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Biomedical subjects

R Everett

Publications and source records attributed to R Everett.

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

Mechanisms for episodes of hypoxemia in preterm infants undergoing mechanical ventilation.

OBJECTIVE: To ascertain possible mechanisms implicated in the development of transient episodes of hypoxemia (oxygen saturation < 85%) frequently observed in preterm infants undergoing mechanical ventilation, even after the acute phase of respiratory failure has passed. STUDY DESIGN: Tidal flow, airway and esophageal pressure, and oxygen saturation were continuously recorded in 10 infants (mean +/- SD, birth weight 733 +/- 149 gm, gestational age 25.5 +/- 2.2 weeks, age 26.3 +/- 11.9 days) who had repeated episodes of hypoxemia without any evident cause. Measurements of minute ventilation (VE) inspiratory compliance (Ci), and inspiratory resistance (Ri) were compared before and during episodes of hypoxemia. RESULTS: All episodes of hypoxemia were preceded by an active exhalation that produced a mean decrease in end-expiratory lung volume of 6.4 +/- 2.8 ml/kg. The reduction in lung volume was immediately followed by a sudden decrease in tidal flow and volume, despite continuation of mechanical ventilation at the same rate and peak pressure. The resulting hypoventilation was associated with a drop in Ci to approximately one half and an increase in Ri to more than double the baseline values. Approximately 30 seconds after the beginning of hypoventilation, the arterial oxygen saturation reached a hypoxemic level (oxygen saturation < 85%)> CONCLUSION: Most hypoxemic episodes were triggered by an expiratory effort that produced a large decrease in lung volume. This reduction in lung volume probably leads to closure of small airways and the development of intrapulmonary shunts, which would explain the rapid development of hypoxemia.

Analysis of Variance↗
PubMed1995

The equine herpesvirus 1 gene 63 RING finger protein partially complements Vmw110, its herpes simplex virus type 1 counterpart.

All alpha herpesviruses of known DNA sequence have been found to encode a protein with similarities to immediate early protein Vmw110 (ICP0) of herpes simplex virus type 1 (HSV-1). The conserved portion of this family of proteins is a characteristic zinc binding module, known as a RING finger or C3HC4 domain. Examples of RING finger domains occur in many other proteins of diverse evolutionary origin and function. Recently, the solution structure of the equine herpesvirus 1 (EHV-1) RING finger protein, encoded by gene 63, has been solved. To investigate whether this structure could be considered to be a paradigm of herpesvirus RING domains, we have constructed a recombinant HSV-1 which expresses the EHV-1 gene 63 protein (EHVg63) in place of Vmw110. Comparison of the growth properties of the recombinant with those of wild-type and Vmw110-defective viruses indicates that EHVg63 is able to fulfil partially, but not completely, the roles of Vmw110 during virus growth in tissue culture.

Animals↗
PubMed1995

Point mutations in the herpes simplex virus type 1 Vmw110 RING finger helix affect activation of gene expression, viral growth, and interaction with PML-containing nuclear structures.

Herpes simplex virus type 1 immediate-early protein Vmw110 (also known as ICP0) has been implicated in the control of the balance between the lytic and latent states, but the precise mechanisms by which it exerts its effects are unknown. Vmw110 includes a characteristic zinc binding domain, termed the C3HC4 domain or RING finger, which is essential for its function. The solution structure of a related herpesvirus RING finger domain suggested that an amphipathic alpha helix might be an important functional component of the RING finger. In this paper, we show that the equivalent region of Vmw110 is important for virus growth in tissue culture and for the normal interaction of Vmw110 with nuclear structures which include the PML protein.

Amino Acid Sequence↗
PubMed1994

Herpes simplex virus type 1 immediate-early protein Vmw110 binds strongly and specifically to a 135-kDa cellular protein.

