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Biomedical subjects

M Kunz

Publications and source records attributed to M Kunz.

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

Influenza virus-induced NF-kappaB-dependent gene expression is mediated by overexpression of viral proteins and involves oxidative radicals and activation of IkappaB kinase.

Influenza A viruses are capable of inducing the expression of a variety of cytokine and proapoptotic genes in infected cells. The promoter regions of most of these genes harbor binding sites for the transcription factor NF-kappaB which is an important mediator of immune and inflammatory responses. Our present study is based on an observation that influenza A virus infection of cells stimulates transcriptional activation of the HIV-1 long terminal repeat (LTR) which harbors two regulatory NF-kappaB elements, and is aimed at identifying the molecular mechanisms involved in this process. We found that the expression of influenza virus hemagglutinin (HA), matrix protein (M), and nucleoprotein (NP), as single factors is sufficient to transcriptionally activate the HIV-1 LTR. This process is mediated by oxidative radicals because treatment of cells with pyrrolidine dithiocarbamate, a scavenger of such radicals, abolished the transactivating ability. Expression of different influenza proteins induces activation of NF-kappaB-dependent gene expression but not transcriptional activation of an AP-1/Ets-dependent promoter, indicating a selectivity for NF-kappaB transactivation. Furthermore, influenza protein expression induces activation of IkappaB kinase (IKK). Accordingly coexpression of a catalytically inactive mutant of IKK abolishes influenza protein induced activation of NF-kappaB as well as HIV-1 LTR-dependent reporter gene expression, suggesting that IKK is an important intermediate within this signaling process. Taken together, our results show that various influenza virus proteins act as viral transactivators to modulate transcriptional activity of kappaB-element harboring promoters such as the HIV-LTR.

Antioxidants↗
PubMed2000

Evidence for a 1,2 Shift of a Hydrogen Atom in a Radical Intermediate of the Methylmalonyl-CoA Mutase Reaction.

An excellent substrate of methylmalonyl-CoA mutase, methylmalonyl-carba-(dethia) coenzyme A (methylmalonyl-CH(2)-CoA), was synthesized by a chemoenzymatic method and its alpha-proton was exchanged with deuterium by long-term incubation in deuterium oxide at pH 6.9. After addition of highly purified epimerase-free methylmalonyl-CoA mutase the enzymatic rearrangement was monitored by 1H NMR spectroscopy. Already in the initial phases of the reaction only 72% of the produced succinyl-CH(2)-CoA was monodeuterated, while unlabeled and geminally dideuterated species, 14% of each, were also formed. After the addition of more enzyme the equilibrium (methylmalonyl-CoA:succinyl-CoA = 1:20) was quickly established, while the proportion of unlabeled succinyl-CH(2)-CoA rose to 30% and the geminally dideuterated species were slowly transformed to vicinally dideuterated ones. After 19 h of incubation the ratio of the unlabeled, monodeuterated, and dideuterated species was roughly 1:1:1 while no appreciable deuterium incorporation from the solvent occurred. The unexpected disproportionation of deuterium can be best explained by a 1,2 shift of a hydrogen atom in the succinyl-CH(2)-CoA radical intermediate competing with the hydrogen transfer from 5'-deoxyadenosine. A precedence for such a hydrogen shift in a radical was previously observed only in the mass spectrometer and was supported by ab initio calculations. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding.

We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach. The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline.

Bacterial Proteins↗
PubMed2000

Mutation reports: epidermolysis bullosa simplex associated with severe mucous membrane involvement and novel mutations in the plectin gene.

