Left ventricular dynamics during recovery from exercise.

Left ventricular dynamics during recovery were measured in dogs, 3 min after brief periods of mild, moderate, and severe treadmill exercise. As compared with resting values, stroke volume was unchanged, and the maximum first derivative of the left ventricular pressure was either unchanged or slightly elevated. Increases in heart rate of 20, 26, and 46 beats/min for mild, moderate, and severe exercise appear to be the major factor in augmenting cardiac output during recovery. With moderate and severe exercise, left ventricular end-diastolic diameter increased and continued to be elevated during recovery, whereas end-systolic diameter decreased during exercise but was elevated above resting values during recovery. Therefore, with strenuous exercise, a sympathetic-mediated increase in contractility recedes promptly during the postexercise period but the Frank-Starling mechanism continues to be a factor.     

Functional tRNAs with altered 3' ends.

The CCA trinucleotide is a universally conserved feature of the 3' end of tRNAs, where it serves as the site of amino acid attachment. Despite this extreme conservation, we have isolated functional mutants of tRNA(His) and tRNA(Val1) with altered CCA ends. A mutant that leads to de-repression of the histidine biosynthetic operon in Salmonella typhimurium has been characterized and found to have the CCA end of the sole tRNA(His) species mutated to UCA. However, constructed mutants of tRNA(His) with ACA or GCA ends appeared to be nonfunctional in vivo. Mutants of Escherichia coli tRNA(Val1) with GCA or ACA ends were isolated on the basis of their ability to promote frameshifting at a specific sequence. These same tRNA(Val1) mutants also caused read-through of stop codons that were one, or in some instances two, codons downstream of the valine codon decoded by the mutant tRNA. A startling implication of these data is that disruption of interactions between the CCA end of the tRNA and the large ribosomal subunit promotes these aberrant codon-anticodon interactions on the small ribosomal subunit.     

Quiescent human diploid fibroblasts. Common mechanism for inhibition of DNA replication in density-inhibited and serum-deprived cells

The mechanism for cessation of proliferation in density-inhibited quiescent human diploid fibroblasts (HDF) and serum-deprived quiescent HDF was compared in two ways. Density-inhibited HDF were fused to either replicating HDF or SV40-transformed HDF and DNA synthesis was measured in the resulting heterokaryons. DNA synthesis was inhibited in the replicating HDF nuclei in heterokaryons in a way that suggested that entry into S phase was blocked, but ongoing DNA synthesis was not inhibited. In contrast, DNA synthesis was induced in the quiescent nuclei in heterokaryons formed with SV40-transformed HDF. Previous experiments had shown that serum-deprived HDF also behave in this way in heterokaryons. To test this similarity further, we examined the inhibitory activity of cell membranes prepared from both types of quiescent HDF. We found that both types of quiescent HDF contain DNA synthesis-inhibitory activity that is (1) effective on replicating HDF; (2) ineffective on SV40-transformed HDF; (3) sensitive to heat and trypsin. Thus, these results support the hypothesis that both density-inhibited HDF and serum-deprived HDF share a common mechanism for arrest in G1 phase. They also suggest that a membrane-bound protein plays a role in the inhibition of DNA synthesis in quiescent HDF.     

The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1-/- and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Delta240-361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Delta240-361 accounted for over 90% of the selenium in the plasma of Sepp1Delta240-361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Delta240-361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1-/- mice but they better maintained their whole body selenium. Sepp1Delta240-361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Delta240-361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1-/- mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.     

Effect of pO(2) during Growth on the Gaseous Diffusional Properties of Nodules of Cowpea (Vigna unguiculata L. Walp.)

Adaptations of nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium CB 756) to growth in pO₂ ranging from 1 to 80% O₂ (volume/volume) involved both readily reversible mechanisms of adjustment and more stable alterations which together resulted in nodules with widely ranging resistance to diffusion of gases. Those grown in subambient pO₂ (1-5% O₂ were altered such that rapid diffusional adjustment was unable to prevent irreversible loss of nitrogenase on their transfer to higher levels of O₂. Those cultured in 80% had adapted to over-supply of O₂ such that their transfer to lower levels of O₂ limited both nitrogenase and respiratory CO₂ release. There was also some evidence for `protective respiration.' Measurement of diffusional properties based on gas exchange kinetics indicated that gaseous permeability values for nodules from 5 to 40% O₂ were relatively constant around 20 × 10⁻³ millimeters per second, while those for nodules from 1% O₂ were as high as 67.7 × 10⁻³ millimeter per second and from 80% as low as 6.8 × 10⁻³ millimeters per second. Estimates of the thickness of the diffusion barrier ranged from 7.5 micrometers for nodules from 1% O₂ to 71.9 micrometers in those from 80% O₂.     

