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C Köhler

Publications and source records attributed to C Köhler.

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

A folding variant of human alpha-lactalbumin induces mitochondrial permeability transition in isolated mitochondria.

A human milk fraction containing multimeric alpha-lactalbumin (MAL) is able to kill cells via apoptosis. MAL is a protein complex of a folding variant of alpha-lactalbumin and lipids. Previous results have shown that upon treatment of transformed cells, MAL localizes to the mitochondria and cytochrome c is released into the cytosol. This is followed by activation of the caspase cascade. In this study, we further investigated the involvement of mitochondria in apoptosis induced by the folding variant of alpha-lactalbumin. Addition of MAL to isolated rat liver mitochondria induced a loss of the mitochondrial membrane potential (Delta Psi(m)), mitochondrial swelling and the release of cytochrome c. These changes were Ca(2+)-dependent and were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. MAL also increased the rate of state 4 respiration in isolated mitochondria by exerting an uncoupling effect. This effect was due to the presence of fatty acids in the MAL complex because it was abolished completely by BSA. BSA delayed, but failed to prevent, mitochondrial swelling as well as dissipation of Delta Psi(m), indicating that the fatty acid content of MAL facilitated, rather than caused, these effects. Similar results were obtained with HAMLET (human alpha-lactalbumin made lethal to tumour cells), which is native alpha-lactalbumin converted in vitro to the apoptosis-inducing folding variant of the protein in complex with oleic acid. Our findings demonstrate that a folding variant of alpha-lactalbumin induces mitochondrial permeability transition with subsequent cytochrome c release, which in transformed cells may lead to activation of the caspase cascade and apoptotic death.

Animals↗
PubMed2001

[New concepts for staging and therapy of cervix cancer by endoscopic surgery].

Non-invasive staging of cervical cancer according to FIGO is out-dated. In contrast to clinical staging exact histopathologic evaluation of tumor extension and tumor biology can be achieved via laparoscopic evaluation. Invasive endoscopic staging is advantageous and should be integrated into the diagnostic armamentarium of gynecologic oncologists and radiotherapists. Through magnification and blood-less surgery minute anatomical structures can be identified laparoscopically. Thus, fibers of autonomic nerves can be visualized and parasympathetic fibres can be preserved. Conservation of these neural structures leads to significant reduction of postoperative morbidity of bladder and rectum. The concept of the sentinel lymph node is also valid for the uterine cervix. Selected biopsy of the sentinel node and identification of single tumor cells leads potentially to higher oncologic safety and may reduce morbidity associated with radical lymphadenectomy. In early stage cervical cancer fertility may be preserved by radical trachelectomy which is combined with laparoscopic parametric and pelvic lymphadenectomy. Following radical trachelectomy a pregnancy rate of 40% is achieved, the recurrence rate following conventional radical hysterectomy is identical.

Cervix Uteri↗
PubMed2001

A framework for improving the quality of health information on the world-wide-web and bettering public (e-)health: the MedCERTAIN approach.

There has been considerable debate about the variable quality of health information on the world-wide-web and its impact on public health. While central authorities to regulate, control, censor, or centrally approve information, in-formation providers or websites are neither realistic nor desirable, public health professionals are interested in making systems available that direct patient streams to the best available information sources. National governments and medical societies have also recognized their responsibility to help users to identify "good quality" information sources. But what constitutes good quality, and how can such a system be implemented in a decentralized and democratic manner? This paper presents a model which combines aspects of consumer education, encouragement of best practices among information providers, self-labeling and external evaluations. The model is currently being implemented and evaluated in the MedCERTAIN project, funded by the European Union under the Action Plan for Safer Use of the Internet. The aim is to develop a technical and organisational infrastructure for a pilot system that allows consumers to access metainformation about web-sites and health information providers, including disclosure information from health providers and opinions of external evaluators. The paper explains the general conceptual framework of the model and presents preliminary experiences including results from an expert consensus meeting, where the framework was discussed.

Databases as Topic↗
PubMed2001

A metadata vocabulary for self- and third-party labeling of health web-sites: Health Information Disclosure, Description and Evaluation Language (HIDDEL).