We report a novel case of epidermolysis bullosa simplex with severe mucous membrane involvement and mutations in the plectin gene (PLEC1). The patient suffered from extensive blistering of the skin and oral and laryngeal mucous membranes. Electron microscopy of a lesional skin biopsy showed cleft formation within the basal cell layer of the epidermis. Antigen mapping displayed entirely negative staining for plectin, a large (>500 kDa) multifunctional adhesion protein present in hemidesmosomes of the basal keratinocytes. Mutation analysis revealed compound heterozygous, previously undisclosed nonsense mutations, Q1713X and R2351X, of paternal and maternal origin, respectively, within exon 32 of PLEC1. Based on earlier reports, plectin deficiency is associated with late onset muscular dystrophy in patients with epidermolysis bullosa. No signs of muscle weakness have been observed during the 4 y follow-up of our patient. This case illustrates the fact that molecular pathological analyses have prognostic implications in identification and evaluation of patients who appear to be at risk for development of muscular dystrophy later in life.

Biopsy↗
PubMed2000

The complement regulators C1 inhibitor and soluble complement receptor 1 attenuate acute lung injury in rabbits.

Because activation of the complement system plays a major role in the pathogenesis of acute lung injury, the availability of new specific complement inhibitors represents a promising therapeutic approach. In the present study we investigated pulmonary edema formation and pulmonary artery pressure (PAP) in acute complement-induced lung injury for possible therapeutic impact of the complement regulators C1 inhibitor and soluble complement receptor 1. Eighteen isolated and ventilated rabbit lungs were perfused with pooled normal human serum (NHS, final concentration 35%) in Krebs-Henseleit buffer in a recirculating system. Lung weight gain and PAP were continuously recorded. Complement activation was blocked by the addition of C1 inhibitor (1.0 U/mL, n = 6) or sCR 1 (2.0 microg/mL, n = 6). Lungs that received NHS without inhibitors served as controls (n = 6). This study was performed according to the Helsinki Declaration and approved by the local government. Application of NHS resulted in an increase of PAP within 20 min from 8+/-2 to 42+/-6 mmHg, which was significantly (P < 0.05) decreased by C1-Inh (25+/-5 mmHg) and sCRI (20 +/-3 mmHg). Moreover, pulmonary edema formation after NHS, as assessed by overall weight gain, was reduced by both C1-Inh and sCR1, compared with controls. These findings were paralleled with significantly decreased thromboxane release rates and reduced tissue deposition of C3c and C5b-9. C1 inhibitor and sCR1 attenuate the complement-induced pulmonary capillary leakage and PAP increase, indicating the protective effect of complement inhibition in isolated perfused rabbit lungs.

Animals↗
PubMed2000

Conformational changes induced in the Tet repressor protein TetR(B) upon operator or anhydrotetracycline binding as revealed by time-resolved fluorescence spectroscopy on single tryptophan mutants.

We have analyzed the tryptophan (trp) fluorescence-decay kinetics of single trp mutants of the Tet repressor protein in the free, the tet operator and anhydrotetracycline (atc)-bound states. The position of the single trp varies between residues 164 and 171, in close proximity to one entrance of the tetracycline-binding pocket. A good fit of the trp fluorescence decay needed generally three exponentials. The decay times vary with detection wavelength, the extent of this variation being correlated to the variation of the emission maximum. Quenching experiments with neutral (acrylamide), cationic (N-methylpyridinium chloride) and anionic quencher (KI) support the interpretation of the three fluorescence components within a conformer model. Operator and atc binding change the ratio of the relative amplitudes of the medium- and long-lived component, thus pointing to structural changes as indicated also by the changes in decay time. Since the fluorescence decay is different between the free, atc- and operator-bound states we conclude that the protein structure is different in each of these three states. The fluorescence quenching constants reflect not only the variation in solvent exposure with position, but also the fact that the net surface charge in this region is negative, because the quenching constants by the cationic quencher are up to 10-fold higher. The atc fluorescence appears to decay monoexponentially with about the same decay time for all mutants, except W170, in which the trp residue sterically interferes with atc.

Bacterial Proteins↗
PubMed1999

[Functional dynamics of impending overt hypothyroidism: spontaneous evolution in patients in the years before the beginning of treatment].

