Voltage-dependent channels permeable to K+ and Na+ in the membrane ofAcer pseudoplatanus vacuoles

Summary The patch-clamp technique in whole-cell configuration was used to study the electrical properties of the tonoplast in isolated vacuoles fromAcer pseudoplatanus cultured cells. In symmetrical KCl or K₂ malate solutions, voltage- and time-dependent inward currents were elicited by hyperpolarizing the tonoplast (inside negative), while in the positive range of potential the conductance was very small. The specific conductance of the tonoplast at –100 mV, in 100mm symmetrical KCl was about 160 S/cm². The reversal potentials (E _rev) of the current, measured in symmetrical or asymmetrical ion concentrations (cation, anion or both) were very close to the values of the K⁺ equilibrium potential. Experiments performed in symmetrical or asymmetrical NaCl indicate that Na⁺ too can flow through the channels. NeitherE _rev nor amplitude and kinetics of the current changed by replacing NaCl with KCl in the external solution. These results indicate the presence of hyperpolarization-activated channels in tonoplasts, which are permeable to K⁺ as well as to Na⁺. Anions such as Cl^– or malate seem to contribute little to the channel current.     

Arsenite Oxidation in Ancylobacter dichloromethanicus As3-1b Strain: Detection of Genes Involved in Arsenite Oxidation and CO2 Fixation

The aim of this study was to characterize a facultative chemolithotrophic arsenite-oxidizing bacterium by evaluating the growth and the rate of arsenite oxidation and to investigate the genetic determinants for arsenic resistance and CO(2) fixation. The strain under study, Ancylobacter dichloromethanicus As3-1b, in a minimal medium containing 3 mM of arsenite as electron donor and 6 mM of CO(2)-bicarbonate as the C source, has a doubling time (t(d)) of 8.1 h. Growth and arsenite oxidation were significantly enhanced by the presence of 0.01 % yeast extract, decreasing the t(d) to 4.3 h. The strain carried arsenite oxidase (aioA) gene highly similar to those of previously reported arsenite-oxidizing Alpha-proteobacteria. The RuBisCO Type-I (cbbL) gene was amplified and sequenced too, underscoring the ability of As3-1b to carry out autotrophic As(III) oxidation. The results suggest that A. dichloromethanicus As3-1b can be a good candidate for the oxidation of arsenite in polluted waters or groundwaters.     

Evaluation of the Prognostic Value of Impaired Renal Function on Clinical Progression in a Large Cohort of HIV-Infected People Seen for Care in Italy

Whilst renal dysfunction, especially mild impairment (60<eGFR<90 ml/min), has been often described in HIV-infected population, its potential contribution to HIV evolution and risk of cerebro-cardiovascular disease (CCVD) has not been clarified. Data from HIV-1 infected patients enrolled in the Italian Cohort of Antiretroviral-Naïve (Icona) Foundation Study collected between January 2000 and February 2014 with at least two creatinine values available. eGFR (CKD-epi) and renal dysfunction defined using a priori cut-offs of 60 (severely impaired) and 90 ml/min/1.73m² (mildly impaired). Characteristics of patients were described after stratification in these groups and compared using chi-square test (categorical variables) or Kruskal Wallis test comparing median values. Follow-up accrued from baseline up to the date of the CCVD or AIDS related events or death or last available visit. Kaplan Meier curves were used to estimate the cumulative probability of occurrence of the events over time. Adjusted analysis was performed using a proportional hazards Cox regression model. We included 7,385 patients, observed for a median follow-up of 43 months (inter-quartile range [IQR]: 21-93 months). Over this time, 130 cerebro-cardiovascular events (including 11 deaths due to CCVD) and 311 AIDS-related events (including 45 deaths) were observed. The rate of CCVD events among patients with eGFR >90, 60-89, <60 ml/min, was 2.91 (95% CI 2.30-3.67), 4.63 (95% CI 3.51-6.11) and 11.9 (95% CI 6.19-22.85) per 1,000 PYFU respectively, with an unadjusted hazard ratio (HR) of 4.14 (95%CI 2.07-8.29) for patients with eGFR <60 ml/min and 1.58 (95%CI 1.10-2.27) for eGFR 60-89 compared to those with eGFR ≥90. Of note, these estimates are adjusted for traditional cardio-vascular risk factors (e.g. smoking, diabetes, hypertension, dyslipidemia). Incidence of AIDS-related events was 9.51 (95%CI 8.35-10.83), 6.04 (95%CI 4.74-7.71) and 25.0 (95%CI 15.96-39.22) per 1,000 PYFU, among patients with eGFR >90, 60-89, <60 ml/min, respectively, with an unadjusted HR of 2.49 (95%CI 1.56-3.97) for patients with eGFR <60 ml/min and 0.68 (95%CI 0.52-0.90) for eGFR 60-89. The risk of AIDS events was significantly lower in mild renal dysfunction group even after adjustment for HIV-related characteristics. Our data confirm that impaired renal function is an important risk marker for CCVD events in the HIV-population; importantly, even those with mild renal impairment (90<eGFR<60) seem to be at increased risk of cerebro-cardiovascular morbidity and mortality.     

