Determinants of anion-proton coupling in mammalian endosomal CLC proteins

Many proteins of the CLC gene family are Cl(-) channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl(-)/H(+) exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl(-) and H(+) transport. Surface expression was unchanged by these mutations. Uncoupled Cl(-) transport could be restored in the ClC-4(E281A) and ClC-5(E268A) proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H(+) donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl(-)/H(+) exchange independently from the transport activity of the neighboring subunit. NO(3)(-) or SCN(-) transport was partially uncoupled from H(+) countertransport but still depended on the proton glutamate. Inserting proton glutamates into CLC channels altered their gating but failed to convert them into Cl(-)/H(+) exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 picosiemens. Our results are consistent with the idea that Cl(-)/H(+) exchange of the endosomal ClC-4 and -5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5) and that the strong rectification of currents arises from the voltage-dependent proton transfer from Glu-268 to Glu-211.     

Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.     

A randomized, double-blind, placebo-controlled, clinical dose–response trial of an extract of Baptisia, Echinacea and Thuja for the treatment of patients with common cold

The aim of this study was to verify the efficacy and safety of an herbal medication containing an extract of a mixture of Baptisiae tinctoriae radix, Echinaceae pallidae/purpureae radix and Thujae occidentalis herba (SB-TOX) in the treatment of upper respiratory tract infections (URIs), and to test whether SB-TOX's clinical efficacy is dose dependent. A total of 91 adults (mean age 42.1±13.0 years) were randomised to receive 19.2 mg of SB-TOX (n=31), 9.6 mg SB-TOX (n=29) or placebo (n=31) three times daily for 3–12 days. Since a “running nose” is the main symptom of a common cold, the total number of facial tissues used throughout the clinical duration of their cold was the primary efficacy parameter. In the intention-to-treat analysis, this total number of tissues decreased with increasing extract dose. The slope across groups according to the Jonckheere test was significant (p=0.0259). In the high-dose group, the standardised effect size Δ/SD was 0.46 compared with placebo. Time to relevant improvement in cold symptoms (measured as the time until less than 30 tissues per day were used) was 1.1 days (95% CI 0.52; 1.67), 0.76 days (95% CI 0.28; 1.24) and 0.52 days (95% CI 0.22; 0.82) in the placebo, low-dose and high-dose groups, respectively (p_LogRank=0.0175). No adverse events were reported. This study demonstrates the efficacy and safety of SB-TOX in the treatment of URIs, and that its efficacy is dose dependent.     

The validation of cervical cytology. Sensitivity, specificity and predictive values

Statistics and methods for the validation of the results of cytologic screening for cervical cancer and its precursors were examined. Many of the methods commonly used, including the calculation of sensitivity and specificity on raw data, contain flaws that undermine their conclusions. Using a large computerized database of 748,871 cytologic screenings of 277,842 women over a ten-year period, the value of screening was examined. Only subsequent histologic examinations within one year were accepted to validate positive initial cytologic diagnoses; only two subsequent cytologic screenings within the next three years were accepted to validate negative initial cytologic diagnoses that had not been followed by a histologic examination. Cases not meeting these criteria were excluded from the initial analysis. From these data, the predictive value of a negative cytologic examination was determined to be 99.8%; the predictive value of a positive cytologic examination was 73.4% for an initial diagnosis of mild-to-moderate dysplasia, 90.6% for a diagnosis of severe dysplasia/carcinoma in situ, 94.5% for a diagnosis of carcinoma in situ or microinvasive carcinoma and 95.5% for an initial diagnosis of invasive carcinoma. Cases with an initial "questionable" cytologic diagnosis had a positive predictive value of only 64.0%. Extrapolation from the validated cases to the entire screened population showed an overall sensitivity of 80% and a specificity of 99.4% for cytologic screening for cervical cancer. The sensitivity was slightly lower for mild and moderate dysplasia (78.1%) and slightly higher for carcinoma in situ and severe dysplasia (81.4%) and invasive carcinoma (82.3%).     

