Development and validation of sensitive methods for determination of sibutramine hydrochloride monohydrate and direct enantiomeric separation on a protein-based chiral stationary phase

Sibutramine hydrochloride monohydrate, chemically 1-(4-chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl) hydrochloride monohydrate (SB center dot HCl center dot H2O), was approved by the U.S. Food and Drug Administration for the treatment of obesity. The objective of this study was to develop, validate, and compare methods using UV-derivative spectrophotometry (UVDS) and reversed-phase high-performance liquid chromatography (HPLC) for the determination of SB center dot HCl center dot H2O in pharmaceutical drug products. The UVDS and HPLC methods were found to be rapid, precise, and accurate. Statistically, there was no significant difference between the proposed UVDS and HPLC methods. The enantiomeric separation of SB was obtained on an alpha-1 acid glycoprotein column. The R- and S-sibutramine were eluted in < 5 min with baseline separation of the chromatographic peaks (alpha = 1.9 and resolution = 1.9).

Sensitivity to halothane and its relationship to the development of PSE (Pale, Soft, Exudative) meat in female lineage broilers

This work aimed to evaluate female lineage broilers for halothane sensitivity and for their susceptibility to the subsequent development of PSE meat. The halothane test was carried out in an anesthetic chamber with 3.0% halothane. The unconscious birds were examined for leg muscle rigidity. If one or both legs became extended and rigid, the birds were classified as halothane sensitive (HAL+), while unresponsive birds were classified as halothane negative (HAL-). The results showed that of 298 birds aged 42 days old, 95.6% were HAL- and 4.4% were HAL+. A sample of pectoralis major muscle was collected from HAL- (n=105) and HAL+ (n=13) birds. The pH and breast fillet color were determined at 4ºC, 24 hours post-mortem. Interestingly, only 2.5% of HAL+ birds displayed PSE meat characteristics compared to 12.7% of HAL- individuals. The halothane test demonstrated that female lineage broilers displayed very little sensitivity towards halothane, indicating that the development of PSE meat is related to other environmental factors.

Development of a Multicommutated Flow System with Chemiluminometric Detection for Quantification of Gentamicin in Pharmaceuticals

A new flow procedure based on multicommutation with chemiluminometric detection was developed to quantify gentamicin sulphate in pharmaceutical formulations. This approach is based on gentamicin's ability to inhibit the chemiluminometric reaction between luminol and hypochlorite in alkaline medium, causing a decrease in the analytical signal. The inhibition of the analytical signal is proportional to the concentration of gentamicin sulphate, within a linear range of 1 to 4 mu g mL(-1) with a coefficient variation <3%. A sample throughput of 55 samples h(-1) was obtained. The developed method is sensitive, simple, with low reagent consumption, reproducible, and inexpensive, and when applied to the analysis of pharmaceutical formulations (eye drops and injections) it gave results with RSD between 1.10 and 4.40%.

Structure of an Odorant-Binding Protein from the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive ""Lid""

Background: The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission. Methodology: Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 angstrom resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date. Conclusion: The structure of AaegOBP1 (= AaegOBP39) shares the common fold of insect OBPs with six alpha-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this ""lid"" may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops. (C) 2010 Elsevier Inc. All rights reserved.

Development and validation of HPLC method for imiquimod determination in skin penetration studies

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mm, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by H-PLC using C(8) column and UV detection at 242 ran. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of uniquimod by in vitro studies. Copyright (C) 2008 John Wiley & Sons, Ltd.

Childhood Trauma and Children`s Emerging Psychotic Symptoms: A Genetically Sensitive Longitudinal Cohort Study

Objective: Using longitudinal and prospective measures of trauma during childhood, the authors assessed the risk of developing psychotic symptoms associated with maltreatment, bullying, and accidents in a nationally representative U. K. cohort of young twins. Method: Data were from the Environmental Risk Longitudinal Twin Study, which follows 2,232 twin children and their families. Mothers were interviewed during home visits when children were ages 5, 7, 10, and 12 on whether the children had experienced maltreatment by an adult, bullying by peers, or involvement in an accident. At age 12, children were asked about bullying experiences and psychotic symptoms. Children`s reports of psychotic symptoms were verified by clinicians. Results: Children who experienced maltreatment by an adult (relative risk=3.16, 95% CI=1.92-5.19) or bullying by peers (relative risk=2.47, 95% CI=1.74-3.52) were more likely to report psychotic symptoms at age 12 than were children who did not experience such traumatic events. The higher risk for psychotic symptoms was observed whether these events occurred early in life or later in childhood. The risk associated with childhood trauma remained significant in analyses controlling for children`s gender, socioeconomic deprivation, and IQ; for children`s early symptoms of internalizing or externalizing problems; and for children`s genetic liability to developing psychosis. In contrast, the risk associated with accidents was small (relative risk=1.47, 95% CI=1.02-2.13) and inconsistent across ages. Conclusions: Trauma characterized by intention to harm is associated with children`s reports of psychotic symptoms. Clinicians working with children who report early symptoms of psychosis should inquire about traumatic events such as maltreatment and bullying.

