In vivo downregulation of protein synthesis in the snail Helix apersa during estivation

Protein synthesis is downregulated during metabolic depression in a number of systems where the metabolic depression is effected by obvious extrinsic cues. The metabolic depression of the estivating land snail Helix apersa occurs in the absence of any obvious physiological stress and has an intrinsic component independent of temperature, pH, O(2) status, or osmolality. We show that this metabolic depression is accompanied by a downregulation of protein synthesis in vivo. The rate of protein synthesis decreases in two major tissues during estivation: to 23% and 53% of the awake rate in hepatopancreas and foot muscle, respectively. We show from calculations of the theoretical contribution of protein synthesis to total O(2) consumption that the depression of protein synthesis must be a significant, obligate, in vivo component of metabolic depression in H. aspersa.     

Incorporation of 8-histaminyl-deoxyadenosine [8-(2-(4-imidazolyl)ethylamino)-2'-deoxyriboadenosine] into oligodeoxyribonucleotides by solid phase phosphoramidite coupling

The 3'phosphoramidite of 8-histaminyl deoxyadenosine has been prepared and successfully incorporated into a short oligodeoxyribonucleotide. The synthetic methodology leading to this preparation is given and the implications for developing new DNAzymes as well as probing unusual nucleic acid structures are discussed.     

Extracellular acidification parallels insulin secretion in INS-1 and HIT-T15 beta-cell lines

As an alternative to manual assays that track insulin secretion, we tested a silicon-based biosensor that allows automated monitoring of extracellular acidification. Glucose stimulation of INS-1 and HIT-T15 cells resulted in a rapid increase in extracellular acidification in a biphasic and concentration-dependent fashion much like insulin secretion (EC(50) INS-1=5 mM and HIT-T15=1 mM). This response was attenuated by verapamil (10 microM) and stimulated by administration of glybenclamide (100 nM) or KCl-induced (40 mM) depolarization. These experiments suggest that automated monitoring of extracellular pH may be a useful assay and support the relevance of linking metabolic activity to insulin secretion.     

Natural variation for seed oil composition in Arabidopsis thaliana

The biochemical pathways involved in the biosynthesis and accumulation of storage lipids in seeds have been extensively studied. However, the regulatory mechanisms of those pathways, their environmental interactions and the ecological implications of variation are poorly understood. We have initiated a new approach: the analysis of natural variation in Arabidopsis thaliana. Three hundred and sixty accessions were surveyed for content of oil, very long chain fatty acids (VLCFAs) and polyunsaturated fatty acids (PUFAs) in their seeds. The results revealed extensive natural variation. A core set of accessions, the seeds of which reproducibly contain extreme amounts of oil, VLCFAs and PUFAs have been identified. Reproducible oil content ranged from 34.6 to 46.0% of seed dry weight. VLCFA content ranged from 13.0 to 21.2% of total fatty acids. PUFA content, ranged from 53.3 to 66.1% of total fatty acids. Interactions were also identified for PUFA and VLCFA content of seeds with vernalisation of plants. Mapping of the regions of the genome involved in controlling the traits was conducted in an F(2) population and indicated that natural variation at the loci FAE1 and FAD3 might be involved in the regulation of VLCFA and PUFA content, respectively. A set of accessions, which capture a broad range of the natural variation for these traits available in A. thaliana, has been selected to form a core set which can be used to further dissect the genetics of the regulation of seed lipid traits and to identify the genes involved.     

Synthesis of polymerized thin films for immobilized ligand display in proteomic analysis

We describe a new material for the display of biomolecular ligands for use in proteomic analysis. We report here on the construction of the first functionalized polymerized diacetylene thin films (PDTFs) for use in displaying immobilized ligands and their application in mass spectral proteomic analysis. Functionalized polymerized thin film surfaces were constructed with diacetylene-containing biotin lipid monomers designed for the capture of proteins (streptavidin) from a complex cellular lysate and detection with mass spectrometry (MS). These materials serve as a prototype for ligand-based spotted arrays amenable to high throughput screening. Functionalized PDTFs can be easily manufactured for customized microarrays and demonstrate high protein specificity and low nonspecific protein adsorption, and the resulting microarrays constructed from these materials are compatible with several different protein analysis platforms. Our results suggest that these materials have broad potential applications for use in mass spectral-based proteomic analysis.     