We describe HIDDEL (Health Information Disclosure, Description and Evaluation Language), formerly known as medPICS (platform for Internet content selection in medicine), a metadata vocabulary designed to enhance transparency, trust and quality of health information on the web. The vocabulary may be used (1) by webmasters to self-describe their contents and policies; (2) by infomediaries (e.g. Healthfinder, NHS Direct/NeLH), e.g. third party evaluators, rating or portal services, to annotate other websites; (3) and by users, to describe their preferences. As an XML application it conforms to the W3C's RDF Specification. The metadata vocabulary is primarily intended to enable descriptions of whole health websites or health information providers. The vocabulary is designed to provide a computer-readable electronic "label" of a health website, telling users who is behind the website, how the website is sponsored, what the con-tent, aim and target audience is, how the information was compiled, what risks the service bears, or what people say about the resource. Client-software can "read" this label automatically, compare it to the user s own set of preferences and needs, and alert and advise users.

Information Services↗
PubMed2000

Interaction of the Arabidopsis polycomb group proteins FIE and MEA mediates their common phenotypes.

Genes of the FERTILISATION INDEPENDENT SEED (FIS) class regulate cell proliferation during reproductive development in Arabidopsis [1-5]. The FIS genes FERTILISATION INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA) encode homologs of animal Polycomb group (Pc-G) proteins, transcriptional regulators that modify chromatin structure and are thought to form multimeric complexes [3-11]. To test whether similarities in fis mutant phenotypes reflect interactions between their protein products, we characterised FIE RNA and protein localisation in vivo, and FIE protein interactions in yeast and in vitro. Expression of FIE mRNA overlaps with that of MEA during embryo sac and seed development and is unaffected in mea mutants. Results from the yeast two-hybrid system and an in vitro pull-down assay indicate that MEA and FIE proteins interact. The relevance of this interaction in vivo is supported by the finding that FIE and MEA co-localise in the nucleus in transfected plant cells. Interaction of MEA and FIE is mediated by the amino-terminal region of MEA. Despite sequence divergence in this domain, MEA can interact with its corresponding animal partner Extrasexcombs (ESC) in the yeast two-hybrid system. We conclude that FIE and MEA act together as part of a multimeric complex and that this accounts for the similarities in mutant phenotypes. We propose that an ancient mechanism for chromatin modification has been independently recruited to different developmental processes in the two kingdoms.

Arabidopsis↗
PubMed2000

Induction of cytochrome P450 (CYP)1A1, CYP1A2, and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes.

Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Characterisation of calmodulin binding to cyclic nucleotide-gated ion channels from Arabidopsis thaliana.

The recently identified cyclic nucleotide-gated ion channels (AtCNGCs) from Arabidopsis thaliana have the ability to bind calmodulin. Using two different methods, we mapped the binding site of AtCNGC1 to the last predicted alpha helix of the cyclic nucleotide binding domain. This is in contrast to CNGCs from animals, where the calmodulin binding site is located in the N-terminus, implying that different mechanisms for CNGC modulation have evolved in animals and plants. Furthermore, we demonstrate that AtCNGC1 and AtCNGC2 have different calmodulin binding affinities and we provide evidence for target specificities among calmodulin isoforms.

Amino Acid Motifs↗
PubMed1999

[Microscopic hematuria in adults--a diagnostic dilemma. Scientific guidelines for management are not available according to a review of the literature].

Red cell urinalysis is a very common laboratory procedure in health care, and microscopic haematuria a common finding in apparently healthy people. Response to a questionnaire sent to all departments of urology in Sweden and to general practitioners in the province of Ostergötland showed both groups to vary considerably in their approach to its management. Accordingly, to determine whether a reliable scientific basis exists for its management, a literature search for the period, 1975-97, was made in MEDLINE, under the headings haematuria, microscopic haematuria and microhaematuria, which yielded a total of 845 items. After scrutiny of the abstracts, 203 reports were selected for closer study in the following respects: study design, series studied, selection criteria, age and gender distribution, test methods used, cut-off values for further investigation, and study results. In 17 series comprising a total of 5,000 patients, urological cancer was diagnosed in less than three per cent of cases. Of almost 200,000 males and females patients tested for haematuria, less than one per cent were found to have disease requiring treatment. In general, the studies were characterised by an absence of control groups, and by differences in study design, selection criteria, test methods, definitions of microscopic haematuria, and investigatory procedures. There were no randomised prospective studies where patients undergoing investigation were compared with those undergoing follow-up only. Thus, as available documentation in professional journals and textbooks shows no consensus to exist as to management in cases of microscopic haematuria, further research is required in this field before scientifically sound guidelines can be issued.

Adult↗
PubMed1999

[Prevalence of newly diagnosed type 2 diabetes, impaired glucose tolerance and abnormal fasting glucose in a high risk population. Data from the RIAD study using new diagnostic criteria for diabetes].

