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Biomedical subjects

M L Schmitz

Publications and source records attributed to M L Schmitz.

At least 37 records · Page 2Linked to original sources
PubMed2000

Mixed-lineage kinase 3 delivers CD3/CD28-derived signals into the IkappaB kinase complex.

The phosphorylation of IkappaB by the multiprotein IkappaB kinase complex (IKC) precedes the activation of transcription factor NF-kappaB, a key regulator of the inflammatory response. Here we identified the mixed-lineage group kinase 3 (MLK3) as an activator of NF-kappaB. Expression of the wild-type form of this mitogen-activated protein kinase kinase kinase (MAPKKK) induced nuclear immigration, DNA binding, and transcriptional activity of NF-kappaB. MLK3 directly phosphorylated and thus activated IkappaB kinase alpha (IKKalpha) and IKKbeta, revealing its function as an IkappaB kinase kinase (IKKK). MLK3 cooperated with the other two IKKKs, MEKK1 and NF-kappaB-inducing kinase, in the induction of IKK activity. MLK3 bound to components of the IKC in vivo. This protein-protein interaction was dependent on the central leucine zipper region of MLK3. A kinase-deficient version of MLK3 strongly impaired NF-kappaB-dependent transcription induced by T-cell costimulation but not in response to tumor necrosis factor alpha or interleukin-1. Accordingly, endogenous MLK3 was phosphorylated and activated by T-cell costimulation but not by treatment of cells with tumor necrosis factor alpha or interleukin-1. A dominant negative version of MLK3 inhibited NF-kappaB- and CD28RE/AP-dependent transcription elicited by the Rho family GTPases Rac and Cdc42, thereby providing a novel link between these GTPases and the IKC.

CD28 Antigens↗
PubMed2000

Inhibitory effect of NF-kappaB on 1,25-dihydroxyvitamin D(3) and retinoid X receptor function.

Responsiveness to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] may be diminished in osteoporosis and inflammatory arthritis. The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is produced in excess in these disorders and has been shown to decrease osteoblast transcriptional responsiveness to vitamin D and to inhibit the binding of the vitamin D receptor (VDR) and its nuclear partner the retinoid X receptor (RXR) to DNA. Previous studies have shown that a vitamin D (VDRE) or retinoid X DNA response element (RXRE) is sufficient to confer TNF-alpha inhibition of vitamin D or retinoid-stimulated transcription in the absence of known TNF-alpha-responsive DNA sequences. We tested the hypothesis that the TNF-alpha-stimulated transcription factor nuclear factor (NF)-kappaB could, in part, mediate TNF-alpha action by inhibiting the transcriptional potency of the VDR and RXR at their cognate cis regulatory sites. Osteoblastic ROS 17/2.8 cells transfected with a dose of NF-kappaB comparable to that stimulated by TNF-alpha decreased 1,25(OH)(2)D(3)-stimulated transcription. This inhibitory effect of NF-kappaB was not observed on basal transcription of a heterologous reporter in the absence of the VDRE. The effects of NF-kappaB and TNF-alpha were comparable but not additive. COS-7 cells were cotransfected with reporters under the regulation of VDRE or RXRE along with vectors expressing VDR, RXR, and NF-kappaB nuclear proteins. Reconstituted NF-kappaB and the NF-kappaB subunit p65 alone, but not p50, dose dependently suppressed basal and ligand-stimulated transcription. p65 overexpression completely abrogated enhanced VDRE-mediated transcriptional activity in response to 1,25(OH)(2)D(3). Electrophoretic mobility shift experiments did not reveal a direct effect of recombinant NF-kappaB or its individual subunits on the binding of heterodimeric VDR-RXR to DNA. These results suggest that TNF-alpha inhibition of hormone-stimulated transcriptional activation may be mediated by activation of NF-kappaB. In contrast, the inhibitory effect of TNF-alpha on binding of receptors to DNA is unlikely to be mediated by NF-kappaB and is not necessary for inhibition of transcription.

Animals↗
PubMed1999

The antiinflammatory sesquiterpene lactone parthenolide inhibits NF-kappa B by targeting the I kappa B kinase complex.