Herpes simplex virus type 1 encodes five immediate-early gene products, at least three of which are required for fully efficient viral gene expression. One of these three, Vmw110 (or ICPO), is a potent and nonspecific activator of gene expression in transfection assays. Viruses which fail to express functional Vmw110 have a cell-type and multiplicity-dependent defect in viral gene expression. In addition, Vmw110 has been implicated in the reactivation of latent virus in mouse and tissue culture model systems. In this report we show that Vmw110 can be isolated as a fast-sedimenting complex from infected cells and that this complex contains a cellular protein which is immune precipitated in association with Vmw110. This association can be reconstructed by adding purified Vmw110 to lysates of several different cell types. By using a GST fusion protein, we have found that the isolated C-terminal portion of Vmw110 can complex strongly and specifically with a similar cellular protein. The relevance of this observation to the roles of Vmw110 in latent and lytic virus infection is discussed.

Animals↗
PubMed1994

Structure of the C3HC4 domain by 1H-nuclear magnetic resonance spectroscopy. A new structural class of zinc-finger.

A recently identified sequence motif, referred to as "C3HC4" (also "RING finger" and "A Box") for its distinctive pattern of putative metal-binding residues, has been found in a wide range of proteins. In a previous paper we described the expression and purification of fragments encompassing this motif from the Vmw110 (IPC0) protein family. We showed that the equine herpes virus protein binds zinc ions and adopts a beta beta alpha beta fold. We now report the tertiary structure of this domain in solution, as determined by two-dimensional 1H-NMR An amphipathic alpha-helix lies along one surface of a triple-stranded beta-sheet. Four pairs of metal-binding residues sequester two zincs at distinct tetrahedral sites. The first and third pairs bind one metal ion, while the second and fourth pairs bind the other, forming an interleaved whole. The first and the fourth pairs are contained within two prominent, well-defined loops related by an approximate dyad symmetry. Conserved residues within the helix, sheet and loops contribute to a compact hydrophobic core. The region comprising the first two beta-strands and the alpha-helix has remarkable structural similarity with a TFIIIA type of zinc finger, even though the C3HC4 domain appears not to bind specifically to DNA or RNA. Using site-directed mutagenesis we demonstrate that exposed polar side-chains of the C3HC4 alpha-helix are essential for trans-activation of gene expression by an intact herpes virus regulatory protein.

Amino Acid Sequence↗
PubMed1993

Epitope mapping identifies an exposed loop between the unique amino- and conserved carboxy-domains of the large subunit of herpes simplex virus type 1 ribonucleotide reductase.

The large subunits of herpes simplex virus types 1 and 2 ribonucleotide reductases contain unique amino-terminal regions comprising 311 and 318 residues respectively, which are not found in ribonucleotide reductases from other sources. We report the mapping of the epitope recognized by monoclonal antibody 1026, which is specific for the large subunit (R1) of HSV-1, and then deduce the structural relationship of the amino-terminal region of R1 with the rest of the protein. A panel of 10 fusion proteins containing sequences spanning the entire R1 subunit were constructed. They were used together with proteolytic fragments of R1 and several synthetic peptides to show that the epitope is discontinuous and appears to be a loop structure centered on a previously located trypsin-sensitive site at residue 305. The existence of the loop was suggested by the observation that reactivity of the antibody with R1 could be blocked by peptides corresponding to residues 289 to 303 and 308 to 313 which flank the trypsin-sensitive site. Our results suggest that the unique amino-terminal region of R1 consists of a structurally distinct domain which is linked to the conserved carboxy region by an exposed loop.

Amino Acid Sequence↗
PubMed1993

An epitope within the DNA-binding domain of the herpes simplex virus immediate early protein Vmw175 is conserved in the varicella-zoster virus gene 62 protein.

We have isolated a panel of monoclonal antibodies that recognize the DNA-binding domain of the herpes simplex virus type 1 (HSV-1) immediate early polypeptide Vmw175. The mice used for the fusions had been immunized with the isolated Vmw175 DNA-binding domain. This had been purified from bacteria that carried a phage T7 expression plasmid with the DNA-binding domain coding region. The epitopes recognized by the monoclonal antibodies were mapped by using a family of truncated versions of the DNA-binding domain, which had also been expressed in the bacterial expression system. The monoclonal antibodies divided into at least four different groups according to this mapping. Several of the monoclonal antibodies recognized Vmw175 expressed in infected BHK cells by HSV-1 strain 17 in Western blots. One of them also recognized the corresponding protein of varicella-zoster virus gene 62. This is further illustration of the relatedness of the two polypeptides.

