Eicosanoid and muscarinic receptor blockade abolishes hyperventilation-induced bronchoconstriction.

This study was designed to test the hypothesis that hyperventilation-induced bronchoconstriction (HIB) results from the combined effects of prostanoid and leukotriene metabolism. A bronchoscope was used in anesthetized dogs to record peripheral airway resistance and HIB before and after combined treatment with inhibitors of cyclooxygenase (indomethacin) and 5-lipoxygenase (MK-0591). Bronchoalveolar lavage fluid (BALF) cells and mediators from hyperventilated and control airways were also measured. Pretreatment with MK-0591 and indomethacin significantly attenuated, but did not abolish, HIB. However, addition of atropine nearly eliminated the residual response. Blockade of eicosanoid metabolism markedly reduced the concentrations of eicosanoids recovered in BALF after hyperventilation. Positive correlations between posthyperventilation BALF prostanoid and epithelial cell concentrations are suggestive of mucosal injury-induced mediator production and release. We conclude that HIB is prevented in the presence of eicosanoid and muscarinic-receptor blockade and that both classes of eicosanoids contribute similarly to the development of HIB.     

Electron spin resonance and electron-spin-echo study of oriented multilayers of L alpha-dipalmitoylphosphatidylcholine water systems.

A detailed electron spin resonance (ESR) study of spin-labeled-oriented multilayers of L alpha-dipalmitoylphosphatidylcholine (DPPC) water systems for low water content (2-10% by weight) is reported with the purpose of characterizing the dynamical and structural properties of model membrane systems. Emphasis is placed on the value of combining such experiments with detailed simulations based on current slow-motional theories. Information is obtained regarding ordering and anisotropic rotational diffusion rates via ESR lineshape analysis over the entire motional range, from the fast motional region through the moderately slow and slow to the rigid limit. This includes the low-temperature gel phase, the liquid crystalline L alpha (1) phase and what appears to be a third high-temperature phase above the L alpha phase. Cholestane (CSL) and spin-labeled DPPC (5-PC, 8-PC, and 16-PC) have been used to probe different depths of the bilayer. While CSL and 5-PC both reflect the high ordering of the bilayer close to the lipid-water interface, CSL appears to be located close enough to the water for the nitroxide to be involved in hydrogen bonding with water molecules. 16-PC reflects the relatively low ordering near the tail of the hydrocarbon chain in the bilayer. Quantitative estimates of ordering and motion are obtained for these cases. The results from CSL indicate that close to the lipid-water interface the DPPC molecule is oriented approximately perpendicular to the bilayer in these low water-content systems. However, all three labeled lipid probes indicate that the hydrocarbon chain of DPPC may be bent away from the bilayer normal by as much as 30 degrees and this evidence is stronger at low temperatures. When cholesterol is added to the DPPC-water system at a concentration greater than or equal to 2.5 mol %, the ordering is greatly increased although the rotational diffusion rate remains almost unaffected in the gel phase. Electron spin echoes (ESE) are observed for the first time from oriented lipid-water multilayers. Results obtained from cw ESR lineshape analysis are correlated with data from ESE experiments, which give a more direct measurement of relaxation times. These results indicate that for detection of very slow motions (close to the rigid limit) ESE experiments are more sensitive to dynamics than continuous wave ESR for which inhomogeneous broadening becomes a major problem.     

Survival of dissociated adrenal chromaffin cells of rat and monkey transplanted into rat brain

Summary Adrenal chromaffin cells from adult rats and monkeys were mechanically dissociated and implanted into the striatum of adult rats by stereotaxic injection. Rat chromaffin cells survived (5%) and showed differentiation by forming processes 1 h-28 days after implantation. Monkey chromaffin cells survived for 48 h but showed very little formation of processes. The method presented allows rapid nonenzymatic dissociation and transplantation of adrenal medullary cells.     

p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease.

