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Biomedical subjects

M K White

Publications and source records attributed to M K White.

At least 19 recordsLinked to original sources
PubMed2006

Interaction of retinoblastoma protein family members with large T-antigen of primate polyomaviruses.

The retinoblastoma gene product pRb and other members of the Rb family of pocket proteins have a central role in the regulation of cell cycle progression. Soon after its discovery, pRb was found to interact with the transforming oncoproteins of DNA tumor viruses and this led to rapid advances in our understanding of the mechanisms of viral transformation and cell cycle progression. DNA viruses of the polyomavirus family have small, circular, double-stranded DNA genomes contained within non-enveloped icosahedral capsids and are highly tumorigenic in experimental animals. At least three types of polyomavirus infect humans: JC virus (JCV), BK virus (BKV) and Simian Vacuolating virus-40. The early region of these viruses encodes the transforming proteins large T-antigen and small t-antigen, which are involved in viral replication and also promote transformation of cells in culture and oncogenesis in vivo. Binding of T-antigen to pRb promotes the activation of the E2F family of transcription factors, which induce the expression of cellular genes required for S phase. In the context of lytic infection, this cell cycle progression is necessary for viral replication because polyomaviruses rely on S phase-specific host factors for their DNA synthesis. In the context of cellular transformation and tumorigenesis, T-antigen/pRB interaction is an indispensable event.

Animals↗
PubMed2006

Human demyelinating disease and the polyomavirus JCV.

Many human neurological diseases involve demyelination of the central and/or peripheral nervous systems. These include the hereditary leukodystrophies--which have a genetic basis; multiple sclerosis (MS)--where the underlying cause of demyelination remains unknown; and progressive multifocal leukoencephalopathy (PML)--where the etiology is well-established as being viral. The human neurotropic polyomavirus--JC virus (JCV)--is the etiologic agent of PML, a fatal demyelinating disease of the central nervous system that occurs mainly in immunosuppressed patients, especially those with HIV/AIDS. JCV belongs to the polyomavirus family of tumor viruses that are characterized by non-enveloped icosahedral capsids containing small, circular, double-stranded DNA genomes. Serological studies have shown that JCV is widespread throughout the human population, but infections are usually restricted by the immune system, particularly cell-mediated immunity, causing the virus to enter a latent phase. An important corollary of this is that situations of severe immunosuppression may permit JCV to replicate and are thus a risk factor for PML.

Demyelinating Diseases↗
PubMed2003

Requirement for the PI3K/Akt pathway in MEK1-mediated growth and prevention of apoptosis: identification of an Achilles heel in leukemia.

The Raf/MEK/ERK kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using DeltaMEK1:ER, a conditionally active form of MEK1 which responds to either beta-estradiol or the estrogen receptor antagonist 4 hydroxy-tamoxifen (4HT), we previously documented the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of human (TF-1) and murine (FDC-P1 and FL5.12) hematopoietic cells lines. Here we demonstrate the ability of DeltaMEK1:ER to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/p70 ribosomal S6 kinase (p70(S6K)) pathway and the importance of this pathway in MEK1-mediated prevention of apoptosis. MEK1-responsive cells can be maintained long term in the presence of beta-estradiol, 4HT or IL-3. Removal of hormone led to the rapid cessation of cell proliferation and the induction of apoptosis in a manner similar to cytokine deprivation of the parental cells. Stimulation of DeltaMEK1:ER by 4HT resulted in ERK, PI3K, Akt and p70(S6K) activation. Treatment with PI3K, Akt and p70(S6K) inhibitors prevented MEK-responsive growth. Furthermore, the apoptotic effects of PI3K/Akt/p70(S6K) inhibitors could be enhanced by cotreatment with MEK inhibitors. Use of a PI3K inhibitor and a constitutively active form of Akt, [DeltaAkt(Myr(+))], indicated that activation of PI3K was necessary for MEK1-responsive growth and survival as activation of Akt alone was unable to compensate for the loss of PI3K activity. Cells transduced by MEK or MEK+Akt displayed different sensitivities to signal transduction inhibitors, which targeted these pathways. These results indicate a requirement for the activation of the PI3K pathway during MEK-mediated transformation of certain hematopoietic cells. These experiments provide important clues as to why the identification of mutant signaling pathways may be the Achilles heel of leukemic cell growth. Leukemia treatment targeting multiple signal transduction pathways may be more efficacious than therapy aimed at inhibiting a single pathway.