The transcription factor NF-kappa B is a key regulator of the cellular inflammatory and immune response. Therefore, components of the NF-kappa B-activating signaling pathways are frequent targets for antiinflammatory agents. This study shows that the sesquiterpene lactone parthenolide inhibits a common step in NF-kappa B activation by preventing the TNF-alpha-induced induction of I kappa B kinase (IKK) and IKK beta, without affecting the activation of p38 and c-Jun N-terminal kinase. Parthenolide impairs NF-kappa B-dependent transcription triggered by expression of TNFR-associated factor-2, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK1), and NF-kappa B-inducing kinase. This compound also prevents activation of both IKKs and DNA binding of NF-kappa B induced by MEKK and NF-kappa B-inducing kinase. Parthenolide targets a component of the I kappa B kinase complex without directly inhibiting IKK alpha, IKK beta, or MEKK1. Therefore, this sesquiterpene lactone could serve as a lead compound for the development of antiinflammatory remedies and is suitable as a molecular tool, allowing the dissection of TNF-alpha-derived signaling pathways leading to the activation of NF-kappa B, c-Jun N-terminal kinase, and p38.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1999

Repression of NF-kappaB impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators.

NF-kappaB is an inducible transcription factor, which is regulated by interaction with inhibitory IkappaB proteins. Previous studies linked the activity of NF-kappaB to the proliferative state of the cell. Here we have analysed the function of NF-kappaB in the cell cycle. Inhibition of NF-kappaB in HeLa cells by stable overexpression of a transdominant negative IkappaB-alpha protein reduced cell growth. A kinetic analysis of the cell cycle revealed a retarded G1/S transition. The IkappaB-alpha overexpressing cell clones showed a decreased percentage of cells in the S phase and an impaired incorporation of bromodeoxyuridine (BrdU). The amounts of cyclins A, B1, D1, D3, and E were unchanged, but the G1-specific proteins cyclin D2 and cdk2 were strongly elevated in the IkappaB-alpha overexpressing cell clones. These cell clones also displayed an increase in cyclin D1-dependent kinase activity, pointing to a cell cycle arrest at the late G1 phase. IkappaB-alpha overexpression crosstalked to cell cycle checkpoints via a reduction of transcription factor p53 and elevation of p21WAF. Surprisingly, the IkappaB-alpha overexpressing cells showed an enrichment of c-Myc in the nucleoli, although the total amount of c-Myc protein was unchanged. These experiments identify an important contribution of the NF-kappaB/IkappaB system for the growth of HeLa cells.

Cell Cycle↗
PubMed1999

Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB.

Reactive oxygen species (ROS) play an important role in cell death induced by many different stimuli. This study shows that hydrogen peroxide-induced apoptosis in T-cells did not require tyrosine kinase p561ck, phosphatase CD45, the CD95 receptor and its associated Caspase-8. H2O2-triggered cell death led to the induced cleavage and activation of Caspase-3. Hydrogen peroxide-treatment of T-cells resulted in the formation of mitochondrial permeability transition pores, a rapid decrease of the mitochondrial transmembrane potential delta psi(m) and the release of Cytochrome C. Inhibition of the mitochondrial permeability transition by bongkrekic acid (BA), or interference with the mitochondrial electron transport system by rotenone or menadione prevented the cytotoxic effect of H2O2. Antimycin A, a mitochondrial inhibitor that increases the release of mitochondrial ROS (MiROS), enhanced apoptosis. Overexpression of Bcl-2 and the viral anti-apoptotic proteins BHRF-1 and E1B 19K counteracted H2O2-induced apoptosis. Pharmacological and genetic inhibition of transcription factor NF-kappaB protected cells from hydrogen peroxide-elicited cell death. This detrimental effect of NF-kappaB mediating hydrogen peroxide-induced cell death presumably relies on the induced expression of death effector genes such as p53, which was NF-kappaB-dependently upregulated in the presence of H2O2.

Adenovirus E1B Proteins↗
PubMed1999

Inhibition of tyrosine phosphatases induces apoptosis independent from the CD95 system.

The inhibition of protein tyrosine phosphatases by pervanadate, a potent activator of B- and T-cells through the induction of tyrosine phosphorylation and downstream signaling events in different activation cascades, efficiently induced apoptosis in lymphoid cell lines. Pervanadate-elicited apoptosis could be blocked by the tyrosine kinase inhibitor herbimycin A. This apoptotic process involved the activation of caspases 3, 8 and 9, the induction of mitochondrial permeability transition, the release of cytochrome C and the fragmentation of chromosomal DNA. T-cells lacking the CD95 receptor or caspase-8 and T-cells stably overexpressing a transdominant negative form of the adaptor protein FADD were still susceptible to pervanadate-induced apoptosis, excluding the involvement of the CD95 system or other FADD-dependent death receptors. The apoptotic program initiated by the inhibition of tyrosine phosphatases did not require the presence of the tyrosine kinase p56lck or phosphatase CD45, whereas Bcl-2 overexpression protected T-cells from pervanadate-induced cytochrome C release, caspase-8 cleavage and apoptosis.

