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Biomedical subjects

M Gale

Publications and source records attributed to M Gale.

At least 19 recordsLinked to original sources
PubMed2001

Rice--the pivotal genome in cereal comparative genetics.

Over the past 15 years rice has been the focus of intense co-ordinated research activity to apply the new molecular biology to this key staple. The fact that rice has a small tractable genome and the development of genetic and genomic tools not available in any other cereal have now ensured the promotion of rice as a favoured research target. However the discovery that gene content and gene order--genome colinearity--have been maintained among all the Poaceae family for some 60 million years of evolution has elevated rice yet further to the status of a 'model' organism. Rice tools can be applied in research on the other major cereals, wheat and maize, and many aspects of rice genetics can be transferred to the many minor economic grass species that have not themselves warranted extensive research and breeding. In this paper we describe some of the applications of the discovery of extensive synteny among the grasses.

Biological Evolution↗
PubMed2001

Molecular clones of hepatitis C virus: applications to animal models.

Hepatitis C virus (HCV) is a global public health problem, with approximately 3% of the world population now infected. The clinical course of HCV often involves chronic infection, which can lead to liver dysfunction and hepatocellular carcinoma. Because HCV cannot be efficiently propagated in cell culture, researchers have relied heavily on animal models to study the physical characteristics of HCV and the course of events associated with HCV infection. The chimpanzee is the only nonhuman primate actually proven to be susceptible to HCV infection and has commonly been used to study viral hepatitis induced by HCV. Molecular cloning of the HCV genome has now allowed HCV transmission studies in chimpanzees to progress from the early work of characterizing infectious serum to a current focus of characterizing infectious HCV molecular clones. Moreover, the cloned HCV genome has paved the way for the development of alternative animal models for HCV, most notably transgenic mouse models for the study of HCV pathogenesis. The authors review these animal model applications of the HCV molecular clones, including construction and transmission of mutant viral genomes. The expression of specific viral protein products in these animal models will provide important insight into the structure-function relation that specific HCV genome sequences impart on virus replication and pathogenesis.

Animals↗
PubMed2000

Tissue specific expression of PKR protein kinase in aging B6D2F1 mice.

A decline in the rate of protein synthesis is a common biochemical change observed with aging in a wide variety of cells and organisms. The double stranded RNA-dependent protein kinase PKR has been shown to phosphorylate eukaryotic initiation factor 2 alpha (eIF-2alpha), a well-characterized factor for down-regulating protein synthesis, in response to environmental stress conditions. Therefore, we were interested in evaluating the role of PKR in the aging process. Tissues from 2- and 20-month-old B6D2F1 male mice were evaluated by Western blot analysis. PKR was detected in all tissues of aging mice confirming its ubiquitous nature. Tissues examined from young mice showed little evidence of PKR expression, suggesting an age-associated up-regulation. P58(IPK), a cellular inhibitor of PKR, was expressed in tissues from both age groups but to a greater extent in tissues of aging mice suggesting an up-regulation to control PKR activity. Hyperphosphorylated eIF-2alpha was increased in selected tissues from older mice compared with tissues from younger mice indicating a possible correlation between PKR expression and kinase function. The data suggest that translational activity is slowing down in a tissue specific manner during the aging process in mice, possibly as the result of increased levels of PKR, and could be a factor in the reduction of the rate of protein synthesis during senescence seen in specific tissues of many organisms.

Aging↗
PubMed2000

UK CropNet: a collection of databases and bioinformatics resources for crop plant genomics.

The UK Crop Plant Bioinformatics Network (UK CropNet) was established in 1996 in order to harness the extensive work in genome mapping in crop plants in the UK. Since this date we have published five databases from our central UK CropNet WWW site (http://synteny.nott.ac.uk/) with a further three to follow shortly. Our resource facilitates the identification and manipulation of agronomically important genes by laying a foundation for comparative analysis among crop plants and model species. In addition, we have developed a number of software tools that facilitate the visualisation and analysis of our data. Many of our tools are made freely available for use with both crop plant data and with data from other species.

Crops, Agricultural↗
PubMed2000

Translational control of viral gene expression in eukaryotes.

As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell.

3' Untranslated Regions↗
PubMed1999

A fluid filtration and clearing technique to assess microleakage associated with three dentine bonding systems.

