Signalling by protein kinase C isoforms in the heart

Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target amino acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues.Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated -, -,and -PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive -, -, -, -, and -PKCs. The kinases that belong to both of these groups display two cystein-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include , , and -PKCs that lack both the C2 and one cystein-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart. evidence that multiple PKC isoforms exist was first provided by Kosaka et al. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates.This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells. (Mol Cell Biochem 157: 65–72, 1996)     

Biotransformation of 4‐fluoro‐N‐(1‐{2‐[(propan‐2‐yl)phenoxy]ethyl}‐8‐azabicyclo[3.2.1]octan‐3‐yl)‐benzenesulfonamide,a novel potent 5‐HT7 receptor antagonist with antidepressant‐like and anxiolytic properties: In vitro and in silico approach

The aim of the study was to investigate the metabolism of 4‐fluoro‐N‐(1‐{2‐[(propan‐2‐yl)phenoxy]ethyl}‐8‐azabicyclo[3.2.1]octan‐3‐yl)‐benzenesulfonamide (PZ‐1150), a novel 5‐HT₇ receptor antagonist with antidepressant‐like and anxiolytic properties, by the following three ways: in vitro with microsomes; in vitro employing Cunninghamella echinulata, and in silico using MetaSite. Biotransformation of PZ‐1150 with microsomes resulted in five metabolites, while transformation with C. echinulata afforded two metabolites. In both models, the predominant metabolite occurred due to hydroxylation of benzene ring. In silico data coincide with in vitro experiments, as three MetaSite metabolites matched compounds identified in microsomal samples. In human liver microsomes PZ‐1150 exhibited in vitro half‐life of 64 min, with microsomal intrinsic clearance of 54.1 μL/min/mg and intrinsic clearance of 48.7 mL/min/kg. Therefore, PZ‐1150 is predicted to be a high‐clearance agent. The study demonstrated the applicability of using microsomal model coupled with microbial model to elucidate the metabolic pathways of compounds and comparison with in silico metabolite predictions.     

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients

Background

Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation.

Methods

Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene.

Results

The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38–76 %, depending on the gene. The highest MI value was found for RASSF1A (52 %) and the lowest for NPRL2/G21 (5 %). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71 % tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = −0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75–92 % NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found.

Conclusions

The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn’t seem to be a critical determinate of its promoter hypermethylation.     

Identification and methicillin resistance of coagulase-negative staphylococci isolated from nasal cavity of healthy horses

The aim of this study was an analysis of the staphylococcal flora of the nasal cavity of 42 healthy horses from 4 farms, along with species identification of CoNS isolates and determination of resistance to 18 antimicrobial agents, particularly phenotypic and genotypic methicillin resistance. From the 81 swabs, 87 staphylococci were isolated. All isolates possessed the gap gene but the coa gene was not detected in any of these isolates. Using PCR-RFLP of the gap gene, 82.8% of CoNS were identified: S. equorum (14.9%), S. warneri (14.9%), S. sciuri (12.6%), S. vitulinus (12.6%), S. xylosus (11.5%), S. felis (5.7%), S. haemolyticus (3.4%), S. simulans (3.4%), S. capitis (1.1%), S. chromogenes (1.1%), and S. cohnii subsp. urealyticus (1.1%). To our knowledge, this was the first isolation of S. felis from a horse. The species identity of the remaining Staphylococcus spp. isolates (17.2%) could not be determined from the gap gene PCR-RFLP analysis and 16S rRNA gene sequencing data. Based on 16S-23S intergenic transcribed spacer PCR, 11 different ITS-PCR profiles were identified for the 87 analyzed isolates. Results of API Staph were consistent with molecular identification of 17 (19.5%) isolates. Resistance was detected to only 1 or 2 of the 18 antimicrobial agents tested in the 17.2% CoNS isolates, including 6.9% MRCoNS. The mecA gene was detected in each of the 5 (5.7%) phenotypically cefoxitin-resistant isolates and in 12 (13.8%) isolates susceptible to cefoxitin. In total, from 12 horses (28.6%), 17 (19.5%) MRCoNS were isolated. The highest percentage of MRCoNS was noted among S. sciuri isolates (100%).     

