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Biomedical subjects

J M Weber

Publications and source records attributed to J M Weber.

At least 55 records · Page 3Linked to original sources
PubMed1996

Inhibition of adenovirus infection with protease inhibitors.

The effect of a series of cysteine and serine protease inhibitors was tested on the growth of human adenovirus type 2 in tissue culture. In accordance with the nature of the adenovirus protease, only the cysteine protease inhibitors were effective in significantly reducing the production of infectious virus. Addition of the inhibitors to the medium 18 h after infection gave IC50 of 30, 40 and 80 nM with N-ethylmaleimide, leupeptin and E64c, respectively. Several lines of evidence suggest that inhibition of infectious virus formation operated through the inhibition of the viral protease rather than cellular toxicity: (a) the yield of physical particles declined only 4-5-fold, while that of infectious virus declined 3-7 orders of magnitude, (b) these particles contained unprocessed precursor proteins and (c) pulse-chase experiments showed that the inhibitors prevented the efficient processing of viral precursor proteins. We conclude that the cysteine protease inhibitors efficiently depress the formation of infectious adenovirus by inhibiting the viral protease.

Adenoviruses, Human↗
PubMed1996

Effect of endurance training on glycerol kinetics during strenuous exercise in humans.

Glycerol kinetics were evaluated during high-intensity exercise in five untrained and five endurance-trained subjects. Glycerol rate of appearance (Ra) in plasma was determined by infusing [2H5]glycerol during rest and 60 minutes of cycle ergometer exercise performed at 70% V02 peak. Mean plasma glycerol concentration was greater in trained than untrained subjects throughout exercise (P<.05). The average glycerol Ra during exercise and the integrated lipolytic response to exercise, expressed as total glycerol Ra above baseline, were both greater in trained (7.85 +/- 0.72 micromol x kg(-1) x min(-1) and 289 +/- 50 micromol x kg(-1) x h(-1), respectively) than in untrained (5.68 +/- 0.90 micromol x kg(-1) x min(-1), and 198 +/- 31 micromol x kg(-1) x h(-1), respectively) subjects (P<.05). We conclude that whole-body lipolytic rates are greater in endurance-trained athletes than in sedentary controls during high-intensity exercise performed at the same relative intensity.

Adult↗
PubMed1996

Adenovirus type 2 endoprotease: isoforms and redox effects.

The cysteine protease encoded by adenovirus type 2 contains eight cysteines, some of which are involved in catalysis and enzyme activation. Here we investigated the effects of oxidation, mercapto-ethanol, dithiothreitol, diamide and protein disulphide isomerase on wild-type and mutant enzymes. Three isoforms of the enzyme were detected in infected cells and a fourth in preparations of purified recombinant enzyme. The latter isoform was absent in preparations of enzyme mutated at any of the three conserved cysteines, C-104, C-122 and C-126. Enzyme activity could be stimulated by agents other than the authentic activating peptide (pVIc), such as cysteamine, though less efficiently. Diamide at low concentrations stimulated the activity of the ts1 enzyme, but inhibited both ts1 and wild-type enzyme at higher concentrations. Protein disulphide isomerase failed to restore enzyme activity to the oxidized isoform. The present studies in combination with previous results using mutants appeared to rule out amino acids C-67, C-122, C-126 and C-127, leaving the two remaining semi-conserved C-17 and C-40 and the conserved C-104 as potential candidates for binding peptide pVIc.

Adenoviruses, Human↗
PubMed1996

Rubella reimmunization: comparative analysis of the immunoglobulin G response to rubella virus vaccine in previously seronegative and seropositive individuals.