BACKGROUND AND OBJECTIVE: In 1997 the American Diabetes Association (ADA) introduced new criteria for the diagnosis of type 2 diabetes mellitus, reducing the upper limit of normal fasting blood sugar from 140 to 126 mg/dl. A level of between 110 and 126 < mg/dl (6.1-7.0 mmol/l) was added as a new category, "impaired fasting glucose" (IFG) as an at-risk factor. It was the aim of this study to determine what effect these new criteria will have on the prevalence of type 2 diabetes and other glucose tolerance stages in the German population. PATIENTS AND METHODS: The analysis was based on data collected in the "Risk-factors in IGT for atherosclerosis and diabetes" (RIAD) study. 1139 persons of an at-risk population group (aged 40-70 years) were investigated. Most of them were relatives of diabetics and/or themselves had obesity and/or dyslipidaemia. All known diabetics were excluded. All subjects underwent an oral glucose test with 75 g glucose, and plasma glucose values were used for classifying them into different glucose tolerance stages. RESULTS: According to the new ADA criteria, confirmed by WHO in 1998, the prevalence of type 2 diabetes mellitus was shifted from 11% to 15.1%, that of IGT from 28.8% to 26.2%. An impaired fasting glucose was found in 27.1% of the population cohort, 9.2% of whom had 2-hour plasma glucose levels corresponding to the diabetes criteria. Men in the first age decade (40-49 years) had a 14% prevalence of previously undetected type 2 diabetes, double that in women. Hyperlipidaemia, hypertension and obesity were significantly more common in diabetics and persons with IGT than in normoglycaemic persons. CONCLUSIONS: The prevalence of previously unknown type 2 diabetes is very high in this at-risk part of the population. Using the criteria for impaired fasting glucose, 9.2% diabetics would remain undetected. We, therefore, support the recommendation that an oral glucose tolerance test be performed in those of the population aged over 45 years and in younger at-risk persons so that any indicated treatment can be initiated at the earliest. Measurement of fasting glucose is adequate in the population aged under 45 years not at special risk.

Adult↗
PubMed1999

Protease activation in apoptosis induced by MAL.

The proteolytic caspase cascade plays a central role in the signaling and execution steps of apoptosis. This study investigated the activation of different caspases in apoptosis induced by MAL (a folding variant of human alpha-lactalbumin) isolated from human milk. Our results show that the caspase-3-like enzymes, and to a lesser extent the caspase-6-like enzymes, were activated in Jurkat and A549 cells exposed to MAL. Activated caspases subsequently cleaved several protein substrates, including PARP, lamin B, and alpha-fodrin. A broad-range caspase inhibitor, zVAD-fmk, blocked the caspase activation, the cleavage of proteins, and DNA fragmentation, indicating an important role for caspase activation in MAL-induced apoptosis. Since an antagonistic anti-CD95 receptor antibody, ZB4, did not influence the MAL-induced killing, we conclude that this process does not involve the CD95-mediated pathway. While MAL did not directly activate caspases in the cytosol, it colocalized with mitochondria and induced the release of cytochrome c. Thus, these results demonstrate that caspases are activated and involved in apoptosis induced by MAL and that direct interaction of MAL with mitochondria leads to the release of cytochrome c, suggesting that this release is an important step in the initiation and/or amplification of the caspase cascade in these cells.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis.

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.

Adenylate Kinase↗
PubMed1999

Characterisation of a novel gene family of putative cyclic nucleotide- and calmodulin-regulated ion channels in Arabidopsis thaliana.

In plants, cyclic GMP is involved in signal transduction in response to light and gibberellic acid. For cyclic AMP, a potential role during the plant cell cycle was recently reported. However, cellular targets for cyclic nucleotides in plants are largely unknown. Here we report on the identification and characterisation of a new gene family in Arabidopsis, which share features with cyclic nucleotide-gated channels from animals and inward-rectifying K+ channels from plants. The identified gene family comprises six members (Arabidopsis thaliana cyclic nucleotide-gated channels, AtCNGC1-6) with significant homology among the deduced proteins. Hydrophobicity analysis predicted six membrane-spanning domains flanked by hydrophilic amino and carboxy termini. A putative cyclic nucleotide binding domain (CNBD) which contains several residues that are invariant in other CNBDs was located in the carboxy terminus. This domain overlaps with a predicted calmodulin (CaM) binding site, suggesting interaction between cyclic nucleotide and CaM regulation. We demonstrated interaction of the carboxy termini of AtCNGC1 and AtCNGC2 with CaM in yeast, indicating that the CaM binding sites are functional. Furthermore, it was shown that both AtCNGC1 and AtCNGC2 can partly complement the K(+)-uptake-deficient yeast mutant CY162. Therefore, we propose that the identified genes constitute a family of plant cyclic nucleotide- and CaM-regulated ion channels.

