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Biomedical subjects

M Rothe

Publications and source records attributed to M Rothe.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cloning of the mistletoe lectin gene and characterization of the recombinant A-chain.

Mistletoe lectin I (MLI) is the major active constituent of mistletoe extracts, which are widely used for adjuvant tumour therapy. The 66-kDa heterodimeric disulphide-linked glycoprotein is classified as type II ribosome-inactivating protein (RIP) due to the rRNA-cleaving enzyme activity of the A-subunit, also referred to as toxic entity. MLI and the close relative ricin both belong to the family of the two-chain plant type II RIP proteins. Isolation of the glycosylated proteins from plant material yield inhomogeneous material probably due to post-translational modifications. The aim of this study was to prepare pure and homogeneous protein as a prerequisite for structural and mechanistic studies in order to gain insight into the mode of action of this cytotoxic plant protein on tumour and immune cells. Of particular interest was to explain whether the differences in toxicity of ML and ricin are the result of variations of their enzymatic activities. By investigating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific amplification of the mistletoe lectin gene. Applying this PCR strategy the full-length 1923 nucleotide DNA sequence coding for the prepro-protein was obtained showing the existence of a single intron-free gene. In order to elucidate the molecular basis for the observed differences in cytotoxicity within the family of RIP the enzymatic A-subunit was expressed in a heterologous system. Expression of the A-chain in E. coli BL21/pT7 resulted in production of insoluble inclusion bodies constituting 20-30% of total protein. Refolding led to a pure and homogeneous protein species with an apparent molecular mass of 27 kDa and a pI value of 6.4. The ribosome-inactivating activity of the unglycosylated recombinant A-chain (IC50 20.5 pM) protein was in the same range as that of the glycosylated plant-derived ML A-chain (IC50 3.7 pM), which was very similar to that of ricin A-chain (IC50 4.9 pM). Thus, the higher cytotoxicity of ricin cannot be accountable for differences in the enzymatic activities of the type II RIP A-chains.

Amino Acid Sequence↗
PubMed1999

Serodiagnosis of Epstein-Barr virus infection by using recombinant viral capsid antigen fragments and autologous gene fusion.

Using recombinant 15- to 30-kDa fragments and fusion with glutathione S-transferase (GST), we investigated the seroreactivity of three large structural proteins of Epstein-Barr virus (EBV), p150 (BcLF1, capsid), p143 (BNRF1, tegument), and gp125 (BALF4, membrane) in Western blots. None of 13 fragments tested, however, was qualified for diagnostic application. In contrast, the two small viral capsid antigens (VCA), p18 (BFRF3) and p23 (BLRF2), demonstrated sensitive (100%) EBV-specific immunoglobulin G (IgG) reactivities. While p18 additionally showed maximum sensitivity for IgM detection, the IgM sensitivity of p23 was restricted (44%). An autologous fusion protein, p23-p18, which consists N-terminally of full-length p23, followed by the carboxy half of p18, was constructed. This antigen was subjected to indirect VCA enzyme-linked immunosorbent assays (ELISAs), for IgG and IgM, as well as to a micro-capture (microc) IgM ELISA. All assays were found to be 100% specific when EBV-negative sera were tested. Using sera from previously infected individuals, the p23-p18 fusion revealed an improved IgG sensitivity of 99% compared to sensitivities of 97 and 93% for the single antigens p18 and p23, respectively. The sensitivity and specificity of the indirect IgM ELISA with samples of primary and past infections, respectively, were 100%. The microc principle for IgM overcame completely the interference by rheumatoid factors. Compared to the specificity of the indirect IgM version, the specificity with sera collected from rheumatoid arthritis patients increased from 48 to 100%. In summary, the p23-p18 IgG and microc IgM ELISAs showed excellent performances and are promising new diagnostic tests for the detection of EBV-specific antiviral capsid antibodies.

Antibodies, Viral↗
PubMed1998

Human toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide.

