Biomedical subjects

U Matern

Publications and source records attributed to U Matern.

At least 37 records · Page 2Linked to original sources

PubMed1997

Characterization and expression of caffeoyl-coenzyme A 3-O-methyltransferase proposed for the induced resistance response of Vitis vinifera L.

Cell-suspension cultures of Vitis vinifera L. cv Pinot Noir accumulated resveratrol upon fungal elicitation, and the activity of S-adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyl-transferase (CCoAOMT), yielding feruloyl-CoA, increased to a transient maximum at 12 to 15 h. CCoAOMT cDNA was cloned from the elicited cells and was shown to encode a polypeptide highly homologous to CCoAOMTs from cells of Petroselinum species or Zinnia species. The expression of the cDNA in Escherichia coli revealed that grapevine CCoAOMT methylates both caffeoyl- and 5-hydroxyferuloyl-coenzyme A and is probably involved in phenolic esterification and lignification. Commercial plant activators induce the disease-resistance response of test plants and are considered to mimic the action of salicylic acid. Among these chemicals, 2,6-dichloroisonicotinic acid and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester provoke systemic acquired resistance (SAR) and were also shown to induce the expression of class III chitinase in grapevine. The SAR response is classified by an unchanged phenotype of tissues, but the mechanistic basis is unknown. Treatment of the cultured V. vinifera cells with either fungal elicitor or low concentrations of salicylic acid and 2,6-dichloroisonicotinic acid, respectively, raised the CCoAOMT or stilbene synthase transcript abundance, suggesting that grapevine is capable of the SAR response, whereas benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester was ineffective. The data imply for the first time (to our knowledge) that the expression of phenyl-propanoid genes in grapevine is induced by SAR activators without phenotypic consequences and suggest a role for CCoAOMT and stilbene synthase in the disease-resistance response leading beyond the level of pathogenesis-related proteins as markers of the SAR.

Amino Acid Sequence↗

PubMed1996

Emergency laparoscopy. Technical support for the laparoscopic diagnosis of intestinal ischemia.

BACKGROUND: Laparoscopic diagnosis of intestinal ischemia is difficult. Dark-colored bowels are not a reliable indicator for infarction, because there is no correlation between color and oxygenation. The same picture is produced by intraluminal blood or feces. False diagnoses are described. METHODS: We analyzed various techniques for assessing intestinal oxygenation and perfusion to support laparoscopic diagnosis. In this study laparoscopy was performed on eight pigs. A 10-cm segment of intestine was fixed to the abdominal wall and rendered ischemic. Measurements of the ischemic segment and normal intestine were taken using laser-Doppler, Doppler ultrasound, spectrophotometer, and pulse oximeter. Doppler ultrasound and pulse oximetry were unsuitable in our model, as was laser-Doppler flowmetry. RESULTS: Only the spectrophotometer proved a highly sensitive means of assessing bowel oxygenation. CONCLUSIONS: This method provides the desired additional information about intestinal oxygenation, thus helping to interpret the laparoscopic picture of dark bowels.

Animals↗

PubMed1996

Effects of the Pringle maneuver on hemodynamics during laparoscopic liver resection in the pig.

The Pringle maneuver (PM) is recognized in conventional liver surgery as a method of controlling bleeding. To determine the hemodynamic effects of the PM during pneumoperitoneum (PP) for laparoscopic liver resection, we measure hemodynamic and blood gas changes in 7 healthy pigs. All variables were recorded 5 min before and 10 and 30 min after employing PP or PM and 10 min after discontinuation of PM. After the induction of PP, cardiac index and arterial carbon dioxide tension significantly increased, accompanied by a significant decrease in pH. After the beginning of PM, cardiac index and mean arterial and central venous pressures decreased significantly, whereas the heart rate rose markedly. After discontinuation of the PM, the systemic vascular resistance index decreased, and the heart rate remained elevated. These results demonstrate severe hemodynamic deterioration with PP and a subsequent PM. The latter should, therefore, be considered only as a last resort for the control of bleeding during laparoscopic liver surgery.

Animals↗

PubMed1995

Molecular cloning and heterologous expression of acridone synthase from elicited Ruta graveolens L. cell suspension cultures.