Rubella virus (RV)-specific immunoglobulin G (IgG) antibodies were studied in military recruits undergoing unselected immunization with live attenuated measles, mumps, and rubella virus (MMR) vaccine. Three different whole-RV enzyme immunoassays (EIAs) and an epitope-specific EIA with a synthetic peptide (BCH-178c) representing a heutralization domain on the RV E1 envelope protein were used. Before vaccination, 84.2, 87.7, and 84.5% of the subjects tested (n = 399) were found to be seropositive (> 10 IU/ml or assay equivalent) by the three whole-RV EIAs, respectively, while only 82.5% were seropositive by the BCH-178c EIA. Although prevaccination seropositivity rates were similar for the whole-RV EIAs (sensitivity, 94 to 100%), many sera considered seropositive by the whole-RV EIAs had E1 peptide EIA antibody levels of < 10 IU/ml (sensitivity, 77.4 to 80.7%). One month after vaccination, 97.8, 97.2, and 93.5% of the subjects who were followed (n = 356) were seropositive by the three whole-RV EIAs, respectively, while 89% had BCH-178c peptide-specific IgG titers of > 10 IU/ml. After vaccination, depending on the assay used, up to 20.6% of initially seropositive individuals exhibited a greater than fourfold increase in RV-specific IgG, while up to 47.3% showed a greater than twofold increase. Increased antibody titers after vaccination (seroboosting) were most frequently associated with low levels of BCH-178c peptide-specific IgG before vaccination. RV protein-specific IgG was also studied by immunoblot assays in a subset (n = 56) of individuals receiving the MMR vaccine. Of these, 89.4 and 91.1% exhibited RV protein (E1, E2, and C protein)-specific IgG before and after vaccination, respectively. Seroboosting (two- to fourfold increase in EIA titers of individuals seropositive by the whole-RV EIA before vaccination) was usually accompanied by a shift in the IgG immunoblot pattern from a single (E1) to multiple (E1-E1, E1-C, or E1-E2-C) specificities, suggesting exposure of new epitopes as a result of viral replication.

Adolescent↗
PubMed1996

Design of the oxygen and substrate pathways. I. Model and strategy to test symmorphosis in a network structure.

This first paper in a series develops a model of structure-function relationships for the oxygen and substrate pathways of oxidative metabolism in working muscle. This will be used in the subsequent experimental papers in asking how biological structures are designed if they serve more than one function and whether one function can be served by more than one structural pathway. We have used the concept of symmorphosis to address this question; in its original form, it postulates that no more structure is built and maintained at each step in a pathway than is required to meet functional demands. The concept of symmorphosis was developed to deal with the problem of modelling the pathway for oxygen from the environment to mitochondria, essentially a single series of interconnected transfer steps. In the present context, the application of this concept is more complex. Both oxygen and substrates are transported directly from the blood to the mitochondria in what appear to be shared steps. The flows along this direct pathway are adjusted during muscular work. However, substrates have an additional option. They can be stored intracellularly as lipid droplets or glycogen, and thus their supply to mitochondria can occur in two steps separated in time: from capillaries to stores during rest, and from stores to mitochondria during work. The integrated pathways have a network structure and the functional flows are partitioned to different branches of the network, and we must ask whether the partitioning of fluxes is related to design constraints. The principle of symmorphosis predicts that the best use is made of the available options and that the design of each step is matched to the specific functional demand in view of a balance to be achieved over the entire network. This will be tested in subsequent papers by determining maximal flows for oxygen, carbohydrates and lipids through each of the transport steps and their respective structural capacities, comparing dogs and goats, animals of the same size whose maximal oxidative capacities differ by more than twofold. Finally, we will ask whether the principle of symmorphosis can be extended to apply to network systems.

Animals↗
PubMed1996

Design of the oxygen and substrate pathways. II. Defining the upper limits of carbohydrate and fat oxidation.