Tricyclic Imipramine Modification of the Circadian Rhythms of Hypothalamic Serotonin, its Precursors and Acid Catabolite in Individually Housed Rats

Circadian rhythms of serotonin (5HT), its precursors tryptophan (TP) and 5-hydroxy-tryptophan (5HTP) and its acid catabolite 5-hydroxy-indoleacetic acid (5HIAA), were determined in the hypothalamus of control rats and rats which had been treated continuously with subcutaneous imipramine (10 mg/kg/day) for 2 weeks.

Rats were individually housed and entrained to LD12:12. Controls showed the 5HT and TP peaks in the light and dark periods respectively, as reported in the literature, but no inverted correlation (antiphase) between SHT and 5HIAA rhythms.

Imipramine significantly modified circadian rhythm characteristics: the 5HT acrophase was advanced, that of TP and 5HIAA was delayed. Imipramine also significantly increased hypothalamic SHT and TP concentrations.     

Glucokinase gene mutations: structural and genotype-phenotype analyses in MODY children from South Italy

Background

Maturity onset diabetes of the young type 2 (or GCK MODY) is a genetic form of diabetes mellitus provoked by mutations in the glucokinase gene (GCK).

Methodology/Principal Findings

We screened the GCK gene by direct sequencing in 30 patients from South Italy with suspected MODY. The mutation-induced structural alterations in the protein were analyzed by molecular modeling. The patients'' biochemical, clinical and anamnestic data were obtained. Mutations were detected in 16/30 patients (53%); 9 of the 12 mutations identified were novel (p.Glu70Asp, p.Phe123Leu, p.Asp132Asn, p.His137Asp, p.Gly162Asp, p.Thr168Ala, p.Arg392Ser, p.Glu290X, p.Gln106_Met107delinsLeu) and are in regions involved in structural rearrangements required for catalysis. The prevalence of mutation sites was higher in the small domain (7/12: ∼59%) than in the large (4/12: 33%) domain or in the connection (1/12: 8%) region of the protein. Mild diabetic phenotypes were detected in almost all patients [mean (SD) OGTT = 7.8 mMol/L (1.8)] and mean triglyceride levels were lower in mutated than in unmutated GCK patients (p = 0.04).

Conclusions

The prevalence of GCK MODY is high in southern Italy, and the GCK small domain is a hot spot for MODY mutations. Both the severity of the GCK mutation and the genetic background seem to play a relevant role in the GCK MODY phenotype. Indeed, a partial genotype-phenotype correlation was identified in related patients (3 pairs of siblings) but not in two unrelated children bearing the same mutation. Thus, the molecular approach allows the physician to confirm the diagnosis and to predict severity of the mutation.     

Circadian variation in stroke onset: identical temporal pattern in ischemic and hemorrhagic events

Stroke is the culmination of a heterogeneous group of cerebrovascular diseases that is manifested as ischemia or hemorrhage of one or more blood vessels of the brain. The occurrence of many acute cardiovascular events—such as myocardial infarction, sudden cardiac death, pulmonary embolism, critical limb ischemia, and aortic aneurysm rupture—exhibits prominent 24 h patterning, with a major morning peak and secondary early evening peak. The incidence of stroke exhibits the same 24 h pattern. Although ischemic and hemorrhagic strokes are different entities and are characterized by different pathophysiological mechanisms, they share an identical double-peak 24 h pattern. A constellation of endogenous circadian rhythms and exogenous cyclic factors are involved. The staging of the circadian rhythms in vascular tone, coagulative balance, and blood pressure plus temporal patterns in posture, physical activity, emotional stress, and medication effects play central and/or triggering roles. Features of the circadian rhythm of blood pressure, in terms of their chronic and acute effects on cerebral vessels, and of coagulation are especially important. Clinical medicine has been most concerned with the prevention of stroke in the morning, when population-based studies show it is of greatest risk during the 24 h; however, improved protection of at-risk patients against stroke in the early evening, the second most vulnerable time of cerebrovascular accidents, has received relatively little attention thus far.     

The GIT–PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells

Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT–PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G‐protein‐coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl‐Met‐Leu‐Phe) peptide, in a rat basophilic leukaemia RBL‐2H3 cell line stably expressing an HA (haemagglutinin)‐tagged receptor for the fMLP peptide. Results. Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1–PIX and GIT2–PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist‐induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist‐enhanced cell spreading was inhibited. Analysis of agonist‐stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans‐well migration. The internalization of the formyl‐peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody‐enhanced agonist‐induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. Conclusions. Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant‐induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G‐protein‐coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist‐induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins.