B-type natriuretic peptide limits infarct size in rat isolated hearts via KATP channel opening

B-type natriuretic peptide (BNP) has been reported to be released from the myocardium during ischemia. We hypothesized that BNP mediates cardioprotection during ischemia-reperfusion and examined whether exogenous BNP limits myocardial infarction and the potential role of ATP-sensitive potassium (K(ATP)) channel opening. Langendorff-perfused rat hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. The control infarct-to-risk ratio was 44.8 +/- 4.4% (means +/- SE). BNP perfused 10 min before ischemia limited infarct size in a concentration-dependent manner, with maximal protection observed at 10(-8) M (infarct-to-risk ratio: 20.1 +/- 5.2%, P < 0.01 vs. control), associated with a 2.5-fold elevation of myocardial cGMP above the control value. To examine the role of K(ATP) channel opening, glibenclamide (10(-6) M), 5-hydroxydecanoate (5-HD; 10(-4) M), or HMR-1098 (10(-5) M) was coperfused with BNP (10(-8) M). Protection afforded by BNP was abolished by glibenclamide or 5-HD but not by HMR-1098, suggesting the involvement of putative mitochondrial but not sarcolemmal K(ATP) channel opening. We conclude that natriuretic peptide/cGMP/K(ATP) channel signaling may constitute an important injury-limiting mechanism in myocardium.     

Role of apoptosis and Fas/FasL system in the oogenesis of the spotted ray Torpedo marmorata

In the present paper we investigated the role played by apoptosis during oogenesis in the cartilaginous fish Torpedo marmorata. TEM, TUNEL and immunohistochemical techniques were employed to specifically reveal morphological and biochemical hallmarks of apoptosis in specimens from birth to sexual maturity. Data obtained demonstrate that apoptosis occurs in prefollicular oocyte selection, in maintaining the homeostasis of granulosa in healthy growing oocyte and in resorbing atretic follicles. In this respect, the involvement of apoptosis in Torpedo marmorata oogenesis closely parallels that found in mammals, thus confirming that strategies of germ cell selection among vertebrates have been evolutionarily preserved.     

Methylation profile of P. lividus sea urchin genes during development

Methylation pattern has been studied in two genes of sea urchin Paracentrotus lividus using sodium bisulfite method to understand the possible role of DNA methylation during invertebrate development. Three regions of the gene for the hatching enzyme have been analyzed and all of them resulted unmethylated in embryos at different stages of development. Four CpG rich regions have been studied in the gene for DNA methyltransferase: upstream, upstream-exon1, intron 1 and exon 20. The upstream-exon 1 region is always unmethylated, while intron 1 and exon 20 are heavy methylated. Only the upstream fragment changed its pattern of methylation during development. For none of the studied regions the reported data show a general direct correlation between gene expression and methylation process during development.     

Fine structure of leydig and sertoli cells in the testis of immature and mature spotted ray Torpedo marmorata

An ultrastructural investigation revealed the presence of true Leydig cells in the testis of sexually mature specimens of Torpedo marmorata. They showed the typical organization of steroid-hormone-producing cells, which, however, changed as spermatocysts approached maturity. In fact, they appeared as active cells among spermatocysts engaged in spermatogenesis, while in regions where spermiation occurred, they progressively regressed resuming the fibroblastic organization typically present in the testis of immature specimens. Such observations strongly suggest that these cells might be engaged in steroidogenesis and actively control spermatogenesis. Sertoli cells, too, appeared to play a role in spermatogenesis control, since, like Leydig cells, they showed the typical aspect of steroidogenic cells. In addition, the presence of gap junctions between Sertoli cells suggests that their activity might be coordinated. After sperm release, most Sertoli cells were modified and, finally, degenerated, but few of them changed into round cells (cytoplasts) or round cell remnants, which continued their steroidogenic activity within the spermatocyst and the genital duct lumen. From the present observations, it can be reasonably concluded that, in T. marmorata, spermatogenesis depends on both Leydig and Sertoli cells, and, as postulated by Callard (1991), in cartilaginous fish, the function of the Leydig cells as producers of steroids might be more recent and subsequent to that of Sertoli cells. In this regard, it is noteworthy that, in immature males, when Leydig cells showed a fibroblastic organization, Sertoli cells already displayed the typical organization of a steroidogenic cell.     

