Different requirements for productive interaction between the active site of HIV-1 proteinase and substrates containing -hydrophobic*hydrophobic- or -aromatic*pro- cleavage sites.

The sequence requirements for HIV-1 proteinase catalyzed cleavage of oligopeptides containing two distinct types of junctions (-hydrophobic*hydrophobic- or -aromatic*Pro-) has been investigated. For the first type of junction (-hydrophobic*hydrophobic-) the optimal residues in the P2 and P2' positions were found to be Val and Glu, respectively, in accord with recent statistical analysis of natural cleavage sites [Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., & Kézdy, F. J. (1991) J. Biol. Chem. 266, 14554-14561]. For the -aromatic*Pro- type of junction, in the specific sequence context studied here, the value of Glu in the P2' position was again observed. An explanation for the inefficient cleavage observed for peptides with the sequence -Val-Tyr*Pro- has been provided from molecular modeling of the putative enzyme-substrate complex. A significant effect upon cleavage rates due to the amino acid in the P5 position has also been documented. While lysine in the P5 position in one sequence of the -hydrophobic*hydrophobic- type produces a peptide cleaved very efficiently (kcat greater than 15 s-1 for Lys-Ala-Arg-Val-Nle*p-nitrophenylalanine-P2'-Ala-Nle-NH2, for P2' = Glu, Gln, Ile, Val, or Ala), for substrates of the -aromatic*Pro- type, the P5 residue can exert either a positive or negative effect on cleavage rates. These results have again been interpreted in light of molecular modeling. We suggest that interaction of the substrate sequence on the periphery of the active site cleft may influence the match of the enzyme-substrate pair and, hence, control the efficiency of catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Psychophysiological responsivity on a laboratory stress task: Methodological implications for a stress-muscle hyperactivity pain model

A stress-muscle hyperactivity-pain (SMP) model has been proposed to explain the etiology of certain musculoskeletal pain disorders. According to this model, subjects should show physiological arousal during periods of stress relative to periods of rest. In a test of this prediction, 31 subjects performed a reaction time task that has been used in previous laboratory studies. Multiple psychophysiological variables were monitored during initial and final 10-minute baselines, during performance on nine 2-minute reaction time tasks, and during 36-second rest intervals following each of the 2-minute tasks. Results showed small but statistically significant differences generally supporting the SMP model when masseter EMG was averaged over time periods of 12 seconds to 2 minutes, but not when masseter EMG was averaged over 10- to 18-minute blocks. These results demonstrated the importance of carefully selecting time intervals for analysis. Additional analyses that compared TMD with symptom-free subjects revealed small differences in EMG that supported the SMP model. Analyses of EMG over shorter time intervals also showed, however, that masseter EMG increased during the 36-second rest interval following performance on a 2-minute stress task; this result suggested that a modification of the SMP model may be necessary.This research was supported in part by Grant 2 S06RR08038-17 funded by the National Institutes of Health.     

The prion protein gene is dispensable for the development of spongiform myeloencephalopathy induced by the neurovirulent Cas-Br-E murine leukemia virus.

The Cas-Br-E murine leukemia virus (MuLV) induces paralysis in susceptible mice that is accompanied by a severe spongiform myeloencephalopathy. These neurodegenerative lesions are very similar to those observed in prion diseases. To determine whether the prion protein gene (Prn-p) product was a downstream effector of this neurovirulent MuLV, we inoculated Prn-p(-/-) knockout homozygote and control heterozygote or wild-type mice with this retrovirus. All groups developed typical paralysis and spongiform encephalopathy, and no differences in clinical or histological phenotypes were observed between these groups. These results indicate that the Cas-Br-E MuLV does not require the prion protein to induce lesions. Thus, MuLV and prion proteins may induce a very similar disease through distinct pathways, or the viral Env protein, which harbors the primary determinant of pathogenicity, may act in a common pathway but downstream of the prion protein.     

Non-reciprocal cross-incompatibility in Trichogramma deion

In non-reciprocal cross-incompatibility (NRCI), the crossing of a female of a strain A with a male of a strain B results in hybrid offspring, whereas the reciprocal cross produces few or no hybrids. Only females are of hybrid origin in Hymenoptera because they arise from fertilized eggs; males arise from unfertilized (haploid) eggs. Crosses between many strains of Trichogramma deion showed some degree of NRCI. Crosses between a T. deion culture collected in Seven Pines, California (SVP) with one from Marysville, California (MRY) showed an extreme form of NRCI in which practically no female offspring was produced when MRY females were crossed with SVP males. The reciprocal cross produced a close to normal proportion of female and male offspring. Detailed studied of this cross indicated that 1) the female offspring produced in the compatible interstrain cross were not the result of parthenogenesis but were true hybrids, 2) the incompatible interstrain cross did not produce female offspring because fertilized eggs died during development, 3) the death of these eggs could not be prevented by either antibiotic or temperature treatment, 4) cytoplasmically inherited factors causing NRCI could be discounted because backcrossed females with the genome of MRY and the cytoplasm of SVP, exhibit the NRCI relationship characteristic of their genome. Therefore the NRCI between these strains appears to be caused by a modification coded for by the nuclear genes of MRY that results in incompatibility when SVP sperm fertilizes MRY eggs. In addition the level of incompatibility in crosses between the SVP females and MRY males is temperature sensitive, the higher the rearing temperature the lower the level of compatibility.     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

Comparison of techniques for measuring pulse-wave velocity in the rat

Mitchell, Gary F., Marc A. Pfeffer, Peter V. Finn, andJanice M. Pfeffer. Comparison of techniques for measuringpulse-wave velocity in the rat. J. Appl.Physiol. 82(1): 203-210, 1997.We evaluatedmethods for measuring average and regional pulse-wave velocity alongthe full length of the aorta in 18-mo-old ether-anesthetized malespontaneously hypertensive rats. Catheter-tip manometers were placed inthe ascending and descending thoracic aorta via the right carotid andleft femoral arteries, respectively. As the distal catheter waswithdrawn at 1-cm intervals, the relationship between distal catheterinsertion distance and distance between transducers was determined fromthe intercept of the insertion distance vs. transmission delayregression line. Methods that assessed the foot-to-foot time delaybetween pressures accurately predicted the separation between catheters(measured distance of 14.3 cm; intercept of 14.0 ± 0.5 cm;P = not significant) were highlyreproducible (coefficient of variation of 2.3% for repeated measurements) and showed minimal variability (range 509 ± 30 to 600 ± 29 cm/s) along the full length of the aorta. Methods that madeuse of the pressure-pressure transfer function were spatially (range ofvalues along the aorta 367 ± 17 to 722 ± 39 cm/s) and temporally more variable, especially during vasoconstriction with methoxamine, due to the effects of reflected waves.