Melusin,a muscle-specific integrin beta1-interacting protein,is required to prevent cardiac failure in response to chronic pressure overload

Cardiac hypertrophy is an adaptive response to a variety of mechanical and hormonal stimuli, and represents an early event in the clinical course leading to heart failure. By gene inactivation, we demonstrate here a crucial role of melusin, a muscle-specific protein that interacts with the integrin beta1 cytoplasmic domain, in the hypertrophic response to mechanical overload. Melusin-null mice showed normal cardiac structure and function in physiological conditions, but when subjected to pressure overload--a condition that induces a hypertrophic response in wild-type controls--they developed an abnormal cardiac remodeling that evolved into dilated cardiomyopathy and contractile dysfunction. In contrast, the hypertrophic response was identical in wild-type and melusin-null mice after chronic administration of angiotensin II or phenylephrine at doses that do not increase blood pressure--that is, in the absence of cardiac biomechanical stress. Analysis of intracellular signaling events induced by pressure overload indicated that phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) was specifically blunted in melusin-null hearts. Thus, melusin prevents cardiac dilation during chronic pressure overload by specifically sensing mechanical stress.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozymes I and II and transmembrane, cancer-associated isozyme IX with lipophilic sulfonamides

A series of new compounds was obtained by reaction of aromatic/heterocyclic sulfonamides incorporating amino groups with N,N-diphenylcarbamoyl chloride and diphenylacetyl chloride. These sulfonamides were assayed for the inhibition of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes: the cytosolic CA I and CA II, and the transmembrane, cancer-associated isozyme CA IX. Good inhibitors against all these isoforms were detected, and the inhibition profile of the newly investigated isozyme IX was observed to be different from that of the cytosolic isozymes, I and II. This may lead to the development of novel anticancer therapies based on the selective inhibition of CA IX.     

The effect of exposure to high flux density static and pulsed magnetic fields on lymphocyte function

We investigated whether a combination of static electromagnetic field (EMF) at a flux density of 4.75 T together with pulsed EMF at a flux density of 0.7 mT generated by an NMR apparatus (NMRF), could promote movements of Ca(2+), cell proliferation, and the eventual production of proinflammatory cytokines in human lymphocytes as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 micro g/ml phytohaemagglutinin (PHA) immediately before the exposure period. Our results clearly demonstrate that NMRF exposure increases the [Ca(2+)](i), without any proliferative, or activating, or proinflammatory effect on both normal and PHA stimulated lymphocytes. Accordingly, the levels of interferon gamma, tumor necrosis factor alpha, interleukin-1beta, interleukin-2, and interleukin-6 remained unvaried after exposure. Exposure of Jurkat cells statistically decreased the [Ca(2+)](i) and the proliferation. This is consistent with the low levels of IL-2 measured in supernatants of these cells after exposure. On the whole our data suggest that static and pulsed NMRF exposure contribute synergistically in the increase of the [Ca(2+)](i) without any activating or proinflammatory effect either in normal or in PHA challenged lymphocytes. In Jurkat cells, by changing the properties of cell membranes, NMRF exposure can influence Ca(2+) transport processes and hence Ca(2+) homeostasis, causing a marked decrease of proliferation.     

Ca2+-independent protein kinase C signalling in mouse eggs during the early phases of fertilization

Protein kinase C (PKC), an enzyme playing a central role in signal transduction pathways, is activated in fertilized mouse eggs downstream of the fertilization Ca2+ signal, to regulate different aspects of egg activation. Given the presence of Ca2+-independent PKC isoforms within the egg, we investigated whether fertilization triggers PKC stimulation in mouse eggs by activating Ca2+-independent signalling pathways. An increase in PKC activity was detected as early as 10 min after the beginning of insemination, when about 90% of eggs had fused with sperm and the first Ca2+ rise was evident in most of the eggs. A similar level of activity was found 20 min later, when about 60% of eggs had resumed meiosis. When the Ca2+ increase was buffered by an intracellular Ca2+ chelating agent, PKC stimulation was not blocked but only slightly reduced. Confocal microscopy analysis revealed that the increase in PKC activity at fertilization coincided with the translocation of PKCdelta, a Ca2+-independent and diacylglycerol-dependent PKC isoform, to the meiotic spindle. When, in the absence of the Ca2+ signal, metaphase-anaphase transition was inhibited, PKCdelta moved to the meiotic spindle but still maintained a sustained cytoplasmic distribution. In summary, our results indicate that: 1) PKC activation is an early event of egg activation; 2) both Ca2+-dependent and Ca2+-independent pathways contribute to increased PKC activity at fertilization; 3) PKCdelta is one of the isoforms participating in this signalling process.     

Degradation of the immunogenic peptide gp100(280-288) by the monocyte-like U937 cell line

The possible degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) in the presence of the monocyte-like line U937, and the effect of degradation on the in vitro-measured immune recognition, were investigated by chromatographic techniques and immunological assays. Results indicate a rapid hydrolysis of the substrate in the presence of the model cells, which is consistent with the hypothesis that degradation of gp100(280-288) is caused by the activity of U937-expressed enzymes, specifically amino- and carboxypeptidases. On the other hand, these results do not support the involvement of other enzymes known to be expressed by U937 cells. From a functional point of view, these data indicate that the degradation of gp100(280-288) severely hampered recognition by specific CTL clones. The results obtained may provide a model for epitope degradation by the antigen-presenting cells found in defined anatomical compartments and may, at least in part, account for the low activity of peptide-based antineoplastic vaccines, as well as for the transience of the effects of subcutaneously administered peptides in general.     