From larval bodies to adult body plans: patterning the development of the presumptive adult ectoderm in the sea urchin larva

Echinoderms are unique among bilaterians for their derived, nonbilateral adult body plan. Their radial symmetry emerges from the bilateral larval body plan by the establishment of a new axis, the adult oral–aboral axis, involving local mesoderm–ectoderm interactions. We examine the mechanisms underlying this transition in the direct-developing sea urchin Heliocidaris erythrogramma. Adult ectoderm arises from vestibular ectoderm in the left vegetal quadrant. Inductive signals from the left coelom are required for adult ectodermal development but not for initial vestibule formation. We surgically removed gastrula archenteron, making whole-ectoderm explants, left-, right-, and animal-half ectoderm explants, and recombinants of these explants with left coelom. Vestibule formation was analyzed morphologically and with radioactive in situ hybridization with HeET-1, an ectodermal marker. Whole ectodermal explants in the absence of coelom developed vestibules on the left side or ventrally but not on the right side, indicating that left–right polarity is ectoderm autonomous by the gastrula stage. However, right-half ectodermal explants robustly formed vestibules that went on to form adult structures when recombined with the left coelom, indicating that the right side retains vestibule-forming potential that is normally suppressed by signals from the left-side ectoderm. Animal-half explants formed vestibules only about half the time, demonstrating that animal–vegetal axis determination occurs earlier. However, when combined with the left coelom, animal-half ectoderm always formed a vestibule, indicating that the left coelom can induce vestibule formation. This suggests that although coelomic signals are not required for vestibule formation, they may play a role in coordinating the coelom–vestibule interaction that establishes the adult oral–aboral axis.     

Alterations in the expression of the Atp7a gene in the early postnatal development of the mosaic mutant mice (Atp7amo-ms) – An animal model for Menkes disease

Copper is a trace element that is essential for the normal growth and development of all living organisms. In mammals, the ATP7A Cu-transporting ATPase is a key protein that is required for the maintenance of copper homeostasis. In both humans and mice, the ATP7A protein is coded by the X-linked ATP7A/Atp7a gene. Disturbances in copper metabolism caused by mutations in the ATP7A/Atp7a gene lead to severe metabolic syndromes Menkes disease in humans and the lethal mottled phenotype in mice. Mosaic is one of numerous mottled mutations and may serve as a model for a severe Menkes disease variant. In Menkes patients, mutations in the ATP7A gene often result in a decreased level of the normal ATP7A protein. The aim of this study was to analyse the expression of the Atp7a gene in mosaic mutants in early postnatal development, a critical period for starting copper supplementation therapy in both Menkes patients and mutant mice. Using real-time quantitative RT-PCR, we analysed the expression of the Atp7a gene in the brain, kidney and liver of newborn (P0.5) and suckling (P14) mice. Our results indicate that in mosaic P0.5 mutants, the Atp7a mRNA level is decreased in all analysed organs in comparison with wild-type animals. In two week-old mutants, a significant decrease was observed only in the kidney. In contrast, their hepatic level of Atp7a tended to be higher than in wild-type mice. We speculate that disturbance in the expression of the Atp7a gene and, consequently, change in the copper concentration of the organs, may contribute to the early fatal outcome of mosaic males.     

Zinc and Iron Concentration and SOD Activity in Human Semen and Seminal Plasma

The aim of the present study was to measure zinc (Zn) and iron (Fe) concentration in human semen and superoxide dismutase (SOD) activity in seminal plasma and correlate the results with sperm quality. Semen samples were obtained from men (N = 168) undergoing routine infertility evaluation. The study design included two groups based on the ejaculate parameters. Group I (n = 39) consisted of males with normal ejaculate (normozoospermia), and group II (n = 129) consisted of males with pathological spermiogram. Seminal Zn and Fe were measured in 162 samples (group I, n = 38; group II, n = 124) and SOD activity in 149 samples (group I, n = 37; group II, n = 112). Correlations were found between SOD activity and Fe and Zn concentration, and between Fe and Zn concentration. SOD activity was negatively associated with volume of semen and positively associated with rapid progressive motility, nonprogressive motility, and concentration. Negative correlation was stated between Fe concentration and normal morphology. Mean SOD activity in seminal plasma of semen from men of group I was higher than in seminal plasma of semen from men of group II. Fe concentration was higher in teratozoospermic males than in males with normal morphology of spermatozoa in group II. Our results suggest that Fe may influence spermatozoa morphology.