Amino Acid Sequence↗
PubMed1999

Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine-rich nuclear export signals in Arabidopsis thaliana.

Transport across the nuclear envelope is mediated by transport receptors from the Importin beta family. We identified Exportin 1 from Arabidopsis (AtXPO1/AtCRM1) as the nuclear export receptor for proteins carrying leucine-rich nuclear export signals (NESs). AtXPO1 shares 42-50% identity with its functional homologues from humans and yeasts. We functionally characterised AtXPO1 by its interaction with NESs of animal and plant proteins, which is inhibited by the cytotoxin leptomycin B (LMB), and also by its interaction with the small GTPase Ran1 in the yeast two-hybrid system. Furthermore, we demonstrated the existence of a nuclear export pathway for proteins in plants. For the characterisation of nuclear export activities, we established an in vivo assay based on the localisation equilibrium of a GFP reporter protein fused to both a nuclear localisation signal (NLS) and an NES motif. Using this in vivo assay we demonstrated that the NES of the heterologous protein Rev is also functional in plants and that its export is inhibited by LMB. In addition, we identified a leucine-rich NES in the Arabidopsis protein AtRanBP1a. The NES, which is located at the carboxy terminus of the protein, is disrupted by mutating three long chain hydrophobic amino acid residues to alanine (L176A, L179A, V181A). In BY-2 protoplasts the NES of AtRanBP1a is functionally indistinguishable from the Rev NES. Our results demonstrate that the machinery for the nuclear export of proteins is functionally conserved in plants.

Amino Acid Sequence↗
PubMed1998

age Mutants of Arabidopsis exhibit altered auxin-regulated gene expression.

An Arabidopsis transgenic line was constructed expressing beta-glucuronidase (GUS) via the auxin-responsive domains (AuxRDs) A and B (BA-GUS) of the PS-IAA4/5 gene in an indoleacetic acid (IAA)-dependent fashion. GUS expression was preferentially enhanced in the root elongation zone after treatment of young seedlings with 10(-7) M IAA. Expression of the BA-GUS gene in the axr1, axr4, and aux1 mutants required 10- to 100-fold higher auxin concentration than that in the wild-type background. GUS expression was nil in the axr 2 and axr 3 mutants. The transgene was used to isolate mutants exhibiting altered auxin-responsive gene expression (age). Two mutants, age1 and age2, were isolated and characterized. age1 showed enhanced sensitivity to IAA, with strong GUS expression localized in the root elongation zone in the presence of 10(-8) M IAA. In contrast, age2 exhibited ectopic GUS expression associated with the root vascular tissue, even in the absence of exogenous IAA. Morphological and molecular analyses indicated that the age1 and age2 alleles are involved in the regulation of gene expression in response to IAA.

Arabidopsis↗
PubMed1998

5-HT2C receptor agonists: pharmacological characteristics and therapeutic potential.

In vitro, (S)-2-(chloro-5-fluoro-indol-1-yl)-1-methylethylamine 1:1 C4H4O4 and (S)-2-(4,4,7-trimethyl-1,4-dihydro-indeno[1, 2-b]pyrrol-1-yl)-1-methylethylamine 1:1 C4H4O4 exhibited high-affinity binding to the serotonin2C (5HT2C) receptors and stimulated turnover of inositol 1,4,5-triphosphate. Affinity to several of the other 5-HT receptor subtypes and to numerous nonserotonergic receptors was much lower. In rats, both compounds elicited behavioral signs of 5-HT2C receptor agonism but not 5-HT2A receptor agonism. Hypomotility induced in rats by high doses of these compounds was reversed by the 5-HT2C receptor antagonist N-(2-naphthyl)-N'-(3-pyridyl)-urea 1:1 HCI. In addition, these compounds were active in tests used to demonstrate anticompulsive effects: reducing schedule-induced polydipsia in rats (prevented by the 5-HT2C/2B receptor antagonist N-(1-methyl-5'-indolyl)-(3-pyridyl)urea 1:1 HCl, reversing increased scratching induced with 8-hydroxy-dipropylaminotetralin 1:1 HCl in squirrel monkeys (no tolerance developed), decreasing responding in the marble-burying task in mice, and decreasing excessive eating of palatable food in rats. In contrast to these compounds, fluoxetine was much less potent, and in some tasks less efficacious, in reducing excessive behavior in these models. These two 5-HT2C receptor agonists do not show anxiogenic effects in the plus-maze in rats. (S)-2-(4,4,7-trimethyl-1,4-dihydro-indeno[1, 2-b]pyrrol-1-yl)-1-methylethylamine 1:1 C4H4O4 reduced the olfactory bulbectomy-induced passive avoidance impairment in rats, a result that indicates antidepressant potential. Similarly, in the differential-reinforcement-of-low rate 72-s operant schedule task in rats, (S)-2-(chloro-5-fluoro-indol-1-yl)-1-methylethylamine 1:1 C4H4O4 increased (and (S)-2-(4,4,7-trimethyl-1,4-dihydro-indeno[1, 2-b]pyrrol-1-yl)-1-methylethylamine 1:1 C4H4O4 showed a tendency to increase) total reinforcements received, which is suggestive of antidepressant activity. The electroencephalography defined sleep-waking pattern in rats produced by these two 5-HT2C agonists, as well as fluoxetine, included increased quiet-waking and decreased rapid-eye-movement sleep, which is characteristic of antidepressant drugs. These results suggest that 5-HT2C receptor agonism is associated with therapeutic potential in obsessive compulsive disorder and depression.