Fluorescent photoaffinity labeling of cytochrome P450 3A4 by lapachenole: identification of modification sites by mass spectrometry

While photoaffinity ligands (PALs) have been widely used to probe the structures of many receptors and transporters, their effective use in the study of membrane-bound cytochrome P450s is less established. Here, lapachenole has been used as an effective photoaffinity ligand of human P450 3A4, and mass spectrometry data demonstrating the efficient and specific photoaffinity labeling of CYP3A4 by this naturally occurring benzochromene compound is presented. Without photolysis, lapachenole is a substrate of CYP3A4 and can be metabolized to hydroxylated products by this enzyme. A high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) procedure was developed to analyze small amounts of intact purified CYP3A4, and analysis of the labeled protein showed the presence of one molecule of lapachenole bound per monomer of protein. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis after proteolytic digestion and isolation of fluorescent photolabeled peptides. Two peptide adducts accounting for >95% of the labeled peptides were isolated by HPLC, and both peptides, ECYSVFTNR (positions 97-105) and VLQNFSFKPCK (positions 459-469), were identified by nano-LC/ESI quadrupole time-of-flight (QTOF) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of modification were further localized to positions Cys-98 and Cys-468 for each peptide by nano-LC/ESI QTOF tandem mass spectrometry (MS/MS). The results provided the first direct evidence for interaction between the PAL and the putative B-B' loop region, which may serve as a substrate access channel or as a part of the CYP3A4 active site. In conclusion, benzochromene analogues are effective PALs, which may be used in the study of other cytochrome P450 structures.     

Adenylate gradients and Ar:O(2) effects on legume nodules: I. Mathematical models

Mathematical models were developed to test the likelihood that large cytosolic adenylate concentration gradients exist across the bacteria-infected cells of legume nodules. Previous studies hypothesized that this may be the case to account for the unusually low adenylate energy charge (AEC; 0.65) measured in the plant fraction of metabolically active nodules (M.M. Kuzma, H. Winter, P. Storer, I. Oresnik, C.A. Atkins, D.B. Layzell [1999] Plant Physiol 119: 399-407). Simulations coupled leghemoglobin-facilitated O(2) diffusion into the infected cell, through bacteroid nitrogenase activity, with the ATP demand for transport and ammonia assimilation in the plant fraction of ureide- and amide-producing nodules. Although large cytosolic adenylate gradients were predicted to exist in both nodule types, amide nodules were predicted to have steeper AEC gradients (0.82-0.52) than ureide nodules (0.82-0.61). The differences were attributed to an additional ATP demand for Asn synthesis in the amide nodule. Simulations for nodules transferred to an Ar:O(2) atmosphere predicted a major reduction in the magnitude of adenylate gradients and an increase in the AEC of the plant fraction. Results were consistent with a number of experimental studies and were used to propose an experimental test of the models.     

Exploration of in vitro pro-drug activation and futile cycling by glutathione S-transferases: thiol ester hydrolysis and inhibitor maturation

In addition to glutathione (GSH) conjugating activity, glutathione S-transferases (GSTs) catalyze "reverse" reactions, such as the hydrolysis of GSH thiol esters. Reverse reactions are of interest as potential tumor-directed pro-drug activation strategies and as mechanisms for tissue redistribution of carboxylate-containing drugs. However, the mechanism and specificity of GST-mediated GSH thiol ester hydrolysis are uncharacterized. Here, the GSH thiol esters of ethacrynic acid (E-SG) and several nonsteroidal antiinflammatory agents have been tested as substrates with human GSTs. The catalytic hydrolysis of these thiol esters appears to be a general property of GSTs. The hydrolysis of the thiol ester of E-SG was studied further with GSTA1-1 and GSTP1-1, as a model pro-drug with several possible fates for the hydrolysis products: competitive inhibition, covalent enzyme adduction, and sequential metabolism. In contrast to hydrolysis rates, significant isoform-dependent differences in the subsequent fate of the products ethacrynic acid and GSH were observed. At low [E-SG], only the GSTP1-1 efficiently catalyzed sequential metabolism, via a dissociative mechanism.