Amino Acid Sequence↗
PubMed1991

A herpes simplex virus type 1 mutant lacking the ICP0 introns reactivates with normal efficiency.

Previous evidence suggests that the latency-associated transcript (LAT) gene of herpes simplex virus type 1 appears to have a role during reactivation of latent virus because viruses which are null mutants in this gene reactivate slowly or less efficiently than wild-type viruses. Mapping studies have shown that the LAT gene covers a region of about 8.5 kb that overlaps the ICP0 gene in the repeat long region of the herpes simplex virus genome. Previously, we had constructed a mutant with a deletion in the region of the LAT gene encoding a stable 2-kb RNA species (that accumulates to high levels in latently infected cells) and had shown that it reactivates normally (T.M. Block, J.G. Spivack, I. Steiner, S. Deshmane, M.T. McIntosh, R.P. Lirette, and N.W. Fraser, J. Virol. 64:3417-3426, 1990). We now show that a mutant which has two deletions downstream of this region (deleted in both ICP0 introns) reactivates normally in explant cocultivation assays. Thus, the slow or inefficient reactivation phenotype of herpes simplex virus type 1 LAT null mutants is not assignable to this region of the LAT gene.

Animals↗
PubMed1986

Structural characterization of human erythropoietin.

Erythropoietin is the primary regulator of red blood cell formation in mammals. Because of its extreme scarcity, very little information is available regarding structural features of this important glycoprotein. We report here the primary structure of human urinary erythropoietin, determined by protein sequencing. In addition, the sites of glycosylation, assignment of disulfide bonds, and the circular dichroism of the hormone analyzed for secondary structure in comparison with the prediction from the sequence are presented.

Amino Acid Sequence↗
PubMed1984

The relationship between flash evoked potentials and evoked amplitude modulation patterns of an applied UHF electromagnetic field in the rat.

Pilot studies demonstrate evidence that the electrophysiological processes associated with flash stimulation of the central nervous system (CNS) of rats, as seen in the recordings of visual-evoked potentials, may also be detectable using an ultrahigh frequency electromagnetic field (UHFEMF). Patterns of amplitude modulation of an applied UHFEMF, when recorded and averaged, show strong correlations with simultaneously recorded evoked potentials. The data support the hypothesis that the UHFEMF amplitude is altered in a dynamic fashion by the tissue's electrophysiological processes that are involved with the generation of CNS electric fields.

Animals↗
PubMed1984

The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks.

The DNA sequence of the F plasmid origin of conjugal DNA transfer, oriT , has been determined. The origin lies in an intercistronic region which contains several inverted repeat sequences and a long AT-rich tract. Introduction of a nick into one of the DNA strands in the oriT region precedes the initiation of conjugal DNA replication, and the position of the strand-specific nicks acquired by a lambda oriT genome upon propagation in Flac-carrying cells has been determined. The nicks were not uniquely positioned, rather there was a cluster of three major and up to 20 minor sites: the biological significance of this observation is not yet fully clear. Nine independent point mutations which inactivate oriT function have been sequenced and found to alter one or other of two nucleotide positions which lie 14 and 19 bp to one side of the rightmost (as drawn) major nick site. These key nucleotides may lie in a recognition sequence for the oriT endonuclease, since mutations at these sites prevent nicking at oriT .

Base Sequence↗
PubMed1984

Application of recombinant DNA technologies to studies on chicken growth hormone.

