Absence of equilibrative nucleoside transporter 1 in ENT1 knockout mice leads to altered nucleoside levels following hypoxic challenge.

Aims: Equilibrative nucleoside transporters (ENT) modulate the flux of adenosine. The ENT1-null (KO) mouse heart is endogenously cardioprotected but the cellular basis of this phenotype is unknown. Therefore, we investigated the cellular mechanisms underlying ENT1-mediated cardioprotection.

Regulation of MT1-MMP and MMP-2 by leptin in cardiac fibroblasts involves Rho/ROCK-dependent actin cytoskeletal reorganization and leads to enhanced cell migration.

Altered leptin action has been implicated in the pathophysiology of heart failure in obesity, a hallmark of which is extracellular matrix remodeling. Here, we characterize the direct influence of leptin on matrix metalloproteinase (MMP) activity in primary adult rat cardiac fibroblasts and focus on elucidating the molecular mechanisms responsible. Leptin increased expression and cell surface localization of membrane type 1 (MT1)-MMP, measured by cell surface biotinylation assay and antibody-based colorimetric detection of an exofacial epitope in intact cells.

Serotonin transporter null male mouse pups have lower ventilation in air and 5% CO2 at postnatal ages P15 and P25.

In Wild Type (WT) and serotonin transporter (5HTT) null mice, we studied oxygen consumption, ventilation and heart rate in air and 5% CO(2) at postnatal (P) days P5, P15, and P25 using either a head-out (younger mice) or whole body plethysmograph (older mice). Body weight and temperature did not differ between the groups. Oxygen consumption differed significantly only in females at P15 when it was reduced in 5HTT nulls (P<0.

Adiponectin increases LPL activity via RhoA/ROCK-mediated actin remodelling in adult rat cardiomyocytes.

Cardiomyocyte substrate utilization is important in maintaining optimal cardiac function. Adiponectin has been shown to confer cardioprotective effects in part via regulating glucose and fatty acid uptake and oxidation in cardiomyocytes. Here we investigated mechanisms whereby adiponectin mediates a particular metabolic effect by focusing on lipoprotein lipase (LPL), an enzyme that increases free fatty acid availability to the heart by breakdown of chylomicrons and very-low-density lipoproteins in circulation.

An APPL1-AMPK signaling axis mediates beneficial metabolic effects of adiponectin in the heart.

Adiponectin promotes cardioprotection by various mechanisms, and this study used primary cardiomyocytes and the isolated working perfused heart to investigate cardiometabolic effects. We show in adult cardiomyocytes that adiponectin increased CD36 translocation and fatty acid uptake as well as insulin-stimulated glucose transport and Akt phosphorylation. Coimmunoprecipitation showed that adiponectin enhanced association of AdipoR1 with APPL1, subsequent binding of APPL1 with AMPKα2, which led to phosphorylation and inhibition of ACC and increased fatty acid oxidation.

Leptin regulates MMP-2, TIMP-1 and collagen synthesis via p38 MAPK in HL-1 murine cardiomyocytes.

A clear association between obesity and heart failure exists and a significant role for leptin, the product of the obese gene, has been suggested. One aspect of myocardial remodeling which characterizes heart failure is a disruption in the balance of extracellular matrix synthesis and degradation. Here we investigated the effects of leptin on matrix metalloproteinase (MMP) activity, tissue inhibitor of metalloproteinase (TIMP) expression, as well as collagen synthesis in HL-1 cardiac muscle cells.

Leptin attenuates hypoxia/reoxygenation-induced activation of the intrinsic pathway of apoptosis in rat H9c2 cells.

Cardiomyocyte apoptosis is a component of cardiac remodeling that can contribute to heart failure in obesity. A role for leptin in mediating this process has been suggested and the objective of this work was to investigate the effect of leptin on apoptosis and associated mechanisms in H9c2 cells which were subjected to hypoxia/reoxygenation (HR) to mimic myocardial ischemia/reperfusion. Qualitative immunofluorescent and quantitative laser scanning cytometry approaches demonstrated that exposure of cells to HR increased DNA fragmentation (TUNEL staining) which was attenuated by leptin (6 nM, 1 h) pretreatment.

Implications of myocardial matrix remodeling by adipokines in obesity-related heart failure.

Owing to the increased incidence of obesity and its association with heart failure, there is now great interest in elucidating the underlying molecular mechanisms linking these pathologies. Since the discovery of adipose-derived hormones and cytokines, their important regulatory role in myocardial function has emerged. The events that these adipokines can regulate include alterations in myocardial metabolism, cardiomyocyte hypertrophy, cell death, and structure and composition of the extracellular matrix.

