Biomedical subjects

Klaus Eschrich

Publications and source records attributed to Klaus Eschrich.

16 recordsLinked to original sources

PubMed2006

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as L-myo-inositol 1-phosphate synthase.

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of D-glucose 6-phosphate to L-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989-998, 2004). One of the forms with an approximately 50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as an approximately 65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified approximately 1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive approximately 65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.

Acetolactate Synthase↗

PubMed2005

Decrease of current responses at human recombinant P2X3 receptors after substitution by Asp of Ser/Thr residues in protein kinase C phosphorylation sites of their ecto-domains.

The whole-cell patch-clamp technique was used to record current responses to nucleotides in HEK 293 cells transiently transfected with the human (h) P2X(3) receptor. When GDP-beta-S was included into the pipette solution, UTP at concentrations which did not alter the holding current, facilitated the alpha,beta-methylene ATP (alpha,beta-meATP)-induced current. The substitution of Ser/Thr residues situated within protein kinase C (PKC) consensus phosphorylation sites of the P2X(3) receptor ecto-domain by the neutral amino acid Ala either abolished (T134A, S178A) or did not alter (T196A, S269A) the UTP-induced potentiation of the alpha,beta-meATP current. The substitution of the same Ser/Thr residues in all four PKC sites by the negatively charged Asp prevented the potentiation by UTP. The Asp mutations abolished the first, fast offset time-constant, but did not alter, or in the case of S269D even increased, the second, slow offset time-constant; at the same time such mutations invariably increased the onset time-constant and massively depressed the peak current amplitude. None of the Ala mutations (with the exception of S269A) influenced the time-course of desensitisation or the peak current amplitude. It is concluded that constitutive activation of PKC sites at the ecto-domain of the hP2X(3) receptor both abolishes the UTP-induced potentiation of the alpha,beta-meATP current and accelerates its rate of desensitisation.

Adenosine Triphosphate↗

PubMed2005

Regulation of human recombinant P2X3 receptors by ecto-protein kinase C.

The whole-cell patch-clamp technique was used to record current responses to nucleotides and nucleosides in human embryonic kidney HEK293 cells transfected with the human purinergic P2X3 receptor. When guanosine 5'-O-(3-thiodiphosphate) was included into the pipette solution, UTP at concentrations that did not alter the holding current facilitated the alpha,beta-methylene ATP (alpha,beta-meATP)-induced current. ATP and GTP, but not UDP or uridine, had an effect similar to that of UTP. Compounds known to activate protein kinase C (PKC) acted like the nucleoside triphosphates investigated, whereas various PKC inhibitors invariably reduced the effects of both PKC activators and UTP. The substitution by Ala of Ser/Thr residues situated within PKC consensus sites of the P2X3 receptor ectodomain either abolished (PKC2 and PKC3; T134A, S178A) or did not alter (PKC4 and PKC6; T196A, S269A) the UTP-induced potentiation of the alpha,beta-meATP current. Both the blockade of ecto-protein kinase C activity and the substitution of Thr-134 or Ser-178 by Ala depressed the maximum of the concentration-response curve for alpha,beta-meATP without altering the EC50 values. Molecular simulation of the P2X3 receptor structure indicated no overlap between assumed nucleotide binding domains and the relevant phosphorylation sites PKC2 and PKC3. alpha,beta-meATP-induced currents through native homomeric P2X3 receptors of rat dorsal root ganglia were also facilitated by UTP. In conclusion, it is suggested that low concentrations of endogenous nucleotides in the extracellular space may prime the sensitivity of P2X3 receptors toward the effect of subsequently applied (released) higher agonistic concentrations. The priming effect of nucleotides might be attributable to a phosphorylation of PKC sites at the ectodomain of P2X3 receptors.

Animals↗

PubMed2005

Characterization of the human P-type 6-phosphofructo-1-kinase gene promoter in neural cell lines.

