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Biomedical subjects

K M Murphy

Publications and source records attributed to K M Murphy.

At least 37 records · Page 2Linked to original sources
PubMed2001

Development of cytochrome oxidase blobs in visual cortex of normal and visually deprived cats.

Cytochrome oxidase (CO) blobs are central to our understanding of the columnar organization and parallel pathways in primate and cat visual cortex. In primates, development of blobs and their relationship with other columnar features of the visual cortex begins pre-natally, before visual experience. In kittens, the supragranular layers differentiate post-natally, after eye opening, raising the possibility that visual experience may influence the development of blobs in cat V1. We have examined the development of blobs in unfolded and flattened sections through the visual cortex of normally reared, dark-reared, monocularly deprived and binocularly deprived kittens. Blobs were found in superficial layers of V1 of normally reared kittens as early as 2 weeks of age, although at this age the overall CO staining in V1 was lighter than in V2. By 6 weeks of age the blobs were adult-like. A patchy pattern of CO staining was also found in V2 of young kittens but not in adults. Visual experience was not necessary for expression of the blobs and monocularly deprived kittens had well developed blobs, indicating that strong Y cell drive is not necessary for the development of blobs in cat V1. CO blobs appear in kitten V1 very early in post-natal development and their expression is independent of visual experience, suggesting that they may be an intrinsic feature of V1 organization.

Age Factors↗
PubMed2001

Structure and specificity of GATA proteins in Th2 development.

Development of Th2 subset of CD4+ T cells involves the interleukin-4 (IL-4)- and Stat6-dependent increase in GATA-3 expression during primary activation. Recently we reported that the phenotypic stability and factor independence of Th2 cells involves acquisition of an intracellular pathway that maintains GATA-3 expression. Evidence from retroviral expression studies implied that this pathway involved an autoactivation of GATA-3 expression, since Stat6-deficient T cells induced endogenous GATA-3 when infected with GATA-3-expressing retroviruses. That study left unresolved the issue of whether GATA-3 autoactivation was direct or indirect. Several other Th2-specific transcription factors have been described, including c-Maf and JunB. We therefore examined the ability of these other transcription factors to induce GATA-3 expression and promote Th2 development. Neither c-Maf nor JunB induced Th2 development in Stat6-deficient CD4+ T cells, in contrast to GATA-3. Consistent with this indication of a possible direct autoactivation pathway, we also observed that heterologous GATA family proteins GATA-1, GATA-2, and GATA-4 were also capable of inducing GATA-3 expression in developing Stat6-deficient T cells and promote Th2 development. Mutational analysis revealed evidence for two distinct mechanisms of GATA-3 action. IL-4 induction by GATA-3 required each of the functional domains to be present, whereas repression of gamma interferon could occur even when mutants of GATA-3 lacking the second transactivation domain, TA2, were expressed. The GATA-dependent induction of the GATA-3 but not the other GATA genes in T cells suggests that T-cell-specific cis elements within the GATA-3 locus likely cooperate with a general GATA recognition motif to allow GATA-3-dependent autoactivation.

Animals↗
PubMed2001

Immune system dysfunction and autoimmune disease in mice lacking Emk (Par-1) protein kinase.

Emk is a serine/threonine protein kinase implicated in regulating polarity, cell cycle progression, and microtubule dynamics. To delineate the role of Emk in development and adult tissues, mice lacking Emk were generated by targeted gene disruption. Emk(-/-) mice displayed growth retardation and immune cell dysfunction. Although B- and T-cell development were normal, CD4(+)T cells lacking Emk exhibited a marked upregulation of the memory marker CD44/pgp-1 and produced more gamma interferon and interleukin-4 on stimulation through the T-cell receptor in vitro. In addition, B-cell responses to T-cell-dependent and -independent antigen challenge were altered in vivo. As Emk(-/-) animals aged, they developed splenomegaly, lymphadenopathy, membranoproliferative glomerulonephritis, and lymphocytic infiltrates in the lungs, parotid glands and kidneys. Taken together, these results demonstrate that the Emk protein kinase is essential for maintaining immune system homeostasis and that loss of Emk may contribute to autoimmune disease in mammals.

Animals↗
PubMed2000

Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation.

Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that activation of the Fas receptor present on a human breast epithelial cell line resulted in a conformational change in the N terminus of the pro-apoptotic protein Bax. This conformational change appeared to occur in the cytosol and precede Bax translocation to the mitochondria. Overexpression of the anti-apoptotic protein Bcl-2 inhibited both the conformational change of Bax as well as its relocalization to the mitochondria. Bcl-2 overexpression did not, however, inhibit Fas-induced cleavage of both procaspase-8 and the pro-apoptotic protein Bid, indicating that Bcl-2 functions downstream of these events. These results suggest that the mechanism by which Bcl-2 inhibits Bax mitochondrial translocation and subsequent amplification of the apoptotic cascade is not by providing a physical barrier to Bax, but rather by inhibiting an upstream event necessary for Bax conformational change.

Apoptosis↗
PubMed2000

Recruitment of Stat4 to the human interferon-alpha/beta receptor requires activated Stat2.

Stat4 activation is involved in differentiation of type 1 helper (Th1) T cells. Although Stat4 is activated by interleukin (IL)-12 in both human and murine T cells, Stat4 is activated by interferon (IFN)-alpha only in human, but not murine, CD4(+) T cells. This species-specific difference in cytokine activation of Stat4 underlies critical differences in Th1 development in response to cytokines and is important to the interpretation of murine models of immunopathogenesis. Here, we sought to determine the mechanism of Stat4 recruitment and activation by the human IFN-alpha receptor. Analysis of phosphopeptide binding analysis suggests that Stat4 does not interact directly with tyrosine-phosphorylated amino acid residues within the cytoplasmic domains of either of the subunits of the IFN-alpha receptor complex. Expression of murine Stat4 in the Stat1-deficient U3A and the Stat2-deficient U6A cell lines shows that IFN-alpha-induced Stat4 phosphorylation requires the presence of activated Stat2 but not Stat1. Thus, in contrast to the direct recruitment of Stat4 by the IL-12 receptor, Stat4 activation by the human IFN-alpha receptor occurs through indirect recruitment by intermediates involving Stat2.

Amino Acid Sequence↗
PubMed2000

Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment.

The initial source of IL-4-inducing Th2 development and the mechanism of stable Th2 commitment remain obscure. We found the reduced level of IL-4 production in Stat6-deficient T cells to be significantly higher than in Th1 controls. Using a novel cell surface affinity matrix technique, we found that IL-4-secreting Stat6-deficient T cells stably expressed GATA-3 and Th2 phenotype. Introducing GATA-3 into Stat6-deficient T cells completely restored Th2 development, inducing c-Maf, Th2-specific DNase I hypersensitive sites in the IL-4 locus, and Th2 cytokine expression. The fact that GATA-3 fully reconstitutes Th2 development in Stat6-deficient T cells indicates it is a master switch in Th2 development. Finally, GATA-3 exerts Stat6-independent autoactivation, creating a feedback pathway stabilizing Th2 commitment.

Animals↗
PubMed2000

Selective loss of type I interferon-induced STAT4 activation caused by a minisatellite insertion in mouse Stat2.

The use of murine systems to model pathogen-induced human diseases presumes that general immune mechanisms between these species are conserved. One important immunoregulatory mechanism involves linkage of innate and adaptive immunity to direct the development of T helper subsets, for example toward subset 1 (TH1) development through STAT4 activation. In analyzing type I interferon signaling, we uncovered a difference between murine and human cells which may affect how these two species control linkage between innate and adaptive immunity. We show that in humans, type I interferons induce TH1 development and can activate STAT4 by recruitment to the IFN-alpha receptor complex specifically via the carboxy-terminus of STAT2. However, the mouse Stat2 gene harbors a minisatellite insertion that has altered the carboxy-terminus and selectively disrupted its capacity to activate STAT4, but not other STATs. This defect in murine Stat2 suggests that the signals leading to STAT4 activation and TH1 development in CD4+ T cells are different between mice and humans.

Animals↗
PubMed2000

Dendritic cell regulation of TH1-TH2 development.

Understanding the control exerted by cytokines on T helper cell subsets 1 and 2 (TH1-TH2) development has progressed to a fairly satisfying knowledge of intracellular signals and transcription factors. Less is understood about the molecular basis of TH1-TH2 development exerted by other parameters, such as how the antigen presenting cell can influence this process. Recent work suggests that dendritic cell subsets contribute significant polarizing influences on T helper differentiation, but how this comes about is less clear. In some cases known pathways may be used, as in the dendritic cell subset 1 exerting TH1 polarization by interleukin 12 (IL-12) production and STAT4 activation. In others, the effects are still in need of explanation.

