A fast sample preparation procedure for mercury speciation in hair samples by high-performance liquid chromatography coupled to ICP-MS

A simple method for mercury speciation in hair samples with a fast sample preparation procedure using high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry is proposed. Prior to analysis, 50 mg of hair samples were accurately weighed into 15 mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCl was added to the samples following sonication for 10 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of inorganic mercury (Ino-Hg), methylmercury (Met-Hg) and ethylmercury (Et-Hg) was accomplished in less than 8 min on a C18 reverse phase column with a mobile phase containing 0.05% v/v mercaptoethanol, 0.4% m/v L-cysteine, 0.06 mol L(-1) ammonium acetate and 5% v/v methanol. The method detection limits were found to be 15 ng g(-1), 10 ng g(-1) and 38 ng g(-1), for inorganic mercury, methylmercury and ethylmercury, respectively. Sample throughput is 4 samples h(-1) (duplicate). A considerable improvement in the time of analysis was achieved when compared to other published methods. Method accuracy is traceable to Certified Reference Materials (CRMs) 85 and 86 human hair from the International Atomic Energy Agency (IAEA). Finally, the proposed method was successfully applied to the speciation of mercury in hair samples collected from fish-eating communities of the Brazilian Amazon.

Direct and Simultaneous Determination of Cd Cu, and Se in Blood Samples by Graphite Furnace Atomic Absorption Spectrometry

A graphite furnace atomic absorption spectrometric method is proposed for the direct and simultaneous determination of Cd, Cu, and Se in human blood. Samples were diluted 1:10 (v/v) in 0.5% (v/v) HNO(3) + 0.5% (v/v) Triton X-100 solution. For 12 mu L injected sample volume + 5 mu L, of 1000 mg L(-1) Pd(NO(3))(2) + 3 mu L of 1000 mg L(-1) Mg(NO(3))(2), the calculated characteristic masses (mo) were 0.9 pg Cd, 16 pg Cu, and 39 pg Se, which are close to those mo values for single-element conditions for THGA furnace (1.3 pg Cd, 17 pg Cu, and 45 pg Se). Calibration curves with linear correlations better than 0.999 were obtained. The limits of detection (LOD) were 0.03 mu g L(-1) Cd, 0.075 mu g L(-1) Cu and 0.3 mu g L(-1) Se, and the relative standard deviations (n= 12) were 2.5%, 0.3%, and 1.5%, respectively. The method was applied for Cd, Cu, and Se determination in 10 human blood samples and the results were in agreement at the 95% confidence level with those obtained by inductively coupled plasma mass spectrometry. Concentrations of analytes in the selected blood samples varied from 1.7 to 3.2 mu g L(-1) Cd, 700 to 921.7 mu g L(-1) Cu, and from 68.6 to 350 mu g L(-1) Se. The accuracy of the proposed method was also evaluated by an addition-recovery experiment and recoveries of Cd, Cu, and Se added to blood samples ranged from 99-109%, 91-103%,and 93-103%, respectively.

BjussuSP-I: A new thrombin-like enzyme isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr = 61,000 under reducing conditions and pI similar to 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated scrine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca2+ and Mg2+). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against I I venom samples of Bothrops, I of Crotalus, and I of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDfNEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom. (C) 2007 Elsevier Inc. All rights reserved.

Evaluation of the genotoxic and antigenotoxic effects after acute and subacute treatments with acai pulp (Euterpe oleracea Mart.) on mice using the erythrocytes micronucleus test and the comet assay

Acai, the fruit of a palm native to the Amazonian basin, is widely distributed in northern South America, where it has considerable economic importance. Whereas individual polyphenolics compounds in Acai have been extensively evaluated, studies of the intact fruit and its biological properties are lacking. Therefore, the present study was undertaken to investigate the in vivo genotoxicity of Acai and its possible antigenotoxicity on doxorubicin (DXR)-induced DNA damage. The Acai pulp doses selected were 3.33, 10.0 and 16.67 g/kg b.w. administered by gavage alone or prior to DXR (16 mg/kg b.w.) administered by intraperitoneal injection. Swiss albino mice were distributed in eight groups for acute treatment with acai pulp (24 h) and eight groups for subacute treatment (daily for 14 consecutive days) before euthanasia. The negative control groups were treated in a similar way. The results of chemical analysis suggested the presence of carotenoids, anthocyanins, phenolic. and flavonoids in Acai pulp. The endpoints analyzed were micronucleus induction in bone marrow and peripheral blood cells polychromatic erythrocytes, and DNA damage in peripheral blood, liver and kidney cells assessed using the alkaline (pH > 13) comet assay. There were no statistically significant differences (p > 0.05) between the negative control and the groups treated with the three doses of Acai pulp alone in all endpoints analyzed, demonstrating the absence of genotoxic effects. The protective effects of Acai pulp were observed in both acute and subacute treatments, when administered prior to DXR. In general, subacute treatment provided greater efficiency in protecting against DXR-induced DNA damage in liver and kidney cells. These protective effects can be explained as the result of the phytochemicals present in Acai pulp. These results will be applied to the developmental of food with functional characteristics, as well as to explore the characteristics of Acai as a health promoter. (C) 2009 Elsevier B.V. All rights reserved.

