Structural plasticity in RNA and its role in the regulation of protein translation in coliphage Q beta

We have analyzed both conformational and functional changes caused by two large cis-acting deletions (delta 159 and delta 549) located within the read-through domain, a 850 nucleotide hairpin, in coliphage Q beta genomic RNA. Studies in vivo show that co-translational regulation of the viral coat and replicase genes has been uncoupled in viral genomes carrying deletion delta 159. Translational regulation is restored in deletion delta 549, a naturally evolved pseudorevertant. Structural analysis by computer modeling shows that structural features within the read-through domain of delta 159 RNA are less well determined than they are in the read-through domain of wild-type RNA, whereas predicted structure in the read-through domain of evolved pseudorevertant delta 549 is unusually well determined. Structural analysis by electron microscopy of the genomic RNAs shows that several long range helices at the base of the read-through domain, that suppress translational initiation of the viral replicase gene in the wild-type genome, have been destabilized in delta 159 RNA. In addition, the structure of local hairpins within the read-through region is more variable in delta 159 RNA than in wild-type RNA. Stable RNA secondary structure is restored in the read-through domain of delta 549 RNA. Our analyses suggest that structure throughout the read-through domain affects the regulation of viral replicase expression by altering the likelihood that long-range interactions at the base of the domain will form. We discuss possible kinetic and equilibrium models that can explain this effect, and argue that observed changes in structural plasticity within the read-through domain of the mutant genomes are key in understanding the process. During the course of these studies, we became aware of the importance of the information contained in the energy dot plot produced by the RNA secondary structure prediction program mfold. As a result, we have improved the graphical representation of this information through the use of color annotation in the predicted optimal folding. The method is presented here for the first time.     

Chronic effects of glucose on insulin signaling in A-10 vascular smooth muscle cells

To examine the effects of hyperglycemia on insulin signaling in A-10 vascular smooth muscle cells, cells were treated with extracellular D-glucose and effects of insulin were studied on the diacylglycerol-protein kinase C signaling system. A-10 cells specifically bound 125I-insulin, and insulin-like growth factor-I did not displace the label. 125I-insulin binding was unaltered under hyperglycemic conditions. To determine if insulin receptors were coupled to other insulin-regulated processes, diacylglycerol, protein kinase C, and glucose transport were evaluated. Insulin increased cellular diacylglycerol (DAG) levels which were also increased following glucose treatment and not further stimulated by insulin. The uptake of 2-[3H]deoxy-D-glucose (2-DOG) was stimulated by insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Insulin- and TPA-stimulated 2-[3H]DOG uptake was inhibited by a protein kinase inhibitor, staurosporine. Preincubation of cells with 500 nM TPA overnight resulted in the inhibition of insulin- and TPA-stimulated 2-[3H]DOG uptake. Protein kinase C activity was translocated from cytosolic to membrane fractions following insulin treatment. Overnight glucose (25 mM) treatment resulted in a 50% decrease in protein kinase C enzyme activity and > 90% decrease in protein kinase C beta immunoreactive levels. Protein kinase C activity and levels were not affected by osmotic control media containing mannitol. A-10 cells express GLUT4-type glucose transporters. Neither insulin-regulatable glucose transporter (GLUT4) mRNA nor GLUT4 protein levels were diminished by glucose. Significant decreases in insulin- and TPA-stimulated 2-[3H]DOG uptake occurred, however, with glucose. The down-regulation of protein kinase C beta and resultant inhibition of 2-[3H]DOG uptake by chronic glucose suggests a biochemical link between hyperglycemia and DAG-protein kinase C signaling in vascular smooth muscle cells.     

