Comparative effects of melatonin and vitamin E in restoring aortic relaxation in pancreatectomized rats

In a previous study we reported the efficacy of melatonin to restore the decreased relaxation response to acetylcholine (ACh) or to sodium nitroprusside (SNP) in aortic rings of rats turned hyperglycemic by subtotal pancreatectomy. The effect was amplified by pre-incubation in a high (44 mmol/l) glucose solution, a situation that resulted in oxidative stress. We hereby compare the effect of another antioxidant, vitamin E, with that of melatonin on ACh response in intact aortic rings or on SNP response in endothelium-denuded aortic rings obtained from pancreatectomized or sham-operated rats. Dose-response curves to ACh or SNP were performed in the presence or absence of melatonin or vitamin E (10-5 mol/1) in 10 or 44 mmol/1 glucose medium. Melatonin was more effective than vitamin E in restoring ACh- or SNP-induced relaxation of aortic rings in a high glucose medium. The differences between the two antioxidants may rely on the ability of melatonin to diffuse readily into intracellular compartments.     

Comparative oxidation studies of methionine residues reflect a structural effect on chemical kinetics in rhG-CSF

The effect of protein conformation on the rate of chemical degradation is poorly understood. To address the role of structure on chemical degradation kinetics, comparative oxidation studies of methionine residues in recombinant human granulocyte colony-stimulating factor (rhG-CSF) were performed. The kinetics of oxidation of methionine residues by hydrogen peroxide (H2O2) in rhG-CSF and corresponding chemically synthesized peptides thereof was measured at different temperatures. To assess structural effects, equilibrium denaturation experiments also were conducted on rhG-CSF, yielding the free energy of unfolding as a function of temperature. A comparison of the relative rates of oxidation of methionine residues in short peptides with those of corresponding methionine residues in rhG-CSF yields an understanding of how protein tertiary structure affects oxidation reactions. For the temperature range that was studied, 4-45 degrees C, the oxidation rate constants followed an Arrhenius equation quite well, suggesting the lack of temperature-induced local structural perturbations that affect chemical degradation rates. One of the four methionine residues, Met 122, exhibited an activation energy significantly different from that of the corresponding peptide. Extrapolation of kinetic data predicts non-Arrhenius behavior around the melting temperature. Three phenomenological models based on different mechanisms are discussed, and an application to shelf life prediction of pharmaceuticals is presented.     

Human polymorphonuclear leukocytes are sensitive in vitro to Helicobacter pylori vaca toxin

BACKGROUND: Interactions between bacterial components and polymorphonuclear leukocytes (PMNL) play a major pathogenic role in Helicobacter pylori-associated diseases. Activation of PMNL can be induced by contact with whole bacteria or by different H. pylori products released in the extracellular space either by active secretion or by bacterial autolysis. Among these products, H. pylori VacA is a secreted toxin inducing vacuolation and apoptosis of epithelial cells. METHODS AND RESULTS: We found that non-opsonic human PMNL were sensitive to the vacuolating effect of VacA+ broth culture filtrate (BCF) and of purified VacA toxin. PMNL incubated with VacA+ BCF showed Rab7-positive large intracytoplasmic vacuoles. PMNL preincubation with H. pylori BCF of different phenotypes dramatically potentialized the oxidative burst induced by zymosan, increased phagocytosis of opsonized fluorescent beads, and up-regulated CD11b cell surface expression, but independently of the BCF VacA phenotype. Moreover, by using purified VacA toxin we showed that vacuolation induced in PMNL did not modify the rate of spontaneous PMNL apoptosis measured by caspase 3 activity. CONCLUSIONS: Taken together, these data showed that human PMNL is a sensitive cell population to H. pylori VacA toxin. However, activation of PMNL (i.e., oxidative burst, phagocytosis, CD11b up-regulation) and PMNL apoptosis are not affected by VacA, raising question about the role of VacA toxin on PMNL in vivo.     

Experimental observations on maternal reproductive rate and offspring characteristics

Starting from a clone of Philodina roseola (Rotifera, Bdelloidea) six homogeneous animals have been reared. From them three different age cohorts have been studied in order to determine their life tables. Cohorts and parental life schedules are compared and discussed. A sort of correlation between offspring fecundity and life span and maternal rate of egg production could be established.     

WT1 CpG islands methylation in human lung cancer: A pilot study

Background

CpG island hypermethylation of gene promoters and regulatory regions is a well-known mechanism of epigenetic silencing of tumor suppressors and is directly linked to carcinogenesis. Wilm’s tumor gene (WT1) is a tumor suppressor protein involved in the regulation of human cell growth and differentiation and a modulator of oncogenic K Ras signaling in lung cancer. Changes in the pattern of methylation of the WT1 gene have not yet been studied in detail in human lung cancer. In this study we compared the methylation profile of WT1 gene in samples of neoplastic and non-neoplastic lung tissue taken from the same patients.

Methods

DNA was extracted from neoplastic and normal lung tissue obtained from 16 patients with non small cell lung cancer (NSCLC). The methylation status of 29 CpG islands in the 5′ region of WT1 was determined by pyrosequencing. Statistical analysis was carried out by T test and Mann Whitney test.

Results

The mean percentage of methylation, considering all CpG islands of WT1 in the neoplastic tissues of the 16 NSCLC patients, was 16.2 ± 3.4, whereas in the normal lung tissue from the same patients it was 5.6 ± 1.7 (p < 0.001). Adenocarcinomas presented higher methylation levels than squamous cell carcinomas (p < 0,001).

Conclusions

Methylation of WT1 gene is significantly increased in NSCLC. Both histotype and exposure to cigarette smoke heavily influence the pattern of CpG islands which undergo hypermethylation.     