Oxidase enzyme immobilisation through electropolymerised films to assemble biosensors for batch and flow injection analysis

Glucose oxidase, lactate oxidase, L-aminoacid oxidase and alcohol oxidase were immobilised on new films based on 2,6-dihydroxynaphthalene (2,6-DHN) copolymerised with 2-(4-aminophenyl)-ethylamine (AP-EA) onto the Pt electrodes. The electropolymerisation was performed by cyclic voltammetry. Different scan rates and scan potential ranges were investigated and selected according to the monomers used. These sensors were tested for hydrogen peroxide, ascorbic acid and acetaminophen by cyclic voltammetry and amperometry. The amperometric studies were carried out in batch as well as in a flow injection analysis (FIA) system. Analytical parameters such as reproducibility, interference rejection, response time, buffer, storage and operational time of the sensors have been studied. These films were also characterised by X-ray photoelectron spectroscopy (XPS). Different strategies for enzyme immobilisation were performed and discussed: enzyme entrapment in the film during the electropolymerisation and covalent attachment of the enzyme to the film via a carbodiimide (1-ethtl-3-(3-dimethylaminopropyl)carbodiimide, EDC) or glutaraldehyde. Different parameters were considered in order to optimise the immobilisation procedures. Results provide a guide to design high sensitive, stable and interference-free biosensors. In addition, studies were performed using these probes in an original FIA based on solenoidal valves. Sensor stability, life time and dynamic range were also optimised in these conditions.     

DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays

The nick translation and terminal transferase assays have been compared to test their relative efficiency in detecting DNA breakage in ejaculated human spermatozoa. The results have been correlated with the percentage of chromomycin A3 positive sperm, a fluorochrome that is indicative of the protamination state of sperm. Examination of the ejaculated sperm of 30 subjects revealed that the percentage of positivity to the nick translation and terminal transferase assays did not differ, even when using different fixatives. It is concluded that the inability of the two assays to distinguish the type of DNA damage, as is possible in somatic nuclei, is most probably linked to the unique nature of sperm chromatin. It is proposed that the presence of the damaged DNA may be the remnants of an imperfect spermiogenesis, probably related to an inadequate protamine deposition. This is supported by the strong correlation between the presence of DNA damage and underprotamination as evidenced by chromomycin A3. © Chapman & Hall     

Spatial patterns in phage-Rhizobium coevolutionary interactions across regions of common bean domestication

Bacteriophages play significant roles in the composition, diversity, and evolution of bacterial communities. Despite their importance, it remains unclear how phage diversity and phage-host interactions are spatially structured. Local adaptation may play a key role. Nitrogen-fixing symbiotic bacteria, known as rhizobia, have been shown to locally adapt to domesticated common bean at its Mesoamerican and Andean sites of origin. This may affect phage-rhizobium interactions. However, knowledge about the diversity and coevolution of phages with their respective Rhizobium populations is lacking. Here, through the study of four phage-Rhizobium communities in Mexico and Argentina, we show that both phage and host diversity is spatially structured. Cross-infection experiments demonstrated that phage infection rates were higher overall in sympatric rhizobia than in allopatric rhizobia except for one Argentinean community, indicating phage local adaptation and host maladaptation. Phage-host interactions were shaped by the genetic identity and geographic origin of both the phage and the host. The phages ranged from specialists to generalists, revealing a nested network of interactions. Our results suggest a key role of local adaptation to resident host bacterial communities in shaping the phage genetic and phenotypic composition, following a similar spatial pattern of diversity and coevolution to that in the host.Subject terms: Microbial ecology, Bacteriophages, Microbial ecology, Biogeography, Microbial communities     

Wild Sicilian Rosemary: Phytochemical and Morphological Screening and Antioxidant Activity Evaluation of Extracts and Essential Oils

To identify the best biotypes, an extensive survey of Sicilian wild rosemary was carried out by collecting 57 samples from various sites, followed by taxonomic characterization from an agronomic perspective. All the biotypes collected were classified as Rosmarinus officinalis L. A cluster analysis based on the morphological characteristics of the plants allowed the division of the biotypes into seven main groups, although the characteristics examined were found to be highly similar and not area‐dependent. Moreover, all samples were analyzed for their phytochemical content, applying an extraction protocol to obtain the nonvolatile components and hydrodistillation to collect the essential oils for the volatile components. The extracts were characterized by LC‐UV‐DAD/ESI‐MS, and the essential oils by GC‐FID and GC/MS analyses. In the nonvolatile fractions, 18 components were identified, namely, 13 flavones, two organic acids, and three diterpenes. In the volatile fractions, a total of 82 components were found, with as predominant components α‐pinene and camphene among the monoterpene hydrocarbons and 1,8‐cineole, camphor, borneol, and verbenone among the oxygenated monoterpenes. Cluster analyses were carried out on both phytochemical profiles, allowing the separation of the rosemary samples into different chemical groups. Finally, the total phenol content and the antioxidant activity of the essential oils and extracts were determined with the Folin–Ciocalteu (FC) colorimetric assay, the UV radiation‐induced peroxidation in liposomal membranes (UV‐IP test), and the scavenging activity of the superoxide radical (O$\rm{{_{2}^{{^\cdot} -}}}$ ). The present study confirmed that the essential oils and organic extracts of the Sicilian rosemary samples analyzed showed a considerable antioxidant/free radical‐scavenging activity.     

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.