Bacterial lipopolysaccharide (LPS) induces activation of the transcription factor nuclear factor kappaB (NF-kappaB) in host cells upon infection. LPS binds to the glycosylphosphatidylinositol (GPI)- anchored membrane protein CD14, which lacks an intracellular signaling domain. Here we investigated the role of mammalian Toll-like receptors (TLRs) as signal transducers for LPS. Overexpression of TLR2, but not TLR1, TLR4, or CD14 conferred LPS inducibility of NF-kappaB activation in mammalian 293 cells. Mutational analysis demonstrated that this LPS response requires the intracellular domain of TLR2. LPS signaling through TLR2 was dependent on serum which contains soluble CD14 (sCD14). Coexpression of CD14 synergistically enhanced LPS signal transmission through TLR2. In addition, purified recombinant sCD14 could substitute for serum to support LPS-induced TLR2 activation. LPS stimulation of TLR2 initiated an interleukin 1 receptor-like NF-kappaB signaling cascade. These findings suggest that TLR2 may be a signaling component of a cellular receptor for LPS.

Cell Line↗
PubMed1998

ASK1 is essential for JNK/SAPK activation by TRAF2.

Tumor necrosis factor (TNF)-induced activation of the c-jun N-terminal kinase (JNK, also known as SAPK; stress-activated protein kinase) requires TNF receptor-associated factor 2 (TRAF2). The apoptosis signal-regulating kinase 1 (ASK1) is activated by TNF and stimulates JNK activation. Here we show that ASK1 interacts with members of the TRAF family and is activated by TRAF2, TRAF5, and TRAF6 overexpression. A truncated derivative of TRAF2, which inhibits JNK activation by TNF, blocks TNF-induced ASK1 activation. A catalytically inactive mutant of ASK1 is a dominant-negative inhibitor of TNF- and TRAF2-induced JNK activation. In untransfected mammalian cells, ASK1 rapidly associates with TRAF2 in a TNF-dependent manner. Thus, ASK1 is a mediator of TRAF2-induced JNK activation.

Animals↗
PubMed1998

Contact with thymic epithelial cells as a prerequisite for cytokine-enhanced human immunodeficiency virus type 1 replication in thymocytes.

We report here that human immunodeficiency virus type 1 (HIV-1)-infected human thymocytes, in the absence of any exogenous stimulus but cocultivated with autologous thymic epithelial cells (TEC), obtained shortly (3 days) after thymus excision produce a high and sustained level of HIV-1 particles. The levels and kinetics of HIV-1 replication were similar for seven distinct viral strains irrespective of their phenotypes and genotypes. Contact of thymocytes with TEC is a critical requirement for optimal viral replication. Rather than an inductive signal resulting from the contact itself, soluble factors produced in the mixed culture are responsible for this effect. Specifically, the synergistic effects of tumor necrosis factor, interleukin-1 (IL-1), IL-6, and granulocyte-macrophage colony-stimulating factor may account by themselves for the high level of HIV-1 replication in thymocytes observed in mixed cultures. In conclusion, the microenvironment generated by TEC-thymocyte interaction might greatly favor optimal HIV-1 replication in the thymus.

Cell Communication↗
PubMed1997

IkappaB kinase-beta: NF-kappaB activation and complex formation with IkappaB kinase-alpha and NIK.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and IkappaB kinase-alpha (IKK-alpha). A widely expressed protein kinase was identified that is 52 percent identical to IKK-alpha. IkappaB kinase-beta (IKK-beta) activated NF-kappaB when overexpressed and phosphorylated serine residues 32 and 36 of IkappaB-alpha and serines 19 and 23 of IkappaB-beta. The activity of IKK-beta was stimulated by tumor necrosis factor and interleukin-1 treatment. IKK-alpha and IKK-beta formed heterodimers that interacted with NIK. Overexpression of a catalytically inactive form of IKK-beta blocked cytokine-induced NF-kappaB activation. Thus, an active IkappaB kinase complex may require three distinct protein kinases.