OBJECTIVES: The aims of this in vitro study were to (a) measure fluid flow through teeth restored with one of three dentine bonding systems and a resin composite restoration; (b) measure the distribution of a silver tracer through the same teeth, and make a comparison with fluid flow; and (c) investigate the effect of thermocycling on both measurement types. METHODS: Coronal segments of 30 premolars, randomly allocated to three equal groups, were assessed by fluid filtration. Each group was restored with a resin composite restoration in conjunction with Fuji Bond LC (FBLC), Scotchbond Multi-Purpose Plus (SMP+) or Prime&Bond 2.1 (P&B2.1). Fluid filtration rates were measured in the intact crown and then after cavity preparation, conditioning, dentine bonding, restoration and at 2 and 24 h, 1 week and 1 month following restoration. Six specimens from each group were thermocycled at 1 week. After final filtration measurements the specimens were perfused with silver nitrate and cleared before scoring tracer penetration. RESULTS: No significant differences (P > 0.05) in fluid filtration rates were found amongst the different bonding systems or at any restoration stage. Thermocycling was not associated with any significant (P > 0.05) increase in fluid filtration. Final fluid filtration and tracer distribution showed a weak and not statistically significant correlation (P > 0.05). The penetration of silver stain indicated a failure of the restorations to seal the cavity and demonstrated a possible pathway by which in vivo post-operative sensitivity could occur. CONCLUSIONS: Although not statistically significant, conditioning of the cavity increased the dentinal permeability but this effect was variable. Thermocycling had no statistically significant effect on microleakage.

Analysis of Variance↗
PubMed1999

Antiapoptotic and oncogenic potentials of hepatitis C virus are linked to interferon resistance by viral repression of the PKR protein kinase.

Hepatitis C virus (HCV) is prevalent worldwide and has become a major cause of liver dysfunction and hepatocellular carcinoma. The high prevalence of HCV reflects the persistent nature of infection and the large frequency of cases that resist the current interferon (IFN)-based anti-HCV therapeutic regimens. HCV resistance to IFN has been attributed, in part, to the function of the viral nonstructural 5A (NS5A) protein. NS5A from IFN-resistant strains of HCV can repress the PKR protein kinase, a mediator of the IFN-induced antiviral and apoptotic responses of the host cell and a tumor suppressor. Here we examined the relationship between HCV persistence and resistance to IFN therapy. When expressed in mammalian cells, NS5A from IFN-resistant HCV conferred IFN resistance to vesicular stomatitis virus (VSV), which normally is sensitive to the antiviral actions of IFN. NS5A blocked viral double-stranded RNA (dsRNA)-induced PKR activation and phosphorylation of eIF-2alpha in IFN-treated cells, resulting in high levels of VSV mRNA translation. Mutations within the PKR-binding domain of NS5A restored PKR function and the IFN-induced block to viral mRNA translation. The effects due to NS5A inhibition of PKR were not limited to the rescue of viral mRNA translation but also included a block in PKR-dependent host signaling pathways. Cells expressing NS5A exhibited defective PKR signaling and were refractory to apoptosis induced by exogenous dsRNA. Resistance to apoptosis was attributed to an NS5A-mediated block in eIF-2alpha phosphorylation. Moreover, cells expressing NS5A exhibited a transformed phenotype and formed solid tumors in vivo. Disruption of apoptosis and tumorogenesis required the PKR-binding function of NS5A, demonstrating that these properties may be linked to the IFN-resistant phenotype of HCV.

Antiviral Agents↗
PubMed1999

Inhibition of double-stranded RNA- and tumor necrosis factor alpha-mediated apoptosis by tetratricopeptide repeat protein and cochaperone P58(IPK).

P58(IPK) is a tetratricopeptide repeat-containing cochaperone that is involved in stress-activated cellular pathways and that inhibits the activity of protein kinase PKR, a primary mediator of the antiviral and antiproliferative properties of interferon. To gain better insight into the molecular actions of P58(IPK), we generated NIH 3T3 cell lines expressing either wild-type P58(IPK) or a P58(IPK) deletion mutant, DeltaTPR6, that does not bind to or inhibit PKR. When treated with double-stranded RNA (dsRNA), DeltaTPR6-expressing cells exhibited a significant increase in eukaryotic initiation factor 2alpha phosphorylation and NF-kappaB activation, indicating a functional PKR. In contrast, both of these PKR-dependent events were blocked by the overexpression of wild-type P58(IPK). In addition, the P58(IPK) cell line, but not the DeltaTPR6 cell line, was resistant to dsRNA-induced apoptosis. Together, these findings demonstrate that P58(IPK) regulates dsRNA signaling pathways by inhibiting multiple PKR-dependent functions. In contrast, both the P58(IPK) and DeltaTPR6 cell lines were resistant to tumor necrosis factor alpha-induced apoptosis, suggesting that P58(IPK) may function as a more general suppressor of programmed cell death independently of its PKR-inhibitory properties. In accordance with this hypothesis, although PKR remained active in DeltaTPR6-expressing cells, the DeltaTPR6 cell line displayed a transformed phenotype and was tumorigenic in nude mice. Thus, the antiapoptotic function of P58(IPK) may be an important factor in its ability to malignantly transform cells.