The human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55Gag, contains at its C-terminal end a proline-rich, 6-kDa domain designated p6. Two functions have been proposed for p6: incorporation of the HIV-1 accessory protein Vpr into virus particles and virus particle production. To characterize the role of p6 in the HIV-1 life cycle and to map functional domains within p6, we introduced a number of nonsense and single and multiple amino acid substitution mutations into p6. Following the introduction of the mutations into the full-length HIV-1 molecular clone pNL4-3, the effects on Gag protein expression and processing, virus particle production, and virus infectivity were analyzed. The production of mutant virus particles was also examined by transmission electron microscopy. The results indicate that (i) p6 is required for efficient virus particle production from a full-length HIV-1 molecular clone; (ii) a Pro-Thr-Ala-Pro sequence, located between residues 7 and 10 of p6, is critical for virus particle production; (iii) mutations outside the Pro-Thr-Ala-Pro motif have little or no effect on virus assembly and release; (iv) the p6 defect is manifested at a late stage in the budding process; and (v) mutations in p6 that severely reduce virion production in HeLa cells also block or significantly delay the establishment of a productive infection in the CEM (12D-7) T-cell line. We further demonstrate that mutational inactivation of the viral protease reverses the p6 defect, suggesting a functional linkage between p6 and the proteolytic processing of the Gag precursor protein during the budding of progeny virions.     

Effects of second-site mutations on dominant interference by a human immunodeficiency virus type 1 envelope glycoprotein mutant.

We have demonstrated previously that a human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein containing a Val-to-Glu substitution at the second amino acid of the transmembrane glycoprotein gp41 (termed the 41.2 mutant) dominantly interferes with wild-type envelope-mediated syncytium formation and virus infectivity. To understand the mechanism by which the 41.2 mutant exerts the dominant interfering phenotype and thereby determine further how the mutant might be used as an inhibitor of viral spread, additional mutations were made in the envelope gene, and the effects of these mutations on interference were determined. It was found that processing of the 41.2 mutant glycoprotein in gp120 and gp41 subunits and a functional CD4-binding domain are necessary for the interfering phenotype to be exhibited fully. However, neither a wild-type V3 loop nor the gp41 cytoplasmic tail is necessary for efficient interference. In addition, it was determined that the dominant interfering phenotype is not conferred exclusively by the glutamate substitution at amino acid 2 of gp41, since a substitution with a basic residue at this position also results in a dominant interfering envelope glycoprotein.     

Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infection.

The matrix domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein contains a highly basic region near its amino terminus. It has been proposed that this basic domain, in conjunction with the HIV-1 accessory protein Vpr, is responsible for the localization of the HIV-1 preintegration complex to the nucleus in nondividing cells. It has also been postulated that the matrix basic domain assists in the targeting of the HIV-1 Gag precursor Pr55Gag to the plasma membrane during virus assembly. To evaluate the role of this highly basic sequence during infection of primary human monocyte-derived macrophages, single- and double-amino-acid-substitution mutations were introduced, and the effects on virus particle production, Gag protein processing, envelope glycoprotein incorporation into virus particles, and virus infectivity in the CEM(12D-7) T-cell line, peripheral blood mononuclear cells, and primary human monocyte-derived macrophages were analyzed. Although modest effects on virus particle production were observed with some of the mutants, none abolished infectivity in primary human monocyte-derived macrophages. In contrast with previously reported studies involving some of the same matrix basic domain mutants, infectivity in monocyte-derived macrophages was retained even when combined with a vpr mutation.     

Characterization of human immunodeficiency virus type 1 matrix revertants: effects on virus assembly, Gag processing, and Env incorporation into virions.

The matrix protein of human immunodeficiency virus type 1 (HIV-1) has been postulated to serve a variety of functions in the virus life cycle. Previously, we introduced a large number of mutations into the HIV-1 matrix and determined the effects on virus replication. These studies identified domains involved in virus assembly and release and envelope glycoprotein incorporation into virions. Here we describe the identification and characterization of viral revertants containing second-site changes in the matrix which compensate for the effects of four of the original mutations on matrix function. Specifically, mutations at matrix residues 4 and 6 severely impaired virus assembly and release; substitutions at residues 4 and 6 reversed the phenotype of the amino acid 4 change while second-site mutations at matrix positions 10, 69, and 97 partially or fully reversed the phenotype of the amino acid 6 substitution. A mutation at matrix residue 62 reversed the effect of a position 34 change which blocks envelope glycoprotein incorporation into virions, and substitutions at residues 27 and 51 reversed the phenotype of a position 86 mutation which redirects virus assembly to the cytoplasm. In addition to determining the effects of the compensatory changes in the context of the original mutations, we also introduced and analyzed the second-site changes alone in the context of the wild-type molecular clone. The data presented here define potential intermolecular and intramolecular interactions which occur in the matrix during the virus life cycle and have implications for our understanding of the relationship between matrix structure and function.