Amino Acid Sequence↗
PubMed1997

Hair concentrations and self-reported abuse history of 20 amphetamine and ecstasy users.

Hair samples of 20 volunteers of the techno-music scene, who more or less regularly consumed ecstasy tablets and speed and anonymously reported their abuse history, were analyzed in one to seven 3 cm segments for amphetamine (A), methamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethamphetamine (MDE) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butylamine (MBDB) by digestion in 1 M NaOH, subsequent extraction with C18 Bond Elut columns, derivatization with pentafluoropropionyl anhydride and GC/MS-SIM measurements using deuterated standards of A, MA, MDA and MDMA. The concentrations were in the regions 0.1 to 4.8 ng/mg for A (17 samples), 0.05 to 0.89 ng/mg for MDA (16 samples), 0.1 to 8.3 ng/mg for MDMA (16 samples), 0.12 to 15 ng/mg for MDE (13 samples) and 0.21 to 1.3 ng/mg for MBDB (2 samples). MA was not detected. For comparison the frequency and the concentration of these drugs in 124 different ecstasy tablets were determined by HPLC. The drug concentration in the hair segments were compared with the volunteers' reports. Despite the enormous interindividual differences qualitatively an increase of the total concentration of MDA, MDMA and MDE in the proximate 3 cm segments with increasing ecstasy abuse frequency during the last three month before sampling is recognized. In the individual comparison with the chronological consumer reports in most cases a longer interruption or a change of the abuse intensity is not clearly seen at the segment concentrations. As a reason the incorporation of the drugs from sweat into elder hair regions and the slow removal by washing are discussed.

Adolescent↗
PubMed1997

Tumor necrosis factor (TNF)-mediated kinase cascades: bifurcation of nuclear factor-kappaB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2.

TNF-induced activation of the transcription factor NF-kappaB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-kappaB-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-kappaB. Herein we show that NIK interacts with additional members of the TRAF family and that this interaction requires the conserved "WKI" motif within the TRAF domain. We also investigated the role of NIK in JNK activation by TNF. Whereas overexpression of NIK potently induced NF-kappaB activation, it failed to stimulate JNK activation. A kinase-inactive mutant of NIK was a dominant negative inhibitor of NF-kappaB activation but did not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-kappaB and JNK, respectively.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Identification and characterization of an IkappaB kinase.

Activation of the transcription factor NF-kappaB by tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the NF-kappaB-inducing kinase (NIK). In a yeast two-hybrid screen for NIK-interacting proteins, we have identified a protein kinase previously known as CHUK. Overexpression of CHUK activates a NF-kappaB-dependent reporter gene. A catalytically inactive mutant of CHUK is a dominant-negative inhibitor of TNF-, IL-1-, TRAF-, and NIK-induced NF-kappaB activation. CHUK associates with the NF-kappaB inhibitory protein, IkappaB-alpha, in mammalian cells. CHUK specifically phosphorylates IkappaB-alpha on both serine 32 and serine 36, modifications that are required for targeted degradation of IkappaB-alpha via the ubiquitin-proteasome pathway. This phosphorylation of IkappaB-alpha is greatly enhanced by NIK costimulation. Thus, CHUK is a NIK-activated IkappaB-alpha kinase that links TNF- and IL-1-induced kinase cascades to NF-kappaB activation.

Cloning, Molecular↗
PubMed1997

Herpesvirus entry mediator, a member of the tumor necrosis factor receptor (TNFR) family, interacts with members of the TNFR-associated factor family and activates the transcription factors NF-kappaB and AP-1.

