Extragastric manifestations of Helicobacter pylori infection

The possible role of Helicobacter pylori as a trigger for some extragastric diseases has been largely investigated in the last year. There are, in fact, several studies concerning cardiovascular diseases, neurological disorders, diabetes mellitus, ear and eyes diseases, immunological and hematological disorders, liver and bile tract diseases, gynecological and respiratory tract pathologies. Among them, idiopathic sideropenic anemia and idiopathic thrombocytopenic purpura still remain the extragastric diseases showing the most convincing results. Concerning ischemic heart disease, there are new interesting data playing in favor of the association, even though there are still some open issues to be clarified. For the other diseases, more studies are needed to clarify the reality of the proposed association.     

Study of mRNA Expression by Real Time PCR of Cpkk1, Cpkk2 and Cpkk3, three MEKs of Cryphonectria parasitica,in Virus‐free and Virus‐infected Isogenic Isolates

Cpkk1 and Cpkk2 are two previously characterized Mitogen‐activated protein kinase kinases (MEK) from Cryphonectria parasitica. For the characterization of the third MEK, primers designed to a conserved region of the known fungal MEK sequences were used in a PCR reaction to amplify genomic DNA from C. parasitica. The sequence of the resulting amplicon was compared to known sequences in the database using a Blast search. Results of the sequence comparison indicated that the initial fragment obtained encoded for a new MEK from C. parasitica, that had highest homology to Pbs2 from Saccharomyces cerevisiae. By inverse PCR we obtained a genomic fragment spanning the entire coding sequence of this MEK, which was named Cpkk3. The cDNA of Cpkk3 was obtained by compiling the sequences of RT‐PCR products resulting from the amplification of purified mRNA. TaqMan^® Probes were designed to analyse the expression of Cpkk1, Cpkk2 and Cpkk3 mRNA through RT‐Real Time PCR. This protocol allowed the expression of Cpkk3 to be successfully compared to the expression of Cpkk1 and Cpkk2, two previously cloned C. parasitica MEKs. No variation in expression was associated with the presence of a virus after 2 days of growth in standard conditions whereas an increase in the expression level of all the three MEKs was shown after 4 days of growth.     

Rapid sexual maturity and short life span in the blue-legged frog and the rainbow frog from the arid Isalo Massif, southern-central Madagascar

Longevity and age at sexual maturity were estimated in two anurans from the arid Isalo Massif (southern-central Madagascar), the blue-legged frog (Mantella expectata) and the rainbow frog (Scaphiophryne gottlebei). Phalanges from 69 individuals of M. expectata and 38 individuals of S. gottlebei were analyzed, using the skeletochronological method, in samples collected during two periods: January-February and November-December 2004. The male gonads of both species were also analyzed in order to better correlate reproductive activity with phenology. The phalangeal diaphysis in adults of both species was composed of two concentric bone layers: an innermost endosteal bone, which was less developed or sometimes lacking in S. gottlebei, and an outermost and broader layer of periosteal bone. Lines of arrested growth (LAGs) were observed in both species, although their recognition was more problematic and their distinctiveness much less evident in S. gottlebei. The results presented here indicate that M. expectata and S. gottlebei have a short life span and attain sexual maturity within the first active season after metamorphosis. Maximum longevity was 3 years in M. expectata and 2 years in S. gottlebei. In S. gottlebei the adult body size is likely attained during the same season in which metamorphosis occurs, but then breeding occurs only after the first latency period. Thus, mature individuals have only one LAG, corresponding to 1 year. The low number of individuals with two LAGs suggests that most animals die before the second latency period.     

Recognition of Smac-mimetic compounds by the BIR domain of cIAP1

Inhibitor of apoptosis proteins (IAPs) are negative regulators of apoptosis. As IAPs are overexpressed in many tumors, where they confer chemoresistance, small molecules inactivating IAPs have been proposed as anticancer agents. Accordingly, a number of IAP-binding pro-apoptotic compounds that mimic the sequence corresponding to the N-terminal tetrapeptide of Smac/DIABLO, the natural endogenous IAPs inhibitor, have been developed. Here, we report the crystal structures of the BIR3 domain of cIAP1 in complex with Smac037, a Smac-mimetic known to bind potently to the XIAP-BIR3 domain and to induce degradation of cIAP1, and in complex with the novel Smac-mimetic compound Smac066. Thermal stability and fluorescence polarization assays show the stabilizing effect and the high affinity of both Smac037 and Smac066 for cIAP1- and cIAP2-BIR3 domains.     

Monitoring liver alterations during hepatic tumorigenesis by NMR profiling and pattern recognition

Human hepatocellular carcinoma (HCC) is the most recurrent malignancy of the liver and represents one of the main causes of cancer death worldwide. Furthermore, the liver is the most frequent site of metastatic colonization, and hepatic metastases are far more common than primary cancers in Western countries. A possible way of investigating liver diseases is to study the tissue metabolic profiles. High-resolution nuclear magnetic resonance (NMR) spectroscopy of hepatic tissue extracts was combined with pattern-recognition and visualization techniques to uncover metabolic differences among analyzed tissue types. Extracts were from primary HCC, chronic hepatitis-C-virus related cirrhotic tissues, hepatic metastases from colorectal carcinomas, and non-cirrhotic normal liver tissues adjacent to metastases as controls. We identified all metabolites present in the NMR spectra, and after statistical evaluation of all spectral classes, we were able to define the metabolic changes underlying the different liver conditions and diseases. In particular, the lactate and the glucose tissue signals were found to primarily discriminate the different histological samples. We followed the biochemical changes of human hepatic lesions through primary (HCC) and secondary (metastases from colorectal carcinoma) liver tumors, cirrhotic tissues, and non-cirrhotic histologically-confirmed normal liver tissues adjacent to metastases, achieving a metabolic differentiation of the various pathological states based upon the variation of the intracellular lactate/glucose ratio. It is suggested that such a signal pattern may act as a potential marker for assessing pathological hepatic lesions.     

