Generation and analysis of expressed sequence tags from the salt-tolerant mangrove species Avicennia marina (Forsk) Vierh

Salinization poses an increasingly serious problem in coastal and agricultural areas with negative effects on plant productivity and yield. Avicennia marina is a pantropical mangrove species that can survive in highly saline conditions. As a first step towards the characterization of genes that contribute to combating salinity stress, the construction of a cDNA library of A. marina genes is reported here. Random expressed sequence tag (EST) sequencing of 1,841 clones produced 1,602 quality reads. These clones were classified into functional categories, and blast comparisons revealed that 113 clones were homologous to genes earlier implicated in stress responses, of which the dehydrins are the most predominant in this category. Of the ESTs analyzed, 30% showed homology to previously uncharacterized genes in the public plant databases. Of these 30%, 52 clones were selected for reverse Northern analysis: 26 were shown to be up-regulated and five shown to be down-regulated. The results obtained by reverse Northern analysis were confirmed by Northern analysis for three clones.     

Protective effects of zinc on lipid peroxidation, antioxidant enzymes and hepatic histoarchitecture in chlorpyrifos-induced toxicity

The present study investigated the hepatoprotective role of zinc in attenuating the toxicity induced by chlorpyrifos in rat liver. Male Sprague-Dawley (SD) rats received either oral chlorpyrifos (13.5mg/kg body weight), zinc alone (227mg/l in drinking water) or combined chlorpyrifos plus zinc treatment for a total duration of 8 weeks. The effects of these treatments were studied on various parameters in rat liver, including lipid peroxidation, antioxidant enzymes, levels of metallothionein (MT) and hepatic histoarchitecture. Chlorpyrifos treatment resulted in a significant increase in hepatic lipid peroxidation and activities of superoxide dismutase (SOD), glutathione peroxidase (G-Px) and glutathione reductase (GR). On the contrary, chlorpyrifos intoxication caused a significant inhibition in the levels of reduced glutathione (GSH), catalase (CAT) and glutathione-S-transferase (GST) activities. However, zinc treatment to chlorpyrifos-intoxicated animals normalized the otherwise raised levels of lipid peroxidation to within normal limits. Moreover, zinc treatment to these animals resulted in an elevation in the levels of GSH, catalase and GST, as well as a significant decrease in the levels of SOD. Levels of MT were also found to be depressed in chlorpyrifos-treated animals, but tended to increase following co-administration of zinc. Additionally, chlorpyrifos-treated animals demonstrated increased vacuolization, necrosis and ballooning of the hepatocytes and dilatation of sinusoids as well as increase in the number of binucleated cells. However, zinc administration to chlorpyrifos-treated animals resulted in overall improvement in the hepatic histoarchitecture, emphasizing the protective potential of zinc. Hence, the present study suggests the protective potential of zinc in alleviating the hepatic toxicity induced by chlorpyrifos.     

Interaction of RKRK Tetrapeptide with Polynucleotides

Abstract

The interactions of RKRK tetrapeptide with polynucleotides using circular dichroism spectroscopy are described.     

Modeling and simulation of liquid–solid circulating fluidized bed ion exchange system for continuous protein recovery

Liquid–solid circulating fluidized bed (LSCFB) is an integrated two‐column (downcomer and riser) system which can accommodate two separate processes (adsorption and desorption) in the same unit with continuous circulation of the solid particles between the two columns. In this study, a mathematical model based on the assumption of homogeneous fluidization was developed considering hydrodynamics, adsorption‐desorption kinetics and liquid–solid mass transfer. The simulation results showed good agreement with the available experimental results for continuous protein recovery. A parametric sensitivity study was performed to better understand the influence of different operating parameters on the BSA adsorption and desorption capacity of the system. The model developed can easily be extended to other applications of LSCFB. Biotechnol. Bioeng. 2009; 104: 111–126 © 2009 Wiley Periodicals, Inc.     

Quantitative characterization of the influence of the nanoscale morphology of nanostructured surfaces on bacterial adhesion and biofilm formation

Bacterial infection of implants and prosthetic devices is one of the most common causes of implant failure. The nanostructured surface of biocompatible materials strongly influences the adhesion and proliferation of mammalian cells on solid substrates. The observation of this phenomenon has led to an increased effort to develop new strategies to prevent bacterial adhesion and biofilm formation, primarily through nanoengineering the topology of the materials used in implantable devices. While several studies have demonstrated the influence of nanoscale surface morphology on prokaryotic cell attachment, none have provided a quantitative understanding of this phenomenon. Using supersonic cluster beam deposition, we produced nanostructured titania thin films with controlled and reproducible nanoscale morphology respectively. We characterized the surface morphology; composition and wettability by means of atomic force microscopy, X-ray photoemission spectroscopy and contact angle measurements. We studied how protein adsorption is influenced by the physico-chemical surface parameters. Lastly, we characterized Escherichia coli and Staphylococcus aureus adhesion on nanostructured titania surfaces. Our results show that the increase in surface pore aspect ratio and volume, related to the increase of surface roughness, improves protein adsorption, which in turn downplays bacterial adhesion and biofilm formation. As roughness increases up to about 20 nm, bacterial adhesion and biofilm formation are enhanced; the further increase of roughness causes a significant decrease of bacterial adhesion and inhibits biofilm formation. We interpret the observed trend in bacterial adhesion as the combined effect of passivation and flattening effects induced by morphology-dependent protein adsorption. Our findings demonstrate that bacterial adhesion and biofilm formation on nanostructured titanium oxide surfaces are significantly influenced by nanoscale morphological features. The quantitative information, provided by this study about the relation between surface nanoscale morphology and bacterial adhesion points towards the rational design of implant surfaces that control or inhibit bacterial adhesion and biofilm formation.     

