An insertion vector for the analysis of gene expression during herpes simplex virus infection

A herpes simplex virus type 1 (HSV-1) insertion vector, pGal8, was designed for analysis of herpesvirus promoters during virus infection. This vector contains a multiple cloning site (MCS) positioned at the 5' end of the lacZ gene for the insertion of promoter sequences. The MCS and lacZ are flanked by sequences from the HSV-1 thymidine kinase encoding gene (tk) to direct homologous recombination into the tk locus of the viral genome. The utility of this vector is demonstrated by construction and comparison of recombinant viruses that express lacZ from the promoters of the genes encoding glycoprotein C, glycoprotein H and glycoprotein E.     

Effects of bicarbonate on fluid and electrolyte transport by the guinea pig gallbladder: A bicarbonate-chloride exchange

Summary Fluid transport and net fluxes of Na, K, Cl and HCO₃ by guinea pig gallbladder were investigatedin vitro. A perfused gallbladder preparation was devised to simultaneously study unidirectional fluxes of²²Na and³⁶Cl. The net Cl flux exceeded the net Na flux during fluid absorption in the presence of HCO₃. This Cl excess was counter-balanced by a net HCO₃ secretion: a HCO₃–Cl exchange. PGE₁ reversed the direction of fluid transport and abolished the net Cl flux. The magnitude of the HCO₃ secretion remained unchanged, but shifted from a HCO₃–Cl exchange to a net secretion of NaHCO₃ and KHCO₃. Furosemide inhibited both the HCO₃–Cl exchange and HCO₃ secretion after PGE₁ without influencing fluid absorption. Ouabain inhibited the HCO₃–Cl exchange as well as fluid absorption; only the effect on the HCO₃ secretion was entirely reversible. Secreted HCO₃ appeared not to be derived from metabolic sources since HCO₃ secretion was abolished in a HCO₃-free bathing medium. HCO₃ secretion was also dependent on the Na concentration of the bathing fluid. Three lines of evidence are presented in favor of an active HCO₃ secretion in guinea pig gallbladder. HCO₃ is secreted against: (i) a chemical gradient, (ii) an electrical gradient and (iii) the direction of fluid movement under control conditions.     

Silicon concentrations and stoichiometry in two agricultural watersheds: implications for management and downstream water quality

Biogeochemistry - Agriculture alters the biogeochemical cycling of nutrients such as nitrogen (N), phosphorus (P), and silicon (Si) which contributes to the stoichiometric imbalance among these...     

Coronavirus genomic and subgenomic minus-strand RNAs copartition in membrane-protected replication complexes.

The majority of porcine transmissible gastroenteritis coronavirus plus-strand RNAs (genome and subgenomic mRNAs), at the time of peak RNA synthesis (5 h postinfection), were not found in membrane-protected complexes in lysates of cells prepared by Dounce homogenization but were found to be susceptible to micrococcal nuclease (85%) or to sediment to a pellet in a cesium chloride gradient (61%). They therefore are probably free molecules in solution or components of easily dissociable complexes. By contrast, the majority of minus-strand RNAs (genome length and subgenomic mRNA length) were found to be resistant to micrococcal nuclease (69%) or to remain suspended in association with membrane-protected complexes following isopycnic sedimentation in a cesium chloride gradient (85%). Furthermore, 35% of the suspended minus strands were in a dense complex (1.20 to 1.24 g/ml) that contained an RNA plus-to-minus-strand molar ratio of approximately 8:1 and viral structural proteins S, M, and N, and 65% were in a light complex (1.15 to 1.17 g/ml) that contained nearly equimolar amounts of plus- and minus-strand RNAs and only trace amounts of proteins M and N. In no instance during fractionation were genome-length minus strands found segregated from sub-genome-length minus strands. These results indicate that all minus-strand species are components of similarly structured membrane-associated replication complexes and support the concept that all are active in the synthesis of plus-strand RNAs.     