Species-specific DNA probe and development of a quantitative PCR assay for the detection of Morganella morganii

Aims: To develop a SYBR Green quantitative PCR assay (qPCR) for the specific detection of Morganella morganii, a fish pathogen responsible for the Histamine Fish Poisoning. Methods and Results: A new primer set, amplifying a 179‐bp fragment of the 16S rRNA gene, was selected for specificity, and 14 M. morganii strains and 32 non‐Morganella strains were evaluated. The melting temperature of 84°C was consistently specific for the amplicon. Two standard curves were constructed: the minimum detection sensitivity was 0·563 pg of pure DNA, corresponding to DNA extracted from nine cells of M. morganii. The qPCR assay was evaluated in experiments with seeded fish samples, and the regression coefficient values were calculated. Conclusions: A highly specific and rapid assay was developed for the detection of M. morganii in tuna fish samples. Significance and Impact of the Study: This method represents the first study about the quantification of pathogenic M. morganii in fish products. This approach can be utilized to prevent the presence of this undesirable species in the food chain.     

Taurine-like GABA aminotransferase inhibitors prevent rabbit brain slices against oxygen–glucose deprivation-induced damage

The activation of the GABAergic system has been shown to protect brain tissues against the damage that occurs after cerebral ischaemia. On the other hand, the taurine analogues (±)Piperidine-3-sulphonic- (PSA), 2-aminoethane phosphonic- (AEP), 2-(N-acetylamino) cyclohexane sulfonic-acids (ATAHS) and 2-aminobenzene sulfonate-acids (ANSA) have been reported to block GABA metabolism by inhibiting rabbit brain GABA aminotransferase and to increase GABA content in rabbit brain slices. The present investigation explored the neuroprotection provided by GABA, Vigabatrin (VIGA) and taurine analogues in the course of oxygen–glucose deprivation and reperfusion induced damage of rabbit brain slices. Tissue damage was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, measured as the index of cell swelling. GABA (30–300?μM) and VIGA (30–300?μM) significantly antagonised LDH and glutamate release, as well as tissue water gain caused by oxygen–glucose deprivation and reperfusion. Lower (1–10?μM) or higher concentrations (up to 3,000?μM) were ineffective. ANSA, PSA and ATAHS significantly reduced glutamate and LDH release and tissue water gain in a range of concentrations between 30 and 300?μM. Lower (0–10?μM) or higher (up to 3,000?μM) concentrations were ineffective. Both mechanisms suggest hormetic (“U-shaped”) effects. These results indicate that the GABAergic system activation performed directly by GABA or indirectly through GABA aminotransferase inhibition is a promising approach for protecting the brain against ischemia and reperfusion-induced damage.     

Mixed acid-base disorders, hydroelectrolyte imbalance and lactate production in hypercapnic respiratory failure: the role of noninvasive ventilation

Background

Hypercapnic Chronic Obstructive Pulmonary Disease (COPD) exacerbation in patients with comorbidities and multidrug therapy is complicated by mixed acid-base, hydro-electrolyte and lactate disorders. Aim of this study was to determine the relationships of these disorders with the requirement for and duration of noninvasive ventilation (NIV) when treating hypercapnic respiratory failure.

Methods

Sixty-seven consecutive patients who were hospitalized for hypercapnic COPD exacerbation had their clinical condition, respiratory function, blood chemistry, arterial blood gases, blood lactate and volemic state assessed. Heart and respiratory rates, pH, PaO₂ and PaCO₂ and blood lactate were checked at the 1st, 2nd, 6th and 24th hours after starting NIV.

Results

Nine patients were transferred to the intensive care unit. NIV was performed in 11/17 (64.7%) mixed respiratory acidosis–metabolic alkalosis, 10/36 (27.8%) respiratory acidosis and 3/5 (60%) mixed respiratory-metabolic acidosis patients (p = 0.026), with durations of 45.1±9.8, 36.2±8.9 and 53.3±4.1 hours, respectively (p = 0.016). The duration of ventilation was associated with higher blood lactate (p<0.001), lower pH (p = 0.016), lower serum sodium (p = 0.014) and lower chloride (p = 0.038). Hyponatremia without hypervolemic hypochloremia occurred in 11 respiratory acidosis patients. Hypovolemic hyponatremia with hypochloremia and hypokalemia occurred in 10 mixed respiratory acidosis–metabolic alkalosis patients, and euvolemic hypochloremia occurred in the other 7 patients with this mixed acid-base disorder.

Conclusions

Mixed acid-base and lactate disorders during hypercapnic COPD exacerbations predict the need for and longer duration of NIV. The combination of mixed acid-base disorders and hydro-electrolyte disturbances should be further investigated.     

Hypertension, cardiovascular risk and polymorphisms in genes controlling the cytochrome P450 pathway of arachidonic acid: A sex-specific relation?

Hypertension is a multifactorial disease in which the interplay of genetic and environmental factors that maintain blood pressure stable throughout life is altered. Cytochrome P450 (CYP)-derived metabolites of arachidonic acid such as epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE), active on vascular tone, endothelial function and renal sodium reapportion, have been identified as candidate mediators in the development of hypertension in several animal models, with remarkable sex-specific effect. Several SNPs, some recognized as functional, in human genes implicated in EETs/20-HETE biosynthesis and metabolism, such as CYP2J2 and CYP4A11, have been tested for association with blood pressure, hypertension and its long-term cardiovascular consequences in different populations, with conflicting results. A sex-specific effect, related to CYP4F2 polymorphisms and expression, has been observed in association studies. This finding indicates that altered 20-HETE bioactivity underlay the excess of hypertension and associated vascular events observed in men with respect to women and is consistent with the results from experimental models. Further epidemiological and mechanistic studies are required to confirm the effect of lipid mediators on blood pressure in humans and define the mechanisms of a putative sex-specific effect.