Apoptosis↗
PubMed1999

Inhibition of tyrosine phosphatases antagonizes CD95-mediated apoptosis.

Ligation of the CD95 receptor resulted in a transient increase of cellular tyrosine phosphorylation. The inhibition of protein tyrosine phosphatases by pervanadate, a potent activator of B cells and T cells through the induction of tyrosine phosphorylation and downstream signaling events in the activation cascade, antagonized CD95-triggered apoptosis. Pervanadate exerted its inhibitory effect only during the early phase of apoptosis prior to the CD95-induced decrease of the mitochondrial transmembrane potential. Inhibition of tyrosine phosphatases delayed the cleavage and activation of caspase-8 and caspase-3 and antagonized the tyrosine dephosphorylation of the CD95 receptor-associated phosphoproteins p61 and p89/92. In contrast, ligation of the tumor necrosis factor (TNF) receptor resulted in a continuous tyrosine dephosphorylation of cellular proteins. Pervanadate-induced tyrosine phosphorylation increased the TNF-alpha-induced cytotoxicity and NF-kappaB activation, suggesting that it stimulates early signaling events prior to the separation of the two signaling pathways.

Apoptosis↗
PubMed1999

Hypericin as a non-antioxidant inhibitor of NF-kappa B.

NF-kappa B is a transcription factor involved in immune and inflammatory responses. Here we show that micromolar concentrations of hypericin inhibited the PMA- and TNF-alpha-induced activation of NF-kappa B in HeLa and TC10 cells, respectively. In contrast, NF-kappa B activated by H2O2 was not influenced by hypericin, indicating a pathway-specificity of hypericin. Hyperforin and a Hypericum perforatum extract standardised on 0.15% hypericin and 5% hyperforin were not active at pharmacologically relevant concentrations. The PMA/TNF-alpha-induced transcription of a reporter gene, which is under the control of the NF-kappa B-dependent IL-6 promoter, was strongly reduced by preincubation with hypericin.

Animals↗
PubMed1999

Pain assessment and treatment in children with cognitive impairment: a survey of nurses' and physicians' beliefs.

OBJECTIVE: To gather information about healthcare providers' beliefs regarding pain and its treatment in children with cognitive impairment. DESIGN: A survey consisting of two acute pain vignettes (a child undergoing surgery and a child undergoing an outpatient medical procedure) was completed by 440 nurses and 146 physicians at Arkansas Children's Hospital. Respondents completed one of four different surveys that systematically varied the child's level of cognitive impairment (none, mild, moderate, severe) in both vignettes. Questions addressed expected pain, pain assessment methods, pharmacologic regimens, and nonpharmacologic interventions. RESULTS: In general, provider discipline and level of cognitive impairment did not significantly influence responses about pain experience and treatment. Potent analgesia, regularly scheduled dosing, and nonpainful administration were selected most frequently. Self-report of pain, patient-controlled analgesia, and behavioral interventions requiring higher cognitive skills were selected less frequently for children with more severe cognitive impairment. CONCLUSIONS: The presence of cognitive impairment appeared to influence provider decisions regarding the appropriateness of specific pain assessment and treatment methods requiring skills on the part of the child. Overall, healthcare provider views regarding analgesia and sedation were similar for all children, regardless of impairment. Whether these beliefs are consistent with clinical practice is yet to be documented.

Analgesics↗
PubMed1999

Children with cognitive impairment: parent report of pain and coping.

Information about pain in children with cognitive impairment is lacking. To gather pain-relevant information in this population, parents of 145 children with borderline to profound cognitive impairment were interviewed regarding their children's pain expression, experience, treatment, and coping behavior. Descriptions of pain expression and coping behavior were associated with the level of cognitive impairment. Children with mild to moderate cognitive impairment were more likely to be described as directly communicating their pain and exhibiting procedural coping strategies similar to those observed in children without cognitive impairments. More than half of the parents reported that their children experienced pain differently than did children without cognitive impairment, with the majority perceiving decreased pain sensitivity and greater pain tolerance. Finally, one third of parents felt that their children's pain was treated differently than that of other children. Half of these parents believed that health care providers had difficulties assessing and treating their children's pain. Results contribute to the developing foundation of information about pain in this special pediatric population.