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22

A unique metabolite with a molecular mass of 119 Da (C(2)H(5)N(3)O(3)) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

Environmental constraints in the study of flexible segments of proteins

The structural problem posed by ill-defined segments in protein structures is similar to those encountered in the study of most peptide hormones, with terminal tracts resembling linear peptides and loops resembling cyclic peptides. The conformational preferences of short linear peptides in solution can be influenced by the use of solvent mixtures of viscosity higher than that of pure water but comparable to that of cytoplasm. In order to check whether it is possible to use these media in the structural study of proteins, we undertook an exploratory study on BPTI in a mixture of dimethylsulfoxide and water. The complete assignment of BPTI in an 80:20 (by volume) DMSO-d ₆/water cryomixture at two temperatures showed that all resonances parallel those in water, hinting at the persistence of the correct protein architecture, which is also confirmed by NOESY experiments. In addition to the NOEs present in the aqueous solution it was possible to detect numerous new cross peaks, in particular from residues belonging to the less-defined regions. The new cross peaks do not originate from spin diffusion and are consistent with the best NMR structure and with the X-ray structures of BPTI.     

The Changes of Lipid Metabolism in Advanced Renal Cell Carcinoma Patients Treated with Everolimus: A New Pharmacodynamic Marker?

Background

Everolimus is a mammalian target of rapamycin (mTOR) inhibitor approved for the treatment of metastatic renal cell carcinoma (mRCC). We aimed to assess the association between the baseline values and treatmentrelated modifications of total serum cholesterol (C), triglycerides (T), body mass index (BMI), fasting blood glucose level (FBG) and blood pressure (BP) levels and the outcome of patients treated with everolimus for mRCC.

Methods

177 patients were included in this retrospective analysis. Time to progression (TTP), clinical benefit (CB) and overall survival (OS) were evaluated.

Results

Basal BMI was significantly higher in patients who experienced a CB (p=0,0145). C,T and C+T raises were significantly associated with baseline BMI (p=0.0412, 0.0283 and 0.0001). Median TTP was significantly longer in patients with T raise compared to patients without T (10 vs 6, p=0.030), C (8 vs 5, p=0.042) and C+T raise (10.9 vs 5.0, p=0.003). At the multivariate analysis, only C+T increase was associated with improved TTP (p=0.005). T raise (21.0 vs 14.0, p=0.002) and C+T increase (21.0 vs 14.0, p=0.006) were correlated with improved OS but were not significant at multivariate analysis.

Conclusion

C+T raise is an early predictor for everolimus efficacy for patients with mRCC.     

Hypoxia induces up-regulation of progranulin in neuroblastoma cell lines

Progranulin (PGRN) is a widely expressed multifunctional protein, involved in regulation of cell growth and cell cycle progression with a possible involvement in neurodegeneration. We looked for PGRN regulation in three different human neuroblastoma cell lines, following exposure to two different stimuli commonly associated to neurodegeneration: hypoxia and oxidative stress. For gene and protein expression analysis we carried out a quantitative RT-PCR and western blotting analysis. We show that PGRN is strongly up-regulated by hypoxia, through the mitogen-actived protein kinase (MAPK)/extracellular signal-regulated kinase (MEK) signaling cascade. PGRN is not up-regulated by H(2)O(2)-induced oxidative stress. These results suggest that PGRN in the brain could exert a protective role against hypoxic stress, one of principal risk factors involved in frontotemporal dementia pathogenesis.     

Nutrient uptake and management under saline conditions in the xerohalophyte: Tecticornia indica (Willd.) subsp. indica

In the present investigation, we studied uptake and management of the major cations in the xerohalophyte, Tecticornia indica (Willd.) subsp. indica as subjected to salinity. Plants were grown under greenhouse conditions at various salinity levels (0, 100, 200 and 400 mM NaCl) over 110 days. At harvest, they were separated into shoots and roots then analyzed for water contents, dry weights (DW), and Na+, K+, Ca2+, and Mg2+ contents. Plants showed a growth optimum at 200 mM NaCl and much better tissue hydration under saline than non-saline conditions. At this salt concentration (200 mM NaCl), shoot Na+ content reached its highest value (7.9 mmol · g-?1 DW). In spite of such stressful conditions, salt-treated plants maintained adequate K+, Ca2+, and Mg2+ status even under severe saline conditions. This was mainly due to their aptitude to selectively acquire these essential cations and efficiently use them for biomass production.