In humans three isoforms of 6-phosphofructo-1-kinase (PFK) exist. Among them platelet-type PFK (PFKP) is highly abundant in the brain. With its distinct allosteric properties PFKP is regarded to be the key enzyme for the regulation of glycolysis in this organ. We cloned 1.7 kb of the 5' upstream promoter of the human PFKP gene and analyzed the promoter activity by deletion and mutation analysis using a luciferase reporter. The transcription start point was determined at 48 bp upstream of the start codon. In deletion studies the region -65 to +48 turned out to be sufficient for promoter activity while fragment -153 to +48 showed the highest promoter activity. Sequence analysis of the region from -153 to +48 revealed a stretch of eight adjacent putative transcription factor binding sites, seven of which are Sp-family specific sites. Sp1 and Sp3 were shown to bind to most if not all of them. Additionally, an NF-Y binding site was identified. Results of deletion and mutation analysis suggest that all of these transcription factors contribute positively to promoter activity. The methylation status of the promoter region was analyzed in different neural tumor cell lines and compared with that in human leukocytes and muscle.

Base Sequence↗

PubMed2005

Pyruvate protects glucose-deprived Müller cells from nitric oxide-induced oxidative stress by radical scavenging.

The cellular defense of Müller cells against oxidative and nitrosative stress was examined after the addition of the nitric oxide donor papanonoate. Glucose concentrations of > or = 550 microM efficiently protected the Müller cells from cell death by maintaining high ATP and glutathione and allowing only a moderate increase of free radicals. Fluorescence-activated cell sorting (FACS) analysis showed that 22% of the cells underwent apoptosis whereas necrosis was strongly suppressed. Under glucose deprivation, the intracellular concentration of ATP declined to 15% after 1 h; glutathione dropped to 50% within 2 h after papanonoate addition. Both the number of cells containing excess free radicals and the mean concentration of free radicals increased twofold at 0.5-2 h of incubation with papanonoate. Cell death switched from prevailing apoptosis to massive necrosis and cell viability decreased drastically. Several metabolites of glycolysis, gluconeogenesis, and the pentose phosphate pathway were tested with respect to their capability to protect the stressed Müller cells. 2 mM pyruvate was found to enhance cell viability 1.6-fold predominantly by reducing the necrotic cell demise. It could be shown that pyruvate did not act by improving the energy status of Müller cells but by scavenging excess free radicals. Inhibition of the monocarboxylate transporters in Müller cells by alpha-cyano-4-hydroxycinnamate abolished this effect. Other 2-ketoacids, like oxalacetate, 2-ketoglutarate and 2-ketobutyrate had a similar protecting effect as pyruvate. Lactate, glutamate, 2-deoxyglucose, and ribose 5-phosphate did not protect Müller cells against oxidative and nitrosative stress.

Adenosine Triphosphate↗

PubMed2005

Lysine 3 acetylation regulates the phosphorylation of yeast 6-phosphofructo-2-kinase under hypo-osmotic stress.

N-terminal acetylation in the yeast Saccharomyces cerevisiae is catalysed by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain the catalytic subunits Ard1p, Nat3p and Mak3p, respectively. Yeast 6-phosphofructo-2-kinase (PFK2) was found to be acetylated at the amino acid lysine 3. The Lys3-Arg mutant was not acetylated and the mutation causes a slight decrease in enzyme activity. PFK2 from yeast cells exposed to hypo-osmotic stress is known to be phosphorylated at Ser8 and Ser652 (Dihazi et al., 2001a). We have taken a mass spectrometric approach to investigate the influence of PFK2 acetylation on its phosphorylation. Wild-type PFK2 and the Lys3-Arg mutant were purified from hypo-osmotically stressed cells and analysed with MALDI-TOF MS for phosphorylation. Wild-type PFK2 without any tag sequence was found to be acetylated and two times phosphorylated at the N-terminal peptide T(1-40) carrying the acetylation. The same results were observed with C-terminally His-tagged PFK2. When the His-tag was added to the N-terminus of the protein PFK2, acetylation was found to be incomplete and only one phosphate was incorporated in the peptide T(1-41). The Lys3-Arg mutant of PFK2 was not at all post-translationally modified at the N-terminal peptide. Our data indicate that Lys3 acetylation affects the N-terminal phosphorylation of PFK2 under hypo-osmotic stress.

Acetylation↗

PubMed2005

In vitro, clinical, and microbiological evaluation of a linear oscillating device for scaling and root planing.