3T3 Cells↗
PubMed1998

Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase.

The interferon (IFN)-induced cellular antiviral response is the first line of defense against viral infection within an animal host. In order to establish a productive infection, eukaryotic viruses must first overcome the IFN-induced blocks imposed on viral replication. The double-stranded RNA-activated protein kinase (PKR) is a key component mediating the antiviral actions of IFN. This IFN-induced protein kinase can restrict viral replication through its ability to phosphorylate the protein synthesis initiation factor eukaryotic initiation factor-2 alpha-subunit and reduce levels of viral protein synthesis. Viruses, therefore, must block the function of PKR in order to avoid these deleterious antiviral effects associated with PKR activity. Indeed, many viruses have developed effective measures to repress PKR activity during infection. This review will focus primarily on an overview of the different molecular mechanisms employed by these viruses to meet a common goal: the inhibition of PKR function, uncompromised viral protein synthesis, and unrestricted virus replication. The past few years have seen exciting new advances in this area. Rather unexpectedly, this area of research has benefited from the use of the yeast system to study PKR. Other recent advances include studies on PKR regulation by the herpes simplex viruses and data from our laboratory on the medically important hepatitis C viruses. We speculate that IFN is ineffective as a therapeutic agent against hepatitis C virus because the virus can effectively repress PKR function. Finally, we will discuss briefly the future directions of this PKR field.

Animals↗
PubMed1998

Regulation of interferon-induced protein kinase PKR: modulation of P58IPK inhibitory function by a novel protein, P52rIPK.

The cellular response to environmental signals is largely dependent upon the induction of responsive protein kinase signaling pathways. Within these pathways, distinct protein-protein interactions play a role in determining the specificity of the response through regulation of kinase function. The interferon-induced serine/threonine protein kinase, PKR, is activated in response to various environmental stimuli. Like many protein kinases, PKR is regulated through direct interactions with activator and inhibitory molecules, including P58IPK, a cellular PKR inhibitor. P58IPK functions to represses PKR-mediated phosphorylation of the eukaryotic initiation factor 2alpha subunit (eIF-2alpha) through a direct interaction, thereby relieving the PKR-imposed block on mRNA translation and cell growth. To further define the molecular mechanism underlying regulation of PKR, we have utilized an interaction cloning strategy to identify a novel cDNA encoding a P58IPK-interacting protein. This protein, designated P52rIPK, possesses limited homology to the charged domain of Hsp90 and is expressed in a wide range of cell lines. P52rIPK and P58IPK interacted in a yeast two-hybrid assay and were recovered as a complex from mammalian cell extracts. When coexpressed with PKR in yeast, P58IPK repressed PKR-mediated eIF-2alpha phosphorylation, inhibiting the normally toxic and growth-suppressive effects associated with PKR function. Conversely, introduction of P52rIPK into these strains resulted in restoration of both PKR activity and eIF-2alpha phosphorylation, concomitant with growth suppression due to inhibition of P58IPK function. Furthermore, P52rIPK inhibited P58IPK function in a reconstituted in vitro PKR-regulatory assay. Our results demonstrate that P58IPK is inhibited through a direct interaction with P52rIPK which, in turn, results in upregulation of PKR activity. Taken together, our data describe a novel protein kinase-regulatory system which encompasses an intersection of interferon-, stress-, and growth-regulatory pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation.

The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2alpha phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

Animals↗
PubMed1997

What happens inside lentivirus or influenza virus infected cells: insights into regulation of cellular and viral protein synthesis.

Efficient manipulation of the regulatory mechanisms controlling host cell gene expression provides the means for productive infection by animal viruses. Upon infecting the host cell, viruses must: (i) bypass the cellular antiviral defense mechanisms to prevent the translational blocks imposed by the interferon pathway; and (ii) effectively "hijack" the host protein synthetic machinery into mass production of virion protein components. The multicomponent regulatory nature of cellular gene expression has provided the means of selecting for a diverse range of mechanisms utilized by animal viruses to ensure that replication efficiency is maintained throughout the virus life cycle. One important research component of the careful examination of gene regulation is those studies that focus on elucidating the mechanisms by which viruses control mRNA translation during host cell infection. Much of the work in our laboratory has focused on elucidating the strategies by which human immunodeficiency virus type 1 and influenza virus regulate protein synthesis during infection. Here we describe the ways in which these two distinctly different RNA viruses ensure the selective and efficient translation of their viral mRNAs in infected cells. These strategies include circumvention of the deleterious effects associated with activation of the interferon-induced protein kinase, PKR. Herein we describe our methodologies designed to elucidate the translational regulation in cells infected by these viruses. We conclude with a brief summary of new directions, utilizing these methods, taken toward understanding the translational control mechanisms imposed by these viral systems, and how our studies of virally infected cells have allowed us to identify growth-regulating components of normal, uninfected cells.