Animals↗
PubMed2001

Natural protection from apoptosis by surfactant protein A in type II pneumocytes.

Surfactant-associated protein A (SP-A) is a component of pulmonary surfactant that binds to a specific receptor (SPAR) on the surface of type II alveolar cells of the lung and regulates gene expression and surfactant secretion. Previously we have shown that activation of SPAR by SP-A binding initiates a signal through pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of phosphatidylinositol 3-kinase (PI3K). In other cell types, cytokines that activate the PI3K signaling pathway promote cell survival. Therefore we investigated whether there was an effect of SP-A on apoptosis as measured by DNA laddering, FACS analysis, TUNEL assay, and annexin V binding. SP-A protected primary cultures of rat type II alveolar cells against the apoptotic effects of etoposide and UV light and also protected the H441 human Clara lung tumor cell line against staurosporine-induced apoptosis. The protective effects of SP-A were abrogated by inhibition of either tyrosine-specific protein kinase activity or PI3K. SP-A/SPAR interaction thus initiates a signaling pathway that regulates apoptosis in type II cells. These findings may be important in understanding the pathogenesis of acute lung injury and pulmonary tumorigenesis and may suggest new therapeutic options.

Animals↗
PubMed2001

Suppression of apoptosis: role in cell growth and neoplasia.

A cell is a potentially dangerous thing. In unicellular organisms, cells divide and multiply in a manner that is chiefly determined by the availability of nutritional substrates. In a multicellular organism, each cell has a distinct growth potential that is designed to subsume a role in the function of the whole body. Departure from this path to one of uncontrolled cellular proliferation leads to cancer. For this reason, evolution has endowed cells with an elaborate set of systems that cause errant cells to self-destruct. This process of cell suicide is known as apoptosis or programmed cell death and it plays a crucial role in the growth of both normal and malignant cells. In this review, we describe the mechanisms whereby programmed cell death is induced and executed. In particular, we concentrate on how anti-apoptotic signals generated by cytokines promote cell survival and how these signal transduction pathways may be involved in the pathogenesis of neoplasia. Understanding how these processes contribute to tumorigenesis may suggest new therapeutic options.

Animals↗
PubMed2001

Interactions between the PI3K and Raf signaling pathways can result in the transformation of hematopoietic cells.

The PI3K/Akt and Raf/MEK/ERK signal transduction cascades are pivotal in transmitting signals from membrane receptors to downstream targets that regulate apoptosis, gene expression, and cell growth. The abilities of activated PI3K, Akt, Raf, and MEK proteins to abrogate the cytokine dependence of three different hematopoietic cell lines were determined. Activated PI3K or Akt expression by themselves did not efficiently annul cytokine dependence. Raf and MEK could abrogate the cytokine dependence of murine FDC-PI and human TF-1 cells; however, the frequency of transformation was dependent on the particular oncogene examined, as more factor-independent cells were isolated after infection with activated retroviruses encoding A-Raf or Raf-1 than were with MEK1 or B-Raf. Cytokine-independent deltaRaf-1-infected cells formed tumors on injection into immunocompromised mice, whereas cytokine-dependent cell lines did not, demonstrating the oncogenic effects of activation of the Raf/MEK/ERK pathway. Overexpression of the antiapoptotic Bcl-2 protein synergized with activation of the Raf/MEK/ERK cascade and increased the efficiency of transformation of FDC-PI and TF-1 cells. In contrast to the results observed with FDC-P1 and TF-I cells, the activated Raf genes did not relieve the cytokine dependence of murine FL5.12 cells. The abilities of the Raf and PI3K pathways to interact and annul the cytokine dependence of FL5.12 cells were determined. The combination of Raf and either PI3K or Akt expression relieved cytokine dependence of some FL5.12 cells, and the efficiency of transformation could be enhanced further by Bcl-2 or Bcl-XL overexpression. Thus, the antiapoptotic PI3K/Akt and Bcl-2/Bcl-XL proteins can interact with the growth-promoting Raf/MEK/ERK pathway and annul the cytokine dependence of certain hematopoietic cells.

