Ultrasonic-assisted derivatization reaction of amino acids prior to their determination in urine by using single-drop microextraction in conjunction with gas chromatography

A derivatization-extraction method that avoids tedious preconcentration steps is established in order to determine amino acids accurately at nanogram levels. The method involves conversion of the analytes of concern to N(O,S)-ethoxycarbonyl amino acid ethyl esters and subsequent extraction by single-drop microextraction (SDME) followed by GC analysis. The reaction proceeds smoothly and rapidly under ultrasonication which removes the bubbles from the bulk solution. Precision is acceptable and 12 non-hydrolyzed amino acids can be determined in urine in this manner. As long as the extraction conditions are consistently applied, quantitative analysis can be performed accurately. The limits of detection were satisfactory in the range 0.010-0.025 microg/ml for GC-FID and 0.26-68 ng/ml for GC-MS(SIM) with 1 ml sample volume.     

Ion channels in cancer: future perspectives and clinical potential

Ion transport across the cell membrane mediated by channels and carriers participate in the regulation of tumour cell survival, death and motility. Moreover, the altered regulation of channels and carriers is part of neoplastic transformation. Experimental modification of channel and transporter activity impacts tumour cell survival, proliferation, malignant progression, invasive behaviour or therapy resistance of tumour cells. A wide variety of distinct Ca²⁺ permeable channels, K⁺ channels, Na⁺ channels and anion channels have been implicated in tumour growth and metastasis. Further experimental information is, however, needed to define the specific role of individual channel isoforms critically important for malignancy. Compelling experimental evidence supports the assumption that the pharmacological inhibition of ion channels or their regulators may be attractive targets to counteract tumour growth, prevent metastasis and overcome therapy resistance of tumour cells. This short review discusses the role of Ca²⁺ permeable channels, K⁺ channels, Na⁺ channels and anion channels in tumour growth and metastasis and the therapeutic potential of respective inhibitors.     

Effect of Plant Species on Persistence of Paecilomyces lilacinus Strain 251 in Soil and on Root Colonization by the Fungus

The effect of 12 plant species on the persistence of Paecilomyces lilacinus strain 251 in soil was investigated. After incorporating formulated conidia into non-sterile soil followed by transplanting different test plants, the population dynamic of the fungus was determined over 100 days. At termination of the experiment, the fungal population in the planted soil was compared to the density of P. lilacinus in the rhizosphere and the percent increase or decrease was calculated for each crop. In addition, the potential of P. lilacinus strain 251 to colonize roots endophytically was investigated. Comparison of the slopes describing the population dynamics of the fungus showed no significant differences between soil without plants and soil from the root zone of the majority of the test plants. Bean was the only plant species consistently exerting a negative effect on the persistence of P. lilacinus strain 251 in the soil. For the first time, P. lilacinus strain 251 was isolated in significant numbers from healthy root tissue of barley plants.     

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

Clostridium perfringens epsilon toxin (Etx) is a pore‐forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx‐H149A), previously reported to have reduced, but not abolished, toxicity. The three‐dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx‐H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx‐H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx‐H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx‐H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx‐H149A identified a glycan (β‐octyl‐glucoside) binding site in domain III of Etx‐H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.     

Vascular Factors and Multiple Measures of Early Brain Health: CARDIA Brain MRI Study

Objective

To identify early changes in brain structure and function that are associated with cardiovascular risk factors (CVRF).

Design

Cross-sectional brain Magnetic Resonance I (MRI) study.

Setting

Community based cohort in three U.S. sites.

Participants

A Caucasian and African-American sub-sample (n= 680; mean age 50.3 yrs) attending the 25 year follow-up exam of the Coronary Artery Risk Development in Young Adults Study.

Primary and Secondary Outcomes

3T brain MR images processed for quantitative estimates of: total brain (TBV) and abnormal white matter (AWM) volume; white matter fractional anisotropy (WM-FA); and gray matter cerebral blood flow (GM-CBF). Total intracranial volume is TBV plus cerebral spinal fluid (TICV). A Global Cognitive Function (GCF) score was derived from tests of speed, memory and executive function.

Results

Adjusting for TICV and demographic factors, current smoking was significantly associated with lower GM-CBF and TBV, and more AWM (all <0.05); SA with lower GM-CBF, WM-FA and TBV (p=0.01); increasing BMI with decreasing GM-CBF (p<0003); hypertension with lower GM-CBF, WM-FA, and TBV and higher AWM (all <0.05); and diabetes with lower TBV (p=0.007). The GCS was lower as TBV decreased, AWM increased, and WM-FA (all p<0.01).

