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.     

Retention of soluble 99mTc-DTPA in the human lung: 24-h postdeposition

Foster, W. Michael, Pamela T. Stetkiewicz, and Arthur N. Freed. Retention of soluble^99mTc-DTPA in the human lung: 24-hpostdeposition. J. Appl. Physiol. 82(4): 1378-1382, 1997.Clearance of low-molecular-weightsolutes, e.g., radiolabeled chelate diethylenetriaminepentaacetate(DTPA), across epithelial surfaces of distal airways and the lungparenchyma is a broadly used technique to assess epithelial integrity.It has been generally assumed that clearance of solute follows a simplefirst-order process and that DTPA clearance through the respiratoryepithelium and into blood and lymphatic channels is complete within afew hours. Using -camera imaging and a radiolabeled aerosol of^99mTc-labeled DTPA, we observed ineight healthy subjects lung retention of radioisotope ~24 hpostdeposition of the ^99mTc-DTPA.Residual lung retention at the 24-h end point averaged 6.0 ± 1.8 (SD)% of the amount of radioisotope initially deposited in the lung.This suggests that for normal healthy subjects a small amount of the^99mTc radioisotope, either in adissociated or chelated form, is nonpermeable or slowly cleared fromrespiratory tisssues.

     

Hyperventilation-induced airway injury and vascular leakage in dogs: effects of alpha 1-adrenergic agonists

Freed, Arthur N., Varsha Taskar, Brian Schofield, andChiharu Omori. Hyperventilation-induced airway injury and vascular leakage in dogs: effects of₁-adrenergic agonists.J. Appl. Physiol. 83(6):1884-1889, 1997.₁-Adrenergic agonistsinhibit hyperventilation-induced bronchoconstriction (HIB) in dogs. Wetested the hypothesis that -agonists inhibit HIB byreducing bronchovascular leakage and edema that theoretically couldcause airway obstruction. Peripheral airways were isolated by using abronchoscope; pretreated with either methoxamine (Mx), norepinephrine(NE), or saline aerosol; and then exposed to a 2,000 ml/min dry-airchallenge (DAC) for 2 min. Colloidal carbon was injected before DAC andused to quantify bronchovascular permeability. Mx-, NE-, andvehicle-treated airways were prepared for morphometric analysis within1 h after DAC. Light microscopy revealed that the 2-min DAC producedminimal bronchovascular leakage and little epithelial damage. However, pretreatment with either Mx or NE significantly enhanced dryair-induced bronchovascular hyperpermeability and mucosal injury. Theincreased damage associated with these₁-agonists implicates aprotective role for the bronchial circulation. The factthat ₁-agonists inhibit HIBsuggests that neither dry air-induced leakage nor injury directlycontributes to the development of airway obstruction. In addition,our data suggest that-agonists attenuate HIB in part byaugmenting hyperventilation-induced bronchovascular leakage and byreplacing airway water lost during a DAC.

     

Herpetomonas megaseliae, Crithidia fasciculata, and Leptomonas collosoma (Kinetoplastida, Trypanosomatidae) in Feces of Lizards Fed Culture Forms of the Flagellates

ABSTRACT. Herpetomonas megaseliae, Crithidia fasciculata , and Leptomonas collosoma from culture survived gut passage in Anolis carolinensis following their ingestion by this lizard. Maximum persistence of H. megaseliae in lizards, as detected by fecal culture, was seven days. No invasion of tissues by H. megaseliae could be detected by means of sectioned material, stained impression slides, or cultures inoculated with material from organs. Crithidia fasciculata was evident in cloacal fluid for up to three days in wet mount preparations. Leptomonas collosoma was observed in feces 24 h after the organisms were fed to lizards. Both C. fasciculata and L. collosoma were cultured from feces of lizards fed the parasites 24 h earlier. Herpetomonas megaseliae was differentiated in lizard feces, with greater than 40% of the forms observed being paramastigotes or opisthomastigotes. Truncate, semispherical forms resembling choanomastigotes were seen, but the kinetoplast was posterior to the nucleus in some of these. Many forms showed extensive coiling of the axoneme within the body of the flagellate. Choanomastigotes and spheromastigotes of C. fasciculata and promastigotes, sphero-mastigotes and amastigotes of L. collosoma were also observed in the feces.