Animals↗
PubMed2000

Surfactant protein A regulates pulmonary surfactant secretion via activation of phosphatidylinositol 3-kinase in type II alveolar cells.

Pulmonary surfactant is secreted by the type II alveolar cells of the lung, and this secretion is induced by secretagogues of several types (e.g., ionomycin, phorbol esters, and terbutaline). Secretagogue-induced secretion is inhibited by surfactant-associated protein A (SP-A), which binds to a specific receptor (SPAR) on the surface of type II cells. The mechanism of SP-A-activated SPAR signaling is completely unknown. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 rescued surfactant secretion from inhibition by SP-A. In order to directly demonstrate a role for PI3K in SPAR signaling, PI3K activity was immunoprecipitated from type II cell extracts. PI3K activity increased rapidly after SP-A addition to type II cells. Since many receptors that activate PI3K do so through tyrosine-specific protein phosphorylation, antisera to phosphotyrosine, insulin-receptor substrate-1 (IRS-1), or SPAR were also examined. These antisera coimmunoprecipitated PI3K activity that was stimulated by SP-A. In addition, the tyrosine-specific protein kinase inhibitors genistein and herbimycin A blocked the action of SP-A on surfactant secretion. We conclude that SP-A signals to regulate surfactant secretion through SPAR, via pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of PI3K. This activation leads to inhibition of secretagogue-induced secretion of pulmonary surfactant.

Animals↗
PubMed2000

Regulation of glucose transport in differentiating HD3 cells.

The chicken erythroblast cell line, HD3, has high glucose transport activity which is lost upon differentiation to the red cell phenotype. HD3 cells, when incubated under conditions where maturation occurs, show substantial loss of GLUT1 and GLUT3 mRNAs. To assess whether cAMP or cellular protein phosphorylation affected GLUT mRNA and protein, the HD3 cells were incubated in the presence of different phosphatase inhibitors. Treatment of HD3 cells with the phosphatase inhibitors okadaic acid, vanadate or with 3-isobutyl-1-methyl-xanthine induced glucose transport and GLUT mRNAs. This suggests that phosphorylation events enhance glucose transport and that their reduction may be involved in the decrease in glucose transport that occurs upon HD3 cells differentiation.

Animals↗
PubMed2000

Erythrocytic differentiation and glyceraldehyde-3-phosphate dehydrogenase expression are regulated by protein phosphorylation and cAMP in HD3 cells.

Utilisation of glucose undergoes a marked decline during erythroblastic differentiation in the chicken. Concomitantly there is a reduction in the expression of glucose transporter proteins and in the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAD). GAD activity declines, after an initial rise, while the level of GAD mRNA decreases rapidly after induction of differentiation. We have employed the temperature-sensitive chicken erythroblast cell line HD3 that differentiates to the erythrocyte phenotype at 42 degrees C in the presence of inducers (hemin and butyric acid). The role of tyrosine and serine/threonine phosphorylation pathways were evaluated with the phosphatase inhibitors sodium vanadate and okadaic acid, respectively. In the presence of phosphatase inhibitors, HD3 cells underwent differentiation and increased their synthesis of hemoglobin which is a marker protein for red blood cells differentiation. The levels of both GAD mRNA and enzymatic activity were increased by phosphatase inhibitors. The role of cAMP in differentiation was also assessed. Differentiation of HD3 cells was associated with an increase in cAMP. However the phosphodiesterase inhibitor IBMX was not a good inducer of hemoglobin synthesis but did induce GAD mRNA and enzymatic activity. Together these results suggest that multiple pathways (including serine/threonine phosphorylation, tyrosine phosphorylation and elevated cAMP) are involved in the regulation of erythroblastic differentiation, hemoglobin synthesis, GAD gene expression and GAD activity in HD3 cells.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Cellular activation by Ca2+ release from stores in the endoplasmic reticulum but not by increased free Ca2+ in the cytosol.