Animals↗
PubMed2000

Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells.

The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADP-ribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis.

Apoptosis↗
PubMed2000

Splenomegaly, hypersplenism and coagulation abnormalities in liver disease.

Splenomegaly is a frequent finding in patients with liver disease. It is usually asymptomatic but may cause hypersplenism. Thrombocytopenia is the most frequent manifestation of hypersplenism and may contribute to portal hypertension related bleeding. A number of therapies are available for treating thrombocytopenia due to hypersplenism including splenectomy, partial splenectomy, partial splenic embolization, TIPS etc. None is entirely satisfactory. Hypersplenism usually improves following liver transplantation. Therapy with cytokines such as thrombopoietin may offer hope for the future. Patients with liver disease also have abnormalities in coagulation. This is not surprising as all coagulation proteins (except for von willebrand factor vWF) and most inhibitors of coagulation are synthesized in the liver. Genetic or acquired abnormalities of coagulation may predispose to thrombosis of the hepatic or portal veins with significant clinical sequelae. An understanding of the mechanisms involved in coagulation and thrombosis is valuable in choosing from the increasing treatment options available. These include clotting factors, haemeostatic drugs and newer therapies such as recombinant factor VIIa. Splenic artery aneurysms are the most common visceral artery aneurysms in man. Rupture is frequently catastrophic. These aneurysms are being increasingly recognized in liver transplant patients and require treatment before or during transplant surgery.

Blood Coagulation Disorders↗
PubMed2000

Role of the Stat4 N domain in receptor proximal tyrosine phosphorylation.

Stat4 is activated by the cytokines interleukin 12 and alpha interferon (IFN-alpha) and plays a significant role in directing development of naïve CD4(+) T cells to the Th1 phenotype. Signal transducers and activators of transcription (STAT) proteins undergo phosphorylation on a conserved tyrosine residue, resulting in homo- and heterodimerization, nuclear translocation, and DNA binding. Stat4 can bind to single IFN-gamma-activated sites (GASs) as a dimer or bind two tandem GASs as a pair of STAT dimers, or tetramer, stabilized through N-terminal domain (N domain) interactions between dimers. We uncovered an unexpected effect of the Stat4 N domain in controlling the proximal activation of Stat4 by tyrosine phosphorylation at activated receptor complexes. Mutation of the N domain at tryptophan residue W37, predicted to interrupt N domain dimer formation, unexpectedly prevented IFN-alpha-induced tyrosine phosphorylation of the Stat4 monomer, blocking dimer formation and nuclear translocation. Furthermore, N domains appear to exert private STAT functions, since interchanging the N domains between Stat1 and Stat4 prevented receptor-mediated tyrosine phosphorylation in one case and interrupted STAT-specific gene activation in another. Finally, replacement of the N domain of Stat1 with that of Stat4 abrogated the normal Stat2 dependence of Stat1 phosphorylation, again suggesting the domains are not equivalent. Thus, in addition to its role in STAT tetramerization, the conserved STAT N domain appears to participate in very proximal steps of receptor-mediated ligand-induced tyrosine phosphorylation.

Amino Acid Sequence↗
PubMed2000

Signaling and transcription in T helper development.

The recognition of polarized T cell subsets defined by cytokine production was followed by a search to define the factors controlling this phenomenon. Suitable in vitro systems allowed the development of cytokine "recipes" that induced rapid polarization of naïve T cells into Th1 or Th2 populations. The next phase of work over the past several years has begun to define the intracellular processes set into motion during Th1/Th2 development, particularly by the strongly polarizing cytokines IL-12 and IL-4. Although somewhat incomplete, what has emerged is a richly detailed tapestry of signaling and transcription, controlling an important T cell developmental switch. In addition several new mediators of control have emerged, including IL-18, the intriguing Th2-selective T1/ST2 product, and heterogeneity in dendritic cells capable of directing cytokine-independent Th development.

Animals↗
PubMed2000

Reporting medical neglect effectively: a guide for nurse practitioners working with child protective services.