This paper quantifies maximal flows of carbohydrates and lipids through the pathways supplying the mitochondria. Maximal flow rates are the main functional parameter used in testing the principle of symmorphosis, which states that structural capacities are quantitatively matched to functional demand. Only under rate-limiting conditions will all of the structural capacity be used. Dogs and goats were compared to obtain large differences in absolute rates. We exercised the animals for long enough to reach steady-state O2 and CO2 exchange rates at intensities eliciting 40%, 60% and 85% of the maximal rate of oxygen consumption (MO2max). We then calculated rates of fat and carbohydrate oxidation from the ratio of CO2 produced to O2 consumed (the respiratory exchange ratio). The dog's Mo2max was more than twice that of the goat (6517 versus 3026 mumol O2 kg-1 min-1). We found the same pattern of fuel selection as a function of exercise intensity in both species, and it appears to be general to mammals. Maximal rates of fat oxidation were reached at 40% exercise intensity, where 77% of the energy was supplied by fat. As exercise intensity increased, all additional energy was supplied by carbohydrates. We conclude that the partitioning of fuel supply to the fat and carbohydrate pathways follows the same pattern in both dogs and goats.

Aerobiosis↗
PubMed1996

Design of the oxygen and substrate pathways. III. Partitioning energy provision from carbohydrates.

This paper quantifies maximal fluxes through the pathway supplying carbohydrates to the mitochondria of muscle cells. Continuous infusions of D-[3-(3)H]glucose together with indirect calorimetry were used to investigate the partitioning of the supply of carbohydrates through the two branches of the pathway: from circulating glucose and from glycogen stores within the muscle cells to the mitochondria. The relative contribution of circulating glucose to total carbohydrate oxidation was small, accounting for only 13% and 23% of the carbohydrate oxidized at exercise intensities approaching MO2max in dogs and goats, respectively. Unexpectedly, maximal rates of circulating glucose oxidation were nearly the same in the two species (when expressed in absolute terms; dog:goat ratio = 1.2), despite the 2.2-fold difference in aerobic capacity. We conclude that the glycogen stores in the muscle cells are the major source of substrates at maximal rates of oxidation, supplying 60-70% of the total energy. Furthermore, it is this branch of the carbohydrate pathway that is adapted to the large difference in aerobic capacity between dogs and goats.

Animals↗
PubMed1996

Design of the oxygen and substrate pathways. IV. Partitioning energy provision from fatty acids.

This paper quantifies the fluxes of fatty acids through the pathways supplying muscle mitochondria with oxidative fuel in exercising dogs and goats. We used continuous infusions of 1-[14C]palmitate and indirect calorimetry to measure fatty acid supply from two sources: the circulation and the triglyceride stores within the muscle cells. Our goal was to determine maximal flux through these two branches of the lipid pathway as key functional parameters for testing the principle of symmorphosis, i.e. that structural capacity is quantitatively matched to functional demand in the oxidative substrate pathways. It is under these rate-limiting conditions that we predict that all of the structural capacity will be used. Maximal rates of fatty acid oxidation were reached at low exercise intensities of 40% Mo2max. Fatty acids from the circulation supplied only a small fraction (15-25%) of the total fat oxidized under these conditions. Although dogs were able to oxidize circulatory fatty acids faster than goats, maximal rates were not in proportion to the 2.2-fold difference in aerobic capacity between the two species. Dogs compensated for their relatively lower use of circulatory fatty acids by oxidizing more triglycerides from lipid droplets in their muscle cells. We conclude that fatty acids from intramuscular triglyceride stores are a major source of fuel during maximal rates of lipid oxidation. Furthermore, it is this branch of the fatty acid pathway that is adapted to the large difference in aerobic capacity between dogs and goats.

Animals↗
PubMed1996

Design of the oxygen and substrate pathways. V. Structural basis of vascular substrate supply to muscle cells.

