Electrical and mechanical responses of rat middle cerebral arteries to reduced PO2 and prostacyclin.

Isolated rat middle cerebral arteries were perfused and superfused with physiological salt solution equilibrated with a control (approximately 140 mmHg) or reduced (approximately 35-40 mmHg) PO2. In other experiments, cerebral arteries were isolated and prostacyclin release was determined by radioimmunoassay for 6-ketoprostaglandin F1alpha. Equilibration of the vessels with reduced PO2 (35 mmHg) solution caused a significant increase in prostacyclin release relative to control PO2 (140 mmHg) conditions. Exposure of middle cerebral arteries to reduced PO2 caused vascular smooth muscle (VSM) hyperpolarization and vessel relaxation, which could be blocked by 1 microM glibenclamide, an inhibitor of the ATP-sensitive K+ channel, but not by 1 mM tetraethylammonium (TEA), an inhibitor of the Ca2+-activated K+ channel. Glibenclamide also inhibited VSM hyperpolarization and vasodilation in response to the stable prostacyclin analog iloprost, but TEA did not affect iloprost-induced dilation of the vessel. Endothelial removal eliminated the electrical and mechanical responses of the arteries to reduced PO2, but vessel responses to iloprost were similar to those of intact vessels. The results of this study are consistent with the hypothesis that hypoxic dilation of rat middle cerebral arteries is due to VSM hyperpolarization mediated by prostacyclin-induced activation of glibenclamide-sensitive K+ channels.

Hypoxia-induced hyperpolarization is not associated with vasodilation of bovine coronary resistance arteries.

The effect of reduced PO2 on the transmembrane potential and diameter of small cannulated coronary resistance arteries was evaluated by microelectrode and videomicroscopic methods. Bovine coronary resistance arteries (158 +/- 8 microm ID) were cannulated with glass micropipettes and perfused and superfused with physiological salt solution. Lowering the PO2 of the physiological salt solution from 140 +/- 4 to 36 +/- 2 mmHg increased the smooth muscle cell transmembrane potential from -51 +/- 2 to -62 +/- 2 mV in both endothelium-intact and -denuded coronary resistance arteries. This hyperpolarization was blocked by superfusion with the K+-channel blocker glibenclamide (1 microM). However, low PO2 did not significantly dilate either endothelium-intact or -denuded coronary resistance arteries, although superfusion with 1 microM cromakalim, a K+-channel activator, induced a 6-mV hyperpolarization and increased the diameter by 33 +/- 10 microm. These results suggest that reduced PO2 directly hyperpolarizes the vascular smooth muscle of coronary resistance arteries by activation of glibenclamide-sensitive K+ channels, but other nonvascular mechanisms may mediate the vasodilation response to low PO2.

Mutations that alter the activity of the Rous sarcoma virus protease.

Mutations designed by analysis of the Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV)-1 protease (PR) crystal structures were introduced into 1) the substrate binding pocket, 2) the substrate enclosing "flaps," and 3) surface loops of RSV PR. Each mutant PR was expressed in Escherichia coli. Changes in activity were detected by following cleavage of a truncated (NC-PR) precursor polypeptide in E. coli and cleavage of synthetic peptide substrates representing RSV and HIV-1 PR cleavage sites in vitro. Mutations in the substrate binding pocket exchanged amino acid residues located close to the substrate in the HIV-1 PR for structurally equivalent residues in the RSV PR. Changing histidine 65 to glycine (H65G) gave an inactive enzyme, while a double mutant R105P,G106V, as well as the triple mutant, H65G,R105P,G106V, produced enzymes which showed significant activity toward a substrate that represented a HIV-1 cleavage site. Mutating the catalytic aspartate (D37S) or an adjacent conserved alanine to threonine (A40T), produced inactive enzymes. In contrast, the substitution A40S was active, but showed a reduced rate of catalysis. Mutations in the flaps of conserved glycines (G69L, G70L) produced inactive PRs. Two extended RSV PR surface loops were shortened to the size found in HIV-1 PR and resulted in drastically reduced activity. These results have confirmed some of the basic predictions made from structural models but have also revealed unexpected roles and interactions in the protein.