Adaptation, Psychological↗
PubMed1999

Efficacy of parental application of eutectic mixture of local anesthetics for intravenous insertion.

OBJECTIVE: To demonstrate that parent application of eutectic mixture of local anesthetics (EMLA) results in equal reduction of the pain of intravenous (IV) placement compared with clinician application of EMLA, and to assess potential difficulties with parental application. STUDY DESIGN: A 2 x 2 randomized block design was used, with 41 children divided into two age groups (5-12 years vs 13-18 years) and randomized to one of two experimental groups (parent-applied EMLA vs clinician-applied EMLA). METHODS: All children were scheduled to have outpatient gastrointestinal endoscopies with IV sedation. EMLA was placed at least 60 minutes before IV insertion either by the parent or a clinician, depending on the experimental group assignment. Outcome measures were child pain ratings and observed behavioral distress ratings. Parents and children were interviewed to determine parent and child anxiety levels in anticipation of the IV insertion, previous needle stick experience, and previous difficulty coping. Feasibility outcomes included technical difficulty with application of EMLA and appearance of the EMLA cream and occlusive covering. RESULTS: Pain ratings and behavioral distress ratings in the low to moderate range for all groups and ws and were consistent w previous empiric reports of EMLA outcome. There were no significant differences in pain or distress ratings for either the age or the experimental groups. Parent ratings of their child's previous difficulty coping was related to the level of behavioral distress exhibited before (r =.50), during (r =.32) and after (r =.44) the IV insertion. In addition, children's anxiety ratings about IV insertion seemed to differ among groups (although not statistically significant for post hoc comparisons), with the most anxiety reported by the younger children when clinicians applied the EMLA and by older children when parents applied the EMLA. CONCLUSION: Parent application of EMLA appears to be as effective as clinician application in reducing children's pain and distress associated with IV insertion. Permitting parents to apply the EMLA at home can allow children who are having procedures on an outpatient basis to benefit from topical anesthesia without having to arrive early to the clinic or hospital. Additionally, application by parents may result in less anticipatory anxiety for younger children.

Adolescent↗
PubMed1998

Various glucocorticoids differ in their ability to induce gene expression, apoptosis and to repress NF-kappaB-dependent transcription.

Glucocorticoids (GCs) influence a great variety of cellular functions by at least three important modes of action: the activation (or repression) of genes controlled by binding sites for the glucocorticoid receptor (GR), the induction of apoptosis in lymphocytes and the recently discovered cross-talk to other transcription factors such as NF-kappaB. In this study we systematically compared various natural and synthetic steroid hormones frequently used as therapeutic agents on their ability to mediate these three modes of action. Betamethasone, triamcinolone, dexamethasone and clobetasol turned out to be the best inducers of gene expression and apoptosis. All GCs including the antagonistic compound RU486 efficiently reduced NF-kappaB-mediated transactivation to comparable extents, suggesting that ligand-induced nuclear localization of the GR is sufficient for transrepression. Glucocorticoid treatment of cells did not result in elevated IkappaB-alpha expression, but impaired the tumor necrosis factor (TNF)-alpha-induced degradation of IkappaB-alpha without affecting DNA binding of NF-kappaB. The structural requirements for the various functions of glucocorticoids are discussed.

Animals↗
PubMed1998

Tumor necrosis factor-alpha-induced cell killing and activation of transcription factor NF-kappaB are uncoupled in L929 cells.

The induction of transcription factor NF-kappaB has been shown to counteract tumor necrosis factor (TNF)-alpha-induced cell death in various cell types. In this study, we investigated the role of NF-kappaB for TNF-alpha-triggered cell death in the widely used mouse cell line L929 by various approaches. Inhibition of the mitochondrial permeability transition by bongkrekic acid impaired TNF-alpha-induced cell death without affecting the activity of NF-kappaB. The reduction of NF-kappaB-mediated gene expression by the synthetic steroid dexamethasone was associated with a decrease in TNF-alpha-mediated cell killing, suggesting that NF-kappaB does not protect L929 cells from TNF-alpha-induced cell death. This concept was reinforced by experiments employing L929 cell lines stably overexpressing a transdominant negative form of IkappaB-alpha. These cell lines were unable to activate NF-kappaB and to inducibly express the IL-6 gene, but they showed the same susceptibility toward TNF-alpha-mediated cell death as L929 wild-type cells.

Animals↗
PubMed1998

p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappaB p65 transactivation mediated by tumor necrosis factor.