The syndrome of subclinical hypothyroidism, with its clinical and metabolic consequences, is frequent. Only a minority of these patients will eventually become overtly hypothyroid. It was the aim of the present study to analyse the spontaneous evolution over 10 years of 27 patients with subclinical hypothyroidism who became overtly hypothyroid (group A). For comparison, 27 patients remaining subclinically hypothyroid and matched for their TSH concentrations were characterised (group B). In group A, continuous increase of TSH concentrations was observed over the whole observation period (p = 0.002; ANOVA). The concentrations of fT4 remained initially stable, and only fell at a late stage in the three years before overt hypothyroidism (p = 0.0001; ANOVA). The concentrations of total T3 remained normal throughout the observation period. Thyroid reserve was already impaired at the beginning and during the whole study period. In contrast, these thyroid parameters of patients of group B remained unchanged over 10 years, and thyroid reserve remained normal. The pathogenesis of overt hypothyroidism is a graded process. Increasing concentrations of TSH, a decrease of fT4 and impaired thyroid reserve are predictors of overt thyroid failure.

Analysis of Variance↗
PubMed1999

Long-range Coulomb forces and localized bonds.

The ionic model is shown to be applicable to all compounds in which the atoms carry a net charge and their electron density is spherically symmetric regardless of the covalent character of the bonding. By examining the electric field generated by an array of point charges placed at the positions of the ions in over 40 inorganic compounds, we show that the Coulomb field naturally partitions itself into localized regions (bonds) which are characterized by the electric flux that links neighbouring ions of opposite charge. This flux is identified with the bond valence, and Gauss' law with the valence-sum rule, providing a secure theoretical foundation for the bond-valence model. The localization of the Coulomb field provides an unambiguous definition of coordination number and our calculations show that, in addition to the expected primary coordination sphere, there are a number of weak bonds between cations and the anions in the second coordination sphere. Long-range Coulomb interactions are transmitted through the crystal by the application of Gauss' law at each of the intermediate atoms. Bond fluxes have also been calculated for compounds containing ions with non-spherical electron densities (e.g. cations with stereoactive lone electron pairs). In these cases the point-charge model continues to describe the distant field, but multipoles must be added to the point charges to give the correct local field.

Journal Article↗
PubMed1999

Hypoxia-induced up-regulation of angiogenin in human malignant melanoma.

Angiogenesis is essential for tumor progression and metastasis, however, the angiogenesis regulators that are biologically relevant for human melanoma are still unknown. In this study, we analyzed the expression of the potent angiogenic factor angiogenin (ANG) in human melanoma in vitro and in vivo. Four different human melanoma cell lines and two normal melanocytes were kept either under normoxic or hypoxic conditions. After 24 h of hypoxic culture conditions, ANG was up-regulated in the melanoma cell lines but not in normal melanocytes. Induction levels correlated with the metastatic potential of the cell lines. These data were confirmed by Northern blot analysis. In contrast, induction of vascular endothelial growth factor by hypoxia was equally strong in the examined highly aggressive melanoma cell lines and in one nonaggressive cell line. Other angiogenic factors tested as well as the melanoma growth stimulatory activity (Gro-alpha) showed no up-regulation. Thus, in the present study, hypoxia-induced up-regulation in melanoma cells was only observed for ANG and vascular endothelial growth factor. Immunohistochemical studies showed that 8 of 10 melanomas and all 15 metastases were positive for ANG, particularly in the vicinity of small vessels, whereas all benign nevi were negative. Reverse transcription-PCR detected only weak ANG mRNA in nevi but strong signals in primary melanomas and metastases. In conclusion, we demonstrate for the first time enhanced expression of ANG in highly metastatic cell lines as well as in melanomas and metastases in vivo, suggesting that ANG expression is associated with the metastatic potential.

Angiogenesis Inducing Agents↗
PubMed1999

Different mitogen-activated protein kinase signaling pathways cooperate to regulate tumor necrosis factor alpha gene expression in T lymphocytes.