Insulin propeptides in conditions associated with insulin resistance in humans and their relevance to insulin measurements

Routine insulin assays measure not only biologically active insulin but also the relatively inactive propeptides, proinsulin and desdipeptide proinsulin. Such measurements may be misleading if insulin propeptide levels are increased, as has been reported in patients with non-insulin-dependent diabetes mellitus (NIDDM). Inferences regarding insulin resistance, based on hyperinsulinemia, could thus be invalidated where routine insulin assays have been used. We have measured plasma insulin levels using a routine assay, together with measurements of the major circulating insulin propeptides, intact proinsulin and des 31,32proinsulin, in various clinical situations associated with apparently increased insulin levels and insulin resistance. Major increases of insulin propeptide levels relative to insulin levels were not seen in obese subjects or in patients taking oral contraceptives or danazol, or in obese subjects compared with non-obese controls. Although the insulinemic responses observed with routine radioimmunoassay in these situations associated with insulin resistance are not confounded by major changes in the proportion of circulating insulin propeptides, further studies will be necessary to validate investigations in other insulin-resistant states.     

True digestibility of amino acids and protein in pigs with 13C as a label to determine endogenous amino acid excretion

The objective of this study was to determine whether differential labeling of 13C occurs in pigs fed diets with different 13C abundances and, if so, to use 13C as a label to determine true amino acid digestibility. Forty-eight pigs averaging 10.5 kg BW were fed dietary treatments consisting of a corn-corn gluten meal-crystalline amino acid diet (C-CGM) and a wheat-soybean meal diet (W-SBM). The 13C abundance of the amino acid fraction (AAF) of the C-CGM and W-SBM diets averaged delta 13C -14.19 and -26.36/1000, respectively. Three pigs/treatment group were killed when groups averaged 10.5 (initial), 22.9, and 46.6 kg BW, and AAF of organs were analyzed for 13C abundance. Carbon 13 in empty body AAF increased (-18.14, -13.98, and -12.66/1000) with increasing body weight in pigs fed the C-CGM diet and decreased (-18.06, -22.78, and -24.76/1000) in pigs fed the W-SBM diet. Liver, small intestine, and longissimus muscle tissues showed similar trends. Each tissue had dietary treatment effects (P < .001) and dietary treatment x weight group (P < .001) interactions. Ten pigs averaging 55.0 kg BW from each treatment group were assigned to metabolism cages and fed at 0700 and 1900. Six of these pigs from each treatment group were implanted with T-cannulas in the ileum and given a 17-d recovery period. At 1900 on d 0 of the collection phase, pigs were switched to the opposite diet that contained chromic oxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebral blood flow relationships associated with a difficult tone recognition task in trained normal volunteers

Tone recognition is partially subserved by neural activity in the right frontal and primary auditory cortices. First we determined the brain areas associated with tone perception and recognition. This study then examined how regional cerebral blood flow (rCBF) in these and other brain regions correlates with the behavioral characteristics of a difficult tone recognition task. rCBF changes were assessed using H2(15)O positron emission tomography. Subtraction procedures were used to localize significant change regions and correlational analyses were applied to determine how response times (RT) predicted rCBF patterns. Twelve trained normal volunteers were studied in three conditions: REST, sensory motor control (SMC) and decision (DEC). The SMC-REST contrast revealed bilateral activation of primary auditory cortices, cerebellum and bilateral inferior frontal gyri. DEC-SMC produced significant clusters in the right middle and inferior frontal gyri, insula and claustrum; the anterior cingulate gyrus and supplementary motor area; the left insula/claustrum; and the left cerebellum. Correlational analyses, RT versus rCBF from DEC scans, showed a positive correlation in right inferior and middle frontal cortex; rCBF in bilateral auditory cortices and cerebellum exhibited significant negative correlations with RT These changes suggest that neural activity in the right frontal, superior temporal and cerebellar regions shifts back and forth in magnitude depending on whether tone recognition RT is relatively fast or slow, during a difficult, accurate assessment.     

Neuropeptides in the insect brain: a review

Thirty-seven plants are listed, which are used by traditional healers in the central and southern parts of Somalia. For each species are listed: the botanical name with synonyms, collection number, vernacular name, medicinal use, preparation of remedy and dosage. Results of a literature survey are also reported including medicinal use, substances isolated and pharmacological effects. Three plants which should have been included in Part I of the series have been added and some corrections to that paper have been made. With these additions the series comprises 180 different plant species, distributed in 59 plant families.