Increased expression and cell surface localization of MT1-MMP plays a role in stimulation of MMP-2 activity by leptin in neonatal rat cardiac myofibroblasts.

Myocardial matrix remodeling is a well-recognized disease modifier in the pathogenesis of heart failure, although the precise underlying molecular mechanisms remain to be elucidated. Here we investigated the effects of leptin, circulating levels of which are typically increased in obese individuals, on MMP and collagen expression and MMP activity in isolated cardiac myofibroblasts. Neonatal rat myofibroblasts were treated with 6 nM recombinant leptin and the collected supernatant analyzed for MMP-2 activity via gelatin zymography.

Identification of Arcobacter species using phospholipid and total fatty acid profiles.

High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) were used to analyze the phospholipids and fatty acids of four Arcobacter species (becoming routinely isolated from a wide variety of food sources, especially of animal origin) to provide information for the identification within these species. Phospholipid differences were observed in the HPLC profiles. GC-MS analysis provided a complete fatty acid composition for each arcobacter that after pattern recognition analysis allows taxonomic classification of each species.

Evaluation of a hydrogen laser vacuum ultraviolet source for photoionization mass spectrometry of pharmaceuticals.

A photoionization hydrogen laser time-of-flight mass spectrometer system (H2-TOFMS) has been evaluated for the rapid analysis of drugs of abuse and pharmaceutical agents extracted from prescription tablets and spiked urine samples. The spectra obtained using the H2-TOFMS showed primarily intact molecular ions (M+*) after introduction by a heated probe and irradiation with vacuum ultraviolet (VUV) photons from the laser. Samples analyzed by this technique required only a simple solid-phase extraction step; no chromatographic separation or derivatization was necessary to identify the drugs of abuse or pharmaceutical agents.

Mass spectrometry of 1,3- and 1,5-dicaffeoylquinic acids.

Samples of 1,3- (1) and 1,5-dicaffeoylquinic acid (2) and their hexaacetate derivatives were examined using positive and negative electrospray ionization mass spectrometry and tandem mass spectrometry. Differences in the various spectra allow the discrimination of each of the isomers. Specific losses in the spectra of 2 also permit the identification of the site of substitution of one of the caffeic acid moieties as being at the 5-position.

Cognitive consequences and central nervous system injury following treatment for childhood leukemia.

Objectives: To determine the relationship between membrane damage and intellectual and academic abilities in children with acute lymphoblastic leukemia (ALL) and pilot test a math intervention for children with ALL who were affected.

Data Sources: Research studies and review articles.

Conclusions: Despite the prophylactic central nervous system (CNS) treatment for long-term disease-free survival, many children with ALL subsequently experience declines in intellectual and academic skills.

Increased eumelanin expression and tanning is induced by a superpotent melanotropin [Nle4-D-Phe7]-alpha-MSH in humans.

Seven normal volunteers (six males and one female) with tanning skin types III or IV (Fitzpatrick scale) were given 10 daily subcutaneous injections of a superpotent synthetic analog of alpha-melanocyte stimulating hormone (alpha-MSH) over two weeks. This agent, [Nle4-D-Phe7]alpha-MSH, also called Melanotan-I (MT-I), was administered at a dose of 0.16 mg/kg/day (Monday-Friday), over a two week period.

Tandem mass spectrometry studies of green tea catechins. Identification of three minor components in the polyphenolic extract of green tea.

Liquid chromatography/electrospray ionization mass and tandem mass spectrometry (MS/MS) techniques were used to identify two minor components and one new compound in the polyphenolic extract of green tea (Camellia sinensis). Identification and structure assignments were based on previously reported sub-structural features in the MS/MS product, precursor and neutral loss scans of reference samples. The structures of two minor components, related to the known green tea components epicatechin gallate (ECG, 5) and epigallocatechin gallate (EGCG, 6), are formed by methylation at the 3"-O-position of the gallic acid moiety.

Design and synthesis of conformationally constrained glucagon analogues.

Glucagon was systematically modified by forming lactam bridges within the central region of the molecule to give conformationally constrained cyclic analogues. Six cyclic glucagon analogues have been designed and synthesized. They are c[Asp(9),Lys(12)][Lys(17,18), Glu(21)]glucagon-NH(2) (1), c[Asp(9),Lys(12)]glucagon-NH(2) (2), c[Lys(12),Asp(15)]glucagon-NH(2) (3), c[Asp(15), Lys(18)]glucagon-NH(2) (4), [Lys(17)-c[Lys(18), Glu(21)]glucagon-NH(2) (5), and c[Lys(12),Asp(21)]glucagon-NH(2) (6).