BACKGROUND: The purpose of this study was to conduct an in vitro and short-term clinical and microbiological evaluation of a linear oscillating device for scaling and root planing (SRP). A comparison was made between conventional ultrasonic scaling (US) and hand scaling (HS) with and without chlorhexidine. METHODS: In vitro, SRP was carried out on human teeth with calculus. Roots and cross-sections thereof were microscopically examined for the efficacy of calculus removal, hard tissue loss, and surface smoothness. In vivo, 11 patients with chronic periodontitis and single-rooted teeth in all quadrants with probing depths of > or =5 mm were selected. One quadrant was treated with linear oscillation and compared to US with chlorhexidine irrigation in the contralateral site. The other arch was treated with HS and compared to HS followed by laser disinfection. One hundred twenty teeth were assessed for clinical attachment level, probing depth, bleeding on probing, and suppuration at baseline and 7, 28, 90, and 180 days. Microbiologically, total numbers of bacteria and six specific periodontal pathogens were determined by quantitative polymerase chain reaction prior to and 1 and 28 days after SRP. Clinical and microbiological data were analyzed statistically with respect to the SRP method, patient specificity, and time effect. RESULTS: In vitro, linear oscillation preserved more root tissues but left more calculus (P <0.05). Significant improvements of all clinical and microbiological parameters were observed for all groups. However, 21 out of 24 tests demonstrated that the clinical microbiological correlations between linear oscillation and control groups did not differ (P <0.05). CONCLUSION: Linear oscillation scaling was clinically acceptable and microbiologically comparable to the control groups despite microscopic remnants of calculus observed in vitro.

Adult↗

PubMed2004

High osmolarity glycerol (HOG) pathway-induced phosphorylation and activation of 6-phosphofructo-2-kinase are essential for glycerol accumulation and yeast cell proliferation under hyperosmotic stress.

In response to changes in the environment, yeast cells coordinate intracellular activities to optimize survival and proliferation. The transductions of diverse extracellular stimuli are exerted through multiple mitogen-activated protein kinase (MAPK) cascades. The high osmolarity glycerol (HOG) MAPK pathway is activated by increased environmental osmolarity and results in a rise of the cellular glycerol concentration to adapt the intracellular osmotic pressure. We studied the importance of the short time regulation of glycolysis under hyperosmotic stress for the survival and proliferation of yeast cells. A stimulation of the HOG-MAPK pathway by increasing the medium osmolarity through addition of salt or glucose to cultivated yeast leads to an activation of 6-phosphofructo-2-kinase (PFK2), which is accompanied by a complex phosphorylation pattern of the enzyme. An increase in medium osmolarity with 5% NaCl activates PFK2 3-fold over the initial value. This change in the activity is the result of a 4-fold phosphorylation of the enzyme mediated by protein kinases from the HOG-MAPK pathway. In the case of hyperosmolar glucose a 5-fold PFK2 activation was achieved by a single phosphorylation with protein kinase A near the carboxyl terminus of the protein on Ser(644) and an additional 5-fold phosphorylation within the same amino-terminal fragment as in the presence of salt. The effect of hyperosmolar glucose is the result of an activation of the Ras-cAMP pathway together with the HOG-MAPK pathway. The activation of PFK2 leads to an activation of the upper part of glycolysis, which is a precondition for glycerol accumulation. Yeast cells containing PFK2 accumulate three times more glycerol than cells lacking PFK2, which are not able to grow under hypertonic stress.

Blotting, Western↗

PubMed2003

Glucose-induced stimulation of the Ras-cAMP pathway in yeast leads to multiple phosphorylations and activation of 6-phosphofructo-2-kinase.

Yeast cells respond to changes of the environment by complex modifications of the metabolism. An increase of the extracellular glucose concentration activates the Ras-cAMP pathway. Via a production of cAMP this pathway stimulates the cAMP-dependent protein kinase (PKA) which is involved in the posttranslational regulation of the key enzymes of gluconeogenesis and glycolysis. 6-Phosphofructo-2-kinase (PFK2) catalyzes the synthesis of fructose 2,6-bisphosphate, the most potent activator of the glycolytic key enzyme 6-phosphofructo-1-kinase. We investigated the molecular mechanism of the glucose-induced phosphorylation and activation of PFK2 in Saccharomyces cerevisiae. After an incubation of PFK2 with ATP and PKA in vitro, two amino acid residues, Thr157 and Ser644, are phosphorylated and the enzyme is activated. A stimulation of the Ras-cAMP pathway by glucose addition to cultivated yeast cells leads to an in vivo activation of PFK2 which is accompanied by a more complex phosphorylation pattern of the enzyme. The phosphorylation of the protein on Ser644 is the result of PKA stimulation while the protein kinase(s) catalyzing the 5-fold phosphorylation of the peptide fragment T(67)(-)(101) is (are) still unknown. The functional significance of T(67)(-)(101) and its phosphorylation is supported by the finding that PFK2 lacking this peptide is inactive.