Cell Division↗
PubMed1996

Interaction of the interferon-induced PKR protein kinase with inhibitory proteins P58IPK and vaccinia virus K3L is mediated by unique domains: implications for kinase regulation.

Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58IPK, a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58IPK- and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58IPK-interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58IPK but was dependent on the presence of the eukaryotic initiation factor 2-alpha homology region, mapping to the 34 aa within the sixth P58IPK TPR motif. Consistent with other TPR proteins, P58IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58IPK , K3L interacted exclusively with the PKR protein kinase catalytic domain. In contrast, however, the K3L-binding region of PKR was localized to within aa 367 to 551, demonstrating that each inhibitor bound PKR in unique, nonoverlapping domains. These data, taken together, suggest that P58IPK and K3L may mediate PKR inhibition by distinct mechanisms. Finally, we will propose a model of PKR inhibition in which P58IPK or a P58IPK complex binds PKR and interferes with nucleotide binding and autoregulation, while formation of a PKR-K3L complex interferes with active-site function and/or substrate association.

Base Sequence↗
PubMed1996

PPM-X: a new X-linked mental retardation syndrome with psychosis, pyramidal signs, and macroorchidism maps to Xq28.

We report a three-generation family manifesting a previously undescribed X-linked mental retardation syndrome. Four of the six moderately retarded males have had episodes of manic-depressive psychosis. The phenotype also includes pyramidal signs, Parkinsonian features, and macroorchidism, but there are no characteristic dysmorphic facial features. Affected males do not show fragile sites at distal Xq on cytogenetic analysis, nor do they have expansions of the CGG repeats at the FRAXA, FRAXE, or FRAXF loci. Linkage analyses were undertaken, and a maximal LOD score of 3.311 at theta = .0 was observed with the microsatellite marker DXS1123 in Xq28. A recombination was detected in one of the affected males with DXS1691 (Xq28), which gives the proximal boundary of the localization. No distal recombination has been detected at any of the loci tested.

Adolescent↗
PubMed1995

Sequential assembly of collagen revealed by atomic force microscopy.

Most polymers which comprise biological filaments assemble by two mechanisms: nucleation and elongation or a sequential, stepwise process involving a hierarchy of intermediate species. We report the application of atomic force microscopy (AFM) to the study of the early events in the sequential or stepwise mode of assembly of a macromolecular filament. Collagen monomers were assembled in vitro and the early structural intermediates of the assembly process were examined by AFM and correlated with turbidimetric alterations in the assembly mixture. The assembly of collagen involved a sequence of distinctive filamentous species which increased in both diameter and length over the time course of assembly. The first discrete population of collagen oligomers were 1-2 nm in diameter (300-500 nm in length); at later time points, filaments approximately 2-6 nm in diameter (> 10 microns in length) many with a conspicuous approximately 67-nm axial period were observed. Occasional mature collagen fibrils with a approximately 67-nm axial repeat were found late in the course of assembly. Our results are consistent with initial end-to-end axial association of monomers to form oligomers followed by lateral association into higher-order filaments. On this basis, there appears to be at least two distinctive types of structural interactions (axial and lateral) which are operative at different levels in the assembly hierarchy of collagen.

Animals↗
PubMed1994

Translational control mediates the developmental regulation of the Trypanosoma brucei Nrk protein kinase.

The expression and function of eukaryotic protein kinases is highly regulated, primarily through transcriptional and post-translational processes. In this report we demonstrate an unusual mechanism for controlling protein kinase function, translational control. The Trypanosoma brucei Nrk loci encode predicted protein kinases. Here we show that Nrk has protein serine-threonine kinase activity and examine the expression and activity of Nrk during parasite development. While Nrk transcripts were previously found to be constitutively expressed throughout the life cycle, we now find that expression of Nrk protein is highly stage-regulated. Immunoblot analysis revealed that Nrk expression dramatically increased as the parasites differentiated from proliferative slender bloodforms to the non-proliferative stumpy bloodforms. Procyclic form organisms expressed moderate levels of Nrk. Analysis of Nrk activity demonstrated that it too was highest in stumpy bloodforms. Metabolic labeling and pulse-chase analysis demonstrated that Nrk accumulation was highest in stumpy bloodforms and indicated that Nrk abundance is primarily controlled at the level of biosynthesis rather than turnover. All Nrk mRNA was contained in the poly(A)+ fraction, and the 5' ends of the transcript were the same in each developmental stage. Thus, Nrk is under translational control. The strict developmental regulation of the Nrk enzymes within the trypanosome life cycle suggests that the Nrk protein kinase may play a role in parasite differentiation.

Animals↗