Cell suspension cultures of Ruta graveolens L. produce a variety of acridone alkaloids, and the accumulation can be stimulated by the addition of fungal elicitors. Acridone synthase, the enzyme catalyzing the synthesis of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA, had been isolated from these cells, and the partial enzyme polypeptide sequence, elucidated from six tryptic fragments, revealed homology to heterologous chalcone synthases. Poly(A)+ RNA was isolated from Ruta cells that had been treated for 6 h with a crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea, and a cDNA library was constructed in lambda 2AP. Clones harboring acridone synthase cDNA were isolated from the library by screening with a synthetic oligonucleotide probe complementary to a short stretch of sequence of the enzyme peptide with negligible homology to chalcone synthases. The identity of the clones was substantiated by DNA sequencing and by recognition of five additional peptides, determined previously from tryptic acridone synthase digests, in the translated sequence. An insert of roughly 1.4 kb encoded the complete acridone synthase, and alignments at both DNA and protein levels corroborated the high degree of homology to chalcone synthases. Expression of the enzyme in vector pET-11c in the Escherichia coli pLysS host strain proved the identity of the cloned cDNA. The heterologous enzyme in the crude E. coli extract exhibit high acridone but no chalcone synthase activity. The results were fully supported by northern blot hybridizations which revealed that the specific transcript abundance did not increase but rather decreased upon white light irradiation of cultured Ruta graveolens L. cells, a condition that commonly induces the abundance of chalcone synthase transcripts.

Acyltransferases↗

PubMed1994

Molecular characterization and cloning of an esterase which inactivates the macrolide toxin brefeldin A.

The macrolide antibiotic brefeldin A (BFA) was described as a phytotoxin and pathogenicity factor from Alternaria carthami Chowdhury, the causal agent of a devastating blight disease in safflower (Carthamus tinctorius L.). The toxin is known to inhibit the endoplasmic reticulum-Golgi flux and processing. Conventional breeding of safflower for resistance to the Alternaria blight disease has failed, and in situ detoxification of brefeldin A is a novel approach for the protection of field-grown safflower plants from the blight disease. As a first step towards this goal, a strain of Bacillus subtilis has been isolated which is capable of hydrolyzing brefeldin A to a non-toxic metabolite, brefeldin A acid. The BFA esterase was purified to homogeneity from B. subtilis extracts and shown to consist of a monomeric peptide of approximately 40 kDa. Besides brefeldin A, the esterase hydrolyzed ethyl valerate, which structurally resembles the lactone portion in the brefeldin A molecule, but failed to accept other macrolides such as erythromycin or zearalenone. Roughly 12% of the esterase sequence was identified by microsequencing tryptic peptides and the N terminus, which lacked a methionine leader residue. Corresponding oligonucleotide probes were employed to clone the esterase gene in pUC18. One of seven clones was sequenced and shown to code for the full size esterase protein of 372 amino acid residues. The esterase gene was subcloned in pT7-7 and expressed in Escherichia coli yielding a fusion protein with a specific esterase activity 3-fold over that of the enzyme purified from B. subtilis. The cloned esterase provides the basis for the generation of transgenic safflower plants as a valuable asset in the research on nonspecific phytotoxins and in supporting breeding for Alternaria blight resistance.

Amino Acid Sequence↗

PubMed1994

An alternative methylation pathway in lignin biosynthesis in Zinnia.

S-Adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is implicated in disease resistant response, but whether it is involved in lignin biosynthesis is not known. We isolated a cDNA clone for CCoAOMT in differentiating tracheary elements (TEs) induced from Zinnia-isolated mesophyll cells. RNA gel blot analysis showed that the expression of the CCoAOMT gene was markedly induced during TE differentiation from the isolated mesophyll cells. Tissue print hybridization showed that the expression of the CCoAOMT gene is temporally and spatially regulated and that it is associated with lignification in xylem and in phloem fibers in Zinnia organs. Both CCoAOMT and caffeic acid O-methyltransferase (COMT) activities increased when the isolated Zinnia mesophyll cells were cultured, whereas only CCoAOMT activity was markedly enhanced during lignification in the in vitro-differentiating TEs. The induction pattern of the OMT activity using 5-hydroxyferuloyl CoA as substrate during lignification was the same as that using caffeoyl CoA. Taken together, the results indicate that CCoAOMT is associated with lignification during xylogenesis both in vitro and in the plant, whereas COMT is only involved in a stress response in vitro. We propose that CCoAOMT is involved in an alternative methylation pathway in lignin biosynthesis. In Zinnia in vitro-differentiating TEs, the CCoAOMT mediated methylation pathway is dominant.

Acyl Coenzyme A↗

PubMed1994

Experience with TEM in Germany.