BRCAness profile of sporadic ovarian cancer predicts disease recurrence

Background

The consequences of defective homologous recombination (HR) are not understood in sporadic ovarian cancer, nor have the potential role of HR proteins other than BRCA1 and BRCA2 been clearly defined. However, it is clear that defects in HR and other DNA repair pathways are important to the effectiveness of current therapies. We hypothesize that a subset of sporadic ovarian carcinomas may harbor anomalies in HR pathways, and that a BRCAness profile (defects in HR or other DNA repair pathways) could influence response rate and survival after treatment with platinum drugs. Clinical availability of a BRCAness profile in patients and/or tumors should improve treatment outcomes.

Objective

To define the BRCAness profile of sporadic ovarian carcinoma and determine whether BRCA1, PARP, FANCD2, PTEN, H2AX, ATM, and P53 protein expression correlates with response to treatment, disease recurrence, and recurrence-free survival.

Materials and Methods

Protein microarray analysis of ovarian cancer tissue was used to determine protein expression levels for defined DNA repair proteins. Correlation with clinical and pathologic parameters in 186 patients with advanced stage III–IV and grade 3 ovarian cancer was analyzed using Chi square, Kaplan-Meier method, Cox proportional hazard model, and cumulative incidence function.

Results

High PARP, FANCD2 and BRCA1 expressions were significantly correlated with each other; however, elevated p53 expression was associated only with high PARP and FANCD2. Of all patients, 9% recurred within the first year. Among early recurring patients, 41% had high levels of PARP, FANCD2 and P53, compared to 19.5% of patients without early recurrence (p = 0.04). Women with high levels of PARP, FANCD2 and/or P53 had first year cumulative cancer incidence of 17% compared with 7% for the other groups (P = 0.03).

Conclusions

Patients with concomitantly high levels of PARP, FANCD2 and P53 protein expression are at increased risk of early ovarian cancer recurrence and platinum resistance.     

Automated liquid-liquid extraction based on 96-well plate format in conjunction with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the quantitation of methoxsalen in human plasma

A sensitive, specific and high throughput bioanalytical method using automated sample processing via 96-well plate liquid-liquid extraction and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of methoxsalen in human plasma. Plasma samples with ketoconazole as internal standard (IS) were prepared by employing 0.2mL human plasma in ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column using isocratic mobile phase, consisting of 10mM ammonium formate and acetonitrile (60:40, v/v), at a flow rate of 0.5mL/min. The linear dynamic range was established over the concentration range 1.1-213.1ng/mL for methoxsalen. The method was rugged and rapid with a total run time of 1.5min. It was successfully applied to a pivotal bioequivalence study in 12 healthy human subjects after oral administration of 10mg extended release methoxsalen formulation under fasting condition.     

A cross-sectional study of pain sensitivity,disease-activity assessment,mental health,and fibromyalgia status in rheumatoid arthritis

IntroductionPain remains the most important problem for people with rheumatoid arthritis (RA). Active inflammatory disease contributes to pain, but pain due to non-inflammatory mechanisms can confound the assessment of disease activity. We hypothesize that augmented pain processing, fibromyalgic features, poorer mental health, and patient-reported 28-joint disease activity score (DAS28) components are associated in RA.MethodsIn total, 50 people with stable, long-standing RA recruited from a rheumatology outpatient clinic were assessed for pain-pressure thresholds (PPTs) at three separate sites (knee, tibia, and sternum), DAS28, fibromyalgia, and mental health status. Multivariable analysis was performed to assess the association between PPT and DAS28 components, DAS28-P (the proportion of DAS28 derived from the patient-reported components of visual analogue score and tender joint count), or fibromyalgia status.ResultsMore-sensitive PPTs at sites over or distant from joints were each associated with greater reported pain, higher patient-reported DAS28 components, and poorer mental health. A high proportion of participants (48%) satisfied classification criteria for fibromyalgia, and fibromyalgia classification or characteristics were each associated with more sensitive PPTs, higher patient-reported DAS28 components, and poorer mental health.ConclusionsWidespread sensitivity to pressure-induced pain, a high prevalence of fibromyalgic features, higher patient-reported DAS28 components, and poorer mental health are all linked in established RA. The increased sensitivity at nonjoint sites (sternum and anterior tibia), as well as over joints, indicates that central mechanisms may contribute to pain sensitivity in RA. The contribution of patient-reported components to high DAS28 should inform decisions on disease-modifying or pain-management approaches in the treatment of RA when inflammation may be well controlled.     

Mechanism of carnitine acylcarnitine translocase-catalyzed import of acylcarnitines into mitochondria

Mitochondrial imports of acylcarnitine and carnitine have been measured by new methods based on the monitoring of deacylation of acylcarnitines and the acetylation of carnitine in the matrix, subsequent to their entry. These methods have shown higher import rates than those calculated from the uptake of radioactive carnitines into mitochondria as employed until presently. This new approach has permitted the import of long chain acylcarnitine to be followed unambiguously; the results have confirmed that the carnitine acylcarnitine translocase is indeed involved in this import which also proceeds by a mole to mole exchange-diffusion against internal carnitine. Depletion of matrix carnitine greatly decreased the substrate import rates based on their uptake assay but much less so when the deacylation and acylation techniques were employed to monitor imports. These results have revealed that there is a small pool of carnitine in the matrix which readily equilibrates with the medium carnitine through the translocase but which equilibrates with the larger matrix carnitine pool slowly. This finding has necessitated reinterpretation of several previous observations on the translocase that were based on the assumption of a single matrix carnitine pool and in which the translocase was assumed to constitute the rate-limiting step in the activity measurements.