Characterization of intracellular Ca(2+) stores in gallbladder smooth muscle

The existence of functionally distinct intracellular Ca(2+) stores has been proposed in some types of smooth muscle. In this study, we sought to examine Ca(2+) stores in the gallbladder by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura 2-loaded isolated myocytes, membrane potential in intact smooth muscle, and isometric contractions in whole mount preparations. Exposure of isolated myocytes to 10 nM CCK caused a transient elevation in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was inhibited by 2-aminoethoxydiphenylborane (2-APB). Application of caffeine induced a rapid spike-like elevation in [Ca(2+)](i) that was insensitive to 2-APB but was abolished by pretreatment with 10 muM ryanodine. These data support the idea that both inositol trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) are present in this tissue. When caffeine was applied in Ca(2+)-free solution, the [Ca(2+)](i) transients decreased as the interval between Ca(2+) removal and caffeine application was increased, indicating a possible leakage of Ca(2+) in these stores. The refilling of caffeine-sensitive stores involved sarcoendoplasmic reticulum Ca(2+)-ATPase activation, similar to IP(3)-sensitive stores. The moderate Ca(2+) elevation caused by CCK was associated with a gallbladder contraction, but caffeine or ryanodine failed to induce gallbladder contraction. Nevertheless, caffeine caused a concentration-dependent relaxation in gallbladder strips either under resting tone conditions or precontracted with 1 muM CCK. Taken together, these results suggest that, in gallbladder smooth muscle, multiple pharmacologically distinct Ca(2+) pools do not exist, but IP(3)R and RyR must be spatially separated because Ca(2+) release via these pathways leads to opposite responses.     

Molecular characterization of gilthead sea bream (Sparus aurata) lipoprotein lipase. Transcriptional regulation by season and nutritional condition in skeletal muscle and fat storage tissues

Lipoprotein lipase (LPL) of gilthead sea bream (Sparus aurata) was cloned and sequenced using a RT-PCR approach completed by 3' and 5'RACE assays. The nucleotide sequence covered 1669 bp with an open reading frame of 525 amino acids, including a putative signal peptide of 23 amino acids long. Sequence alignment and phylogenetic analysis revealed a high degree of conservation among most fish and higher vertebrates, retaining the consensus sequence the polypeptide "lid", the catalytic triad and eight cysteine residues at the N-terminal region. A tissue-specific regulation of LPL was also found on the basis of changes in season and nutritional condition as a result of different dietary protein sources. First, the expression of LPL in mesenteric adipose tissue was several times higher than in liver and skeletal muscle. Secondly, the spring up-regulation of LPL expression in the mesenteric adipose tissue was coincident with a pronounced increase of whole body fat content. Thirdly, the highest expression of LPL in the skeletal muscle was found in summer, which may serve to cover the increased energy demands for muscle growth and protein accretion. Further, in fish fed plant-protein-based diets, hepatic LPL expression was up-regulated whereas an opposite trend was found in the mesenteric adipose tissue, which may contribute to drive dietary lipids towards liver fat storage. Finally, it is of interest that changes in circulating triglyceride (TG) levels support the key role of LPL in the clearance of TG-rich lipoproteins. This study is the first report in fish of a co-regulated expression of LPL in oxidative and fat storage tissues under different physiological conditions.     

Epidermal growth factor: Characteristics of specific binding in membranes from liver,placenta, and other target tissues

Epidermal growth factor, a 6,400-dalton polypeptide from the mouse submaxillary gland, binds specifically to cells and membranes derived from a variety of human, rat, mouse, and bovine tissues. Liver, placenta, skin, cornea, and cultured chondrocytes, Hela cells, and Chang liver cells bind large amounts of epidermal growth factor, whereas fat cells, resting and lectin-stimulated human peripheral lymphocytes, mouse thymocytes, cultured rat hepatoma cells, and mammary cells from virgin and pregnant mice bind little or no epidermal growth factor. The binding site for epidermal growth factor is distinct from receptors for other anabolic peptides such as insulin, nerve growth factor, and growth hormone. The binding of epidermal growth factor is rapid and reversible. The rate constant of association is approximately 10⁶ mole^?1 sec^?1, the rate constant of dissociation is about 6 × 10^?4 sec^?1, and the apparent equilibrium dissociation constant is about 10^?9m. Trypsin at low concentrations (50–200 μg/ml) destroys the receptor site for epidermal growth factor. The binding of epidermal growth factor by membranes is not accompanied by appreciable degradation of the peptide present in the medium or of that bound to the membranes. Use can be made of the high affinity and specificity of membranes for epidermal growth factor to measure by a competitive binding assay as little as 200 pg of EGF per ml (3 × 10^?11m).     