Abdominal cysticercosis in a rhesus macaque (Macaca mulatta)

A mid-abdominal mass was discovered during routine physical examination of a rhesus macaque (Macaca mulatta). Further diagnostics and exploratory laparotomy were performed, revealing a fluid-filled cyst attached to the caudal free margin of the greater omentum. Formation and pulsatile movement of white-colored circumferential bands within the wall of the cyst were observed during surgery. The cyst was removed and later was dissected. The discovery of a single invaginated scolex identified the cyst as a cysticercus. The location and characteristics of the cysticercus were consistent with the larval form of Taenia hydatigena.     

Reciprocal regulation of cGMP-mediated vasorelaxation by soluble and particulate guanylate cyclases

Nitric oxide (NO) and atrial natriuretic peptides (ANP) activate soluble (sGC) and particulate guanylate cyclase (pGC), respectively, and play important roles in the maintenance of cardiovascular homeostasis. However, little is known about potential interactions between these two cGMP-generating pathways. Here we demonstrate that sGC and pGC cooperatively regulate cGMP-mediated relaxation in human and murine vascular tissue. In human vessels, the potency of spermine-NONOate (SPER-NO) and ANP was increased after inhibition of endogenous NO synthesis and decreased by prior exposure to glyceryl trinitrate (GTN). Aortas from endothelial NO synthase (eNOS) knockout (KO) mice were more sensitive to ANP than tissues from wild-type (WT) animals. However, in aortas from WT mice, the potency of ANP was increased after pretreatment with NOS or sGC inhibitor. Vessels from eNOS KO animals were less sensitive to ANP after GTN pretreatment, an effect that was reversed in the presence of an sGC inhibitor. cGMP production in response to SPER-NO and ANP was significantly greater in vessels from eNOS KO animals compared with WT animals. This cooperative interaction between NO and ANP may have important implications for human pathophysiologies involving deficiency in either mediator and the clinical use of nitrovasodilators.     

Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome

Down syndrome is a complex genetic and metabolic disorder attributed to the presence of three copies of chromosome 21. The extra chromosome derives from the mother in 93% of cases and is due to abnormal chromosome segregation during meiosis (nondisjunction). Except for advanced age at conception, maternal risk factors for meiotic nondisjunction are not well established. A recent preliminary study suggested that abnormal folate metabolism and the 677C-->T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene may be maternal risk factors for Down syndrome. The present study was undertaken with a larger sample size to determine whether the MTHFR 677C-->T polymorphism was associated with increased risk of having a child with Down syndrome. Methionine synthase reductase (MTRR) is another enzyme essential for normal folate metabolism. A common polymorphism in this gene was recently associated with increased risk of neural tube defects and might also contribute to increased risk for Down syndrome. The frequencies of the MTHFR 677C-->T and MTRR 66A-->G mutations were evaluated in DNA samples from 157 mothers of children with Down syndrome and 144 control mothers. Odds ratios were calculated for each genotype separately and for potential gene-gene interactions. The results are consistent with the preliminary observation that the MTHFR 677C-->T polymorphism is more prevalent among mothers of children with Down syndrome than among control mothers, with an odds ratio of 1.91 (95% confidence interval [CI] 1.19-3.05). In addition, the homozygous MTRR 66A-->G polymorphism was independently associated with a 2. 57-fold increase in estimated risk (95% CI 1.33-4.99). The combined presence of both polymorphisms was associated with a greater risk of Down syndrome than was the presence of either alone, with an odds ratio of 4.08 (95% CI 1.94-8.56). The two polymorphisms appear to act without a multiplicative interaction.