Amino Acid Sequence↗

PubMed2003

Oral Actinomyces isolates forming red colonies on brain heart blood agar can bee unambiguously classified as A odontolyticus by macroscopic examination.

The accurate classification of oral Actinomyces isolates as one species is difficult. Out of 18 Actinomyces isolates forming red colonies on brain heart blood agar, 12 could be straightforwardly assigned as Actinomyces odontolyticus by biochemical, morphological, and chemotaxonomic characteristics. For the remaining six isolates, the results of the different identification methods were inconsistent. By sequencing a 16S ribosomal DNA fragment by a rapid mass spectrometric method, all isolates could be identified unambiguously as A. odontolyticus. This result proves the importance of red colony pigmentation on brain heart blood agar together with the characteristic cell morphology for unequivocal assignment of oral Actinomyces isolates to the species A. odontolyticus.

Actinomyces↗

PubMed2003

Different sensitivities of mutants and chimeric forms of human muscle and liver fructose-1,6-bisphosphatases towards AMP.

AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 microM and 4.8 microM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the N-terminal region of the muscle enzyme towards liver FBPase (Lys20-->Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20-->Lys and double mutation Glu19-->Asp/Glu20-->Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 microM). The decrease of sensitivity for AMP of the muscle mutant Lys20-->Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20-->Lys and Glu19-->Asp/Glu20-->Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.

Adenosine Monophosphate↗

PubMed2003

The effects of extracts from periodontopathic bacteria on human periodontal fibroblasts stimulated with mineralization supplements.

Bacterial effects on in vitro mineralization of human periodontal fibroblasts (HPF) have not yet been examined in great detail. In our study, we investigated the effects of soluble extracts of the periodontopathic bacteria Porphyromonas gingivalis, Bacteroides forsythus and, Treponema denticola on cell proliferation, mineralization, as well as on osteoblastic markers present in HPF cultured in vitro, such as alkaline phosphatase (ALP) activity and collagen content. Periodontal fibroblasts stimulated by B-glycerophosphate, ascorbic acid and dexamethasone (BAD) or by dexamethasone and ascorbic acid (DA) were compared to unstimulated cells. During the cultivation period, the stimulation of HPF by combined dexamethasone and ascorbic acid (DA) had a strong inductive effect on proliferation, ALP activity and collagen formation. The extracts obtained from the periodontal pathogens had a suppressing effect on the proliferation rate of HPF. The extracts from P. gingivalis, B. forsythus and T. denticola caused a decrease in ALP activity within 24 h of application. While extracts obtained from P. gingivalis and B. forsythus induced a reduction in collagen content in BAD- and DA-stimulated HPF cells, T. denticola extracts led to an increase in collagen. Our data suggest that specific periodontopathic bacteria may suppress tissue regeneration in vivo not only by activating host defense mechanisms but also directly via a suppression of growth and differentiation of HPF and a reduction in the extracellular collagen matrix. For the process of pocket formation, not even the direct influence of viable bacteria seems to be necessary. Additionally, long-distance effects of bacteria harboured in periodontal pockets or in root canals may be of importance.

Ascorbic Acid↗

PubMed2002

Simultaneous determination of different DNA sequences by mass spectrometric evaluation of Sanger sequencing reactions.

All currently available DNA sequencing protocols rest fundamentally upon the homogeneity of the template. In this paper we describe the parallel DNA sequencing of various templates in one sample by a combination of the Sanger method and MALDI-TOF mass spectrometric analysis of the products. PCR-amplified hypervariable 16S rDNA fragments of the bacterium Escherichia coli DF1020 and cDNA of the 6-phosphofructo-1-kinase isoenzymes (PFK-1, EC 2.7.1.11) in rat brain were chosen as model systems for essentially heterogeneous templates. Avoiding cloning of the inhomogeneous PCR products we were able to read three sequences for both the 16S rDNA fragment of E.coli DF1020 and the cDNA of 6-phosphofructo-1-kinase from the peak lists of the Sanger sequencing reactions. Short sequences with a length between 21 and 25 nt were sufficient to reflect the heterogeneity of the 16S rDNA genes in E.coli and the existence of three isoenzymes of PFK-1 in rat brain.

Animals↗

PubMed2002

Fructose-1,6-bisphosphatase genes in animals.

