Respiratory activities of intralaryngeal branches of the recurrent laryngeal nerve

To distinguish experimentally between motor nerve activity destined for vocal cord abductor muscles and that bound for muscles that adduct the cords, we recorded efferent activities of intralaryngeal branches of the recurrent laryngeal nerve (RLN) in decerebrate, vagotomized, paralyzed, ventilated cats. Activities of the whole RLN and phrenic nerve were also recorded. Nerve activities were assessed at several steady-state end-tidal O2 and CO2 concentrations. The nerve to the thyroarytenoid (TA) muscle, a vocal cord adductor, was only slightly active under base-line (normocapnic, hyperoxic) conditions but in most cats developed strong activity during expiration in hypocapnia or hypoxia. In severe hypocapnia, phasic expiratory TA activity persisted even during phrenic apnea, indicating continuing activity of the respiratory rhythm generator. The nerve to the posterior cricoarytenoid (PCA) muscle, the vocal cord abductor, was always active in inspiration but often showed expiratory activity as well. This expiratory activity was usually enhanced by hypercapnia and often inhibited by hypoxia. The results are consistent with previous electromyographic findings and emphasize the importance of distinguishing abductor from adductor activity in studies of laryngeal control.     

Cloning and characterization of a tandemly repeated DNA sequence in the crane family (Gruidae)

A tandemly repeated DNA sequence possessing a unique PstI site has been characterized in several species of the crane family. The "Pst family" comprises at least 8800 monomer units 187 base pairs (bp) in length and constitutes 0.14% of the genome of the sarus crane (Grus antigone). The array is located in the centromeric heterochromatin of chromosome 2 in the two species where in situ hybridizations of a cloned monomer to metaphase chromosome spreads were carried out. DNA sequence comparisons between five monomer units from G. antigone revealed a high degree of homology between four of the individual repeats, while the fifth was somewhat divergent. The G + C content deduced from the DNA sequence makes it likely that the Pst family constitutes part of a density satellite seen in profiles of crane DNA centrifuged to equilibrium in CsCl. The common occurrence of tandem arrays such as the Pst family, with repeat lengths close to 200 bp, leads us to an hypothesis implicating nucleosomes in the evolution of such families.     

Improved developmental potential of rabbit oocytes fertilized by sperm microinjection into the perivitelline space enlarged by hypertonic media

The objectives of the present study were: 1) to develop a simple and more efficient technique for sperm microinjection than is currently available, using the rabbit as a model, and 2) to evaluate the development of rabbit oocytes fertilized by single or multiple sperm microinjection. Hyperosmotic sucrose in phosphate-buffered saline (SPBS) was employed to dehydrate oocytes to increase the perivitelline space for sperm microinjection and prevent possible injury to the vitellus. In the first experiment, 58% (n = 29) oocytes treated with 0.5 M SPBS developed to morulae following multiple sperm microinjection compared, respectively, to 47% (n = 34) and 60% (n = 15) for control IVF with or without sucrose exposure (P greater than 0.05). Blastocyst development from microinjected oocytes, however, was much lower (P less than 0.05) than that of controls (14% vs. 42% and 40%, respectively). Sham operation by puncturing the zona pellucida of the sucrose-treated oocytes with the microinjection pipette did not increase parthenogenesis (P greater than 0.05). In Experiment 2 a smaller-size injection pipette and shorter sucrose exposure time after sperm microinjection resulted in 41% (n = 42) of the oocytes developing into blastocysts for the microinjection group, whereas only 21% (n = 24) developed to blastocysts in the control IVF group (P less than 0.05). When relatively older oocytes (17 hr post ovulation injection) were used to test if microinjection could reduce the time to fertilization and cleavage (Expt. 3), an average of 27% (n = 63) blastocysts resulted from microinjection vs. 0% (n = 28) for the control IVF group.     