Bovine lens gamma-glutamylcysteine synthetase. Inhibition by glutathione and adenine nucleotides

Steady-state kinetic analysis shows that glutathione binds reversibly to both Mg . enzyme and Mg . enzyme . L-glutamate forms of gamma-glutamylcysteine synthetase to form inactive complexes. The Ki values for binding to these two species of enzyme are 4 mM and 0.4 mM, respectively; those for S-methyl glutathione are 16 mM and 0.5 mM, respectively. These data suggest that glutathione is an important feedback inhibitor and contributes to the regulation of glutathione synthesis by modulating the synthesis rate of the precursor dipeptide. Adenosine 5'-diphosphate (5'ADP) is also an inhibitor and competes with both ATP and L-beta-chloroalanine for Mg . enzyme . L-glutamate and Mg . enzyme . L-glutamylphosphate, respectively. Under physiological conditions in the lens, 5' ADP competes effectively with L-cysteine for Mg . enzyme . L-glutamylphosphate, owing to the low concentration of L-cysteine, and less effectively with ATP for Mg . enzyme . L-glutamate, because of a high concentration of ATP.     

Minus-strand copies of replicating coronavirus mRNAs contain antileaders.

The 5' leader sequence on mRNAs of the porcine transmissible gastroenteritis coronavirus was determined and found to be 90 nucleotides in length. An oligodeoxynucleotide with a sequence from within the leader was used as a probe in Northern analysis on RNA from infected cells, and an antileader (a minus-strand copy of the leader sequence) was shown to be present on all mRNA minus-strand species. RNase protection analysis showed the antileader to be approximately the same length as the leader. The kinetics of antileader appearance was the same as that for the appearance of minus-strand RNA species. This, along with a demonstration that viral mRNAs become packaged, gives further support to the idea that coronavirus mRNAs can undergo replication via subgenomic mRNA-length replicative intermediates, and that input mRNAs from infecting virions may serve as initial templates for their own replication. In this sense, then, coronaviruses behave in part like RNA viruses with segmented genomes.     

The statistical mechanics of complex signaling networks: nerve growth factor signaling

The inherent complexity of cellular signaling networks and their importance to a wide range of cellular functions necessitates the development of modeling methods that can be applied toward making predictions and highlighting the appropriate experiments to test our understanding of how these systems are designed and function. We use methods of statistical mechanics to extract useful predictions for complex cellular signaling networks. A key difficulty with signaling models is that, while significant effort is being made to experimentally measure the rate constants for individual steps in these networks, many of the parameters required to describe their behavior remain unknown or at best represent estimates. To establish the usefulness of our approach, we have applied our methods toward modeling the nerve growth factor (NGF)-induced differentiation of neuronal cells. In particular, we study the actions of NGF and mitogenic epidermal growth factor (EGF) in rat pheochromocytoma (PC12) cells. Through a network of intermediate signaling proteins, each of these growth factors stimulates extracellular regulated kinase (Erk) phosphorylation with distinct dynamical profiles. Using our modeling approach, we are able to predict the influence of specific signaling modules in determining the integrated cellular response to the two growth factors. Our methods also raise some interesting insights into the design and possible evolution of cellular systems, highlighting an inherent property of these systems that we call 'sloppiness.'     

Chest wall motion of infants during spinal anesthesia

To test the extent to which diaphragmatic contraction moves the rib cage in awake supine infants during quiet breathing, we studied chest wall motion in seven prematurely born infants before and during spinal anesthesia for inguinal hernia repair. Infants were studied at or around term (postconceptional age 43 +/- 8 wk). Spinal anesthesia produced a sensory block at the T2-T4 level, with concomitant motor block at a slightly lower level. This resulted in the loss of most intercostal muscle activity, whereas diaphragmatic function was preserved. Rib cage and abdominal displacements were measured with respiratory inductance plethysmography before and during spinal anesthesia. During the anesthetic, outward inspiratory rib cage motion decreased in six infants (P less than 0.02, paired t test); four of these developed paradoxical inward movement of the rib cage during inspiration. One infant, the most immature in the group, had inward movement of the rib cage both before and during the anesthetic. Abdominal displacements increased during spinal anesthesia in six of seven infants (P less than 0.05), suggesting an increase in diaphragmatic motion. We conclude that, in the group of infants studied, outward rib cage movement during awake tidal breathing requires active, coordinated intercostal muscle activity that is suppressed by spinal anesthesia.