Transanal endoscopic microsurgery (TEM) presents a minimally invasive procedure for local removal of large adenomas as well as early carcinomas of the rectum. 57 surgical departments/clinics, which practice this method in Germany were asked about their experience. 44 questionnaires were evaluated. Indications for the 1,900 recorded operations were 1,411 adenomas, 433 carcinomas (curative intention: 286; palliative intention: 147), and 56 other procedures. The conversion rate to laparotomy was dependent on the experience with this surgical method (1.2%-11.6%). A radical rectal resection as a second step was performed in 5.7% of patients in the case of advanced carcinoma in the histologic examination of the specimen. Complications appeared in 120 patients (6.3%), 77 patients (4.0%) recovered with conservative treatment, and 43 (2.3%) had to undergo surgery. The mortality rate was 0.2% (3/1,900 pts).

Anal Canal↗

PubMed1994

[Laparoscopic techniques in therapy of choledocholithiasis].

Several methods for therapy of common bile duct stones (CBDS) during laparoscopic cholecystectomy are available. After intraoperative cholangiography laparoscopic removal of CBDS can be achieved via the cystic duct or a choledochotomy using a thin cholangioscope, balloon catheters and Dormia baskets. Lithotripsy (electrohydraulic or laser) is necessary in presence of larger stones. Also combined procedures with intraoperative antegrade or retrograde papillotomy have been described. Published studies show that laparoscopic common bile duct exploration can be performed successfully by experienced surgeons. However, these procedures are demanding and time-consuming. Further evaluation is essential.

Cholangiography↗

PubMed1992

Brefeldin A binds to glutathione S-transferase and is secreted as glutathione and cysteine conjugates by Chinese hamster ovary cells.

Radioactively labeled brefeldin A was used to probe for proteins that interact with this metabolite. The most prominent protein labeled after in vivo incubation of Chinese hamster ovary cells with [3H]brefeldin A turned out to have an apparent molecular mass of 26 kDa. Radioactive peptides derived from the [3H]brefeldin A-labeled protein showed sequence identity with glutathione S-transferases, and immunoblotting after two-dimensional gel electrophoresis confirmed this result. In addition, Chinese hamster ovary cells convert the antibiotic to its glutathionyl and cysteinyl derivatives and secrete them rapidly into the medium. From these findings we conclude that detoxification of the antibiotic in mammalian cells occurs via the glutathion S-transferase system. This may explain the often observed reversibility of brefeldin A's action on the steady state of organelles in mammalian cells.

Amino Acid Sequence↗

PubMed1991

Different types of microsomal enzymes catalyze ortho- or para-hydroxylation in the biosynthesis of carnation phytoalexins.

Cell suspension cultures of carnation (Dianthus caryophyllus L.) accumulate, upon challenge with crude fungal elicitor, various dianthramide phytoalexins, all of which derive from N-benzoylanthranilate. In vitro, microsomes from the elicited carnation cells hydroxylated N-benzoylanthranilate in the 4- and/or 2'-positions to yield the hydroxyanthranilate and/or salicyloyl derivatives. 2'-Hydroxylation was shown to precede 4-hydroxylation in the formation of N-salicyloyl-4-hydroxyanthranilate, and both these activities depended strictly on NADPH and molecular oxygen. 4-Hydroxylation was shown to be catalyzed by cytochrome P-450-dependent monooxygenase(s), whereas the 2'-hydroxylating activity appeared to be due to a novel class of enzymes, also responding synergistically to NADH in combination with NADPH and showing apparent inhibition by cytochrome c but not by carbon monoxide. The difference in type of 4- and 2'-hydroxylases was corroborated by the exclusive inhibition of either activity in imidazole vs. MOPS buffers as well as their differential heat sensitivities. In the course of these studies, low concentrations of N-salicyloylanthranilate turned out to inhibit the cytochrome P-450-dependent 4-hydroxylation more strongly than any of the commercial inhibitor chemicals tested, while neither the substrate, N-benzoylanthranilate, nor the final product, N-salicyloyl-4-hydroxyanthranilate, exhibited such significant inhibition. In addition, 2'-hydroxylation activity was affected much less by N-benzoylanthranilate, N-salicyloylanthranilate or by inhibitor chemicals. The results demonstrate the requirement of two different classes of hydroxylase activities that appear to introduce the antimycotic quality to the dianthramides for phytoalexin defense.

Carbon Monoxide↗

PubMed1991

Molecular cloning, induction and taxonomic distribution of caffeoyl-CoA 3-O-methyltransferase, an enzyme involved in disease resistance.

