lesions in the frontal lobes, 16 with left frontal (LF) and 14 with right frontal (RF) lesions, were allocated into three groups, each with 10 participants. The APS treatment was initially compared to two other control conditions, an information/education (IE) approach and treatment-as-usual or traditional rehabilitation (TR), with each of the control groups subsequently receiving the APS intervention in a crossover design. This design allowed for an evaluation of the treatment through assessment before and after treatment and on follow up, six months later. There was an improvement on some executive and functional measures after the implementation of the APS programme in the three groups. Size, and to a lesser extent laterality, of lesion affected baseline performance on measures of executive function, but there was no apparent relationship between size, laterality or site of lesion and level of benefit from the treatment intervention. The results were discussed in terms of models of executive functioning and the effectiveness of domain specific interventions in the rehabilitation of executive dysfunction.