Interleukin-6 (IL-6) is a pleiotropic cytokine, which is involved in inflammatory and immune responses, acute phase reactions, and hematopoiesis. In the mouse fibrosarcoma cell line L929, the nuclear factor (NF)-kappaB plays a crucial role in IL-6 gene expression mediated by tumor necrosis factor (TNF). The levels of the activated factor do not, however, correlate with the variations of IL-6 gene transcription; therefore, other factors and/or regulatory mechanisms presumably modulate the levels of IL-6 mRNA production. Upon analysis of various deletion and point-mutated variants of the human IL-6 gene promoter coupled to a reporter gene, we screened for possible cooperating transcription factors. Even the smallest deletion variant, containing almost exclusively a NF-kappaB-responsive sequence preceding the IL-6 minimal promoter, as well as a recombinant construction containing multiple kappaB-motifs, could still be stimulated with TNF. We observed that the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was able to repress TNF-stimulated expression of the IL-6 gene, as well as of a kappaB-dependent reporter gene construct, without affecting the levels of NF-kappaB binding to DNA. Furthermore, we clearly show that, using a nuclear Gal4 "one-hybrid" system, the MAPK inhibitors SB203580 and PD0980589 have a direct repressive effect on the transactivation potential of the p65 kappaB subunit. Therefore, we conclude that, in addition to cytoplasmic activation and DNA binding of NF-kappaB, the p38 and extracellular signal-regulated kinase MAPK pathways act as necessary cooperative mechanisms to regulate TNF-induced IL-6 gene expression by modulating the transactivation machinery.

Animals↗
PubMed1998

Sesquiterpene lactones specifically inhibit activation of NF-kappa B by preventing the degradation of I kappa B-alpha and I kappa B-beta.

Extracts from certain Mexican Indian medicinal plants used in traditional indigenous medicine for the treatment of inflammations contain sequiterpene lactones (SLs), which specifically inhibit the transcription factor NF-kappa B (Bork, P. M., Schmitz, M. L., Kuhnt, M., Escher, C., and Heinrich, M. (1997) FEBS Lett. 402, 85-90). Here we show that SLs prevented the activation of NF-kappa B by different stimuli such as phorbol esters, tumor necrosis factor-alpha, ligation of the T-cell receptor, and hydrogen peroxide in various cell types. Treatment of cells with SLs prevented the induced degradation of I kappa B-alpha and I kappa B-beta by all these stimuli, suggesting that they interfere with a rather common step in the activation of NF-kappa B. SLs did neither interfere with DNA binding activity of activated NF-kappa B nor with the activity of the protein tyrosine kinases p59fyn and p60arc. Micromolar amounts of SLs prevented the induced expression of the NF-kappa B target gene intracellular adhesion molecule 1. Inhibition of NF-kappa B by SLs resulted in an enhanced cell killing of murine fibroblast cells by tumor necrosis factor-alpha. SLs lacking an exomethylene group in conjugation with the lactone function displayed no inhibitory activity on NF-kappa B. The analysis of the cellular redox state by fluorescence-activated cell sorter showed that the SLs had no direct or indirect anti-oxidant properties.

Animals↗
PubMed1998

Co-stimulatory effect of nitric oxide on endothelial NF-kappaB implies a physiological self-amplifying mechanism.

Here we investigated the effects of the second messenger molecule NO at various concentrations on the activation of transcription factor NF-kappaB, IkappaB-alpha kinase (IKK-alpha), Jun N-terminal kinase (JNK) and apoptosis in murine endothelial cells. Low concentrations of NO alone failed to activate NF-kappaB, IKK-alpha and JNK. When NF-kappaB was prestimulated by TNF-alpha or phorbol 12-myristate 13-acetate, the addition of NO at low concentrations enhanced the activation of NF-kappaB. This provides a mechanism for a self-amplifying signal in the inflammatory response, since the inducible NO synthase in endothelial cells is regulated by NF-kappaB. The co-stimulatory effect of NO on NF-kappaB activation was also evident from IKK-alpha kinase assays and reporter gene experiments in endothelial cells. High doses of NO impaired the TNF-alpha-induced DNA-binding activity of NF-kappaB. Accordingly, these high amounts of NO also repressed the TNF-alpha-induced transactivation by NF-kappaB as efficient as dexamethasone. The doses of NO required for the inhibition of NF-kappaB are not cytotoxic for the endothelial cells, enabling the establishment of an autoregulatory loop for NF-kappaB signaling.

Animals↗