Renal redox-sensitive signaling, but not blood pressure, is attenuated by Nox1 knockout in angiotensin II-dependent chronic hypertension

We demonstrated previously that, in mice with chronic angiotensin II-dependent hypertension, gp91phoxcontaining NADPH oxidase is not involved in the development of high blood pressure, despite being important in redox signaling. Here we sought to determine whether a gp91phox homologue, Nox1, may be important in blood pressure elevation and activation of redox-sensitive pathways in a model in which the renin-angiotensin system is chronically upregulated. Nox1-deficient mice and transgenic mice expressing human renin (TTRhRen) were crossed, and 4 genotypes were generated: control, TTRhRen, Nox1-deficient, and TTRhRen Nox1-deficient. Blood pressure and oxidative stress (systemic and renal) were increased in TTRhRen mice (P < 0.05). This was associated with increased NADPH oxidase activation. Nox1 deficiency had no effect on the development of hypertension in TTRhRen mice. Phosphorylation of c-Src, mitogen-activated protein kinases, and focal adhesion kinase was significantly increased 2-to 3-fold in kidneys from TTRhRen mice. Activation of c-Src, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and focal adhesion kinase but not of extracellular signal regulated kinase 1/2 or extracellular signal regulated kinase 5, was reduced in TTRhRen/Nox1-deficient mice (P < 0.05). Expression of procollagen III was increased in TTRhRen and TTRhRen/Nox1-deficient mice versus control mice, whereas vascular cell adhesion molecule-1 was only increased in TTRhRen mice. Our findings demonstrate that, in Nox1-deficient TTRhRen mice, blood pressure is elevated despite reduced NADPH oxidase activation, decreased oxidative stress, and attenuated redox signaling. Our results suggest that Nox1-containing NADPH oxidase plays a key role in the modulation of systemic and renal oxidative stress and redox-dependent signaling but not in the elevation of blood pressure in a model of chronic angiotensin II-dependent hypertension.

Hydrogen Sulfide Prevents Ethanol-Induced Gastric Damage in Mice: Role of ATP-Sensitive Potassium Channels and Capsaicin-Sensitive Primary Afferent Neurons

The aim of this study was to evaluate the protective effect of hydrogen sulfide (H(2)S) on ethanol-induced gastric lesions in mice and the influence of ATP-sensitive potassium (K(ATP)) channels, capsaicin-sensitive sensory afferent neurons, and transient receptor potential vanilloid (TRPV) 1 receptors on such an effect. Saline and L-cysteine alone or with propargylglycine, sodium hydrogen sulfide (NaHS), or Lawesson`s reagent were administrated for testing purposes. For other experiments, mice were pretreated with glibenclamide, neurotoxic doses of capsaicin, or capsazepine. Afterward, mice received L-cysteine, NaHS, or Lawesson`s reagent. After 30 min, 50% ethanol was administrated by gavage. After 1 h, mice were sacrificed, and gastric damage was evaluated by macroscopic and microscopic analyses. L-Cysteine, NaHS, and Lawesson`s reagent treatment prevented ethanol-induced macroscopic and microscopic gastric damage in a dose-dependent manner. Administration of propargylglycine, an inhibitor of endogenous H(2)S synthesis, reversed gastric protection induced by L-cysteine. Glibenclamide reversed L-cysteine, NaHS, or Lawesson`s reagent gastroprotective effects against ethanol-induced macroscopic damage in a dose-dependent manner. Chemical ablation of sensory afferent neurons by capsaicin reversed gastroprotective effects of L-cysteine or H(2)S donors (NaHS or Lawesson`s reagent) in ethanol-induced macroscopic gastric damage. Likewise, in the presence of the TRPV1 antagonist capsazepine, the gastroprotective effects of L-cysteine, NaHS, or Lawesson`s reagent were also abolished. Our results suggest that H(2)S prevents ethanol-induced gastric damage. Although there are many mechanisms through which this effect can occur, our data support the hypothesis that the activation of K(ATP) channels and afferent neurons/TRPV1 receptors is of primary importance.

Development of an adenoviral vector with robust expression driven by p53

Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx beta containing a p53-responsive element, a minimal promoter and the first intron of the rabbit P-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG served as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities. (c) 2007 Elsevier Inc. All rights reserved.