The mammalian tumor necrosis factor receptor (TNFR) family consists of 10 cell-surface proteins that regulate development and homeostasis of the immune system. Based on an expressed sequence tag, we have cloned a cDNA encoding a novel member of the human TNFR family. A closely related protein, designated HVEM (for herpesvirus entry mediator), was identified independently by another group as a mediator of herpesvirus entry into mammalian cells (Montgomery, R., Warner, M., Lum, B., and Spear, P. (1996) Cell 87, 427-436). HVEM differed from our clone by two amino acid residues, suggesting that the two proteins represent polymorphism of a single HVEM gene. We detected HVEM mRNA expression in several human fetal and adult tissues, although the predominant sites of expression were lymphocyte-rich tissues such as adult spleen and peripheral blood leukocytes. The cytoplasmic region of HVEM bound to several members of the TNFR-associated factor (TRAF) family, namely TRAF1, TRAF2, TRAF3, and TRAF5, but not to TRAF6. Transient transfection of HVEM into human 293 cells caused marked activation of nuclear factor-kappaB (NF-kappaB), a transcriptional regulator of multiple immunomodulatory and inflammatory genes. HVEM transfection induced also marked activation of Jun N-terminal kinase, and of the Jun-containing transcription factor AP-1, a regulator of cellular stress-response genes. These results suggest that HVEM is linked via TRAFs to signal transduction pathways that activate the immune response.

Cell Line↗
PubMed1997

Effect of pigmentation on the drug deposition in hair of grey-haired subjects.

The hair samples of 15 grizzled patients with a permanent medical treatment by amitriptyline, carbamazepine, chlorprothixene, diclofenac, doxepine, indomethacine, maprotiline or metoclopramide, or with a chronic heroin and cocaine abuse were separated into white and pigmented fibers and both fractions were independently investigated by GC-MS. The drugs were found in pigmented fibers as well as in white fibers, but the concentrations in the white fibers were smaller than in the pigmented ones for the most of the samples investigated. The concentration ratio of the drugs or their metabolites in both hair fractions (white/pigmented) was found to be between 0.09 and 1.57 (mean 0.70, 30 concentration pairs). There are large differences in this ratio between different subjects with the same drug, whereas for different drugs in the same subject in many cases similar ratios were measured. As a reason a different grade of pigmentation of the hair and the influence of the drug structure are discussed. From these results it follows that the natural hair colour is an important parameter in the evaluation of drug concentration in hair.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1997

Structural and concentration effects on the deposition of tricyclic antidepressants in human hair.

In order to elucidate the possibility of a long-term compliance analysis the hair samples of 56 patients, who were under a permanent treatment with tricyclic antidepressants, were analyzed for amitriptyline, clomipramine, doxepine, imipramine and maprotiline as well as their nor-metabolites. The hair concentrations varied between 0.4 and 40 ng/mg with no strong difference between the five drugs. In comparison to the therapeutic plasma levels described in literature the nor-metabolites always accumulate less in hair than drugs. No correlation was found between the hair concentration and the daily dose. The investigation of the full hair length in 3-cm sections for several examples of all five drugs shows that they can be detected in normally kept hair at least 1 year after intake.

Adult↗
PubMed1997

Early lethality, functional NF-kappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice.

TRAF2 is an intracellular signal-transducing protein recruited to the TNFR1 and TNFR2 receptors following TNF stimulation. To investigate the physiological role of TRAF2, we generated TRAF2-deficient mice. traf2-/- mice appeared normal at birth but became progressively runted and died prematurely. Atrophy of the thymus and spleen and depletion of B cell precursors also were observed. Thymocytes and other hematopoietic progenitors were highly sensitive to TNF-induced cell death and serum TNF levels were elevated in these TRAF2-deficient animals. Examination of traf2-/- cells revealed a severe reduction in TNF-mediated JNK/SAPK activation but a mild effect on NF-kappaB activation. These results suggest that TRAF2-independent pathways of NF-kappaB activation exist and that TRAF2 is required for an NF-kappaB-independent signal that protects against TNF-induced apoptosis.

Animals↗
PubMed1996

A nonradioactive assay for ribosome-inactivating proteins.

