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T M Gallagher

Publications and source records attributed to T M Gallagher.

At least 37 records · Page 2Linked to original sources
PubMed1995

Overexpression of the MHV receptor. Effect on progeny virus secretion.

The intracellular interaction of the coronavirus mouse hepatitis virus (MHV) with its cellular receptor (MHVR) was investigated. Overexpression of MHVR from vaccinia vectors during an ongoing MHV infection resulted in dramatic inhibition of virus production. Infectivity in both cytoplasmic extracts and supernatants was reduced by over three orders of magnitude relative to control cultures in which a truncated MHVR lacking virus binding activity was expressed. Complete MHV virions were not detectable in supernatants of MHVR expressing cells. In the presence of overexpressed MHVR, the coronavirus spike protein was not cleaved into posttranslation products S1 and S2, nor was it fully processed into a form resistant to endoglycosidase H digestion, indicating that intracellular engagement of spike with receptor prevented spike transport and consequent association with virions.

Animals↗
PubMed1994

Reconstitution of Flock House provirions: a model system for studying structure and assembly.

Assembly of Flock House virus in infected Drosophila cells proceeds through an intermediate, the provirion, which lacks infectivity until the coat precursor protein, alpha, undergoes a spontaneous "maturation" cleavage (A. Schneemann, W. Zhong, T. M. Gallagher, and R. R. Rueckert, J. Virol 6:6728, 1992). We describe here methods for purifying provirions in a state which permitted dissociation and reassembly. Dissociation, to monomeric alpha protein and free RNA, was accomplished by freezing at pH 9.0 in the presence of 0.5 M salt and 0.1 M urea. When dialyzed at low ionic strength and pH 6.5, the dissociation products reassembled spontaneously to form homogeneous provirions with a normal complement of RNA as judged by cosedimentation with authentic virions and by ability to undergo maturation cleavage with acquisition of substantial, though subnormal, infectivity. Reconstitution experiments, i.e., remixing components after separating RNA from capsid protein, generated abnormal particles, suggesting the presence in the unfractionated dissociation products of an unidentified "nucleating" component.

Animals↗
PubMed1993

Dissemination of MHV4 (strain JHM) infection does not require specific coronavirus receptors.

In this report, we demonstrate the syncytial spread of MHV4 (strain JHM) infection through non-murine cell cultures which lack a specific MHV4 receptor and are therefore resistant to infection by free virions. This was achieved by allowing infected murine cells to settle onto confluent monolayers of non-murine cells in a straightforward infectious center assay. Receptor-independent syncytium formation induced by cells expressing the MHV4 spike (S) from recombinant vaccinia viruses (VV) indicated that spread was mediated by this coronavirus glycoprotein. We conclude that the S protein of MHV4 is so potently fusogenic that it does not require prior binding to a virus-specific surface receptor to induce fusion of closely-opposed plasma membranes.

Animals↗
PubMed1993

Verb class distinctions and AAC language-encoding limitations.

This study explored the status of an English grammatical distinction in the language of individuals who have never been able to encode that distinction previously. English past tense marking was used as a context to examine regular and irregular verb class distinctions in the language of two adults with severe congenital physical impairments who rely on augmentative and alternative communication (AAC) systems to communicate. In the subjects' lexically based AAC systems, past tense was marked on regular verbs and irregular verbs using the same strategy. The subjects accessed their AAC displays using four-digit eye gaze number codes. They were shown a novel affixation strategy through manipulation of the four-digit codes that allowed them to mark past tense on regular verbs via an affixation process. Their semantic strategy for marking past tense on irregular verbs was not changed. The subjects' patterns of use of the two strategies on exemplars of each verb class revealed limited evidence of distinctive use of the two strategies based on verb class membership. Theoretical and clinical implications are discussed.

Adult↗
PubMed1992

Cell receptor-independent infection by a neurotropic murine coronavirus.

The cellular receptors for a coronavirus, mouse hepatitis virus (MHV), have been recently identified as one or more members of the carcinoembryonic antigen (CEA) family. The neurotropic JHM strain of MHV (MHV-JHM) possesses a highly fusogenic surface (S) glycoprotein. This protein is now shown to promote the spread of MHV into cells lacking the specific CEA-related MHV receptor. Resistant cells are recruited into MHV-induced syncytium with consequent production of progeny virus. Cell-to-cell spread of virus via membrane fusion without the requirement for specific cell surface receptor offers a novel way for virus to spread within infected hosts.

Animals↗
PubMed1992

Maturation cleavage required for infectivity of a nodavirus.

Nodaviral morphogenesis involves formation of labile precursor particles, called provirions, which mature by autocatalytic cleavage of the 407-residue coat precursor protein between asparagine residue 363 and alanine residue 364. It has previously been demonstrated that maturation results in increased physicochemical stability of the virion. We show here that cleavage of coat protein in purified provirions of Flock House virus was accompanied by a five- to eightfold increase in specific infectivity. Cleavage-negative provirions, produced by site-directed mutagenesis of asparagine residue 363 to aspartate, threonine, or alanine, displayed no infectivity above revertant frequencies as measured by plaque assay. All viable revertants (nine of nine) restored asparagine to the mutated position, suggesting high specificity for asparagine at the cleavage site.

