Cyclophilin A-induced alterations of human immunodeficiency virus type 1 CA protein in vitro.

Cyclophilin A (CyP A), a cellular chaperone with cis-trans prolyl isomerase activity, is required for postassembly events in human immunodeficiency virus type 1 (HIV-1) replication. The requirement for CyP A maps to sequences in the capsid (CA) domain of the structural precursor, Gag. To determine the effects of interaction with CyP A on capsid (CA) protein structure, the binding interaction was investigated in vitro, using recombinant HIV-1 CA protein (which forms oligomers in solution) and human CyP A. The CA and CyP A proteins interacted to form a complex which was detected predominantly as a heterodimer on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Complex formation exhibited a pH optimum of 5. The CA protein in the complex was exclusively in a conformation whereby the N terminus was blocked to Edman degradation. This finding was unexpected since CyP A binds to the central region of the CA protein (residues 85 to 93). Examination of CA protein incubated with CyP A for alterations in structure indicated that CyP A preferentially interacted with the subpopulation of trypsin-susceptible subunits in the oligomers and significantly reduced their sensitivity to proteolysis. Like CA-CyP A complex formation, conversion to trypsin resistance also exhibited a pH optimum of 5. Both complex formation and the changes in tryptic susceptibility were only partially inhibited by cyclosporin A (CsA). This appeared to be due to a CA subpopulation able to bind CyP A despite the presence of CsA. Our results identify specific tryptic sites both proximal and distal to the CyP A binding region that are altered by CyP A binding and/or by CyP A's prolyl isomerase activity. Comparison with the X-ray structure of a complex containing CyP A bound to an amino-terminal fragment of the CA protein (CA1-151) (T.R. Gamble et al., Cell 87:1285-1294, 1996) indicates that the tryptic sites that become inaccessible are among the same residues that lose a significant amount of accessible surface area through CA-CA subunit contacts made in the presence of CyP A.     

Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability.     

Direct and correlated responses to selection for weaning weight,post-weaning weight gain and six-week weight in mice

Summary Four lines of mice were formed from a common base population and selected for 37 generations for either increased 3-week weight (weaning weight), 6-week weight, 3–6 week gain, or maintained as a randomly bred control line. Realised heritability estimates for short-term (long-term) responses were 0.33±0.20 (0.07±0.10), 0.46±0.14 (0.26±0.09), 0.36±0.14 (0.24±0.11) for 3-week weight, 6-week weight and 3–6 week gain, respectively. Realised genetic correlations estimated from short-term (long-term) responses were 0.23±0.08 (0.35±0.10) between 3-week weight and 3–6 week gain; 0.82±0.04 (0.58±0.08) between 3-week weight and 6-week weight; and 0.81±0.04 (0.97±0.04) between 3–6 week gain and 6-week weight. The genetic correlation between 3-week weight and 6-week weight was asymmetric with a greater correlated response for 3-week weight when selecting for 6-week weight (1.06) than vice versa (0.63).     

The blood supply to the abdominal organs of the fetal guinea-pig

Guinea-pigs near term of pregnancy were anaesthetized with diazepam and sodium pentobarbitone. A fetus was exposed and the vitelline artery catheterized to measure blood pressure and heart rate or to render a reference sample of blood for the determination of organ blood flow by the microsphere technique. The radioactive microspheres were injected through a catheter in the right atrium. Mean arterial blood pressure was 4.0 kPa and heart rate was 261 beats min-1. The liver, spleen, pancreas and gut receive most of their blood supply from the same trunk as the vitelline artery. The sample from this vessel was also used to calculate blood flow to the adrenal glands, kidneys, urogenital tract, and placenta, assuming even mixing of microspheres and blood in the abdominal aorta. Umbilical blood flow, corrected to a fetal weight of 100 g, averaged 7.5 ml min-1. The adrenal glands, which are known to increase their cortisol secretion near term, had a very high rate of perfusion. If the microspheres were injected in the umbilical vein, almost all were trapped in the liver, confirming the absence of a ductus venosus in the guinea-pig fetus. Most of these microspheres were found in the quadrate lobe of the liver. Hepatic arterial blood flow was also unequally distributed, with more than two-thirds going to the right lobe of the liver. Although the distribution of portal venous blood flow is not known, it is evident that different areas of the liver are presented with blood of greatly varying oxygen saturation.     

pH titration studies of 13C reductively methylated N-terminal serine of glycophorin AM

The ¹³C resonances of N^α,N-[¹³C]dimethylserine of partially ¹³C reductively methylated glycophorin A^M were monitored as a function of pH at 45°C. For comparison, limited data are also presented for the pH dependence of the ¹³C resonances of N^α,N- [¹³C]dimethylserine of fully ¹³C reductively methylated deglycosylated glycophorin A^M. The ‘major’ component of N^α,N- [¹³C]dimethylserine of glycophorin A^M did not titrate, whereas the ‘minor’ component titrated with a pK_a of 7.80 (Hill coefficient of 0.95). Similar results are also indicated for the N^α,N- [¹³C]dimethylserine resonances of ¹³C reductively methylated deglycosylated glycophorin A^M.     

The mechanism of inhibition of ureogenesis by acidosis

In perfused rat liver a decrease of cytosol pH, determined with pH-sensitive microelectrodes7 from 7.2 to 6.85 is associated with a 50% fall in ureogenesis from ammonium chloride. In isolated rat hepatocytes the fall in ureogenesis due to acidosis is associated with decrease in the mitochondrial and cytosolic concentration of citrulline. Limitation of carbamoyl phosphate synthesis and thus citrulline supply could be responsible for the inhibition of ureogenesis observed.     

Radioimmunoassay of carbonic anhydrase III in rat tissues.

A specific and sensitive radioimmunoassay for the rat carbonic anhydrase III isoenzyme was developed. High concentrations of carbonic anhydrase III were detected in soleus muscle and male liver. Female liver and other skeletal muscles contained significantly lower concentrations, and only trace amounts were found in heart, prostate, kidney, brain, plasma, urine and, possibly, erythrocytes.     

Dynamics of soil microbial biomass N under zero and shallow tillage for spring wheat,using15N urea

Summary Field studies to determine the effect of zero and shallow (10 cm) cultivation on microbial biomass were conducted on several Chernozemic soils in western Canada. Using the CHCl₃ fumigation method, the distribution of microbial biomass N and the immobilization and subsequent release of added¹⁵N (¹⁵N-urea) from the microbial biomass were determined in the A horizon, at the 0 to 5 and 5 to 10 cm depth, during the growing season for spring wheat.Temporal variation in microbial biomass N, associated with the development of the rhizosphere, was characterized by an increase between Feekes stage 1 and 5 or 10 and decrease at Feekes stage 11.4. Over the long term, the variation in biomass N between tillage systems corresponded with crop residue distribution. Immobilization of fertilizer N was related to the increase in biomass N from Feekes stage 1, which in turn, was associated with the incorporation of recent crop residues or levels of labile organic matter in the surface soil. The study demonstrated the relatively rapid remineralization of immobilized fertilizer N under field conditions and emphasized the role of the microbial biomass N as both a sink and source of mineral N.