Animals↗
PubMed1997

Differential regulation of indoleamine 2,3-dioxygenase expression by nitric oxide and inflammatory mediators in IFN-gamma-activated murine macrophages and microglial cells.

Induction of indoleamine 2,3-dioxygenase (IDO) and nitric oxide synthase (NOS) is involved in the immunomodulatory roles of IFN-gamma and evidence suggests that these pathways are functionally cross-regulated. We report here that nitric oxide (NO) negatively modulates the expression of IDO activity in IFN-gamma-primed macrophages, but not in microglial cells from mouse. In MT2 macrophages, the induction of IDO activity by IFN-gamma was further increased by the presence of NOS inhibitors, whereas culturing of IFN-gamma-activated MT2 cells with NO generators produced a marked reduction of IDO activity expression. Conversely, neither NOS inhibitors nor exogenous NO affected the induction of the enzyme activity in N11 microglial cells after IFN-gamma activation. LPS and picolinic acid, two costimulatory agents that up-regulate inducible NOS in activated cells, regulated IDO induction differently in the two cell lines. LPS and picolinic acid caused a significant decrease of IDO activity in IFN-gamma-activated MT2 cells. This effect, however, did not appear to be mediated by the ability of LPS and picolinic acid to stimulate NO production. In N11 cells, LPS further stimulated the enzyme activity and picolinic acid had no effect. Northern blot analysis revealed that, in MT2 macrophages, NOS inhibitors increased the levels of IDO mRNA, while a reduction was observed with picolinic acid. No changes in IDO mRNA levels were detected in N11 cells. Consistent with the functional heterogeneity of phagocytes, the reported results indicate the existence of marked differences in the regulation of IDO expression between murine macrophages and microglial cells.

Animals↗
PubMed1996

Isolation and expression of a cDNA clone encoding human kynureninase.

Kynureninase (L-kynurenine hydrolase), a pyridoxal-5'-phosphate-(pyridoxal-P)-dependent enzyme, catalyses the cleavage of L-kynurenine and L-3-hydroxykynurenine into anthranilic and 3-hydroxyanthranilic acids, respectively. In this report, we describe the isolation of a cDNA clone encoding human kynureninase. Degenerate oligonucleotides designed from the amino acid sequences of peptides from rat liver kynureninase, were used as primers for reverse-transcription PCR of rat kidney RNA. The resulting rat cDNA product was then used to screen a human hepatoma cell line (Hep G2) cDNA library. Analysis of a positive cDNA clone showed the presence of an insert of 1651 nucleotides containing an open reading frame coding for a protein of 456 amino acids (theoretical molecular mass = 52357 Da). The predicted amino acid sequence of human kynureninase displayed high similarity to that reported for the rat enzyme and to a Saccharomyces cerevisiae gene product putatively ascribed to kynureninase. Profile analysis of kynureninase primary structure indicated the presence of a pyridoxal-P-binding site consensus sequence assigned to class-V aminotransferases, with Lys276 being the residue binding the cofactor. RNA blot analysis of human tissues, including brain, showed the presence of an approximately 2.0-kb mRNA species in all tissues tested. A second mRNA species (approximately 2.6 kb) was also detected in some tissues. After transfection of HEK-293 cells with the cDNA coding for kynureninase, the K(m) values of L-kynurenine and DL-3-hydroxykynurenine for the recombinant enzyme were 671 +/- 37 microM and 13.2 +/- 2.0 microM, respectively.

Amino Acid Sequence↗