The critical power function is dependent on the duration of the predictive exercise tests chosen

The linear relationship between work accomplished (W-lim) and time to exhaustion (t(lim)) can be described by the equation: W-lim = a + CP.t(lim). Critical power (CP) is the slope of this line and is thought to represent a maximum rate of ATP synthesis without exhaustion, presumably an inherent characteristic of the aerobic energy system. The present investigation determined whether the choice of predictive tests would elicit significant differences in the estimated CP. Ten female physical education students completed, in random order and on consecutive days, five art-out predictive tests at preselected constant-power outputs. Predictive tests were performed on an electrically-braked cycle ergometer and power loadings were individually chosen so as to induce fatigue within approximately 1-10 mins. CP was derived by fitting the linear W-lim-t(lim) regression and calculated three ways: 1) using the first, third and fifth W-lim-t(lim) coordinates (I-135), 2) using coordinates from the three highest power outputs (I-123; mean t(lim) = 68-193 s) and 3) using coordinates from the lowest power outputs (I-345; mean t(lim) = 193-485 s). Repeated measures ANOVA revealed that CPI123 (201.0 +/- 37.9W) > CPI135 (176.1 +/- 27.6W) > CPI345 (164.0 +/- 22.8W) (P < 0.05). When the three sets of data were used to fit the hyperbolic Power-t(lim) regression, statistically significant differences between each CP were also found (P < 0.05). The shorter the predictive trials, the greater the slope of the W-lim-t(lim) regression; possibly because of the greater influence of 'aerobic inertia' on these trials. This may explain why CP has failed to represent a maximal, sustainable work rate. The present findings suggest that if CP is to represent the highest power output that an individual can maintain for a very long time without fatigue then CP should be calculated over a range of predictive tests in which the influence of aerobic inertia is minimised.

Australian doctors' beliefs and practice regarding Helicobacter pylori

Objective: To determine beliefs and behaviours of Australian doctors regarding Helicobacter pylori. Design: Anonymous reply-paid postal survey mailed in December 1995 and again in March 1996. Subjects: All members on the mailing lists of the Gastroenterological Society of Australia Endoscopy Section (n = 397) and the Australian Society of Infectious Diseases (n = 264; those without medical qualifications were asked not to reply), and 400 general practitioners (GPs) randomly selected from the Royal Australian College of General Practitioners. Main outcome measures: Differences between specialist groups in belief in a causative association between H. pylori and peptic disease and in use of eradication therapy and pre- and post-treatment testing for H. pylori. Results: 92.6% of doctors believed H. pylori causes duodenal ulcer, with GPs significantly less likely to believe than gastroenterologists (odds ratio [OR], 0.22; 95% confidence interval [CI], 0.00-0.81). In duodenal ulcer, 93.4% of doctors believed H. pylori eradication therapy should be given, but fewer (83.4%) claimed to give it always or mostly, with GPs less likely to report giving it than gastroenterologists (OR, 0.06; 95% CI, 0.02-0.19). For non-ulcer dyspepsia, gastrointestinal surgeons were more likely than gastroenterologists to believe in a causative link with H. pylori (OR, 5.6; 95% CI, 3.0-10.7) and in a need for eradication therapy (OR, 3.6; 95% CI, 1.7-7.7). Most doctors (79.3%) believed in confirming the presence of H. pylori before eradication therapy in duodenal ulcer. Only 51.6% believed post-eradication testing necessary (45.5%), yet 79.1% reported performing it. Conclusions: Significant differences exist between specialist groups in beliefs and self-reported behaviours regarding H. pylori.