Animals↗
PubMed1991

Alteration of the pH dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein.

Infection of susceptible murine cells with the coronavirus mouse hepatitis virus type 4 (MHV4) results in extensive cell-cell fusion at pHs from 5.5 to 8.5. The endosomotropic weak bases chloroquine and ammonium chloride do not prevent MHV4 infection. In marked contrast, we have selected variants from a neural cell line persistently infected with MHV4 which are entirely dependent on acid pH to fuse host cells and are strongly inhibited by endosomotropic weak bases. Wild-type and variant viruses were compared at the level of the fusion-active surface (S) glycoprotein gene. Cloning and sequencing of each 4,131-base open reading frame predicted a total of eight amino acid differences which fell into three distinct clusters. Each S glycoprotein, when expressed from cDNA, was synthesized in equivalent amounts, and similar proportions were transported to the cell surface. Wild-type S induced cell-cell fusion at neutral pH, whereas variant S required prolonged exposure to acidic pH to induce fusion. Expression of hybrid S genes prepared by exchange of restriction fragments between wild-type and variant cDNAs revealed that elimination of neutral pH fusion was solely dependent on amino acid alterations at positions 1067 (Q to H), 1094 (Q to H), and 1114 (L to R). These changes lie within a predicted heptad repeat region of the transmembrane cleavage fragment of S (S2). These findings demonstrate that the pH dependence of coronavirus fusion is highly variable and that this variability can be determined by as few as three amino acid residues.

Amino Acid Sequence↗
PubMed1990

Monoclonal antibody-selected variants of MHV-4 contain substitutions and deletions in the E2 spike glycoprotein.

Selection and analysis of MHV-4 (strain JHM) variants resistant to E2-specific neutralizing MAbs was performed. Two types of variation in the E2 spike glycoprotein were found. From minimally passaged stocks of MHV-4, putative point mutants were obtained. These mutants were resistant only to the MAb used to select them. In contrast, multiply passaged stocks were found to harbor variants uniformly resistant to two selecting MAbs. Northern and Western blot analysis of the viral RNAs and proteins synthesized by these doubly-resistant variants showed that they contained large deletions in both mRNA 3 and its E2 translation product, localized to a 15 kilodalton region within the amino terminal 90B post-translational fragment. The selective advantage of this second class of variants lacking sequences within E2 90B was a result of their reduced cytopathology, thereby allowing cultures to support virus production for prolonged periods.

Animals↗
PubMed1990

The minimum alveolar concentration (MAC) and hemodynamic effects of halothane, isoflurane, and sevoflurane in newborn swine.

To determine the minimum alveolar concentration (MAC) and hemodynamic responses to halothane, isoflurane, and sevoflurane in newborn swine, 36 fasting swine 4-10 days of age were anesthetized with one of the three volatile anesthetics in 100% oxygen. MAC was determined for each swine. Carotid artery and internal jugular catheters were inserted and each swine was allowed to recover for 48 h. After recovery, heart rate (HR), systemic systolic arterial pressure (SAP), and cardiac index (CI) were measured awake and then at 0.5, 1.0, and 1.5 MAC of the designated anesthetic in random sequence. The (mean +/- SD) MAC for halothane was 0.90 +/- 0.12%; the MAC for isoflurane was 1.48 +/- 0.21%; and the MAC for sevoflurane was 2.12 +/- 0.39%. Awake (mean +/- SD) measurements of HR, SAP, and CI did not differ significantly among the three groups. Compared to the awake HR, the mean HR decreased 35% at 1.5 MAC halothane (P less than 0.001), 19% at 1.5 MAC isoflurane (P less than 0.005), and 31% at 1.5 MAC sevoflurane (P less than 0.005). Compared to awake SAP, mean SAP measurements decreased 46% at 1.5 MAC halothane (P less than 0.001), 43% at 1.5 MAC isoflurane (P less than 0.001), and 36% at 1.5 MAC sevoflurane (P less than 0.005). Mean SAP at 1.0 and 1.5 MAC halothane and isoflurane were significantly less than those measured at equipotent concentrations of sevoflurane (P less than 0.005). Compared to awake CI, mean CI measurements decreased 53% at 1.5 MAC halothane (P less than 0.001) and 43% at 1.5 MAC isoflurane (P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, Inhalation↗
PubMed1990

Neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the amino-terminal half of the spike glycoprotein.