Phylogenetic distribution of the novel avian endogenous provirus family EAV-0

A new family of related endogenous proviruses, existing at 50 to 100 copies per haploid genome and distinguishable by remarkably short long terminal repeats, has been described for domestic chickens (Gallus gallus subsp domesticus). In this communication, by using Southern blot analysis and probes derived from both internal viral sequences and locus-specific, cellular flanking sequences, we studied the genetic distribution of this family of moderately repetitive avian endogenous retroviruses within the genomes of four Gallus species. Eight inbred lines of domestic chickens, the evolutionary progenitor to the domestic chicken (red jungle fowl), and two more distantly related species (grey and green jungle fowl) were studied. All Gallus species harbored this class of elements, although the different lines of domestic chickens and different species of jungle fowl bore distinguishable complements of the proviral loci. Jungle fowl appeared to have fewer copies than domestic chickens. For three randomly isolated proviral loci, domestic chickens (G. gallus subsp. domesticus) and red jungle fowl (G. gallus subsp. gallus) showed only a proviral state, whereas the most primitive and divergent of the jungle fowl, the green jungle fowl (G. varius), consistently demonstrated only preintegration states or disparate alleles. The presence of this family in all Gallus species and of related sequences in other genera suggests that a primordial founding integration event occurred prior to the evolutionary separation of Gallus species and possibly related genera. Additionally, at least one proviral locus has been acquired subsequent to speciation, indicating that this family was actively infectious after the primary founding event. This conserved, repetitive proviral family appears to represent the vestigial remnant of an avian retrovirus class related to and evolutionarily more ancient than the Rous-associated virus-0 family of avian endogenous retroviruses.     

The viral envelope gene is involved in macrophage tropism of a human immunodeficiency virus type 1 strain isolated from brain tissue.

Human immunodeficiency virus type 1 (HIV-1) strains isolated from the central nervous system (CNS) may represent a subgroup that displays a host cell tropism different from those isolated from peripheral blood and lymph nodes. One CNS-derived isolate, HIV-1SF128A, which can be propagated efficiently in primary macrophage culture but not in any T-cell lines, was molecularly cloned and characterized. Recombinant viruses between HIV-1SF128A and the peripheral blood isolate HIV-1SF2 were generated in order to map the viral gene(s) responsible for the macrophage tropism. The env gene sequences of the two isolates are about 91.1% homologous, with variations scattered mainly in the hypervariable regions of gp120. Recombinant viruses that have acquired the HIV-1SF128A env gene display HIV-1SF128A tropism for macrophages. Furthermore, the gp120 variable domains, V1, V2, V4, and V5, the CD4-binding domain, and the gp41 fusion domain are not directly involved in determining macrophage tropism.     

Generation of seal influenza virus variants pathogenic for chickens, because of hemagglutinin cleavage site changes.

Influenza virus A/seal/Mass/1/80 (H7N7) was adapted to grow in MDCK cells and chicken embryo cells (CEC) in the absence of exogenous protease. The biological properties of the virus variants obtained coincided with intracellular activation of the hemagglutinin (HA) by posttranslational proteolytic cleavage and depended on the cell type used for adaptation. MDCK cell-adapted variants contained point mutations in regions of the HA more distant from the cleavage site. It is proposed that these mutations are probably responsible, through an unknown mechanism, for enhanced cleavability of HA in MDCK cells. Such virus variants were apathogenic in chickens. CEC-adapted variants, on the other hand, contained an insertion of basic amino acids at the HA cleavage site, in addition to scattered point mutations. The insertions converted the cleavage sites in the variant virus HAs so that they came to resemble the cleavage site found in highly pathogenic avian influenza viruses. CEC variants with such cleavage site modifications were highly pathogenic for chickens. The lethal outcome of the infection in chickens demonstrated for the first time that an influenza virus derived from a mammalian species can be modified during adaptation to a new cell type to such an extent that the resulting virus variant becomes pathogenic for an avian species.     

Combined infection by Moloney murine leukemia virus and a mink cell focus-forming virus recombinant induces cytopathic effects in fibroblasts or in long-term bone marrow cultures from preleukemic mice.

We described previously a preleukemic state in mice inoculated with Moloney murine leukemia virus (M-MuLV) characterized by generalized hematopoietic hyperplasia in the spleen. To investigate this further, long-term bone marrow cultures (LTBMC) from preleukemic mice were established. Surprisingly, LTBMC from M-MuLV-inoculated preleukemic mice showed less hematopoiesis than LTBMC from control mice. This resulted from a quantitative defect in establishment of bone marrow stromal cells in the LTBMC. This phenomenon could also be observed in LTBMC from normal mice infected in vitro with a stock of M-MuLV containing a mink cell focus-forming virus (MCF) derivative (M-MCF), but not in LTBMC infected with M-MuLV alone. This implicated MCF derivatives in the reduction in bone marrow stromal cells. The phenomenon could also be detected in infected NIH 3T3 cells. Combined infection of M-MuLV plus M-MCF resulted in fewer cells, in comparison to uninfected cells or cells infected with either virus alone. Further studies indicated that this was predominantly due to an inhibition in cell growth rather than to cell lysis. The cytopathic effect did not appear to result from overreplication of viral DNA, as measured by Southern blots. Thus, combined infection with M-MuLV and an MCF derivative had cytostatic effects on cell growth. This phenomenon might also contribute to the leukemogenic process in vivo.     