Processing of avian retroviral gag polyprotein precursors is blocked by a mutation at the NC-PR cleavage site.

The avian sarcoma and leukosis viruses (ASLV) encode a protease (PR) at the C terminus of gag which in vivo catalyzes the processing of both gag and gag-pol precursors. The studies reported here were undertaken to determine whether PR is able to cleave these polyproteins while it is still part of the gag precursor or whether the release of its N terminus to form free PR is necessary for full proteolytic activity. To address this question, we created a mutation that disrupts the PR cleavage site between the NC and PR coding regions of the gag gene. This mutation was introduced into a eukaryotic vector that expresses only the gag precursor and into an otherwise infectious clone of ASLV that carries the neo gene as a selectable marker. These constructs were expressed in monkey COS cells or in quail QT35 cells, respectively. Processing was impaired in both systems. Mutant particles were formed, but they contained no mature processed gag proteins. We observed only the uncleaved gag precursor polypeptide Pr76 in one case or Pr76 and a cleaved product of about 60 kDa in the other. Processing of the mutant gag precursor could be complemented in trans by from a wild-type construct, suggesting that the mutation did not induce gross structural alterations in its precursor. Our results suggest that the PR first must be released from its precursor before it can attack other sites in the gag and gag-pol polyproteins and that cleavage at the NC-PR boundary is a prerequisite for the initiation of the PR-directed processing.

Assembly and processing of avian retroviral gag polyproteins containing linked protease dimers.

Assembly and maturation of retroviral particles requires the aggregation and controlled proteolytic cleavage of polyprotein core precursors by a precursor-encoded protease (PR). Active, mature retroviral PR is a dimer, and the accumulation of precursors at sites of assembly may facilitate subunit interaction and subsequent activation of this enzyme. In addition, it has been suggested that cellular cytoplasmic components act as inhibitors of PR activity, so that processing is delayed until the nascent virions leave this compartment and separate from the surface of host cells. To investigate the mechanisms that control PR activity during virus assembly, we studied the in vivo processing of retroviral gag precursors that contain tandemly linked PR subunits in which dimerization is concentration independent. Sequences encoding four different linked protease dimers were independently joined to the end of the Rous sarcoma virus (RSV) gag gene in a simian virus 40-based plasmid vector which expresses a myristoylated gag precursor upon transfection of COS-1 cells. Three of these plasmids produced gag precursors that were incorporated into viruslike particles and proteolytically cleaved by the dimers to mature core proteins that were indistinguishable from the processed products of wild-type gag. The amount of viral gag protein that was assembled and packaged in these transfections was inversely related to the relative proteolytic activities of the linked PR dimers. The fourth gag precursor, which contained the most active linked PR dimer, underwent rapid intracellular processing and did not form viruslike particles. In the absence of the plasma membrane targeting signal, processing of all four linked PR dimer-containing gag precursors was completed entirely within the cell. From these results, we conclude that the delay in polyprotein core precursor processing that occurs during normal virion assembly does not depend on a cytoplasmic inhibitor of PR activity. We suggest that dimer formation is not only necessary but may be sufficient for the initiation of PR-directed maturation of gag and gag-pol precursors.

A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains.

