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Biomedical subjects

U Matern

Publications and source records attributed to U Matern.

At least 19 recordsLinked to original sources

Instruments for minimally invasive surgery: principles of ergonomic handles.

Although the advantages of minimally invasive surgery (MIS) have been clearly established for the patient, the surgeon must cope with disadvantages caused by unergonomic instrument handles. Pressure areas and persisting nerve lesions have been described in the literature. The shape of the instrument handles has been identified as the reason for these disorders. To prevent these, it is necessary to use ergonomically designed handles for MIS instruments. Anatomic, physiologic, and ergonomic facts as well as the results of the authors' own experiences and tests are presented. On this basis, an ideal ergonomic working posture for the laparoscopic surgeon and an optimal grasp for manipulating the instruments' functional elements are recommended. To enable the surgeon to evaluate ergonomic handles for MIS instruments according to his own needs, 14 criteria for genuine "ergonomic handles" are established. On the basis of these criteria, deficiencies of handles currently available (ring and shank handles at an angle or with axial extension to the instrument shaft, and pistol handles) are discussed. Furthermore, new handles, developed by the authors according to the criteria for genuine ergonomic handles, are presented.

Equipment Design

Differential regulation and distribution of acridone synthase in Ruta graveolens.

Cell suspension cultures of Ruta graveolens L. accumulate polyketide metabolites such as acridone alkaloids and flavonoid pigments. Whereas flavonoid synthesis is induced by light, the production of alkaloids can be enhanced in dark-cultured cells by treatment with fungal elicitors. Acridone synthase (ACS) catalyzes the committed condensing reaction of acridone biosynthesis yielding 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl- and malonyl-CoAs. The reaction proceeds in a manner analogous to that of chalcone synthase (CHS) which catalyzes the first committed step in flavonoid biosynthesis and cDNA and protein sequences of Ruta ACS possess a high degree of sequence homology to heterologous CHSs. ACS transcript abundance and specific activity were monitored in cultured R. graveolens cells irradiated either continuously with white light or treated with fungal elicitor over a period of 24 h and found to increase transiently upon elicitor treatment and to decrease upon light irradiation. Immunodetection with a rabbit polyclonal ACS antiserum revealed that the amounts of ACS polypeptide decreased slightly in light-irradiated cells but increased in elicitor-treated Ruta cells. Fluorescence microscopy and tissue print hybridizations were employed to aid in localizing the sites of storage and biosynthesis of acridone alkaloids in Ruta plants. Yellow fluorescing alkaloids were detected particularly in root tissue adjacent to the rhizodermis, but also in the endodermis and vascular tissue of the hypocotyl. ACS transcript abundance in situ followed the same spatial pattern, indicating that the synthesis of acridones likely proceeds at all sites of deposition rather than exclusively in the root. Expression in planta and the induction response of ACS suggest that the alkaloids serve as phytoanticipins or phytoalexins in the defense of Ruta particularly to soil-borne pathogens or as feeding deterrents.

Acridines

Anthranilate N-hydroxycinnamoyl/benzoyltransferase gene from carnation: rapid elicitation of transcription and promoter analysis.

Hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) catalyses the committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.). Three HCBT cDNAs were cloned previously from suspension-cultured carnation cells that had been induced with fungal elicitor. A rapid, transient induction of hcbt transcripts upon elicitation, reaching maximal abundances within about 0.5 h and returning to basal levels within 4 h, suggested the involvement of unusual cis elements. A DNA fragment of 3.8 kb, spanning the hcbt2 gene with the 5'-flanking region of roughly 1.8 kb, was cloned from carnation plants. The gene encodes one long open reading frame lacking introns. The DNA sequence revealed a conserved TATA box, three elicitor response elements (EREs) and a 9 bp direct repeat as well as an interrupted direct repeat of 11 bp in the TATA distal region. EMSA revealed the binding of elicitor-inducible nuclear factors to the promoter region from -377 to -326 spanning two of the EREs, and their functional relevance was confirmed by transient expression assays of hcbt2 promoter-GUS reporter gene constructs in parsley protoplasts. Furthermore, an oligo(A) segment was present immediately preceding the start of translation (+140 to +150). Transient expression analysis demonstrated that the sequence upstream to -1157 at least is required in context with the 5'-UTR, particularly including the poly(A) segment, for strong expression and full elicitor induction of the hcbt2 gene. The results suggested that several sequence motifs scattered over a wide range of the 5'-flanking region and into the exonic sequence are responsible for the full elicitor regulation of the hcbt2 gene.

Acyltransferases

Structure of the parsley caffeoyl-CoA O-methyltransferase gene, harbouring a novel elicitor responsive cis-acting element.