Characterization of acyl-phosphatidylinositol from the opportunistic pathogen Corynebacterium amycolatum

The aim of the present study was to characterize a new lipid detected in the opportunistic pathogen Corynebacterium amycolatum. It was identified as acyl-phosphatidylinositol (acyl-PI), and revealed as a mixture of homologues compounds by electrospray ionization mass spectrometry, with pseudomolecular ions, (M-H)-, observed at 1099 (the major one) 1113, and 1127. Acyl-PI exclusively contained octadecenoyl on the inositol moiety (as 3-O-acyl), an unsaturated fatty acyl (mostly octadecenoyl) at sn-1 position of the glycerol and a saturated fatty acyl (mainly hexadecanoyl) at the sn-2 position. Acyl-PI constitutes a new natural substance and seems to be unique among the phospholipids of C. amycolatum. Other more complex molecules, previously undetected, and assigned in this work to several acyl forms of phosphatidylinositol trimannosides, lacked octadecenoyl in their polar heads. The present study reveals the existence of acyl-PI in C. amycolatum as rather unexpected finding and, additionally, gives evidence for the ability of this species to synthesize a great variety of inositol-containing phospholipids.     

Contaminant yeast detection in industrial ethanol fermentation must by rDNA-PCR

AIMS: The present work focuses on the possibility to use conserved primers that amplify yeast ITS1-5.8S-ITS2 ribosomal DNA locus (rDNA) to detect the presence of non-Saccharomyces cerevisiae yeast in fermentation must of bioethanol fermentation process. METHODS AND RESULTS: Total DNA was extracted from pure or mixed yeast cultures containing different cell concentrations and different contaminant/fermenting yeast concentrations and submitted to PCR. Upon improvement of detection limits and DNA extraction protocol, must samples of distillery were checked for the presence of contaminant yeast. Contaminant rDNA bands were detected only in industrial samples during contamination episodes, but not in noncontaminated must. CONCLUSIONS: The method described here could detect the presence of contaminant yeast from industrial must in eight hours after sampling. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved procedure may help to avoid severe contamination episodes at fermentation industries by decreasing the detection time from 5 days to 8 h and possible quantification of contaminant yeasts that can impose economical loss to the process.     

Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs)

The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.     

Shell Beds as paleoecological puzzles: A case study from the Upper Permian of the Paraná Basin, Brazil

Summary Microstratigraphic, sedimentological, and taphonomic features of the Ferraz Shell Bed, from the Upper Permian (Kazanian-Tatarian?) Corumbataí Formation of Rio Claro Region (the Paraná Basin, Brazil), indicate that the bed consists of four distinct microstratigraphic units. They include, from bottom to top, a lag concentration (Unit 1), a partly reworked storm deposit (Unit 2), a rapidly deposited sandstone unit with three thin horizons recording episodes of reworking (Unit 3), and a shell-rich horizon generated by reworking/winnowing that was subsequently buried by storm-induced obrution deposit (Unit 4). The bioclasts of the Ferraz Shell Bed represent exclusively bivalve mollusks.Pinzonellaillusa andTerraia aequilateralis are the dominant species. Taphonomic analysis indicates that mollusks are heavily time-averaged (except for some parts of Unit 3). Moreover, different species are time-averaged to a different degree (disharmonious time-averaging). The units differ statistically from one another in their taxonomic and ecological composition, in their taphonomic pattern, and in the size-frequency distributions of the two most common species. Other Permian shell beds of the Paraná Basin are simílar to the Ferraz Shell Bed in their faunal composition (they typically contain similar sets of 5 to 10 bivalve species) and in their taphonomic, sedimentologic, and microstratigraphic characteristics. However, rare shell beds that include 2–3 species only and are dominated by articulated shells preserved in life position also occur. Diversity levels in the Permian benthic associations of the Paraná Basin were very low, with the point diversity of 2–3 species and with the within-habitat and basin-wide (alpha and gamma) diversities of 10 species, at most. The Paraná Basin benthic communities may have thus been analogous to low-diversity bivalve-dominated associations of the present-day Baltic Sea. The ‘Ferraz-type’ shell beds of the Paraná Basin represent genetically complex and highly heterogeneous sources of paleontological data. They are cumulative records of spectra of benthic ecosystems time-averaged over long periods of time (10²–10⁴ years judging from actualistic research). Detailed biostratinomic reconstructions of shell beds can not only offer useful insights into their depositional histories, but may also allow paleoecologists to optimize their sampling designs, and consequently, refine paleoecological and paleoenvironmental interpretations.     