Molecular modeling based on the crystal structure of the Rous sarcoma virus (RSV) protease dimer has been used to link the two identical subunits of this enzyme into a functional, single polypeptide chain resembling the nonviral aspartic proteases. Six different linkages were selected to test the importance of different interactions between the amino acids at the amino and carboxyl termini of the two subunits. These linkages were introduced into molecular clones of fused protease genes and the linked protease dimers were expressed in Escherichia coli and purified. Catalytically active proteins were obtained from the inclusion body fraction after renaturation. The linked protease dimers exhibited a 10-20-fold range in catalytic efficiencies (Vmax/Km) on peptide substrates. Both flexibility and ionic interactions in the linkage region affect catalytic efficiency. Some of the linked protease dimers were 2-3-fold more active than the nonlinked enzyme purified from bacteria, although substrate specificities were unchanged. Similar relative efficiencies were observed using a polyprotein precursor as substrate. Mutation of one catalytic Asp in the most active linked protease dimer inactivated the enzyme, demonstrating that these proteins function as single polypeptide chains rather than as multimers.

Detection of transforming ras proteins containing leucine at position 61 by a new mouse monoclonal antibody, ras(53-69)Leu61.

A monoclonal antibody (mAb) was prepared after immunization of mice with a peptide that corresponds to amino acids 53 to 69 of a transforming ras protein. The amino acid sequence in this region is conserved among all members of the ras protooncogene family in rodent, rabbit, and human cells. The peptide used for immunization differs from the normal sequence by a single residue; Leu replaces Gln at a site corresponding to amino acid 61. A bacterial expression vector was constructed to synthesize H-ras transforming protein that contains this change (rasLeu61). In immunoblotting experiments, the affinity purified mAb, ras(53-69)Leu61, reacts specifically with the purified, bacterially produced rasLeu61 protein and does not react with bacterially produced normal H-ras protein. In immunoblotting experiments with cell lysates, the mAb recognizes the transforming protein in NIH3T3 cells transformed by the c-rasHLeu61 oncogene but fails to react with normal H-ras protein in the same cells or cells which produce 100 times more normal protein than NIH3T3. The mAb immunoprecipitates [35S]methionine-labeled H- and N-rasLeu61 proteins from transformed NIH3T3 cells under conditions in which the cells produce basal levels of the transforming protein, equivalent to the low amount of the normal protein ordinarily present in nontransformed NIH3T3 cells. The antibody fails to immunoprecipitate normal H-ras protein, even when present at high levels, or N-ras protein containing Lys as amino acid 61. Affinity purified mAb ras(53-69)Leu61 also recognizes the transforming ras protein specifically in immunohistochemical staining of tissue culture cells, and this staining is abolished by preincubating the antibody with the corresponding peptide. Staining was not observed with control NIH3T3 cells or cells that produce 100 times more normal H-ras protein than NIH3T3. However, in thin sections of normal human or rabbit skin the antibody reacted strongly with an unknown antigen, in cells of the basal layer of the epidermis, that is neither normal nor transforming ras protein. This new immunological reagent should be useful for the selective identification of Leu61 containing H-, K-, and N-ras transforming proteins in in vitro studies and analyses using rodent, rabbit, or human tissue culture cells. Its utility for direct staining of tissues may be limited to situations in which the presence of transforming protein can be verified by another method such as immunoblotting after gel electrophoresis.

Correlation between H-ras p21TLeu61 protein content and tumorigenicity of NIH3T3 cells.

We have prepared a number of NIH3T3 clonal cell lines that contain an H-ras transforming gene with an A----T transversion at the 61st codon. The clonal lines contain 1 to 3 cell equivalents of the transforming oncogene and some lines look more morphologically transformed than others. Using Y13-238, a rat monoclonal antibody that recognizes H-ras p21 but not Ki- or N-ras in rodent cells, we found that the degree of morphological change is correlated with the relative amount of transforming protein in the selected clonal lines. Nude mice were injected with cells from lines containing different amounts of the transforming protein, ranging from approximately 1 to 10 times the level of normal H-ras protein present in NIH3T3 cells. Tumors arose in all mice that received cells containing the transforming protein. Their time of appearance (tumor latency) was correlated with the number of cells injected and the amount of transforming protein present in each clonal line; however, the subsequent rate of growth and ultimate size of the tumors were similar. Thus, it appears that the transforming protein has a significant effect on some early step in tumor development. Our results also show that relatively low amounts of transforming ras protein are sufficient to cause tumorigenicity in NIH3T3 cells and that higher amounts of the transforming protein cause proportionately faster responses.