This paper quantifies the structural capacity of the transport steps for oxygen, glucose and fatty acids from the blood in capillaries to the cytosol of muscle cells and compares it with maximal rates of oxygen and substrate transport measured in the same animals and reported in the preceding papers of this series. Dogs have relatively more muscle per unit body mass than goats (37 versus 26%), but the maximal rate of oxidation per gram of muscle is still larger in the dog by a factor of 1.55. The maximal rates of substrate supply from the circulation are similar in both species. We predict that these differences in physiological parameters should be matched by proportional differences in structural capacity. We find that capillary volume and surface area are matched to maximal oxygen demand. The rate of vascular substrate supply is proportional neither to the capillary surface area nor to the length of intercellular junctions. The sarcolemmal surface area per gram of muscle is the same in both species. Using the physiological data presented in the companion papers of this series, we have calculated the maximal flux densities of circulatory glucose and fatty acids across the capillary wall and the sarcolemma. We find, for both substrates, that the flux densities across the sarcolemma reach a maximum at nearly the same level and at low exercise intensities in both species. In contrast, the flux densities across the capillary surface and the endothelial junctions are higher in goats than in dogs. We conclude that the capillaries are designed for O2 supply up to maximal rates of oxidation but not for the supply of the substrates (glucose and fatty acids) at the rates required at high exercise intensities. These are limited by the transport capacities of the sarcolemma.

Animals↗
PubMed1996

Design of the oxygen and substrate pathways. VI. structural basis of intracellular substrate supply to mitochondria in muscle cells.

This paper quantifies the structures involved in the transport and oxidation of carbohydrates and fatty acids within the muscle cell. The structural capacity is measured on whole-body random samples of the musculature of dogs and pygmy goats and compared with maximal rates of oxygen consumption and substrate oxidation. Comparing dogs and goats of the same body size provided a 1.55-fold difference in the maximal rate of oxidation when related to muscle mass. As in previous studies, we found that the volume of mitochondria was approximately proportional to aerobic capacity. The maximal glucose flux from intracellular stores to mitochondria is 1.6 times greater in the dog than in the goat; we find that the amount of glycogen stored in the muscle cells is 4.2 times as great in the dog, but part of the intracellular glycogen pool is used for anaerobic rather than for oxidative metabolism. The maximal fatty acid flux from intracellular stores to mitochondria is 1.5 times larger in the dog, and the amount of lipid stored is 2.3 times as great in the dog. Every lipid droplet is in direct contact with the outer membrane of a mitochondrion and the contact surface area is 3.6 times greater in the dog than in the goat. Additional measurements are needed to investigate the role of structural limitation at this step. The amount of substrates stored intracellularly in the muscle cells of the dog is about twice as much as would match the differences in the maximal rates of utilization. This allows the endurance-specialized dogs to run for longer periods at higher rates of oxidation.

Animals↗
PubMed1996

Design of the oxygen and substrate pathways. VII. Different structural limits for oxygen and substrate supply to muscle mitochondria.

This paper integrates the results of a series of studies on the supply of O2 and substrates for oxidative muscle metabolism and draws conclusions on the role of structural design in partitioning and limiting substrate supply. The studies compared dogs and goats exercising at different intensities and combined physiological, biochemical and morphometric investigations. In both species, the rate of fatty acid oxidation reached an upper limit at low exercise intensities, and only glucose consumption was increased at higher exercise intensities. The supply of both glucose and fatty acids from the capillaries reached maximal rates at low exercise intensities; this limitation is related to the design of the sarcolemma as calculations suggest that the endothelium introduces only a small resistance to substrate flux. From these findings, it appears that the capillaries are designed to satisfy O2 supply up to maximal O2 demand. The increase in substrate supply to the mitochondria at higher exercise intensities is achieved by drawing on intracellular stores of glycogen and lipids. The size of these stores is larger in dogs than in goats, providing the athletic species with twice the fuel reserves. These findings are interpreted on the basis of a network model with fluxes partitioned between direct and indirect pathways and with some structures shared by more than one function. Whereas O2 is supplied through a direct pathway, the supply of both substrates is split temporally to allow, during exercise, immediate fuel supply to the mitochondria from intracellular stores; these are replaced from the vasculature, during periods of rest, to a size commensurate with high rates of combustion. Considering this complexity, we conclude that the results are compatible with the principle of symmorphosis applied to a network structure and that the adjustment of design to functional demand involves different structures for O2 and for substrates.