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X<Subscript>7</Subscript>-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X₇-invalidated (KO) mice was tested. Low concentrations (1–100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca²⁺]_i) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X₇ receptors. One millimolar ATP provoked a sustained increase in the [Ca²⁺]_i only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K⁺]_i) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X₄ receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K⁺]_i triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X₄ receptors. Higher concentrations of ATP elicited similar responses after binding to P2X₇ receptors. The expression of the P2X₇ receptors was also coupled to a better response to P2Y receptors.     

Trace Elements and Metallothionein in Liver and Kidney of <Emphasis Type="Italic">Felis catus</Emphasis>

Trace metals such as Zn, Cu, and Fe are essential for life; differently, no biochemical function is known for Cd. Changes in dietary metal concentrations can cause deficiency or toxicity. Studies on trace elements in cat are lacking. This paper aimed to analyze Zn, Cu, Fe and Cd concentrations in liver and kidney of pathological domestic cat and to isolate metallothionein (MT) in these tissues. It was not possible to explore a possible correlation between metal concentrations and pathologies because the incidence for each of them was too low. Fe was the most abundant metal; in particular, the liver accumulates average Fe concentrations one order of magnitude higher than Zn and Cu, ranging from 66.75 and 1,444.23 μg/g. Significantly, higher levels of Fe were found in the liver of elder animals. Zn concentrations varied between 26.31 and 84.78 μg/g in the liver whereas in the kidney, ranged between 7.69 and 71.15 μg/g. Cu concentrations were between 2.37 and 112.91 μg/g in liver and between 2.12 and 9.85 μg/g in kidney. Cd was the least abundant metal with the exception of the kidney of the oldest cats where it reached a maximum of 13.71 μg/g. Gel-filtration metal distribution profiles from cytosolic extracts revealed the presence of Cd, Cu, Zn thioneins either in the liver or in the kidney. Because tissue samples were taken from pathological cats from different breed and age, care must be taken to use these data as a baseline profile of trace elements in healthy animals. Our results are indicative that for some specimens the feed levels of Fe and Cu could be higher than the optimal dietary intake and in few cats, there was also an exposure to Cd that was counteracted by MT biosynthesis.     

Proteomics and transcriptomics investigation on longissimus muscles in Large White and Casertana pig breeds

Consumer complaints against the blandness of modern lean meat and the frequent reference to the more strongly flavored meat that was available years ago have prompted reconsideration of high fat-depositing typical pig breeds. Casertana and Large White pig breeds are characterized by a different tendency toward fat accumulation as they exhibit opposite genetic and physiological traits with respect to the energy metabolism. These physiological differences were investigated in longissimus lumborum muscles through proteomics (2-DE, MS/MS) and microarray approaches. Data were analyzed for pathway and network analyses, as well as GO term enrichment of biological functions. As a result, Casertana showed a greater amount of proteins involved in glycolitic metabolism and mainly rely on fast-mobilizable energy sources. Large White overexpressed cell cycle and skeletal muscle growth related genes. Metabolic behavior and other implications are discussed.     

Involvement of a quorum-sensing-regulated lipase secreted by a clinical isolate of Burkholderia glumae in severe disease symptoms in rice

Burkholderia glumae is an emerging rice pathogen in several areas around the world. Closely related Burkholderia species are important opportunistic human pathogens for specific groups of patients, such as patients with cystic fibrosis and patients with chronic granulomatous disease. Here we report that the first clinical isolate of B. glumae, strain AU6208, has retained its capability to be very pathogenic to rice. As previously reported for rice isolate B. glumae BGR1 (and also for the clinical isolate AU6208), TofI or TofR acyl homoserine lactone (AHL) quorum sensing played a pivotal role in rice virulence. We report that AHL quorum sensing in B. glumae AU6208 regulates secreted LipA lipase and toxoflavin, the phytotoxin produced by B. glumae. B. glumae AU6208 lipA mutants were no longer pathogenic to rice, indicating that the lipase is an important virulence factor. It was also established that type strain B. glumae ATCC 33617 did not produce toxoflavin and lipase and was nonpathogenic to rice. It was determined that in strain ATCC 33617 the LuxR family quorum-sensing sensor/regulator TofR was inactive. Introducing the tofR gene of B. glumae AU6208 in strain ATCC 33617 restored its ability to produce toxoflavin and the LipA lipase. This study extends the role of AHL quorum sensing in rice pathogenicity through the regulation of a lipase which was demonstrated to be a virulence factor. It is the first report of a clinical B. glumae isolate retaining strong rice pathogenicity and finally determined that B. glumae can undergo phenotypic conversion through a spontaneous mutation in the tofR regulator.