Optimal Induction of Hepatitis C Virus Envelope-Specific Immunity by Bicistronic Plasmid DNA Inoculation with the Granulocyte-Macrophage Colony-Stimulating Factor Gene

In this study, we have constructed various DNA vaccine vectors that carried hepatitis C virus (HCV) envelope genes without and with the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in several different ways. In Buffalo rats that received plasmids carrying the HCV envelope genes, which encode envelope proteins E1 and E2, both antibody and lymphoproliferative responses against these proteins were induced. These responses were greatly enhanced by the codelivery of the GM-CSF gene. In particular, inoculation with a bicistronic plasmid that independently expressed the GM-CSF gene and the envelope genes in the same construct generated the highest antibody titers and significantly increased lymphoproliferative responses against these proteins. Moreover, strong antibody responses to homologous and heterologous hypervariable region 1 peptides were elicited in the immunized rats.     

The dynamic regulation of myocardial oxidative phosphorylation: Analysis of the response time of oxygen consumption

Although usually steady-state fluxes and metabolite levels are assessed for the study of metabolic regulation, much can be learned from studying the transient response during quick changes of an input to the system. To this end we study the transient response of O₂ consumption in the heart during steps in heart rate. The time course is characterized by the mean response time of O₂ consumption which is the first statistical moment of the impulse response function of the system (for mono-exponential responses equal to the time constant). The time course of O₂ uptake during quick changes is measured with O₂ electrodes in the arterial perfusate and venous effluent of the heart, but the venous signal is delayed with respect to O₂ consumption in the mitochondria due to O₂ diffusion and vascular transport. We correct for this transport delay by using the mass balance of O₂, with all terms (e.g. O₂ consumption and vascular O₂ transport) taken as function of time. Integration of this mass balance over the duration of the response yields a relation between the mean transit time for O₂ and changes in cardiac O₂ content. Experimental data on the response times of venous [O₂] during step changes in arterial [O₂] or in perfusion flow are used to calculate the transport time between mitochondria and the venous O₂ electrode. By subtracting the transport time from the response time measured in the venous outflow the mean response time of mitochondrial O₂ consumption (t_mito) to the step in heart rate is obtained.In isolated rabbit heart we found that t_mito to heart rate steps is 4-12 s at 37°C. This means that oxidative phosphorylation responds to changing ATP hydrolysis with some delay, so that the phosphocreatine levels in the heart must be decreased, at least in the early stages after an increase in cardiac ATP hydrolysis. Changes in ADP and inorganic phosphate (P_i) thus play a role in regulating the dynamic adaptation of oxidative phosphorylation, although most steady state NMR measurements in the heart had suggested that ADP and P_i do not change. Indeed, we found with ³¹P-NMR spectroscopy that phosphocreatine (PCr) and P_i change in the first seconds after a quick change in ATP hydrolysis, but remarkably they do this significantly faster (time constant ~2.5 s) than mitochondrial O₂ consumption (time constant 12 s). Although it is quite likely that other factors besides ADP and P_i regulate cardiac oxidative phosphorylation, a fascinating alternative explanation is that the first changes in PCr measured with NMR spectroscopy took exclusively place in or near the myofibrils, and that a metabolic wave must then travel with some delay to the mitochondria to stimulate oxidative phosphorylation. The t_mito slows with falling temperature, intracellular acidosis, and sometimes also during reperfusion following ischemia and with decreased mitochondrial aerobic capacity. In conclusion, the study of the dynamic adaptation of cardiac oxidative phosphorylation to demand using the mean response time of cardiac mitochondrial O₂ consumption is a very valuable tool to investigate the regulation of cardiac mitochondrial energy metabolism in health and disease.     

Age-associated increase in PGE2 synthesis and COX activity in murine macrophages is reversed by vitamin E

We previouslyshowed that increased macrophage andPGE₂ production with age is due toenhanced cyclooxygenase (COX) activity and COX-2 expression. This studydetermined the effect of vitamin E supplementation on macrophagePGE₂ synthesis in young and old mice and its underlying mechanism. Mice were fed 30 or 500 parts permillion vitamin E for 30 days. Lipopolysaccharide (LPS)-stimulated macrophages from old mice produced significantly morePGE₂ than those from young mice.Vitamin E supplementation reversed the increasedPGE₂ production in old mice buthad no effect on macrophage PGE₂production in young mice. In both LPS-stimulated and unstimulated macrophages, COX activity was significantly higher in old than in youngmice at all intervals. Vitamin E supplementation completely reversedthe increased COX activity in old mice to levels comparable to those ofyoung mice but had no effect on macrophage COX activity of young miceor on COX-1 and COX-2 protein or COX-2 mRNA expression in young or oldmice. Thus vitamin E reverses the age-associated increase in macrophagePGE₂ production and COX activity.Vitamin E exerts its effect posttranslationally, by inhibiting COXactivity.

