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C Köhler

Publications and source records attributed to C Köhler.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1996

Regulation of the kynurenine metabolic pathway by interferon-gamma in murine cloned macrophages and microglial cells.

Several pieces of evidence suggest a major role for brain macrophages in the overproduction of neuroactive kynurenines, including quinolinic acid, in brain inflammatory conditions. In the present work, the regulation of kynurenine pathway enzymes by interferon-gamma (IFN-gamma) was studied in immortalized murine macrophages (MT2) and microglial (N11) cells. In both cell lines, IFN-gamma induced the expression of indoleamine 2,3-dioxygenase (IDO) activity. Whereas tumor necrosis factor-alpha did not affect enzyme induction by IFN-gamma, lipopolysaccharide modulated IDO activity differently in the two IFN-gamma-activated cell lines, causing a reduction of IDO expression in MT2 cells and an enhancement of IDO activity in N11 cells. Kynurenine aminotransferase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilic acid dioxygenase appeared to be constitutively expressed in both cell lines. Kynurenine 3-hydroxylase activity was stimulated by IFN-gamma. It was notable that basal kynureninase activity was much higher in MT2 macrophages than in N11 microglial cells. In addition, IFN-gamma markedly stimulated the activity of this enzyme only in MT2 cells. IFN-gamma-treated MT2 cells, but not N11 cells, were able to produce detectable amounts of radiolabeled 3-hydroxyanthranilic and quinolinic acids from L-[5-3H] tryptophan. These results support the notion that activated invading macrophages may constitute one of the major sources of cerebral quinolinic acid during inflammation.

3-Hydroxyanthranilate 3,4-Dioxygenase↗
PubMed1996

Cytochrome P4502D4 in the brain: specific neuronal regulation by clozapine and toluene.

Twenty-four hr after a single dose of the neuroleptic drug clozapine, cytochrome P4502D4 (P4502D4) immunoreactivity, which was barely detectable in the brains of untreated rats, was clearly evident in neurons of the substantia nigra pars compacta, ventral tegmental area, granular neurons of the olfactory bulb, and Purkinje and granular neurons of the cerebellum. Induction was maintained with daily administration for 3 weeks. The mRNA for P4502D4 was detected by Northern blotting and localized by in situ hybridization in neurons throughout the brain and in the Bergman glia in the cerebellum. There were no detectable changes in the distribution or quantity of P4502D4 mRNA after treatment with clozapine. The overall P450 content of the brain increased with daily administration to a approximately 7-fold induction by 3 weeks of clozapine treatment. No induction of 2D4 was observed with the dopamine D2 receptor blockers haloperidol, chlorpromazine, and sulpiride or with the serotonin receptor blocker mianserin. A clozapine-like induction of P4502D4 was obtained on administration of toluene to rats. The specificity of the induction of P4502D4 in the brain with respect to both the drugs that induce it and the cells in which it is induced suggests that induction of this enzyme could be involved in the therapeutic action of clozapine. The similarity of induction of P4502D4 elicited by clozapine and by the neurotoxin toluene suggests that more information is needed before a beneficial or toxicological role can be assigned to this isozyme.

Animals↗
PubMed1995

Characterization of two members (ACS1 and ACS3) of the 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thaliana.

The nucleotide sequences of two highly homologous 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14)-encoding genes, ACS1 and ACS3, of Arabidopsis thaliana (At) have been determined. The sequence analysis shows that ACS3 is a pseudogene representing a truncated version of ACS1. The missing region of ACS3 corresponding to the fourth exon of ACS1 has been shown by Southern analysis to be absent in the At genome. The chromosomal locations of the five members of the At ACS multigene family have been determined. The results show that each family member resides on a different chromosome. This observation suggests that the ACS3 pseudogene originated by a partial inter-chromosomal gene duplication. The ACS1 polypeptide contains all the conserved and characteristic domains found in the ACC synthase isoenzymes from various plant species, but is unable to express ACS activity in Escherichia coli and yeast. The predicted amino-acid sequence of ACS1 is missing the highly conserved tripeptide, Thr-Asn-Pro (TNP), between Ile204 and Ser205. Introduction of TNP into ACS1 restores the ACS activity, whereas its removal from the enzymatically active ACS2 results in a loss of activity. The results suggest that TNP is crucial for expression of ACS activity in E. coli.

Amino Acid Sequence↗
PubMed1995

Cloning and functional expression of a soluble form of kynurenine/alpha-aminoadipate aminotransferase from rat kidney.

Several aminotransferases with kynurenine aminotransferase (KAT) activity are able to convert L-kynurenine into kynurenic acid, a putative endogenous modulator of glutamatergic neurotransmission. In the rat, one of the described KAT isoforms has been found to correspond to glutamine transaminase K. In addition, rat kidney alpha-aminoadipate aminotransferase (AadAT) also shows KAT activity. In this report, we describe the isolation of a cDNA clone encoding the soluble form of this aminotransferase isoenzyme from rat (KAT/AadAT). Degenerate oligonucleotides were designed from the amino acid sequences of rat kidney KAT/AadAT tryptic peptides for use as primers for reverse transcription-polymerase chain reaction of rat kidney RNA. The resulting polymerase chain reaction fragment was used to screen a rat kidney cDNA library and to isolate a cDNA clone encoding KAT/AadAT. Analysis of the combined DNA sequences indicated the presence of a single 1275-base pair open reading frame coding for a soluble protein of 425 amino acid residues. KAT/AadAT appears to be structurally homologous to aspartate aminotransferase in the pyridoxal 5'-phosphate binding domain. RNA blot analysis of rat tissues, including brain, revealed a single species of KAT/AadAT mRNA of approximately 2.1 kilobases. HEK-293 cells transfected with the KAT/AadAT cDNA exhibited both KAT and AadAT activities with enzymatic properties similar to those reported for the rat native protein.