Conclusion

In middle age adults, CVRF are associated with brain health, reflected in MRI measures of structure and perfusion, and cognitive functioning. These findings suggest markers of mid-life cardiovascular and brain health should be considered as indication for early intervention and future risk of late-life cerebrovascular disease and dementia.     

Bombesin and neurotensin exert antiproliferative effects on oval cells and augment the regenerative response of the cholestatic rat liver

The regenerative capacity of the cholestatic liver is significantly attenuated. Oval cells are hepatic stem cells involved in liver's regeneration following diverse types of injury. The present study investigated the effect of the neuropeptides bombesin (BBS) and neurotensin (NT) on oval cell proliferation as well as on hepatocyte and cholangiocyte proliferation and apoptosis in the cholestatic rat liver. Seventy male Wistar rats were randomly divided into five groups: controls, sham operated, bile duct ligated (BDL), BDL + BBS (30 μg/kg/d), BDL + NT (300 μg/kg/d). Ten days later, alpha-fetoprotein (AFP) mRNA (in situ hybridization), cytokeratin-19 and Ki67 antigen expression (immunohistochemistry) and apoptosis (TUNEL) were evaluated on liver tissue samples. Cells with morphologic features of oval cells that were cytokeratin-19(+) and AFP mRNA(+) were scored in morphometric analysis and their proliferation was recorded. In addition, the proliferation and apoptotic rates of hepatocytes and cholangiocytes were determined. Alanine aminotransferase (ALT) levels and hepatic oxidative stress (lipid peroxidation and glutathione redox state) were also estimated. The neuropeptides BBS and NT significantly reduced ALT levels and hepatic oxidative stress. Both agents exerted similar and cell type-specific effects on oval cells, hepatocytes and cholangiocytes: (a) oval cell proliferation and accumulation in the cholestatic liver was attenuated, (b) hepatocyte proliferation was increased along with a decreased rate of their apoptosis and (c) cholangiocyte proliferation was attenuated and their apoptosis was increased. These observations might be of potential value in patients with extrahepatic cholestasis.     

Does mixed-species flocking influence how birds respond to a gradient of land-use intensity?

Conservation biology is increasingly concerned with preserving interactions among species such as mutualisms in landscapes facing anthropogenic change. We investigated how one kind of mutualism, mixed-species bird flocks, influences the way in which birds respond to different habitat types of varying land-use intensity. We use data from a well-replicated, large-scale study in Sri Lanka and the Western Ghats of India, in which flocks were observed inside forest reserves, in ‘buffer zones'' of degraded forest or timber plantations, and in areas of intensive agriculture. We find flocks affected the responses of birds in three ways: (i) species with high propensity to flock were more sensitive to land use; (ii) different flock types, dominated by different flock leaders, varied in their sensitivity to land use and because following species have distinct preferences for leaders, this can have a cascading effect on followers'' habitat selection; and (iii) those forest-interior species that remain outside of forests were found more inside flocks than would be expected by chance, as they may use flocks more in suboptimal habitat. We conclude that designing policies to protect flocks and their leading species may be an effective way to conserve multiple bird species in mixed forest and agricultural landscapes.     

“Once upon a Time in the Mediterranean” Long Term Trends of Mediterranean Fisheries Resources Based on Fishers’ Traditional Ecological Knowledge

We investigate long-term changes in the Mediterranean marine resources driving the trawl fisheries by analysing fishers’ perceptions (Traditional Ecological Knowledge, TEK) throughout the Mediterranean Sea during the last 80 years. To this end, we conducted an extended set of interviews with experienced fishers that enabled us to classify species (or taxa) as ‘decreasing’ or ‘increasing’ both in terms of abundance, as well as average size in the catch. The aspect that most clearly emerged in all the investigated areas over time was the notable increase of fishing capacity indicators, such as engine power and fishing depth range. Atlantic mackerel, poor cod, scorpionfishes, striped seabream, and John Dory demonstrated a decreasing trend in the fishers’ perceived abundance, while Mediterranean parrotfish, common pandora, cuttlefish, blue and red shrimp, and mullets gave indications of an increasing temporal trend. Although, as a rule, trawler captains did not report any cataclysmic changes (e.g. extinctions), when they were invited to estimate total catches, a clear decreasing pattern emerged; this being a notable finding taking into account the steep escalation of fishing efficiency during the past century. The overall deteriorating status of stocks in most Mediterranean regions calls for responsible management and design of rebuilding plans. This should include historical information accounting for past exploitation patterns that could help defining a baseline of fish abundance prior to heavy industrial fisheries exploitation.