A sensitive nonradioactive method to determine the activity of ribosome-inactivating proteins (RIPs) based on a combined transcription/translation in vitro assay was established. Using this assay we investigated the RIP activities of the heterodimeric toxic plant lectins ricin and mistletoe lectin I (ML-I). The enzymatic activities of the holoproteins were compared to that of the RIP-active chain of ML-I (ML-I A-chain) and recombinant ML-I A-chain expressed in Escherichia coli. The IC50 values determined for the plant toxins showed that the translation-inactivating activity of ML-I (39.8 pM) is about four times higher than that of ricin (176.0 pM). The plant-derived ML-I A-chain is more toxic (3.4 pM) in the cell-free translation system than the respective holoprotein. The recombinant ML-I A-chain was found to be about three times less active (IC50 10.6 pM) than the A-chain from plant. The in vitro assay described here is a convenient method for the fast determination of RIP activity with a 1000-fold lower detection limit than that of commonly used RIP assays.

Animals↗
PubMed1996

Anatomy of TRAF2. Distinct domains for nuclear factor-kappaB activation and association with tumor necrosis factor signaling proteins.

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family of proteins interact with and transduce signals for members of the TNF receptor superfamily. TRAF1, TRAF2, and TRAF3 share a conserved C-terminal TRAF domain. TRAF2 plays a key role in transducing signals for activation of the transcription factor nuclear factor-kappaB (NF-kappaB). We have performed extensive mutational analysis on TRAF2, examining the requirements for NF-kappaB activation, self-association, and interaction with other molecules involved in TNF signaling. Examination of point mutants and TRAF2-TRAF3 chimeric proteins indicates that the N-terminal RING finger and two adjacent zinc fingers of TRAF2 are required for NF-kappaB activation. The two distinct TRAF-N and TRAF-C subdomains of the TRAF domain appear to independently mediate self-association and interaction with TRAF1. Interaction of TRAF2 with TNF-R2 and TRADD requires sequences at the C terminus of the TRAF-C domain, whereas interaction with the protein kinase receptor-interacting protein V(RIP) occurs via sequences at the N terminus of the TRAF-C domain. Thus, distinct domains of TRAF2 are involved in recruitment and signaling functions.

Antigens, CD↗
PubMed1996

I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins associate with and transduce signals from TNF receptor 2, CD40, and presumably other members of the TNF receptor superfamily. TRAF2 is required for CD40- and TNF-mediated activation of the transcription factor NF-kappa B. Here we describe the isolation and characterization of a novel TRAF-interacting protein, I-TRAF, that binds to the conserved TRAF-C domain of the three known TRAFs. Overexpression of I-TRAF inhibits TRAF2-mediated NF-kappa B activation signaled by CD40 and both TNF receptors. Thus, I-TRAF appears as a natural regulator of TRAF function that may act by maintaining TRAFs in a latent state.

Adaptor Proteins, Signal Transducing↗
PubMed1996

The tumor necrosis factor-inducible zinc finger protein A20 interacts with TRAF1/TRAF2 and inhibits NF-kappaB activation.

TRAF1 and TRAF2 form an oligomeric complex that associates with the cytoplasmic domains of various members of the tumor necrosis factor (TNF) receptor superfamily. TRAF2 action is required for activation of the transcription factor NF-kappaB triggered by TNF and the CD40 ligand. Here we show that TRAF1 and TRAF2 interact with A20, a zinc finger protein, whose expression is induced by agents that activate NF-kappaB. Mutational analysis revealed that the N-terminal half of A20 interacts with the conserved C-terminal TRAF domain of TRAF1 and TRAF2. In cotransfection experiments, A20 blocked TRAF2-mediated NF-kappaB activation. A20 also inhibited TNF and IL-1-induced NF-kappaB activation, suggesting that it may inhibit NF-kappaB activation signaled by diverse stimuli. The ability of A20 to block NF-kappaB activation was mapped to its C-terminal zinc finger domain. Thus, A20 is composed of two functionally distinct domains, an N-terminal TRAF binding domain that recruits A20 to the TRAF2-TRAF1 complex and a C-terminal domain that mediates inhibition of NF-kappaB activation. Our findings suggest a possible molecular mechanism that could explain A20's ability to negatively regulate its own TNF-inducible expression.

Cell Line↗