Trans-Caffeoyl-CoA 3-O-methyltransferase is involved in the reinforcement of the plant cell wall under conditions that trigger the disease resistance response (Pakusch, A.-E., Kneusel, R.E., and Matern, U. (1989) Arch. Biochem. Biophys. 271, 488-494). Partial amino acid sequences of the enzyme from cultured parsley cells that had been treated with a crude elicitor were identified (Pakusch, A.-E., Matern, U., and Schiltz, E. (1991) Plant Physiol. 95, 137-143), and corresponding degenerated oligonucleotides of 29- and 30-nucleotide length were synthesized. Northern hybridizations with these probes revealed one specific RNA band, and the amount of this RNA appeared to be transiently induced upon elicitation of the cells. De novo enzyme synthesis was confirmed by Western blotting experiments using a specific antiserum. The time course of induction closely followed the pattern observed for phenylalanine ammonia-lyase and suggested the operational coordination of the methyltransferase with the general phenylpropanoid pathway in vivo. Full size cDNA of 1.258 kilobases was isolated in lambda gt11, sequenced and found to contain a remarkably long 5'-untranslated leader sequence followed by an open reading frame that codes for a 241-residue polypeptide representing the 27-kDa subunit of the native, dimeric parsley enzyme. Almost no homology was found to protein sequences filed in data banks. Southern hybridization with genomic DNA suggested that only one or two copies of the respective gene(s) are present in the parsley genome. Caffeoyl-CoA-specific methyltransferase activity was demonstrated in taxonomically widely diverse plants such as Dianthus caryophyllus (Caryophyllaceae), Carthamus tinctorius (Asteraceae) or Daucus carota, and Ammi majus (Apiaceae) where it is commonly induced by elicitor treatment. In Northern blots with RNA from Ammi or Daucus, parsley cDNA hybridized specifically to one band comparable in size to the parsley RNA, whereas Dianthus and Carthamus appear to code for slightly larger RNAs (roughly 1.45 and 1.3 kilobases, respectively). Slot-blot hybridizations revealed in all instances the rapid and transient increase of mRNA levels in response to elicitation. This emphasizes the integral role of the enzyme in disease resistance expression in plants far beyond parsley and also illustrates a new physiological context for the induction of 4-coumarate:CoA ligase.

Amino Acid Sequence↗

PubMed1990

Detoxification of the macrolide toxin brefeldin A by Bacillus subtilis.

The macrolide toxin brefeldin A is a determinant of Alternaria leaf blight disease in safflower, which causes severe economic losses worldwide. Soilborne bacteria, classified as Bacillus subtilis spp., were isolated and shown to readily metabolize brefeldin A in laboratory culture to one major product. This product was identified by high resolution 2D 1H NMR and FAB mass spectroscopies as the acid resulting from hydrolysis of the macrolide ring in brefeldin A . In contrast to brefeldin A, the acid completely lacked phytotoxic activity in the standard leaf bioassay. Detoxification of brefeldin A by the lactonase activity from Bacillus subtilis may be exploited in the future to introduce resistance to Alternaria leaf blight in safflower.

Bacillus subtilis↗

PubMed1990

Rapid purification of the peptide toxins microcystin-LR and nodularin.

An isolation procedure was developed to provide within one day microcystin-LR, a cyclic heptapeptide toxin from Microcystis aeruginosa PCC 7806. After ODS (octadecylsilyl) solid phase extraction, the crude toxin fraction was chromatographed using a strong anion exchange column. The toxin was eluted with 0.02 M ammonium bicarbonate. An at least 95% purity was revealed on HPLC separation by monitoring at 214 nm. Application of the procedure to the cyclic pentapeptide toxin nodularin from Nodularia spumigena AV2 was examined.

Chromatography, Gel↗

PubMed1990

Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures.

Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.

Alkyl and Aryl Transferases↗

PubMed1989

The biosynthesis of phytoalexins in Dianthus caryophyllus L. cell cultures: induction of benzoyl-CoA:anthranilate N-benzoyltransferase activity.

