Biochemical and genetic studies on alkaline phosphatase ofCeratitis capitata

Two forms of alkaline phosphatase exist in the integument of the “white pupae” (wp) and dark pupae (dp) mutant strains ofCeratitis capitata, during transition from larvae to pupae. They were separated by DEAE-cellulose chromatography. Both isoenzymes have a molecular weight of approximately 180,000 and two pH optima, at 9.4 and at 11.0. The isoenzymes of the “dark pupae” mutant catalyze the hydrolysis of phosphotyrosine and β-glycerophosphate but not phosphoserine, phosphothreonine, ATP, and AMP. In contrast, the isoenzymes of the white pupae mutant hydrolyze all the substrates tested. The ALPase 1 of the dark pupae mutant was inhibited byL-tyrosine, butL-phenylalanine had no effect on either isoenzyme. The effects of divalent cations, EDTA, temperature, urea, and 2-mercaptoethanol were also investigated. Electrophoretic analysis did not reveal any variants of the larval and pupal isoenzymes, but ALPase A, an adult stage-specific isoenzyme, was found to be polymorphic. The electrophoretic variants were shown to be controlled by three codominant alleles located on the third chromosome ofCeratitis capitata. Since we found no hybrid enzyme, we conclude that ALPase A is monomeric.     

Protective effect of adenosine against a calcium paradox in the isolated frog heart.

The effect of adenosine on the calcium paradox in the isolated frog heart was studied. Addition of adenosine during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein and creatine kinase release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that adenosine protected frog myocardial cells by reducing the massive calcium influx during reperfusion possibly through an action on calcium channels. Adenosine exerted its action in a dose-dependent manner; a concentration of 10 microM adenosine provided maximum protection of myocardial cells against the calcium paradox damage. Higher concentrations of adenosine produced side effects on both electrical and mechanical activity. These results are discussed in terms of the possible mechanism involved in the protective effect of adenosine.     

Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples

Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.     

Seasonal variations of cellular stress response of the gilthead sea bream (Sparus aurata)

The present study aimed to investigate the seasonal cellular stress response in vital organs, like the heart, the liver, the whole blood and the skeletal (red and white) muscles of the Mediterranean fish Sparus aurata during a 1-year acclimatization period in the field, in two examined depths (0–2 m and 10–12 m). Processes studied included heat shock protein expression and protein kinase activation. Molecular responses were addressed through the expression of Hsp70 and Hsp90, the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK), the extracellular signal-regulated kinases (ERK-1/2) and c-Jun N-terminal kinases (JNK1/2/3). The induction of Hsp70 and Hsp90 and the phosphorylation of p38 MAPK, JNKs and ERKs in the examined five tissues of the gilthead sea bream indicated a cellular stress response under the prism of a seasonal pattern which was characterized by distinct tissue specificity. Specifically, Hsp induction and MAPK activation occurred before peak summer water temperatures, with no further increases in their levels despite increases in water temperatures. Moreover, although water temperature did not vary significantly with depth of immersion, significant effects of depth on cellular stress response were observed, probably caused by different light regime. The expression and the activation of these certain proteins can be used as tools to define the extreme thermal limits of the gilthead sea bream.     

Characterization of mapacalcine-sensitive Ca(2+) channels in rat kidney

Mapacalcine receptors have been found to be associated with a Ca(2+) permeability insensitive to all known calcium blockers. Recently, high densities of mapacalcine receptors have been detected in the choroid plexus of rat brain. To determine a possible role for these channels, we have investigated their presence on other structures which, like choroid plexus, are involved in the secretion of biological fluids. Our data demonstrate that there are specific mapacalcine receptors on kidney membranes and glomeruli preparations. The mapacalcine receptors were present in all structures of the kidney. However, autoradiographic data demonstrated that superficial part of the cortex was more labeled than the other part of the kidney. These data would suggest that mapacalcine receptors could play a role in calcium homeostasis.     

Use of green fluorescent protein in visualisation of pneumococcal invasion of broncho-epithelial cells in vivo

The pneumococcus is the principle cause of bacterial pneumonia and also a major cause of bacterial meningitis. The mechanisms and sites of pneumococcal adherence and invasion of the respiratory tract in vivo are not clear however. We have made pneumococci expressing green fluorescent protein (GFP) and used it to trace pneumococcal adherence and invasion in vivo. By using GFP pneumococci we have shown bacterial adherence and invasion of broncho-epithelial cells in vivo by 4 h post-infection, with increases in pneumococcal invasiveness by 24 h. Using confocal image analysis we have shown varying levels of pneumococcal penetration and internalisation into host cells, as well as translocation through epithelial layers. To our knowledge this is the first report of pneumococcal invasion and cellular translocation in vivo.     

Identification of alpha1-adrenergic receptors and their involvement in phosphoinositide hydrolysis in the frog heart

The aim of this study was to characterize alpha(1)-adrenergic receptors in frog heart and to examine their related signal transduction pathway. alpha(1)-Adrenergic binding sites were studied in purified heart membranes using the specific alpha(1)-adrenergic antagonist [(3)H]prazosin. Analysis of the binding data indicated one class of binding sites displaying a K(d) of 4.19 +/- 0.56 nM and a B(max) of 14.66 +/- 1.61 fmol/mg original wet weight. Adrenaline, noradrenaline, or phenylephrine, in the presence of propranolol, competed with [(3)H]prazosin binding with a similar potency and a K(i) value of about 10 microM. The kinetics of adrenaline binding was closely related to its biological effect. Adrenaline concentration dependently increased the production of inositol phosphates in the heart in the presence or absence of propranolol. Maximal stimulation was about 8.5-fold, and the half-maximum effective concentration was 30 and 21 microM in the absence and presence of propranolol, respectively. These data clearly show that alpha(1)-adrenergic receptors are coupled to the phosphoinositide hydrolysis in frog heart. To our knowledge, this is the first direct evidence supporting the presence of functional alpha(1)-adrenergic receptors in the frog heart.     

Non-genomic effects of thyroid hormone in adult cardiac myocytes: relevance to gene expression and cell growth

Besides the well-characterized genomic action of thyroid hormone (TH), mediated by thyroid hormone receptors (TRs), accumulating data support the so-called non-genomic action of TH, which is often related to activation of signalling pathways. In this study, we sought to determine whether TH activates intracellular signalling pathways in the adult cardiac myocytes and whether such activation modulates cell growth and the expression of target proteins important in cardiac function. We demonstrate that TH promotes a rapid increase in the phosphorylation of several kinases, ERK1/2, PKCδ, p38-MAPK and Akt. This activation is inhibited by triiodothyroacetic acid (triac), which is a TH analogue known to displace the hormone from membrane bound receptors, indicating that this TH effect is mediated through a cell membrane-initiated mechanism. Furthermore, using specific inhibitors of the TH-activated kinases, we show that the long-term effects of TH on the expression of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), α- and β-myosin heavy chain (MHC) and cell growth are reverted, implying that what is initiated as a non-genomic action of the hormone interfaces with genomic effects. These data provide further insights into the underlying mechanisms of TH action in the heart with potentially important implications in the management of cardiac pathology.