Animals↗
PubMed1995

The adenovirus protease is required for virus entry into host cells.

We have analyzed the mechanisms used by adenovirus to gain entry into the host cell. Using both virus infection and the ability of adenovirus particles to enhance polylysine/DNA uptake as a measure of virus entry, we have demonstrated that the adenovirus-encoded 23K protease is required for two functions in the infection process. A proteolytic processing of the capsid is required to generate a virus capsid that can increase membrane interactions at pH 5. We have found that forms of adenovirus capsid that have not undergone the processing reactions (immature capsids) are deficient in their ability to disrupt membranes at pH 5 and are unable to enhance the entry of polylysine/DNA complexes. A second role of the protease was revealed by experiments using inhibitors of the protease. Mature virus capsids lose their ability to enhance gene delivery and become noninfectious after exposure to inhibitors of the protease (1 microM N-ethylmaleimide, 100-300 microM copper chloride, 1 microM MDL28170, or anti-protease antiserum), suggesting that the viral protease activity is required during the cellular entry process.

Adenoviruses, Human↗
PubMed1995

Electrophoretic and spectral characterization of wild type and mutant adenovirus protease.

The P137L mutation in the adenovirus type 2 protease results in a temperature-sensitive protein-trafficking phenotype expressed during infection but not in vitro. Homology-derived secondary structure prediction placed the mutation within an externally disposed loop. Circular dichroism and urea gradient gel electrophoresis suggested that, unlike other thiol proteases, the Ad2 protease is comprised of a single conformational domain. The -0.32-kcal difference in the free energy of folding and the temperature-independent CD spectra of the mutant and wild type enzymes point to a very subtle structural change as the cause of the in vivo phenotype.

Adenoviruses, Human↗
PubMed1995

Proline 137 is critical for adenovirus protease encapsidation and activation but not enzyme activity.

Infection with the adenovirus type 2 ts1 mutant at the nonpermissive temperature resulted in the production of noninfectious virions. This has been ascribed to the P137L mutation in the virus-encoded cysteine protease which causes a defect in protease activity. Here we have examined the ts1 defect in detail as a means of learning more about the viral protease. The ts1 protease accumulated in the nucleus normally and was found associated with incomplete particles as was the case with wt. This enzyme was active in both wt and ts1 incomplete particles produced at 39 degrees (ts1-39 TCs), provided they were dissociated with 4 M urea. While the wt protease was packaged into complete particles, the ts1-39 particles were totally devoid of protease. This defect was nearly completely corrected by addition of the 11-residue activating peptide PVIc (GVQSLKRRRCF) to the medium late in infection. Rescue of ts1 occurred via restoration of enzyme activity and packaging of the ts1 enzyme into complete virions. Recombinant ts1 enzyme was not temperature sensitive. The P137L mutation responsible for the ts1 defect appeared therefore to be an in vivo phenotype involving apparently linked events of protease packaging and activation mediated by the PVI protein.

Adenoviridae↗
PubMed1995

Predicted structure of the adenovirus DNA binding protein.

The DNA sequence of a portion of the MAV1 SmaI-D fragment coding for the C-terminal 147 amino acids of the adenoviral DNA-binding protein (DBP) has been determined. A multiple sequence alignment was constructed of the MAV1 fragment and the DBPs of Ad.2, 4, 5, 7, 12, 40, and 41 to examine the degree of conservation of features that have been mapped on the Ad.2 DBP and to identify further conserved features. The less conserved N-terminal segment of the protein contains two nuclear localization signals and two acidic regions, the host range region, and all of the 11 phosphorylation sites. The highly conserved C-terminal segment contains a potential leucine zipper and zinc finger motifs. These sequence features were mapped onto a predicted secondary structure of the Ad.2 DBP.

Adenoviridae↗