Mortality from Congenital Heart Disease in Mexico: A Problem on the Rise

Background and Objectives

Temporal trends in mortality from congenital heart disease (CHD) vary among regions. It is therefore necessary to study this problem in each country. In Mexico, congenital anomalies were responsible for 24% of infant mortality in 2013 and CHD represented 55% of total deaths from congenital anomalies among children under 1 year of age. The objectives of this study were to analyze the trends in infant mortality from CHD in Mexico (1998 to 2013), its specific causes, age at death and associated socio-demographic factors.

Methods

Population-based study which calculated the compounded annual growth rate of death rom CHD between 1998 and 2013. Specific causes, age at which death from CHD occurred and risk factors associated with mortality were analyzed for the year 2013.

Results

Infant mortality from CHD increased 24.8% from 1998 to 2013 (114.4 to 146.4/ 100,000 live births). A total of 3,593 CHD deaths occurred in 2013; the main causes were CHD with left-to-right shunt (n = 487; 19.8/100,000 live births) and cyanotic heart disease (n = 410; 16.7/100,000). A total of 1,049 (29.2%) deaths from CHD occurred during the first week of life. Risk factors associated with mortality from CHD were, in order of magnitude: non-institutional birth, rural area, birth in a public hospital and male sex.

Conclusions

Mortality from CHD has increased in Mexico. The main causes were CHD with left-to-right shunt, which are not necessarily fatal if treated promptly. Populations vulnerable to death from CHD were identified. Approximately one-third of the CHD occurred during the first week of life. It is important to promote early diagnosis, especially for non-institutional births.     

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated hepatocytes

Hepatocytes, isolated from rats fed a low-protein diet, were incubated with [32P]Pi and the phosphoproteins analysed. Immunoprecipitation using antibody against El of branched-chain 2-oxo acid dehydrogenase complex demonstrated phosphorylation of the alpha-subunit of El. Analysis of the tryptic phosphopeptides from the alpha-subunit indicated that two sites were phosphorylated. 4-methyl 2-oxopentanoate and DL-2-chloro 4-methylpentanoate decreased labelling of both sites. No major direct effects of several hormones on phosphorylation of branched-chain 2-oxo acid dehydrogenase was observed.     

Cloning and expression of two novel aldo-keto reductases from Digitalis purpurea leaves.

The aldo-keto reductase (AKR) superfamily comprises proteins that catalyse mainly the reduction of carbonyl groups or carbon-carbon double bonds of a wide variety of substrates including steroids. Such types of reactions have been proposed to occur in the biosynthetic pathway of the cardiac glycosides produced by Digitalis plants. Two cDNAs encoding leaf-specific AKR proteins (DpAR1 and DpAR2) were isolated from a D. purpurea cDNA library using the rat Delta4-3-ketosteroid 5beta-reductase clone. Both cDNAs encode 315 amino acid proteins showing 98.4% identity. DpAR proteins present high identities (68-80%) with four Arabidopsis clones and a 67% identity with the aldose/aldehyde reductase from Medicago sativa. A molecular phylogenetic tree suggests that these seven proteins belong to a new subfamily of the AKR superfamily. Southern analysis indicated that DpARs are encoded by a family of at most five genes. RNA-blot analyses demonstrated that the expression of DpAR genes is developmentally regulated and is restricted to leaves. The expression of DpAR genes has also been induced by wounding, elevated salt concentrations, drought stress and heat-shock treatment. The isolated cDNAs were expressed in Escherichia coli and the recombinant proteins purified. The expressed enzymes present reductase activity not only for various sugars but also for steroids, preferring NADH as a cofactor. These studies indicate the presence of plant AKR proteins with ketosteroid reductase activity. The function of the enzymes in cardenolide biosynthesis is discussed.