Mass spectrometry of 3,5- and 4,5-dicaffeoylquinic acids and selected derivatives.

The mass spectral properties of 3,5- and 4,5-dicaffeoylquinic acids (DCQAs) and selected derivatives were examined using electron ionization (EI), fast atom bombardment (FAB) and electrospray ionization (ESI). EI analysis of the trimethylsilyl derivatives provides molecular mass (M(r)) information, but the spectrum is dominated by fragment ions of the caffeic acid group; isomers cannot be differentiated using EI. FAB analysis, in both the positive and negative ion detection modes, provides M(r) information on the free compounds, but little fragmentation is observed using normal scan conditions.

Urinary nucleosides.

The methods of analysis, origins, and clinical significance of urinary nucleosides are reviewed through 1997. Structures, chromatographic and mass spectral data and references to the clinical literature are presented for each of the 57 nucleosides currently identified in normal and pathogenic human urine samples. Data from the HPLC separation and GC/MS analysis of 37 individual HPLC fractions are presented and discussed.

Mass spectrometry of selected components of biological interest in green tea extracts.

Mass spectrometric methods including EIMS, FABMS, and LC/ESIMS have been surveyed as tools for the detection of catechins in extracts of green tea (Camellia sinensis). EIMS provide both molecular weight and structure information, including epimer differentiation, on compounds 1, 2, and 4 and some structural information with compounds 5 and 6. FABMS gives both molecular weight and structure information, by an retro-Diels-Alder mechanism, on all compounds.

Mass spectrometry of nucleotides and oligonucleotides.

Recent advances in the use of mass spectrometry for the determination of the molecular weight and sequencing of oligonucleotides are discussed. Matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) mass spectrometry have been shown to be especially important techniques for both molecular weight assignment and sequencing of oligonucleotides, and are the focus of this article which covers the literature through early 1996.

Isolation of isoflavones from soy-based fermentations of the erythromycin-producing bacterium Saccharopolyspora erythraea.

A search for an abundant and economical source of isoflavones, particularly genistein, led to the discovery that the erythromycin-producing organism Saccharopolyspora erythraea also produces this promising new cancer-prevention agent. Erythromycin fermentation is a large-scale, soybean-based process used world-wide for the commercial production of this medically important antibiotic. Results from this study indicate that genistin (the glucoside form of genistein), which is added to the fermentation in the soybean media, was converted to genistein through the action of a beta-glucosidase produced by the organism.

Evaluation of mass spectrometric techniques for characterization of engineered proteins.

Mass spectrometric characterization of engineered proteins has been examined using bovine recombinant Acyl-CoA-Binding Protein (rACBP), [15N]-labeled rACBP, and a number of sequence variants of ACBP produced by site-directed mutagenesis. The mass spectrometric techniques include ESIMS and MALDIMS for analysis of the intact protein. Peptide maps have been obtained either by direct analysis of enzymatically derived mixtures by PDMS, ESIMS, and MALDIMS or by off- and on-line HPLC-mass spectrometry.

Synthesis of thiazole-4-carboxamide-adenine difluoromethylenediphosphonates substituted with fluorine at C-2' of the adenosine.

Synthesis of an analogue 3 of thiazole-4-carboxamide adenine-dinucleotide (TAD) in which the beta-oxygen atom of the pyrophosphate bridge is replaced by a difluoromethylene group has been achieved. Likewise, 2'-deoxy-2'-fluoroadenosine containing analogues of TAD (4) and its difluoromethylenediphosphonate congener (5) have been synthesized. Adenosine 5'-difluoromethylenediphosphonate (8) was prepared from 5'-O-tosyladenosine (6) and tris(tetra-n-butylammonium)difluoromethylenediphosphonate (7) by a modified procedure of Poulter's.

Urinary excretion of modified nucleosides as biological marker of RNA turnover in patients with cancer and AIDS.

Using boronate gel affinity chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC), a method for the simultaneous determination of 12 urinary modified nucleosides has been developed. The RP-HPLC fractions were identified by gas chromatography/mass spectrometry analysis. The HPLC quantitation of urinary nucleoside levels before and after surgery of cancer patients suggested that urinary 5'-deoxy-5'-methylthioadenosine and N-[(9-beta-D-ribofuranosyl-9H-purine-6-yl) carbamoyl]-L-threonine (t6A) levels were helpful in monitoring therapeutic effects in cancer patients.