     

Follow-Up Study of Hypervariable Region Sequences of the Hepatitis C Virus (HCV) Genome in an Infant with Delayed Anti-HCV Antibody Responses

An infant born prematurely and infected with hepatitis C virus (HCV) one month after birth was followed for 4.5 years. The patient did not produce detectable anti-HCV antibodies until two years after the onset of hepatitis. Before seroconversion, a single clone of HCV, as determined by quasispecies of the hypervariable region (HVR) of the HCV genome, was almost exclusively found in the serum. After seroconversion, however, another distinct lineage of HCV clones replaced it within half a year. As HCV infection persisted further in the presence of anti-HCV antibodies, many derivatives of both sequence lineages emerged to exhibit the typical quasispecies feature of HVR sequences. Neither seroconversion nor the changes in HVR sequences influenced the serum aminotransferase titers.     

Enrichment of Vibrio parahaemolyticus in a Simple Medium

A medium which contained 3% NaCl and 0.2% Teepol in 1/15 M phosphate buffer was prepared and was evaluated to be a useful enrichment medium for the isolation of Vibrio parahaemolyticus in marine specimens. Glucose salt Teepol broth produced a poorer result than direct culture.     

The molecular weight and thiol residues of acetyl-coenzyme A synthetase from ox heart mitochondria

1. A constant molecular weight of 57000 was obtained by gel filtration of highly purified acetyl-CoA synthetase over a 1000-fold range of enzyme concentrations. The amino acid analysis is reported. 2. With native enzyme at 20 degrees C the relatively rapid reaction of four thiol residues with p-hydroxymercuribenzoate caused an immediate inhibition reversible by either CoA or mercaptoethanol. Other substrates did not protect against this rapid inhibition. 3. The much slower reaction of the remaining four thiol residues was independent of the concentration of the mercurial, first-order with respect to enzyme, and had a large energy of activation (+136kJ/mol), suggesting that a conformation change in the protein was rate-limiting. This slow phase of the reaction was accompanied by an irreversible inactivation of the enzyme. 4. The effects of substrates on this irreversible inactivation at pH7.0 in 5 mm-MgCl(2) indicated strong binding of ATP and pyrophosphate by the enzyme (concentrations for half-maximal effects, K((1/2)), were <30mum and <10mum respectively) and weaker binding of acetyl-CoA (K((1/2)) about 1 mm), AMP (K((1/2)) about 2mm) and acetate. In the presence of acetate, MgCl(2) and p-hydroxymercuribenzoate, titration of the enzyme with ATP revealed at least two ATP binding sites/mol. 5. The experiments suggest that reaction of the thiol residues with mercurial causes loss of enzymic activity by altering the structure of the enzyme, rather than that the thiol residues play a direct role in the catalysis.     

Highly Disordered Amyloid-β Monomer Probed by Single-Molecule FRET and MD Simulation

Monomers of amyloid-β (Aβ) protein are known to be disordered, but there is considerable controversy over the existence of residual or transient conformations that can potentially promote oligomerization and fibril formation. We employed single-molecule Förster resonance energy transfer (FRET) spectroscopy with site-specific dye labeling using an unnatural amino acid and molecular dynamics simulations to investigate conformations and dynamics of Aβ isoforms with 40 (Aβ40) and 42 residues (Aβ42). The FRET efficiency distributions of both proteins measured in phosphate-buffered saline at room temperature show a single peak with very similar FRET efficiencies, indicating there is apparently only one state. 2D FRET efficiency-donor lifetime analysis reveals, however, that there is a broad distribution of rapidly interconverting conformations. Using nanosecond fluorescence correlation spectroscopy, we measured the timescale of the fluctuations between these conformations to be ～35 ns, similar to that of disordered proteins. These results suggest that both Aβ40 and Aβ42 populate an ensemble of rapidly reconfiguring unfolded states, with no long-lived conformational state distinguishable from that of the disordered ensemble. To gain molecular-level insights into these observations, we performed molecular dynamics simulations with a force field optimized to describe disordered proteins. We find, as in experiments, that both peptides populate configurations consistent with random polymer chains, with the vast majority of conformations lacking significant secondary structure, giving rise to very similar ensemble-averaged FRET efficiencies.