Ca(2+) release from intracellular stores and/or transmembrane influx can increase the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). Such changes in [Ca(2+)](i) might transduce signals regulating transcription, motility, secretion, and so on. Surfactant secretagogues such as ATP and ionomycin stimulate the release and transmembrane influx of Ca(2+), both of which increase [Ca(2+)](i). The addition of surfactant protein A (SP-A) or depleting cellular Ca(2+) inhibited both surfactant secretion and Ca(2+) transients. Current results suggest that Ca(2+) signalling stimulates surfactant secretion by type II pneumocytes, but not via increased [Ca(2+)](i). Treatment of cells with a Ca(2+) chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM), stimulated secretion but decreased [Ca(2+)](i). Adding SP-A or depleting Ca(2+) inhibited BAPTA-AM-induced secretion. When studied directly, Ca(2+) in the endoplasmic reticulum store ([Ca(2+)](l)) decreased in response to BAPTA, ionomycin and thapsigargin, and increased in response to SP-A. Phorbol ester (PMA) induced surfactant secretion without altering [Ca(2+)](i) or [Ca(2+)](l) and was unaffected by Ca(2+) depletion. The addition of PMA to Ca(2+)-releasing secretagogues increased secretion, but combining two Ca(2+)-releasing secretagogues did not. These results suggest that (1) Ca(2+) signalling of type II cell surfactant secretion reflects changes in [Ca(2+)](l), not [Ca(2+)](i), (2) PMA elicits secretion differently from Ca(2+)-releasing secretagogues, and (3) SP-A inhibits secretion by enhancing Ca(2+) sequestration within endoplasmic reticulum stores. Whether other cell types signal via changes in [Ca(2+)](l) is unknown.

Animals↗
PubMed1998

Regulation of a heterologous glucose transporter promoter in chicken embryo fibroblasts.

GLUT1 is a glucose transporter responsible for increased cellular glucose uptake upon oncogenic transformation or mitogenic stimulation. This is associated with transcriptional activation of the GLUT1 gene. Three regions in the mouse GLUT1 gene mediate this regulation (the promoter and two enhancers). In contrast, chicken GLUT1 transcription is completely uninducible, suggesting either avian signal transduction events are different or the chicken GLUT1 gene lacks these transcriptional control elements. To distinguish between these possibilities, reporter plasmids containing mouse GLUT1 control elements in rodent and avian fibroblasts were compared. These elements function within chicken cells, suggesting that signaling pathways are similar to mouse cells but that the avian GLUT1 gene lacks the control elements necessary to respond to them.

3T3 Cells↗
PubMed1998

Molecular analysis of the interactions between protein kinase C-epsilon and filamentous actin.

Protein kinase C-epsilon (PKC-epsilon) contains a putative actin binding motif that is unique to this individual member of the PKC gene family. We have used deletion mutagenesis to determine whether this hexapeptide motif is required for the physical association of PKC-epsilon and actin. Full-length recombinant PKC-epsilon, but not PKC-betaII, -delta, -eta, or -zeta, bound to filamentous actin in a phorbol ester-dependent manner. Deletion of PKC-epsilon amino acids 222-230, encompassing a putative actin binding motif, completely abrogated this binding activity. When NIH 3T3 cells overexpressing either PKC-epsilon or the deletion mutant of this isozyme were treated with phorbol ester only wild-type PKC-epsilon colocalized with actin in zones of cell adhesion. In binary reactions, it was possible to demonstrate that purified filamentous actin is capable of directly stimulating PKC-epsilon phosphotransferase activity. These and other findings support the hypothesis that a conformationally hidden actin binding motif in the PKC-epsilon sequence becomes exposed upon activation of this isozyme and functions as a dominant localization signal in NIH 3T3 fibroblasts. This protein-protein interaction is sufficient to maintain PKC-epsilon in a catalytically active conformation.