Tumor necrosis factor a (TNF-alpha) is a potent proinflammatory cytokine and plays a crucial role in early events of inflammation. TNF-alpha is primarily produced by monocytes and T lymphocytes. In particular, T-cell-derived TNF-alpha plays a critical role in autoimmune inflammation and superantigen-induced septic shock. However, little is known about the intracellular signaling pathways that regulate TNF expression in T cells. Here we show that extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38-mitogen-activated protein kinase (MAPK) pathways control the transcription and synthesis of TNF-alpha in A3.01 T cells that produce the cytokine upon T cell activation by costimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin. Selective activation of each of the distinct MAPK pathways by expression of constitutively active kinases is sufficient for TNF-alpha promoter induction. Furthermore, blockage of all three pathways almost abolishes TPA/ionomycin-induced transcriptional activation of the TNF-alpha promoter. Selective inhibition of one or more MAPK pathways impairs TNF-alpha induction by TPA/ionomycin, indicating a cooperation between these signal transduction pathways. Our approach revealed that the MAPK kinase 6 (MKK6)/p38 pathway is involved in both transcriptional and posttranscriptional regulation of TNF expression. Moreover, analysis of the progressive 5' deletion mutants of the TNF-alpha promoter indicates that distinct promoter regions are targeted by either ERK-, JNK-, or p38-activating pathways. Thus, unlike what has been reported for other TNF-alpha-producing cells, all three MAPK pathways are critical and cooperate to regulate transcription of the TNF-alpha gene in T lymphocytes, suggesting a T-cell-specific regulation of the cytokine.

Adaptor Proteins, Signal Transducing↗
PubMed1999

The MKK6/p38 stress kinase cascade is critical for tumor necrosis factor-alpha-induced expression of monocyte-chemoattractant protein-1 in endothelial cells.

Monocyte chemoattractant protein-1 (MCP-1), a member of the C-C subfamily of chemokines, is important for the local recruitment of leukocytes to sites of inflammatory challenge. Here, we investigated endothelial signaling pathways involving members of the mitogen-activated protein (MAP) kinase superfamily and studied their role for MCP-1 expression in endothelium. We show that tumor necrosis factor-alpha (TNF-alpha), a potent inflammatory activator of endothelium, leads to activation of MAP kinases ERK, p38, and JNK in human umbilical vein endothelial cells (HUVEC). Contribution of MAP kinase pathways to TNF-alpha-induced synthesis of endothelial MCP-1 was then studied by pharmacologic inhibition and transient expression of dominant negative or constitutively active kinase mutants using flow cytometry, Northern blot, and luciferase reporter gene assays. Inhibition of Raf/MEK/ERK or SEK/JNK pathways had no significant effect on MCP-1 levels, whereas blocking the MKK6/p38 pathway by p38 inhibitors SB203580 or SB202190 or by a dominant negative mutant of MKK6, the upstream activator of p38, strongly inhibited TNF-alpha-induced expression of MCP-1. Consistent with that finding, expression of wild-type or constitutively active MKK6 significantly enhanced the effect of limiting TNF-alpha concentrations on MCP-1 synthesis. These data suggest a crucial role for the MKK6/p38 stress kinase cascade in TNF-alpha-mediated endothelial MCP-1 expression.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Strong expression of the lymphoattractant C-X-C chemokine Mig is associated with heavy infiltration of T cells in human malignant melanoma.

