Structure-Based Alignment and Consensus Secondary Structures for Three HIV-Related RNA Genomes

HIV and related primate lentiviruses possess single-stranded RNA genomes. Multiple regions of these genomes participate in critical steps in the viral replication cycle, and the functions of many RNA elements are dependent on the formation of defined structures. The structures of these elements are still not fully understood, and additional functional elements likely exist that have not been identified. In this work, we compared three full-length HIV-related viral genomes: HIV-1_NL4-3, SIVcpz, and SIVmac (the latter two strains are progenitors for all HIV-1 and HIV-2 strains, respectively). Model-free RNA structure comparisons were performed using whole-genome structure information experimentally derived from nucleotide-resolution SHAPE reactivities. Consensus secondary structures were constructed for strongly correlated regions by taking into account both SHAPE probing structural data and nucleotide covariation information from structure-based alignments. In these consensus models, all known functional RNA elements were recapitulated with high accuracy. In addition, we identified multiple previously unannotated structural elements in the HIV-1 genome likely to function in translation, splicing and other replication cycle processes; these are compelling targets for future functional analyses. The structure-informed alignment strategy developed here will be broadly useful for efficient RNA motif discovery.     

Effect of an infusion of Gn-RH upon levels of sex hormones in prepubertal and pubertal girls: evidence for relative ovarian insensitivity.

Changes in levels of sex steroids and gonadotropins were measured in 16 normal prepubertal and 15 pubertal girls prior to and after a 3 hour infusion of 100 microgrm synthetic gonadotropin releasing hormone (Gn-RH). Plasa estradiol (E2) concentrations rose significantly (p less than 0.02) from 29.7 +/- 4.6 (SE) pg/ml in the basal period to to 46.8 +/- 7.1 at the end of the infusion in the pubertal girls but were unchanged in the prepubertal girls. Estrone (E1), progesterone (P), 17-HYDROXYPROGESTERONE (17OHP), TESTOSTERONE (T), DIHYDROTESTOSTERONE (DHT), and androstenedione (A), dehydroepiandrosterone (DHA) and dehydroepiandrosterone sulfate (DHAS) levels were not altered in either maturity group. Basal plasma E2, E1, T, DHT, DHA and DHAS concentrations significantly correlated with the releasable pool of LH evoked by Gn-RH from the pituitary gonadotropes. We conclude: 1) The ovary is not highly and rapidly responsive to transient elevations of endogenous gonadotropin, and 2) Adrenal androgens may to some extent modulate the maturation of the hypothalamic-pituitary-gonadal system, at least as reflected by the pituitary response to exogenous Gn-RH.     

Functional Genomics Identifies Metabolic Vulnerabilities in Pancreatic Cancer

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Plasma glucose and insulin levels in monkeys anticipating feeding

To see whether plasma glucose or insulin changed in anticipation of feeding, we provided seven rhesus monkeys with four-hour access to food every other day. Blood was sampled before and during a 30-minute signal which ended with food availability and before and during a 30-minute signal which was not closely and reliably linked with food availability. Plasma insulin showed no evidence of conditioning. Plasma glucose was higher during the signal than prior to the signal in both experiments. This probably reflects the arousing nature of the signal rather than appetitive-associated learning. However, the differences, while statistically significant, were probably biologically trivial because they fall within the normal fluctuations of meal-fed monkeys. Under the conditions of this experiments, it appears that conditional changes in glucose and insulin do not reliably occur in monkeys anticipating access to food.     

Guinea pig pancreatic acini prepared with purified collagenase

Dispersed guinea pig pancreatic acinar cells have been used to investigate several aspects of stimulus-secretion coupling but possess the disadvantage that they are less sensitive and less responsive to secretagogues than in vitro preparations of intact pancreatic tissue (lobules). To overcome the poor responsiveness of isolated acinar cells, we have developed a new procedure for preparing dispersed, intact pancreatic acini whose sensitivity to secretagogues and morphological characteristics are similar to those of pancreatic lobules. Dispersed acini can be manipulated as suspensions of cells and full access of macromolecular probes to apical and basolateral plasmalemmal domains is obtained. Acini were prepared in good yields (~70% on a DNA basis) using only purified collagenase and mild mechanical shear in medium containing 2.0 mM Ca²⁺. Morphologically, acinar cells in the preparations retained intact junctional complexes, asymmetrical distribution of intramembranous particles between apical and basolateral plasmalemmal domains, and polarized distribution of intracellular organelles as found in intact pancreas. Dose-response curves of acini and mechanically prepared lobules to caerulein, carbachol, and bombesin were similar though acini were more sensitive to the C-terminal octapeptide of cholecystokinin. Net stimulated secretory protein discharge was ~36% over 2 h. Crude collagenase was purified for use in preparation of acini by Sephadex G-75 column chromatography which resolved collagenase from clostripain and a non-sulfhydryl-requiring protease. The purified collagenase contained at least four proteins with molecular weights between 85 000 and 110 000. Collagenase with <0.14 units of protease per unit of collagenase produced highly responsive acini; collagenase with >0.9 units of protease per unit of collagenase yielded unresponsive acini. Acini incubated with crude collagenase, chymotrypsin, or the non-sulfhydryl-requiring protease showed depressed secretory response to caerulein. Freeze-fracture electron microscopy of protease-treated acini indicated that the intramembranous particles aggregated and that many of the tight junctions had undergone a proliferation of non-cross-linked sealing strands which extended far down the basolateral plasma membrane and encircled gap junctions. Acini incubated with purified collagenase or with a clostripain-containing fraction from the Sephadex G-75 column appeared unaltered. This procedure produces acini which are morphologically and biochemically similar to the in situ pancreas and overcomes the poor response to secretagogues by isolated pancreatic acinar cells.