Antisera to the variable region of ras oncogene proteins, and specific detection of H-ras expression in an experimental model of chemical carcinogenesis.

Antisera were prepared in mice, rats and rabbits by immunization with peptides corresponding to regions of highest variability, located near the C-termini of four ras proteins. Two of these, H-ras (171-189) and K-rasB (171-186), react uniquely with H-ras and K-rasB gene products in immunoblots and immunoprecipitation reactions. Affinity-purified rabbit H-ras (171-189) antibody detects H-ras p21 in tissue culture cells and in tissue sections. Epithelial cells in normal mouse skin and cells in papillomas and carcinomas, in a mouse model system of chemical carcinogenesis in which mutational activation of H-ras occurs with high frequency, express high levels of H-ras p21 protein. These results suggest an hypothesis to explain the mechanism and preferential activation of particular ras loci in certain neoplasia.

Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons.

The importance of mutational activation of the Ha-ras protooncogene in polycyclic aromatic hydrocarbon-induced mouse skin tumors was investigated in a complete carcinogenesis model using repetitive applications of 7,12-dimethylbenz[a]anthracene (DMBA), or in an initiation-promotion model using a single application of dibenz[c,h]acridine (DB[c,h]ACR) or benzo[a]pyrene (B[a]BP) followed by chronic treatment with phorbol 12-myristate 13-acetate. DNA isolated from carcinomas induced by DMBA or DB[c,h]ACR, but not by B[a]P, efficiently transformed NIH 3T3 cells, and a high percentage of the transformed foci had an amplified Ha-ras gene. Restriction enzyme Southern blot analysis and DNA sequencing revealed that the amplified Ha-ras genes of the transformants had an A----T transversion in the second position of the 61st codon. The same mutation was also detected in primary tumor DNA in a high percentage of the DMBA- or DB[c,h]ACR-induced carcinomas. Identification of the mutation in NIH 3T3 cells transformed with DNA from DB[c,h]ACR-induced benign skin papillomas suggests that it is an early event in skin carcinogenesis. Thus, mutation of the 61st codon of the Ha-ras-1 gene appears to be a critical step in the formation of mouse skin tumors induced in both of the two models tested. Our analyses also delineate two other classes of hydrocarbon-induced carcinomas--namely, tumors whose DNAs efficiently transform 3T3 cells but do not contain mutated ras genes and tumors whose DNAs do not transform 3T3 cells.

Characterization of a solitary long terminal repeat of avian endogenous virus origin.

A recombinant lambda phage library constructed with a partial EcoR1 digest of DNA from a normal RPRL line 15B chicken was screened using 32P-labeled plasmid containing Rous-associated virus (pRAV-2). Nucleotide sequence analyses of a fragment of one subclone revealed the presence of a solitary long terminal repeat (LTR) that is similar to the LTRs of avian endogenous retroviruses ev1 and ev2. This LTR is flanked by unique 6 bp direct repeats characteristic of the target site for duplication of avian leukosis viruses.

Nucleotide sequence of noncoding regions in Rous-associated virus-2: comparisons delineate conserved regions important in replication and oncogenesis.

The nucleotide sequence of the regions flanking the long terminal repeat of Rous-associated virus-2 has been determined. The region analyzed spans the ends of the viral genome and includes the terminus of the env gene, the 3' noncoding region, the 5' noncoding region, and the beginning of the gag gene. These data have been compared with sequences available from other avian retroviruses. The comparisons reveal sections which are highly conserved and others which are quite variable. Sequence homologies within the conserved regions suggest details concerning the mode of origin of the src-transducing viruses. Included in the variable section is a region (XSR) found only in certain strains of Rous-derived virus. Its absence from other oncogenic viruses indicates that these sequences are not required to elicit disease.