Health care providers who suspect or observe medical neglect of chronic health problems in children are obligated to report the findings to Child Protective Services (CPS). Such reporting results in uneasiness for clinicians, who may jeopardize their therapeutic relationships with the patient and family, or worse, provoke them to withdraw from treatment. To the contrary, reporting medical neglect and discussing it openly and empathetically with the family can foster more cohesive teamwork and prompt families to address their problems. Knowing your state's child abuse laws, how to effectively report medical neglect, and how to communicate and collaborate with CPS caseworkers may result in the provision of therapeutic services to families. Enhancing family functioning can result in increased efforts toward caring for their child's medical problem.

Child↗
PubMed1999

The human T-cell leukemia virus type-1 Tax protein regulates the activity of the IkappaB kinase complex.

Two cytokine-inducible kinases, IKKalpha and IKKbeta, are components of a 700-kDa kinase complex that specifically phosphorylates IkappaB. Phosphorylation of IkappaB by IKK leads to its ubiquitination and subsequent degradation, resulting in the nuclear translocation of NF-kappaB. The oncogenic protein Tax, encoded by human T-cell leukemia virus type-1 (HTLV-1), stimulates IKK activity to result in constitutive nuclear levels of NF-kappaB. In an attempt to gain insights into the mechanism by which Tax mediates constitutive activation of the NF-kappaB pathway, we analyzed the chromatographic distribution of IKK proteins using cellular extracts prepared from three T lymphocytes either lacking or containing Tax. IKK kinase activity and the distribution of proteins in the IKK complex were characterized. In extracts prepared from cells containing Tax, the activity of both IKKalpha and IKKbeta present in the 700-kDa IKK complex were increased. Surprisingly, cell lines expressing Tax also contained an additional peak of IKKbeta, but not IKKalpha activity, that migrated at 300 kDa rather than at 700 kDa. We noted that extracts containing Tax had extremely low levels of IkappaBbeta, but not IkappaBalpha, and contained predominantly a truncated form of the MAP3K MEKK1. These results suggest that Tax may target several components of the NF-kappaB pathway leading to constitutive activation of this important regulator of cellular gene expression.

Animals↗
PubMed1999

Bax membrane insertion during Fas(CD95)-induced apoptosis precedes cytochrome c release and is inhibited by Bcl-2.

Ligation of the Fas cell surface receptor leads to activation of caspases and subsequent apoptosis. Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that the pro-apoptotic protein, Bax, translocates from the cytosol specifically to the mitochondria following Fas ligation in MCF10A1 breast epithelial cells. Bax translocation was dependent on caspase activation, and preceded the release of cytochrome c and loss of mitochondrial respiratory activity. Bax translocation occurred in concert with activation of downstream caspases as determined by cleavage of a synthetic substrate, proteolysis of poly(ADP-ribose) polymerase, and processing of procaspase-3 and -7. Overexpression of the anti-apoptotic protein, Bcl-2, prevented Bax insertion, cytochrome c release, complete processing of caspase-3 and -7, and full activation of DEVD-specific cleavage activity. These data establish a role for Bax mitochondrial insertion during Fas-mediated apoptosis, and support a model in which Bax insertion amplifies the Fas apoptotic cascade through cytochrome c release and complete processing of caspases-3 and -7. In addition, our findings indicate that prevention of Bax insertion into the mitochondria represents a novel mechanism by which Bcl-2 inhibits Fas-induced apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Bullous systemic lupus erythematosus (type I) in a dog.

In human patients with systemic lupus erythematosus, cutaneous subepidermal blistering can occur because of the production of antibodies specific for basement membrane antigens. This condition is referred to as bullous systemic lupus erythematosus (BSLE). A dog was diagnosed with BSLE because it fulfilled the following criteria: (i) a diagnosis of systemic lupus erythematosus by standard methods; (ii) an acquired, vesicular, erosive and ulcerative eruption; (iii) microscopical subepidermal vesicles with neutrophil-predominant inflammation at the dermo-epidermal junction; (iv) deposition of IgG at the epidermal basement membrane zone; and (v) circulating IgG autoantibodies against type VII collagen. Anti-collagen VII type I-BSLE therefore needs to be considered as a possible differential diagnosis for canine autoimmune subepidermal blistering diseases.

Animals↗