It has been shown that cell cultures of Dianthus caryophyllus L. c.v. Eleganz accumulate N-benzoyl-4-methoxyanthranilic acid, previously identified as the phytoalexin methoxydianthramide B, in response to treatment either with a crude elicitor isolated from the cell walls of Phytophthora megasperma f.sp. glycinea or with a commercial yeast extract. Cell-free extracts from the induced cells efficiently catalyzed the N-benzoylation of anthranilate in the presence of benzoyl-CoA. The partially purified transferase was shown to be specific for anthranilate with almost no activity toward 4-hydroxyanthranilate, whereas acyl donors other than benzoyl-CoA such as salicyloyl-, cinnamoyl-, or 4-coumaroyl-CoA were also accepted. Elicitor treatment of the cells additionally induced an S-adenosyl-L-methionine:N-benzoyl-4-hydroxyanthranilate 4-O-methyltransferase activity. We propose, therefore, that methoxydianthramide B is derived from N-benzoylanthranilic acid via N-benzoyl-4-hydroxyanthranilic acid. Dark-grown cells contained little N-benzoyltransferase activity (approx 8 mu kat/kg), which increased roughly ninefold within 6 h following the addition of the elicitor. In addition, phenylalanine ammonia-lyase activity of the cells increased about twofold under these conditions to a maximum (approx 40 mu kat/kg) at 5 h. The rapid induction of both enzyme activities suggests that the shikimate pathway is of crucial importance in the disease resistance response of carnation cells.

Acyltransferases↗

PubMed1989

Nontoxic and toxic oligopeptides with D-amino acids and unusual residues in Microcystis aeruginosa PCC 7806.

Toxic and nontoxic peptides were isolated from the cyanobacterium Microcystis aeruginosa PCC 7806 by a procedure including extraction of cells with water-saturated 1-butanol, chromatography of the extract on silica gel plates and high performance liquid chromatography (HPLC) on Partisil-5. The toxin was shown to be only a minor constituent, being negatively charged and thus separable by electrophoresis, within the HPLC-purified fraction. It contained erythro-beta-methyl-D-Asp, D-Glu, D-Ala, L-Leu, and L-Arg known to be part of the Microcystis peptide-toxin with Mr 994. The major part of the HPLC-purified fraction was assigned, however, to a nontoxic peptide with a Mr of 956. Partial hydrolysis studies of the nontoxic peptide(s) revealed amino acid sequences composed of D-Glu, N-methyl-Phe, and 3,4-dehydro-Pro, aside from the common L-amino acids. Cyclic linkage in the nontoxic peptide(s) appears likely.

Amino Acids↗

PubMed1989

Formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA by Zn2+-dependent enzymes in cultured plant cells and its activation by an elicitor-induced pH shift.

A novel hydroxylase activity catalyzing the formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA was identified in crude extracts from cultured parsley cells. The extracts were less active (Vmax/Km) in converting trans-4-coumaric to trans-caffeic acid. Optimal hydroxylase activity was found at pH 6.5 with a steep decline toward both pH 7.4 and pH 5.0. The enzyme activity requires ascorbate and Zn2+ at optimal concentrations of 50 and 0.5 mM, respectively. No other reductant could replace ascorbate, whereas high concentrations of Ca2+ partially substituted for Zn2+. The enzyme is soluble and appears to be located in the cytoplasm. The unusual pH optimum suggests that the hydroxylase is inactive at the normal cytoplasmic pH. Upon treatment of parsley cells with an elicitor derived from Phytophthora megasperma f. sp. glycinea, the cytoplasmic pH dropped by approximately 0.25 pH unit within 55 min as determined by 31P NMR spectroscopy. Our results suggest that this shift in the cytoplasmic pH is sufficient for the activation of the hydroxylase, eventually leading to the formation of caffeoyl and feruloyl esters. Such esters may be a part of a very rapid resistance response of the plant cells, which would leave no time for de novo enzyme synthesis.

Acyl Coenzyme A↗

PubMed1989

S-adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase from elicitor-treated parsley cell suspension cultures.

An S-adenosyl-L-methionine:caffeoyl-CoA 3-O-methyltransferase was purified 82-fold from elicitor-induced parsley cell suspension cultures by ammonium sulfate fractionation, anionic exchange and hydrophobic interaction chromatographies, and chromatofocusing. The enzyme has an apparent pI of 5.7 and a molecular weight of approx 48,000 determined by gel filtration chromatography. Maximal activity was observed at pH 7.5 in 50 mM phosphate or Tris-HCl buffers and the additional presence of 0.5 M NaCl. The methyltransferase activity was dependent on Mg2+, whereas EDTA, Mn2+, and Ca2+ inhibited the reaction. The partially purified enzyme efficiently catalyzed the methylation of caffeoyl-CoA, but also accepted with low affinity various other caffeic esters as substrates. Dark-grown parsley cells contained considerable methyltransferase activity which was nevertheless increased approx threefold within 12 h following the addition of a crude fungal elicitor to the cell suspensions. We propose that the O-methyltransferase activity is an important component in the rapid resistance response of the cells, which depends on the formation of cell wall-bound ferulic polymers.

Acyl Coenzyme A↗