Perception in attention deficit hyperactivity disorder

A large body of research demonstrated that individuals with attention deficit hyperactivity disorder (ADHD) suffer from various neuropsychological deficits. In contrast, less is known and only divergent evidence exists on perceptual functions of individuals with ADHD. This is problematic as neuropsychological and perceptual functions are closely interrelated and are often difficult to disentangle in behavioral assessments. This study presents the conduct and results of a systematic literature review on perceptual functions in children and adults with ADHD. This review considers studies using psychophysical methods (objective measurements) and self- and informant reports (subjective measurements). Results indicate that individuals with ADHD have altered perceptual functions in various domains as compared to typically developing individuals. Increased perceptual functions in individuals with ADHD were found with regard to olfactory detection thresholds, whereas reduced perceptual functions were evident for aspects of visual and speech perception. Moreover, individuals with ADHD were found to experience discomfort to sensory stimuli at a lower level than typically developing individuals. Alterations of perceptual functions in individuals with ADHD were shown to be moderated by various factors, such as pharmacological treatment, cognitive functions, and symptom severity. We conclude by giving implications for daily life functioning and clinical practice.     

Chemical speciation of some heavy metals and human health risk assessment in soil around two municipal dumpsites in Sagamu,Ogun state,Nigeria

Environmental and health risk posed by heavy metals from municipal landfill cannot be over emphasized. However, the toxicity and fate of metal in the soil is dependent on its chemical form and therefore quantification of the different forms of metal is more meaningful than the estimation of its total concentration. This study investigated the chemical form and potential hazards of heavy metal pollution at two municipal landfills in Sagamu, Ogun state, Nigeria. Soil samples were collected around the landfills and chemical form of Cu, Zn, Cd, Pb, and Fe were studied, using the Tessier Five-step sequential chemical extraction procedure. The results showed that Cu and Fe were speciated into residual fractions with averages of 23.9 and 31.3% respectively, while Cd and Zn were associated with Carbonate fractions with respective averages of 20.3 and 20.6%. The order of mobility and bioavailability of these metals are: Cd > Pb > Cu > Zn > Fe. A comparison of the result of total extractible metals with standard set by USEPA reveals that Cd and Cu level in the dumpsite soils are far above the critical permissible limit of 3.0 and 250 mg kg^?1, respectively which potent a health risk. Assessment of soil pollution level using geoaccumulation index (I_geo) revealed that the landfill was extremely polluted by Cd (I_geo > 5). Pearson correlation and principal component analysis showed that there were no significant correlations (p < 0.05) among all the metals, suggesting that they are all from different anthropogenic sources. The cancer risk ranged from 1.36E?01 to 2.18E?04 and 5.82E?01 to 9.35E?04 for Children and Adult respectively. The level of cancer risk falls above the threshold values (10^?4–10^?6) which US Environmental Protection Agency considered as unacceptable risk. Based on the above findings, it was suggested that environmental management policy should be implemented to decrease the environmental risks.     

Expression of the glyceraldehyde-3-phosphate dehydrogenase gene from the extremely thermophilic archaebacterium Methanothermus fervidus in E. coli. Enzyme purification, crystallization, and preliminary crystal data

The gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extremely thermophilic archaebacterium Methanothermus fervidus (growth optimum 82 degrees C) was cloned in vector pJF118EH and expressed in E. coli cells. As shown by molecular mass determination, protein sequencing, heat stability, and substrate saturation kinetics, the enzyme synthesized in E. coli is identical to the original enzyme from M. fervidus. The high thermostability of the E. coli-produced M. fervidus GAPDH allows rapid purification to homogeneity. From this enzyme protein crystals were grown which proved to be suitable for X-ray analysis. The crystals are of tetragonal space group P4(1)22 and contain a dimer per asymmetric unit.