The sequence of the S-adenosyl-L-methionine:trans-caffeoyl-CoA O-methyltransferase (CCoAOMT, EC2.1.1.104) gene, including the 5'-flanking region of 5 kb, was determined from parsley (Petroselinum crispum) plants. The enzyme appears to be encoded by one or two genes, and the ORF is arranged in five exons spaced by introns from 107 to 263 bp in length. The genomic sequence matches the ORF of the cDNA previously reported from elicited parsley cell cultures, showing only three base changes that do not affect the enzyme polypeptide sequence. S1 nuclease protection assays and primer extension analyses with genomic and cDNA templates revealed the transcription start site 67 bp upstream of the translation start codon, indicating a shorter 5'-UTR than reported previously for the transcript. Promoter regulatory consensus elements such as two 'CAAT' boxes and one 'TATA' box were identified at -196, -127 and -31, respectively, relative to the transcription start site, and an SV 40-like enhancer element is located 347 bp upstream. Most notably, three putative cis-regulatory elements were recognized by sequence alignments, which represent motifs recurring in the promoters of several genes of the stress-inducible phenylpropanoid pathway (boxes P, A and L). Transient expression assays with a set of 5'-truncated promoter-GUS fusions show that significant promoter activity is retained in a 354 bp promoter fragment. In vitro DNase 1 footprint experiments and electrophoretic mobilty shift assays (EMSA) identified in this fragment a unique sequence motif with elicitor-inducible trans-factor binding activity, which was unrelated to boxes P, A, or L. This novel cis-regulatory element, designated box E, appears to be conserved in the TATA-proximal regions of other stress-inducible phenylpropanoid genes, and in vitro binding of nuclear protein was confirmed in EMSA assays for such an element from the PAL-1 promoter (-54 to -45). Moreover, the deletion of box E reduced the activity and erased the elicitor-responsiveness of the CCoAOMT promoter in transient expression assays. The results corroborate the proposed physiological function of CCoAOMT in elicited plant cells and may shed new light on the sequential action of trans-active factors in the regulation of phenylpropanoid genes.

Amino Acid Sequence

Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L.

Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.), and the product N-benzoylanthranilate is the precursor of several sets of dianthramides. The transferase activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract. The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa. Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of these sequences differed in a few amino acid residues only suggesting the presence of isoenzymes. A specific 0.8 kb cDNA probe was generated by RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)+ RNA from elicited carnation cells. Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1-3, were sequenced and shown to encode full-size N-benzoyltransferases. The translated peptide sequences revealed more than 95% identity among these three clones. The additional two clones harbored insert sequences mostly homologous with pchcbt 1 but differing in the 3'-flanking regions due to variable usage of poly(A) addition sites. The identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequences as well as by the activity of the benzoyltransferase expressed in Escherichia coli. Therefore, carnation encodes a small family of anthranilate N-benzoyltransferase genes. In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Thus the cDNAs described represent also the first hydroxycinnamoyl-transferases cloned from plants, which classifies the enzymes as hydroxycinnamoyl/benzoyltransferases.

Acyltransferases

Differential expression of chitinases in Vitis vinifera L. responding to systemic acquired resistance activators or fungal challenge.

The concept of systemic acquired resistance (SAR) enables a novel approach to crop protection, and particular pathogenesis-related proteins, i.e. an acidic chitinase, have been classified as markers of the SAR response. Basic class I (VCHIT1b) and a class III (VCH3) chitinase cDNAs were cloned from cultured Vitis vinifera L. cv Pinot Noir cells and used to probe the induction response of grapevine cells to salicylic acid or yeast elicitor. Furthermore, the cells were treated with the commercial SAR activators 2,6-dichloroiso-nicotinic acid or benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester. Elicitor or salicylic acid induced both VCHIT1b and VCH3 transcript abundances, whereas 2,6-dichloroiso-nicotinic acid or benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester enhanced exclusively the expression of VCH3. To assess the systemic sensation of chitinase expression, single leaves of Vitis vinifera L. cv Pinot Noir or Vitis rupestris plants were inoculated with Plasmopara viticola spore suspensions, and the VCH3 and VCHIT1b mRNA amounts in the infected versus the adjacent, healthy leaf were monitored. Two VCH3 mRNA maxima were observed 2 and 6 d postinoculation in the infected, susceptible V. vinifera tissue, whereas in the healthy leaf the transcript increased from low levels d 2 postinoculation to prominent levels d 6 to 8 postinoculation. The level of VCH3 mRNA increased also over 4 d in the inoculated, resistant V. rupestris tissue. However, necrotic spots rapidly limited the infection, and the VCH3 transcript was undetectable in the upper-stage, healthy leaf. The expression of VCHIT1b remained negligible under either experimental condition. Overall, the results suggest that the selective expression of VCH3 might be a reliable indicator of the SAR response in V. vinifera L.

Amino Acid Sequence

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

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

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

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

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

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

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

[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

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

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

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

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