Neuroattenuated variants of mouse hepatitis virus type 4 (MHV-4) selected for resistance to neutralizing monoclonal antibodies (R.G. Dalziel, P.W. Lampert, P. J. Talbot, and M. J. Buchmeier, J. Virol. 59:463-471, 1986) were found to harbor large deletions in both mRNA 3 and its protein product, the 180-kilodalton viron spike (S) glycoprotein. By using antipeptide antibodies directed against selected portions of the chain, deletions were mapped to the middle of the amino-terminal S1 fragment, one of the two posttranslational cleavage products of S, and involved omission of 15 kilodaltons of protein. Deletion mutants could be selected only after multiple passage of virus through cultured cell lines; minimally passaged MHV-4 stocks contained putative point mutants selectable by neutralizing monoclonal antibodies but no deletions. Enhanced growth of deletion mutants relative to wild-type virus was observed in four cell lines used for virus propagation and was attributed to delayed and diminished cytopathic effects that allowed cultures to support virus production for prolonged periods. This hypothesis was reinforced by the finding that no selective advantage for the deletion mutants was observed in two cell lines resistant to virus-induced cytopathic effects. These results indicate that the passaging of MHV-4 in culture generates heterogeneity in S structure and eventually selects for rare neutralization-resistant deletion mutants with decreased virulence properties.

Amino Acid Sequence↗
PubMed1989

Sequence analysis reveals extensive polymorphism and evidence of deletions within the E2 glycoprotein gene of several strains of murine hepatitis virus.

Direct RNA sequence analysis of the E2 gene of wild-type MHV-4 and of neutralization resistant, neuroattenuated variants has identified a polymorphic region with respect to deletions. These variants had large deletions of 142 to 159 amino acids mapping to a localized region in the amino-terminal domain of the peplomer glycoprotein. The nucleotide sequence of the E2 gene for wild-type strain MHV-4 was found to be very similar to that of MHV-JHM but had an insertion of 423 nucleotides resulting in the addition of a stretch of 141 unique amino acids in the amino-terminal domain of E2. We propose that deletions reflect a major source of heterogeneity in the E2 protein of MHV.

Amino Acid Sequence↗
PubMed1989

Communication mode use of two hearing-impaired adolescents in conversation.

Communication mode use in the dyadic conversational speech of adolescent simultaneous communication (SC)-trained hearing-impaired twin boys was investigated. Proportional frequencies of modes and the English structural characteristics of the spoken components of utterances produced in each mode were examined. The results indicated that these adolescents were using an integrated bimodal form of English with a grammatical base that did not vary as a function of the presence or absence of simultaneous signs either in their speech or their partner's speech. Implications of the results are discussed.

Child↗
PubMed1989

Spinal anaesthesia in infants born prematurely.

Spinal anaesthesia was used for 25 infants who were born prematurely and who subsequently required inguinal herniotomy. Hyperbaric bupivacaine 0.5% was administered in a dose of 0.3 mg/kg. Satisfactory surgical anaesthesia was achieved in 22 patients and there were no episodes of haemodynamic instability. The technique was not associated with postoperative apnoea. Spinal anaesthesia is a safe and effective alternative to general anaesthesia in these high-risk infants. However, postoperative apnoea monitoring is still essential for all infants born prematurely.

Anesthesia, Spinal↗
PubMed1988

Assembly-dependent maturation cleavage in provirions of a small icosahedral insect ribovirus.

Extracts from nodavirus-infected Drosophila cells contained detergent-labile 140S "young" particles much richer than mature virions in their content of protein alpha, a precursor of coat proteins beta and gamma. Incorporation studies in infected cells showed that most newly synthesized alpha protein was assembled into young particles within a few minutes. Incubation of the particles, either in cytoplasmic extracts or after purification, resulted in spontaneous first-order cleavage of alpha protein to form beta-plus-gamma chains. Alpha protein that was not associated with particles failed to cleave. Cleavage was accompanied by a marked increase in detergent stability of the particles and was unaffected by a broad spectrum of protease inhibitors or by coating with precipitating antibody. We conclude (i) that alpha chains are cleaved only after assembly into provirions, (ii) that cleavage occurs internally and is likely therefore autocatalytic, and (iii) that cleavage stabilizes the mature virus particles.

Animals↗
PubMed1987

Structure of an insect virus at 3.0 A resolution.

We report the first atomic resolution structure of an insect virus determined by single crystal X-ray diffraction. Black beetle virus has a bipartite RNA genome encapsulated in a single particle. The capsid contains 180 protomers arranged on a T = 3 surface lattice. The quaternary organization of the protomers is similar to that observed in the T = 3 plant virus structures. The protomers consist of a basic, crystallographically disordered amino terminus (64 residues), a beta-barrel as seen in other animal and plant virus subunits, an outer protrusion composed predominantly of beta-sheet and formed by three large insertions between strands of the barrel, and a carboxy terminal domain composed of two distorted helices lying inside the shell. The outer surfaces of quasi-threefold related protomers form trigonal pyramidyl protrusions. A cleavage site, located 44 residues from the carboxy terminus, lies within the central cavity of the protein shell. The structural motif observed in BBV (a shell composed of 180 eight-stranded antiparallel beta-barrels) is common to all nonsatellite spherical viruses whose structures have so far been solved. This highly conserved shell architecture suggests a common origin for the coat protein of spherical viruses, while the primitive genome structure of BBV suggests that this insect virus represents an early stage in the evolution of spherical viruses from cellular genes.

Biological Evolution↗