Human malignant melanoma (MM) is a highly aggressive tumour which is particularly prone to specific local immune responses. To determine the microanatomical location and the species of chemokines possibly involved in the intricate control of cell migration and positioning of immune effector cells in primary and metastatic MM lesions, the expression of those chemokines with lymphocyte and/or macrophage chemoattractant properties was analysed by in situ hybridization. GROalpha (growth-related oncogene) and IL-8 (interleukin 8) were expressed at low levels by single melanoma cells, adjacent keratinocytes, and infiltrating leukocytes. In contrast, the lymphocyte-specific chemokine Mig (monokine induced by interferon-gamma) was strongly expressed by mononuclear cells (mainly macrophages) infiltrating the tumour margin in primary MM lesions, whereas expression was less intense in MM metastasis. IP-10 (interferon-gamma inducible protein 10) was expressed in the same loci at lower intensity. Marked infiltration of T cells was exclusively detected in those areas which exhibited strong Mig expression, whereas areas in the vicinity of tumour cells devoid of Mig expression were not infiltrated. In contrast to Mig, expression of MCP-1 (macrophage chemotactic protein-1) was weaker and mainly detected in lesional basal keratinocytes, occasionally at sites of macrophage infiltration, as well as in single melanoma cells. MIP-1alpha (macrophage inflammatory protein 1alpha) showed similar, albeit weaker expression compared with MCP-1. Other chemokines relevant for the recruitment of monocytes and lymphocytes, such as RANTES (regulated on activation, normal T cells expressed and secreted) and MIP-1beta, were barely detectable. In summary, the chemokine expression profiles support the notion that particularly in heavily infiltrated primary MM lesions, Mig and to a lesser extent IP-10 are important mediators of an IFN-gamma-dependent pathway. Due to their lymphoattractant properties and the known inhibitory effects on the tumour vasculature, both chemokines may be critical for the control of local melanoma tumour growth.

Chemokine CXCL10↗
PubMed1999

Anoxia-induced up-regulation of interleukin-8 in human malignant melanoma. A potential mechanism for high tumor aggressiveness.

Besides its proinflammatory properties, interleukin-8 (IL-8) has been suggested as an important promoter for melanoma growth. To study the role of IL-8 in melanoma biology, we determined the in vivo expression of IL-8 mRNA by in situ hybridization in primary melanoma lesions and metastases. High levels of melanoma cell-associated IL-8-specific transcripts were exclusively detected in close vicinity of necrotic/hypoxic areas of melanoma metastases, whereas both in primary melanomas and in non-necrotic metastases IL-8 expression was low or absent. To analyze further the up-regulation of IL-8 mRNA expression in necrotic/hypoxic tumor areas, human melanoma cell lines of different aggressiveness exposed to severe hypoxic stress (anoxia) were used as an in vitro model. Anoxia induced IL-8 mRNA and protein expression in the highly aggressive/metastatic cell lines MV3 and BLM but not in the low aggressive cell lines IF6 and 530. As shown by IL-8 promoter-dependent reporter gene analysis and mRNA stability assays, elevated mRNA levels in melanoma cells were due to both enhanced transcriptional activation and enhanced IL-8 mRNA stability. Interestingly, transcriptional activation was abolished by mutations in the AP-1 and the NF-kappaB-like binding motifs, indicating that both sites are critical for IL-8 induction. Concomitantly, anoxia induced an enhanced binding activity of AP-1 and NF-kappaB transcription factors only in the highly aggressive cells. From our in vitro and in vivo data we suggest that anoxia-induced regulation of IL-8 might be a characteristic feature of aggressive tumor cells, thus indicating that IL-8 might play a critical role for tumor progression in human malignant melanoma.

Antigens, CD↗
PubMed1999

Flow measurements through aortocoronary and intraluminal coronary shunts.

