Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites.     

Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2+-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria.

1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2--3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.     

Endothelium specific Weibel-Palade bodies in a continuous human cell line,EA.hy926

Summary Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor. Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific organelle in a continuous, vigorously replicating human cell line.     

NO mediates effects of estrogen on central regulation of blood pressure in restrained,ovariectomized rats

We tested the hypotheses that estrogen replacement in ovariectomized (OVX) rats attenuates cardiovascular responses to psychological stress and that nitric oxide (NO) in the brain mediates these effects. Female rats were OVX; one group received 17beta-estradiol (OVX-E) for 11-12 days and the other received vehicle (OVX-V). Seven days after OVX, OVX-E and OVX-V rats were chronically instrumented for arterial pressure measurements and intracerebroventricular injections. Later (4-5 days), OVX-E and OVX-V rats received intracerebroventricular injections of NG-nitro-l-arginine (88 microg/kg), an inhibitor of constitutive NO production, or vehicle. Mean arterial pressure (MAP) and heart rate responses were then measured in conscious rats exposed to two cycles of 1-h restraint/1-h rest. We show that MAP responses in restrained OVX-E rats were attenuated both during restraint and during rest. Although inhibition of NO production in the brain had no effect on MAP responses to restraint in OVX-V rats, it augmented responses in restrained OVX-E rats, especially during periods of rest, so that MAPs in restrained OVX-E and OVX-V rats were indistinguishable. Finally, NO levels in hypothalami and brain stems were elevated in restrained OVX-E, but not OVX-V, rats compared with their respective unrestrained controls. These results show that estrogen replacement in OVX rats reduces arterial pressure responses to psychological stress and that these effects are mediated, at least in part, by NO.     

The DNA sequence on bacteriophage G4 recognized by the Escherichia coli B restriction enzyme.

Bacteriophage G4 possesses a single EcoB site located in the overlap between restriction fragments HinfI-12 and HaeIII-6. The sequence 5′-T-G-A … 8N … T-G-C-T occurs once in this segment and nowhere else in the DNA sequence of G4. Four independent G4 mutants that were not restricted by Escherichia coli B possessed the sequence 5′-T-G-A … 8N … T-G-C-C. The common sequence shared by the previously mapped EcoB sites on φXsB1, simian virus 40, f1, and fd DNAs is 5′-T-G-A … 8N … T-G-C-T … 9N … T. However, the sequence in the region of the G4 EcoB site contains an A instead of the final T conserved in these other examples. When the G4 EcoB site is aligned with the other EcoB sites, there are no conserved residues within 50 bases of the common sequence, 5′-T-G-A … 8N … T-G-C-T, except for those seven residues. The analysis of the EcoB site on G4 provides further evidence that only those seven bases are recognized by the E. coli B restriction enzyme.     

High-precision,high-throughput stability determinations facilitated by robotics and a semiautomated titrating fluorometer

The use of statistical modeling to test hypotheses concerning the determinants of protein structure requires stability data (e.g., the free energy of denaturation in H(2)O, DeltaG(HOH)) from hundreds of protein mutants. Fluorescence-monitored chemical denaturation provides a convenient method for high-precision, high-throughput DeltaG(HOH) determination. For eglin c we find that a throughput of about 20 min per protein can be attained in a two-channel semiautomated titrating fluorometer. We find also that the use of robotics for protein purification and preparation of the solutions for chemical denaturation gives highly precise DeltaG(HOH) values in which the standard deviation of values from multiple preparations (+/-0.051 kcal/mol) differs very little from multiple measurements from a single preparation (+/-0.040 kcal/mol). Since the variance introduced into model fitting by DeltaG(HOH) increases as the square of measurement error, there is a premium on precision. In fact, the fraction of stability behavior explicable by otherwise perfect models goes from 98% to only 50% over the error range commonly reported for chemical denaturation measurements (0.1-0.6 kcal/mol). We have found that the precision of chemical denaturation DeltaG(HOH) measurements depends most heavily on the precision of the instrument used, followed by protein purity and the capacity to precisely prepare the solutions used for titrations.     

High capacity gel preparative electrophoresis for purification of fragments of genomic DNA

We have designed a high-capacity gel electrophoretic device for the purification of large amounts of restriction endonuclease fragments of genomic DNA. This device exploits the high resolution of gel electrophoresis in conjunction with an electronic system permitting discontinuous sample elution over a large gel surface area. This feature preserves resolution and greatly increases capacity and yield. The resulting DNA fractions may be used in restriction endonuclease, ligation, transfection, and transformation reactions without further extensive manipulation. Furthermore, DNA fragments of a broad size range are recovered with high efficiency from the gel.     

Transposition of a long member of the L1 major interspersed DNA family into the mouse beta globin gene locus.

A long member of the highly repeated long interspersed DNA family L1Md (for L1 in Mus domesticus) has integrated by transposition into a target site which lies between the two adult beta globin genes of mouse. DNA hybridization and nucleotide sequence analysis show that this target site, which is part of the single copy DNA flanking the globin genes, is interrupted by the L1 element in one chromosome but is uninterrupted in both allelic and ancestral chromosomes. Other large DNA rearrangements of the region between the two adult beta globin genes are also associated with these allelic chromosomes, and include insertions or deletions of both single copy DNA and simple and complex repetitive DNA. This has caused extensive reorganization of this intergenic region. However, the distance between the two genes flanking this region remains conserved, suggesting that the spacing of the globin genes may be subject to conservative selection.