A comparison of the amino acid sequences of the liver and muscle fructose-1,6-bisphosphatase (FbPase) isoforms in primates and rodents suggested an ancient duplication event leading to the corresponding genes. We investigated the presence of both genes in the rabbit (order lagomorphs) and in species belonging to further distantly related metazoan taxa. By an analysis of the available complete genomes and proteomes of the nematode Caenorhabditis elegans and of Drosophila melanogaster only one sequence homologous to known FbPases was found in each species. The corresponding mRNAs were characterized by cDNA sequencing. We then carried out reverse transcription-polymerase chain reactions to amplify central fragments of the FbPase cDNAs from liver and muscle of Gallus gallus, Xenopus laevis, and Esox lucius, respectively. Their sequencing revealed that (i) the livers of chicken, frog, and fish contain mRNAs which are closely related to mammalian liver FbPase mRNAs, (ii) chicken muscle contains an mRNA which is most homologous to mammalian muscle FbPase mRNAs, (iii) frog muscle contains both a liver-type and a muscle-type FbPase mRNA, while (iv) in fish muscle no FbPase mRNA could be detected by our approach despite the doubtless presence of the enzyme in this organ. An alignment of the partial amino acid sequences of the different FbPases showed that the residues that are thought to be in contact with the substrate, fructose-2,6-bisphosphate, and Mg(2+) are totally conserved, while some amino acids having contact with adenosine monophosphate were found to vary among several species. The question of what might be the advantage of having more than one gene coding for FbPase per haploid genome is discussed.

Amino Acid Sequence↗

PubMed2002

Fructose inhibits apoptosis induced by reoxygenation in rat hepatocytes by decreasing reactive oxygen species via stabilization of the glutathione pool.

Oxidative stress induces apoptosis in liver parenchymal cells. The present study demonstrates that the substitution of fructose for glucose as sole carbon source in the incubation medium reduced apoptosis due to reoxygenation up to 50% in cultured rat hepatocytes. This anti-apoptotic action of fructose cannot be explained by the effects of this sugar on the intracellular ATP concentration and the ATP/ADP ratio. Rather, the suppression of apoptosis by fructose seems to be a consequence of remarkably higher intracellular levels of glutathione observed during reoxygenation in fructose-fed hepatocytes in contrast to glucose-fed ones. With fructose as substrate, the generation of excess reactive oxygen species (ROS) during the initial phase of reoxygenation was strongly reduced. With respect to ROS reduction and stabilization of the cellular glutathione pool fructose was found as efficient as a pretreatment of glucose fed cells with N-acetyl-L-cysteine. The enhanced metabolization of ROS by the glutathione/glutathione peroxidase system in fructose-cultured hepatocytes under reoxygenation was expected to improve their mitochondrial status so that late events in the apoptotic pathway are suppressed. This could be confirmed by the reduced release of cytochrome c from mitochondria into the cytosol as well as by the observed decrease of caspase-3 activity during reoxygenation.

Adenosine Diphosphate↗

PubMed2002

Changes in activity and expression of phosphofructokinase in different rat brain regions after basal forebrain cholinergic lesion.

Selective lesion of rat basal forebrain by the cholinergic immunotoxin 192IgG-saporin was used as an animal model to address the question of whether the changes in cortical glucose metabolism observed in patients with Alzheimer's disease may be related to impaired cholinergic transmission. At different times after creating the immunolesion, the isoenzyme pattern and steady-state mRNA levels of the key glycolytic enzyme phosphofructokinase were determined in cortex, hippocampus, basal forebrain and nucleus caudatus. The loss of cholinergic input was accompanied by a persistent decrease in choline acetytransferase and acetylcholine esterase activities in the cortical target areas similar to the cholinergic malfunction seen in Alzheimer's dementia. The basal forebrain lesion induced by the immunotoxin resulted in a transient increase in phosphofructokinase activity peaking on day 7 after inducing the lesion in cortical areas. In parallel, an increased steady-state level of phosphofructokinase mRNA was determined by RT/real-time PCR and in situ hybridization. In contrast, analysis by western blotting and quantitative PCR revealed no changes in the phosphofructokinase isoenzyme pattern after immunolesion. It is concluded that common metabolic mechanisms may underlie the degenerative and repair processes in denervated rat brain and in the diseased Alzheimer's brain.

Acetylcholinesterase↗