AIMS: Temporary insertion of shunts during coronary artery bypass grafting (CABG) on the beating heart may provide the minimal flow required for adequate myocardial protection (40 to 60 ml/min). We investigated the flow as a function of the pressure head over three aortocoronary shunts and one intraluminal coronary shunt. METHODS: The aortocoronary shunts (VS-01590 with bulb size 2, 3 and 4 mm) and the intraluminal shunt (IVS-4030, bulb size 4 mm) were perfused with 47% glycerin solution at 37 degrees C. The preload was raised in 5 mmHg steps from 35 to 80 mmHg. The afterload was set at 12 mmHg. A regression analysis of the flow on the pressure head was performed. RESULTS: For maximal preload the flow through the aortocoronary shunts was 15.9 +/- 1.3, 46.2 +/- 2.2 and 75.4 +/- 3.3 ml/min, for the intraluminal shunt it was 76.1 +/- 3.4 ml/min. To provide a flow of 40 ml/min a preload of 70, 50 and 45 mmHg was necessary for the 3 mm and 4 mm aortocoronary shunt and the intraluminal shunt respectively. For the aortocoronary and the intraluminal shunts the beta-coefficients were 0.27, 0.66, 1.13 and 1.02 ml/(min*mmHg) respectively with all p < 0.0001. CONCLUSIONS: For adequate pressure head the 3 mm and 4 mm aortocoronary shunt and the intraluminal shunt provide myocardial protection. In case of severe proximal coronary stenosis the intraluminal shunt will not guarantee myocardial protection and main benefit reduces to working in a bloodless field. The insertion of shunts is a cheap and simple method to optimize CABG on the beating heart.

Arteriovenous Shunt, Surgical↗
PubMed1999

Leucocyte activation in erythema nodosum.

As a neutrophilic vascular reaction of small vessels in the subcutaneous tissue and a neutrophilic inflammatory infiltrate are common histopathological features found in initial erythema nodosum (EN) lesions, we asked the question whether activated polymorphonuclear neutrophils (PMN) might play a role in the pathogenesis of EN. We analysed the production of reactive oxygen intermediates (ROI) as a marker for activated neutrophils in the peripheral blood of patients suffering from EN (n = 10) and healthy volunteers (n = 10). FACS analysis of dihydrorhodamine 123-stained PMN was used to detect ROI production due to respiratory burst activity. Patients suffering from EN had a fourfold higher percentage of preactivated ('primed') ROI-producing PMN compared with healthy volunteers. Activation of PMN with formylated methionyl-leucyl-phenylalanine and phorbol 12-myristate 13-acetate led to an increase of ROI production per cell but had no influence on the percentage of activated cells. The percentage of ROI-producing cells in patients with EN correlated with the clinical severity. From these data, we conclude that ROI might play a role in the pathogenesis of EN. ROI might exert their effects by oxidative tissue damage and by promoting tissue inflammation.

Adolescent↗
PubMed1999

Epicardial, biatrial synchronous pacing for prevention of atrial fibrillation after cardiac surgery.

About 30% of patients develop AF after open heart surgery. Biatrial synchronous pacing (BSP) has been shown to promote sinus rhythm in patients with paroxysmal AF refractory to drug therapy. We conducted a prospective, randomized study to test the effect of BSP via epicardial electrodes on the incidence of AF after heart surgery, as compared to conventional therapy. To apply BSP, we attached two epicardial electrodes to the right and one to the left atrium. Immediately following surgery, BSP was initiated in the AAI-Mode at a rate of 10 beats/min above the underlying rhythm (maximum 110 beats/min) and continued for 3 days, during which the rhythm was continually monitored. After 21 (age 63 +/- 9 years) of the planned 200 patients, the study was prematurely aborted because of the proarrhythmic effect of BSP: 6 of the 12 patients treated with BSP developed sensing failure (P amplitude < 1 mV), which provoked AF in 5 of these 6 patients. BSP was discontinued due to diaphragmal stimulation in two patients and due to ventricular stimulation by a dislocated left atrial electrode in one patient. Two patients in the control group (n = 9) developed AF. Using the available standard technology, BSP via epicardial electrodes is not suitable to suppress AF after heart surgery, primarily due to postoperative deterioration of atrial sensing and its profibrillatory effect. In patients requiring atrial pacing after heart surgery, sensing thresholds must be closely monitored to prevent induction of AF.

Atrial Fibrillation↗