Development of impedimetric and optical calcium biosensor by using modified gold electrode with porcine S100A12 protein

We describe the development of a label free method to analyze the interactions between Ca(2+) and the porcine S100A12 protein immobilized on polyvinyl butyral (PVB). The modified gold electrodes were characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and surface plasmon resonance (SPR) techniques. SEM analyses of PVB and PVB-S100A12 showed a heterogeneous distribution of PVB spherules on gold surface. EIS and CV measurements have shown that redox probe reactions on the modified gold electrodes were partially blocked due the adsorption of PVB-S100A12, and confirm the existence of a positive response of the immobilized S100Al2 to the presence of calcium ions. The biosensor exhibited a wide linear response to Ca(2+) concentrations ranging from 12.5 to 200 mM. The PVB-S100A12 seems to be bound to the gold electrode surface by physical adsorption: we observed an increase of 1184.32 m degrees in the SPR angle after the adsorption of the protein on the PVB surface (in an indication that 9.84 ng of S100A12 are adsorbed per mm(2) of the Au-PVB electrode), followed by a further increase of 581.66 m degrees after attachment of the Ca(2+) ions. In addition, no SPR response is obtained for non-specific ions. These studies might be useful as a platform for the design of new reusable and sensitive biosensing devices that could find use in the clinical applications. (C) 2010 Elsevier B.V. All rights reserved.

The Arabidopsis bZIP Gene AtbZIP63 Is a Sensitive Integrator of Transient Abscisic Acid and Glucose Signals

Glucose modulates plant metabolism, growth, and development. In Arabidopsis (Arabidopsis thaliana), Hexokinase1 (HXK1) is a glucose sensor that may trigger abscisic acid (ABA) synthesis and sensitivity to mediate glucose-induced inhibition of seedling development. Here, we show that the intensity of short-term responses to glucose can vary with ABA activity. We report that the transient (2 h/4 h) repression by 2% glucose of AtbZIP63, a gene encoding a basic-leucine zipper (bZIP) transcription factor partially involved in the Snf1-related kinase KIN10-induced responses to energy limitation, is independent of HXK1 and is not mediated by changes in ABA levels. However, high-concentration (6%) glucose-mediated repression appears to be modulated by ABA, since full repression of AtbZIP63 requires a functional ABA biosynthetic pathway. Furthermore, the combination of glucose and ABA was able to trigger a synergistic repression of AtbZIP63 and its homologue AtbZIP3, revealing a shared regulatory feature consisting of the modulation of glucose sensitivity by ABA. The synergistic regulation of AtbZIP63 was not reproduced by an AtbZIP63 promoter-5`-untranslated region:beta-glucuronidase fusion, thus suggesting possible posttranscriptional control. A transcriptional inhibition assay with cordycepin provided further evidence for the regulation of mRNA decay in response to glucose plus ABA. Overall, these results indicate that AtbZIP63 is an important node of the glucose-ABA interaction network. The mechanisms by which AtbZIP63 may participate in the fine-tuning of ABA-mediated abiotic stress responses according to sugar availability (i.e., energy status) are discussed.

Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K(+) channel activity and reactive oxygen species generation

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H(2)O(2) release using different substrates and ATP-sensitive K(+) transport activities are increased in mitochondria from animals on high fat diets. The increase in H(2)O(2) release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K(+) channels, indicating it was not related to an observed increase in K(+) transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K(+) transport in mitochondria can be modulated by diet.

CDK9 a potential target for drug development

The family of Cyclin-Dependent Kinases (CDKs) can be subdivided into two major functional groups based on their roles in cell cycle and/or transcriptional control. CDK9 is the catalytic subunit of positive transcription elongation factor b (P-TEFb). CDK9 is the kinase of the TAK complex (Tat-associated kinase complex), and binds to Tat protein of HIV, suggesting a possible role for CDK9 in AIDS progression. CDK9 complexed with its regulatory partner cyclin T1, serves as a cellular mediator of the transactivation function of the HIV Tat protein. P-TEFb is responsible for the phosphorylation of the carboxyl-terminal domain of RNA Pol II, resulting in stimulation of transcription. Furthermore, the complexes containing CDK9 induce the differentiation in distinct tissue. The CDK9/cyclin T1 complex is expressed at higher level in more differentiated primary neuroectodermal and neuroblastoma tumors, showing a correlation between the kinase expression and tumor differentiation grade. This may have clinical and therapeutical implications for these tumor types. Among the CDK inhibitors two have shown to be effective against CDK9: Roscovitine and Flavopiridol. These two inhibitors prevented the replication of human immunodeficiency virus (HIV) type 1 by blocking Tat transactivation of the HIV type 1 promoter. These compounds inhibit CDKs by binding to the catalytic domain in place of ATP, preventing transfer of a phosphate group to the substrate. More sensitive therapeutic agents of CDK9 can be designed, and structural studies can add information in the understanding of this kinase. The major features related to CDK9 inhibition will be reviewed in this article.