Altered adhesion, proliferation and death in neural cultures from adults with schizophrenia

The causes of schizophrenia are unknown, but there is evidence linking subtle deviations in neural development with schizophrenia. Embryonic brain development cannot be studied in an adult with schizophrenia, but neurogenesis and early events in neuronal differentiation can be investigated throughout adult life in the human olfactory epithelium. Our past research has demonstrated that neuronal cultures can be derived from biopsy of the human adult olfactory epithelium. In the present study, we examined mechanisms related to neurogenesis and neuronal differentiation in adults with schizophrenia versus well controls. Forty biopsies were collected under local anaesthesia from ten individuals with DSM III-R schizophrenia and ten age- and sex-matched well controls. All patients, except one, were receiving antipsychotic medication at the time of the biopsy, Immunostaining for neuronal markers indicated that neurogenesis occurred in the biopsies from both patients and controls since all contained cells expressing tubulin and/or olfactory marker protein. The major findings of this study are: 1. biopsies from patients with schizophrenia showed a significantly reduced ability to attach to the culture slide: 29.9% of patient biopsies attached compared to 73.5% of control biopsies; 2. biopsies from patients with schizophrenia had a significantly greater proportion of cells undergoing mitosis: 0.69% in the patients compared to 0.29% in the controls; and 3. dopamine (10 mu M) significantly increased the proportion of apoptotic cells in the control cultures but significantly decreased the proportion in patients' cultures. (C) 1999 Elsevier Science B.V. All rights reserved.

The binding of closantel to ovine serum albumin, and homogenate fractions of Haemonchus contortus

Closantel binds to the serum proteins of the host and affects blood sucking parasites when they ingest the brood of treated hosts. Closantel binds specifically to ovine serum albumin (K-a of 9.3 x 10(6)M(-1)) at site I, the warfarin/phenylbutazone binding site of albumin Closantel also binds to invertebrate haemocyanin and haemolymph. The strongest binding of closantel in homogenates of H. contortus is found in fractions containing soluble proteins. This binding is of low affinity and, because the site itself is not fully denaturable, it may not be proteinaceous. There is no detectable difference in binding affinity between homogenate fractions from closantel susceptible and resistant isolates of adult or larval worms suggesting that closantel resistance is not due to changes in the closantel receptor or carrier. (C) 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Comparative analysis of dendritic cell density and total number in commonly transplanted organs: morphometric estimation in normal mice

Dendritic cells (DC) are considered to be the major cell type responsible for induction of primary immune responses. While they have been shown to play a critical role in eliciting allosensitization via the direct pathway, there is evidence that maturational and/or activational heterogeneity between DC in different donor organs may be crucial to allograft outcome. Despite such an important perceived role for DC, no accurate estimates of their number in commonly transplanted organs have been reported. Therefore, leukocytes and DC were visualized and enumerated in cryostat sections of normal mouse (C57BL/10, B10.BR, C3H) liver, heart, kidney and pancreas by immunohistochemistry (CD45 and MHC class II staining, respectively). Total immunopositive cell number and MHC class II+ cell density (C57BL/10 mice only) were estimated using established morphometric techniques - the fractionator and disector principles, respectively. Liver contained considerably more leukocytes (similar to 5-20 x 10(6)) and DC (similar to 1-3 x 10(6)) than the other organs examined (pancreas: similar to 0.6 x 10(6) and similar to 0.35 x 10(6): heart: similar to 0.8 x 10(6) and similar to 0.4 x 10(6); kidney similar to 1.2 x 10(6) and 0.65 x 10(6), respectively). In liver, DC comprised a lower proportion of all leukocytes (similar to 15-25%) than in the other parenchymal organs examined (similar to 40-60%). Comparatively, DC density in C57BL/10 mice was heart > kidney > pancreas much greater than liver (similar to 6.6 x 10(6), 5 x 10(6), 4.5 x 10(6) and 1.1 x 10(6) cells/cm(3), respectively). When compared to previously published data on allograft survival, the results indicate that the absolute number of MHC class II+ DC present in a donor organ is a poor predictor of graft outcome. Survival of solid organ allografts is more closely related to the density of the donor DC network within the graft. (C) 2000 Elsevier Science B.V. All rights reserved.

Determination of the stability constants of mercury(II) complexes of mixed donor macrobicyclic encapsulating ligands

The reactions of mercury(II) with the mixed donor encapsulating ligands 3,6,16-trithia-6,11,19-triazabicyclo[6.6.6]icosane (AMN(3)S(3)sar) and 1-amino-8-methyl-6,19-dithia-3,10,13,16-tetraazabicyclo[6.6.6]icosane (AMN(4)S(2)sar) have been studied. NMR ligand-ligand competition experiments with the ligands 1,4,8,11-tetraazaeyclotetradecane ([14]aneN(4)), 1-thia-4,7,10-triazacyclododecane ([12]aneN(3)S) and ethylenediaminetetraacetic acid (EDTA) with AMN(3)S(3)sar and Hg(II) indicated that [14]aneN(4) would be an appropriate competing ligand for the, determination of the Hg(II) stability constant. Calculations indicated the ratio of concentrations of AMN3S3sar, [14]aneN(4) and Hg(II) required for the determination of the stability constant ranged from 1:1:1 to 1:5:1. Refinement of the titration curves yielded log(10)K[Hg(AMN(3)S(3)sar)](2+) = 17.7. A similar competition titration resulted in the determination of the stability constant for the AMN(4)S(2)sar system as log(10)K[Hg(AMN(4)S(2)sar)](2+) = 19.5. The observed binding constants for the mixed N/S donor systems and the hexaaza analogues sar (3,6,10,13,16,19-hexaazabicyclo [6.6.6]icosane) and diamsar (1,8-diamino-3,6,10,13,16,19 -hexazabicyclo [6.6.6] icosane (log(10)K-[Hg(diamsar)](2+) = 26.4; log(10)K[Hg(sar)](2+) = 28.1) differ by approximately ten orders of magnitude. The difference is ascribed not to a cryptate effect but to a mismatch in the Hg-N and Hg-S bond lengths in the N/S systems.