2-Aminoadipate Transaminase↗
PubMed1995

Identification of a mitochondrial form of kynurenine aminotransferase/glutamine transaminase K from rat brain.

A soluble aminotransferase with kynurenine aminotransferase (KAT) activity has been recently isolated from rat brain. This enzyme corresponds to a cytosolic form of glutamine transaminase K (GTK). In addition to the cytosolic enzyme, a mitochondrial-associated form of this KAT/GTK also exists. In the present work we have isolated a rat brain cDNA clone encoding a KAT/GTK enzyme identical to the soluble form but carrying an additional stretch of 32 amino acids at its NH2-terminus. Several structural features of this sequence resemble those of leader peptides for mitochondrial import. Evidence that the isolated cDNA encoded for mitochondrial KAT/GTK was obtained after transfection of HEK-293 cells with the cDNA coding for this new KAT/GTK isoenzyme. In fact, a significant enrichment of both KAT and GTK enzymatic activities was found in the crude mitochondrial fraction of the transfected cells.

Amino Acid Sequence↗
PubMed1995

Hypercholesterolemia increases manganese superoxide dismutase immunoreactive macrophages in myocardium.

The effect of hypercholesterolemia on manganese superoxide dismutase (MnSOD)-containing macrophages was investigated in male New Zealand white rabbits. Macrophages from control animals, which were marked with the RAM-11 antibody, demonstrated co-localization with MnSOD immunoreactivity, e.g. in the peri- and paravascular space within the myocardium, but not in the bone marrow. In rabbits fed a 0.5% cholesterol-enriched diet for 42 days, a significant increase (P < 0.01) of MnSOD-immunoreactive macrophages within the myocardium was found concomitant to the drastic elevation of serum cholesterol level. In the bone marrow, MnSOD immunoreactivity did not change after cholesterol feeding. Thus in cholesterol-fed rabbits, the increase of MnSOD-containing macrophages seems to parallel that of lipoproteins. MnSOD is considered as being protective against the cytotoxic effects of those superoxide anions, possibly generated in macrophages, which are involved in the metabolism of modified lipoproteins.

Animals↗
PubMed1995

Cloning and characterization of a soluble kynurenine aminotransferase from rat brain: identity with kidney cysteine conjugate beta-lyase.

In this study, we describe the cloning and characterization of a soluble form of kynurenine aminotransferase (KAT, EC 2.6.1.7) present in rat brain. Soluble KAT was purified from rat kidney and the amino acid sequences of four tryptic peptides determined. These peptides were found to belong to the amino acid sequence reported for rat kidney soluble cysteine conjugate beta-lyase, indicating that rat kidney KAT and beta-lyase represent the same molecular entity. Oligonucleotide probes derived from the beta-lyase cDNA were then used as primers for PCR of reverse-transcribed rat brain poly(A)+ RNA. After subcloning of the resulting PCR fragment and sequencing of the isolated rat brain clone, its oligonucleotide sequence was found to be identical to that reported for the beta-lyase cDNA. Further evidence that the isolated rat brain clone encoded for KAT was obtained by transfecting HEK-293 cells with a construct containing the coding sequence for the enzyme. The transfected cells exhibited KAT activity and, in the presence of 2 mM pyruvate and 2-oxoglutarate, the Km values for L-kynurenine were 1.2 mM and 86.3 microM, respectively. Northern blot analysis of rat kidney, liver, and brain RNA revealed a single species of KAT/beta-lyase mRNA of approximately 2.1 kb.

Amino Acid Sequence↗
PubMed1995

Preferential neuronal loss in layer III of the medial entorhinal cortex in rat models of temporal lobe epilepsy.

We recently described a pronounced neuronal loss in layer III of the entorhinal cortex (EC) in patients with intractable temporal lobe epilepsy (Du et al., 1993a). To explore the pathophysiology underlying this distinct neuropathology, we examined the EC in three established rat models of epilepsy using Nissl staining and parvalbumin immunohistochemistry. Adult male rats were either electrically stimulated in the ventral hippocampus for 90 min or injected with kainic acid or lithium/pilocarpine. Animals were observed for behavioral changes for up to 6 hr and were killed 24 hr or 4 weeks after the experimental treatments. At 24 hr, all animals that had exhibited a bout of acute status epilepticus showed a consistent pattern of neuronal loss in the EC in Nissl-stained sections. Neurodegeneration was most pronounced in layer III of the medial Ec at all dorsoventral levels. A few surviving neurons were frequently present in the lesioned area. An identical pattern of nerve cell loss was also seen in the EC of rats killed 4 weeks following the treatments. This lesion was completely prevented by an injection of diazepam and pentobarbital, given 1 hr after kainic acid administration. Immunohistochemistry demonstrated a relative resistance of parvalbumin-positive neurons in layer III of the medial EC. Taken together, these experiments indicate that prolonged seizures cause a preferential neuronal loss in layer III of the medial EC and that this lesion may be related to a pathological elevation of intracellular calcium ion concentrations.

Animals↗