Messenger RNA isolated from chicken pituitaries was used to construct a chicken pituitary cDNA library. A chicken growth hormone cDNA clone was isolated using 32P-labeled mammalian growth hormone cDNA probes. The amino acid sequence (derived from the DNA sequence) of the mature form of chicken growth hormone shows 77% homology with that of bovine growth hormone. The chicken growth hormone cDNA clone was used to generate a vector capable of producing chicken growth hormone in Escherichia coli. The recombinant E. coli-derived chicken growth hormone was similar to pituitary chicken growth hormone in several biochemical and immunological properties. The recombinant-derived hormone has been used to establish a sensitive radioimmunoassay for growth hormone determinations made from chicken sera. The chicken growth hormone gene has also been introduced into a retroviral vector capable of establishing productive infections of chicken cells both in in vitro and in vivo. The resulting infections are accompanied by the production of radioimmunoassay-detectable growth hormone. The concentrations of growth hormone in sera of Leghorn chickens infected with the recombinant retrovirus are three- to tenfold higher than in control animals.

Amino Acid Sequence↗
PubMed1984

Factors affecting spontaneous tumor incidence rates in mice: a literature review.

The recent increased use of mice for lifetime oncogenicity testing has resulted in concern for the modulating effects on spontaneous tumor incidence rates by factors other than the test article and the high variability of spontaneous tumor incidence rates in control mice. The various factors shown to affect spontaneous tumor incidence rates in mice must be considered in the planning and conduct of oncogenicity studies with this species if biologically meaningful results are to be achieved. This review reports the results of investigations conducted during the past 50 years to determine the effects of various environmental, dietary, hormonal, viral, and genetic factors on spontaneous tumor incidence rates for mice.

Altitude↗
PubMed1982

Cloning, mutation, and location of the F origin of conjugal transfer.

pED806 , a pBR322 derivative carrying the origin of transfer ( oriT ) of F, was rapidly lost from cells carrying an F tra+ plasmid. Instability was increased in a RecA- host, and depended in particular upon the Ftra YZ genes that produce the nick at oriT at which transfer is initiated. Instability was also correlated with the orientation of the oriT fragment in the vector plasmid. Mutants of pED806 selected as being stable in the presence of Flac proved to carry cis-dominant oriT mutations. The oriT site was subcloned from pED806 on a HaeII fragment including a HaeII-Bg/II segment of F DNA approximately 385 base pair (bp) long into the 2.25 kilobase (kb) vector plasmid pED825 , giving pED822 . pED822 was fully proficient for oriT function, and recircularised in recipient cells by a recA- and tra-independent oriT -specific ligation/recombination event. ' Phasmids ' constructed by cloning pED806 or an oriT - mutant into a lambda vector were used to confirm that the nick site in lambda oriT phages grown in the presence of Flac tra+ is indeed at oriT . The nick site in a further lambda oriT phage (ED lambda 102) was then located 140 +/- 20 bp from the Bg/II site forming one terminus of the F fragment cloned in pED806 and pED822 .

Base Sequence↗
PubMed1981

The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants.

By introduction of recombinant plasmids into monkey CV1 cells, we have unambiguously demonstrated that sequences entirely within the 72 bp repeat, which is located upstream of the SV40 early region, are crucial for T-antigen expression in vivo. We have also shown that a DNA fragment containing the 72 bp repeat, inserted directly before chicken conalbumin or adenovirus-2 major late promoter sequences in chimeric plasmids where these promoters replace that of the SV40 early genes, caused a dramatic increase in the expression of T-antigen in vivo. This effect was independent of the orientation of the 72 bp repeat, but was sensitive to its location within the plasmid, when the 72 bp repeat was separated from the promoter sequences, T-antigen expression was reduced. Insertion of the 72 bp repeat into equivalent plasmids containing no known eukaryotic promoter sequences (plasmids which were not detectably expressed in vivo) gave rise to a measurable, but smaller level of expression. The stimulation of expression by the 72 bp repeat is cis-acting : it required covalent linkage to the recombinant. We discuss the possibility that the 72 bp repeat region in SV40 may act as a bi-directional entry site for RNA polymerase B such that promoter sequences linked to the repeat are more efficiently utilised.

Animals↗