Screening for leprosy in immigrants: A decision analysis model

Almost all leprosy cases reported in industrialized countries occur amongst immigrants or refugees from developing countries where leprosy continues to be an important health issue. Screening for leprosy is an important question for governments in countries with immigration and refugee programmes. A decision analysis framework is used to evaluate leprosy screening. The analysis uses a set of criteria and parameters regarding leprosy screening, and available data to estimate the number of cases which would be detected by a leprosy screening programme of immigrants from countries with different leprosy prevalences, compared with a policy of waiting for immigrants who develop symptomatic clinical diseases to present for health care. In a cohort of 100,000 immigrants from high leprosy prevalence regions (3.6/10,000), screening would detect 32 of the 42 cases which would arise in the destination country over the 14 years after migration; from medium prevalence areas (0.7/10,000) 6.3 of the total 8.1 cases would be detected, and from low prevalence regions (0.2/10,600) 1.8 of 2.3 cases. Using Australian data, the migrant mix would produce 74 leprosy cases from 10 years intake; screening would detect 54, and 19 would be diagnosed subsequently after migration. Screening would only produce significant case-yield amongst immigrants from regions or social groups with high leprosy prevalence. Since the number of immigrants to Australia from countries of higher endemnicity is not large routine leprosy screening would have a small impact on case incidence.

Mass of the milky way

We present a new estimate of the mass of the Milky Way based on the escape velocity of a sample of distant stars, about 12 kpc from the Galactic centre and about 5 kpc from the plane of the Galaxy. Our sample is very different from previous escape-velocity studies, being compiled from an all-object spectroscopic survey of a region of sky. The derived mass within 12 kpc of the Galactic centre is (1.3 +/- 0.3) x 10(11) M-circle dot.

Developmental characterization, function and regulation of a Laccase2 encoding gene in the honey bee, Apis mellifera (Hymenoptera, Apinae)

In insects, exoskeleton (cuticle) formation at each molt cycle includes complex biochemical pathways wherein the laccase enzymes (EC 1.10.3.2) may have a key role. We identified an Amlac2 gene that encodes a laccase2 in the honey bee, Apis mellifera, and investigated its function in exoskeleton differentiation. The Amlac2 gene consists of nine exons resulting in an ORE of 2193 nucleotides. The deduced translation product is a 731 amino acid protein of 81.5 kDa and a pl of 6.05. Amlac2 is highly expressed in the integument of pharate adults, and the expression precedes the onset of cuticle pigmentation and the intensification of sclerotization. In accordance with the temporal sequence of exoskeleton differentiation from anterior to posterior direction, the levels of Amlac2 transcript increase earlier in the thoracic than in the abdominal integument. The gene expression lasts even after the bees emerge from brood cells and begin activities in the nest, but declines after the transition to foraging stage, suggesting that maturation of the exoskeleton is completed at this stage. Post-transcriptional knockdown of Amlac2 gene expression resulted in structural abnormalities in the exoskeleton and drastically affected adult eclosion. By setting a ligature between the thorax and abdomen of early pupae we could delay the increase in hemolymph ecdysteroid levels in the abdomen. This severely impaired the increase in Amlac2 transcript levels and also the differentiation of the abdominal exoskeleton. Taken together, these results indicate that Amlac2 expression is controlled by ecdysteroids and has a critical role in the differentiation of the adult exoskeleton of honey bees. (C) 2010 Elsevier Ltd. All rights reserved.

Process development for a recombinant Chinese Hamster Ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system

The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 muM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 107 cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system. (C) 2004 Wiley Periodicals, Inc.

Synthesis of homoallylic oxygenated alpha-methylene-gamma-butyrolactones: a model for preparing biologically active natural lactones

In this Letter we describe a 12% overall yield synthesis of a model for homoallylic oxygenated alpha-methylene-gamma-butyrolactones with relative stereochemistry defined by selective hydrogenation with Rh/Al(2)O(3). The synthesis was realized in 9 steps involving simple reactions. (C) 2008 Elsevier Ltd. All rights reserved.