3T3 Cells↗
PubMed1998

Loss of glucose transport in developing avian red cells.

Although red cells are generally associated with significant glucose transport and dependence on glycolysis, the mature red cells of some species (e.g. pig) show very low glucose transport. The generally low level of glucose transport in mature mammalian red cells is the result of maturational development, since it has been shown that even in red cells which have negligible glucose transport (e.g. pig red cells) the corresponding reticulocytes have significant glucose transport activity. The reticulocytes of the chicken, however, show minimal glucose transport activity. But this also is the result of maturational development, since chicken bone marrow red cells do transport glucose which diminishes upon cell maturation in vitro. The erythroblast chicken cell line, HD3, has high glucose transport activity which is lost upon induction to the red cell phenotype. Growing HD3 cells have much higher levels of transport than native chicken bone marrow cells and this is associated in part with elevation of glucose transporter (GLUT) mRNAs as a consequence of the expression of the v-erbA and v-erbB oncogenes. Both native bone marrow red cells and HD3 cells, when incubated in vitro under conditions where maturation occurs, show substantial losses of GLUT mRNA and GLUT proteins. To assess whether the inducers of maturation (hemin and butyrate) affect only the normally expressed GLUTs, chicken GLUT3 expressed from a different promoter was introduced into the HD3 cell by retroviral infection. Both the endogenous and exogenous transporters were lost upon cell differentiation and maturation, leaving a cell with low glucose transport activity. Conversely, in growing cells, butyrate had a pronounced effect on the elevation of the GLUT3 mRNA, especially on the exogenous GLUT3 mRNA, and elevated glucose transport prior to differentiation. These results are consistent with the conclusion that chicken red cell development involves a requirement to reduce glucose transport activity. The near absence of glucose transport in the embryonic chicken red cell is thus due to a loss of this transporter during early development which occurs at an earlier developmental stage in the chicken red cell than in the mammalian red cell.

Animals↗
PubMed1997

Phylogenetic analysis of membrane trafficking proteins: a family reunion and secondary structure predictions.

The realization that a highly conserved family of membrane proteins are localized to transport vesicles and selectively interact with proteins anchored at appropriate target sites of membrane fusion inspired a simple and compelling explanation of how proteins might be transferred and segregated within the cell, the "SNARE hypothesis". This model holds that vesicle and target membrane proteins (designated as v-SNARE and t-SNARE proteins, respectively) wind around one another to form a three-stranded coiled coil structure, termed the prefusion complex. While the molecular topology of the prefusion complex has not been established, the concept that phylogenetically diverse SNARE proteins may become interlocked in a stable coiled coil is particularly attractive, because such a tertiary fold would only be permitted between strictly matched binding partners. For this reason, we have performed a phenetic analysis of all known SNARE sequences to assess the evolutionary and structural relatedness of these ancient protein families. Our phylogenetic analysis and consensus structure predictions revealed that syntaxin and SNAP-25 homologs are significantly related and constitute a superfamily of t-SNARE proteins that fall naturally into four major classes with distinct architectural motifs. The synaptobrevins sorted into three different classes of v-SNARE proteins. Comparison of the consensus structure predictions within each lineage or class of SNARE proteins strongly implied that coiled coil domains may not be required for fusion complex assembly in simple eukaryotic cells. It is our hypothesis that SNARE proteins in the late secretory pathway of mammalian cells may have elaborated more complex secondary structures (coiled coils), at about the time metazoan organisms diverged from yeast, that provide a sterically rigid foundation for positioning a conserved binding domain, the amphipathic alpha-helix.

Animals↗
PubMed1996

Vocally disruptive behavior.

Vocally disruptive behavior (VDB) is intelligible or unintelligible noise making that may be goal-directed or purposeless. VDB can be conceptualized as a cyclic phenomenon. VDB has been linked to cognitive impairment, poor sleep, requiring assistance with ADLs, and being at high risk for falling. General nursing interventions for VDB include remaining calm, using gentle touch, creating a familiar, home-like environment, and using diversions during ADLs.

Aged↗