Identification of distinct T cell receptor (TCR)-gamma delta heterodimers using an anti-TCR-gamma variable region serum

T cell hybridomas were generated from CD3+, CD4-, CD8- splenocytes and fetal thymocytes. V gamma 1-expressing proteins present on these murine TCR-gamma delta hybridomas were identified by using an anti-TCR V gamma 1 peptide serum. This antiserum specifically immunoprecipitated 41-kDa TCR V gamma-C gamma 4 chains and 31-kDa TCR V gamma-C gamma 1/2 chains from distinct heterodimers expressed on the TCR-gamma delta T cell hybridomas. The RNA from a hybridoma with a 31-kDa TCR-gamma chain hybridized with a V gamma 1 probe but failed to hybridize with a V gamma 2 probe. In contrast, the RNA from a hybridoma with a 32-kDa TCR-gamma chain hybridized with a V gamma 2 probe. This 32-kDa TCR-gamma chain was not immunoprecipitated by the anti-V gamma 1 serum. These data were consistent with the conclusion that the 31-kDa protein was the product of a V gamma 1 to C gamma 2 rearrangement, whereas the 32-kDa protein was the product of a V gamma 2 to C gamma 1 rearrangement. Furthermore, Southern analyses confirmed that the 32-kDa protein was the product of a V gamma 1.2-J gamma 2 rearrangement, and all three of the 41-kDa TCR-gamma chains were the results of V gamma 1.1-J gamma 4 rearrangements. This was the first demonstration at the clonal level of TCR-gamma proteins which use members of the V gamma 1 gene family, as well as the C gamma 2 constant region. Additional biochemical analyses of the TCR-gamma and -delta proteins from three independently derived C gamma 4-bearing T cell hybridomas suggested that most of the molecular mass diversity observed in the bulk subpopulation of peripheral C gamma 4-containing heterodimers may be contributed by the TCR-delta chains.     

Effect of limited trypsin digestion on the renal Na+−H+ exchanger and its regulation by cAMP-Dependent protein kinase

Summary The Na⁺–H⁺ exchanger from solubilized rabbit renal brush border membranes is inhibited by cAMP-dependent protein kinase (PKA) mediated protein phosphorylation. To characterize this inhibitory response and its sensitivity to limited proteolysis, the activity of the transporter was assayed after reconstitution of the proteins into artificial lipid vesicles. Limited trypsin digestion increased the basal rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake in reconstituted proteoliposomes and blocked the inhibitory response to PKA-mediated protein phosphorylation. To determine if the inhibitory response to PKA-mediated protein phosphorylation could be restored to the trypsin-treated solubilized proteins, nontrypsinized solubilized brush border membrane proteins were separated by column chromatography. The addition of small molecular weight polypeptides, fractionated on Superose-12 FPLC (V _e=0.7), to trypsinized solubilized brush border membrane proteins restored the inhibitory response to PKA-mediated protein phosphorylation. Similarly, the addition of the 0.1m NaCl fraction from an anion exchange column, Mono Q-FPLC, also restored the inhibitory response to PKA. Both protein fractions contained a common 42–43 kDa protein which was preferentially phosphorylated by PKA.These results indicate that limited trypsin digestion dissociates the activity of the renal Na⁺–H⁺ exchanger from its regulation by PKA. It is suggested that trypsin cleaves an inhibitory component of the transporter and that this component is the site of PKA-mediated regulation. Phosphoprotein analysis of fractions that restored PKA regulation raises the possibility that a polypeptide of 42–43 kDa is involved in the inhibition of the renal Na⁺–H⁺ exchanger by PKA-mediated, protein phosphorylation.     

Molecular genetic, biochemical and nuclear magnetic resonance studies on the role of the tryptophan residues of glutamine-binding protein from Escherichia coli

The results of molecular genetic, biochemical and nuclear magnetic resonance studies on glutamine-binding protein of Escherichia coli suggest that the only two tryptophan residues, at positions 32 and 220, in the protein molecule are likely to be involved in (or sensitive to) interactions with the membrane-bound protein components of the glutamine transport system. It has been found that both tryptophan residues have limited motional freedom, are located away from the surface of the protein molecule and are not close to the ligand-binding site. Their presence, however, is required for the optimal transport of L-glutamine across the cytoplasmic membrane, though not essential for the ligand-binding process. The relevance of these results to the structure and function of the glutamine-binding protein in the glutamine transport system is discussed.