Biomedical subjects

U Matern

Publications and source records attributed to U Matern.

At least 37 records · Page 2Linked to original sources

PubMed1999

MIS instruments. An experimental comparison of various ergonomic handles and their design.

BACKGROUND: Various handles are available for use in minimally invasive surgery. Nonergonomic positioning of the hand and fingers can lead to pressure areas, nerve irritation, and rapid fatigue. This study was designed to compare the ergonomic efficiency of several different handles, along with the concepts underlying them, in an attempt to find the best one for laparoscopic surgery. We also consider whether the position of the surgeon in relation to the patient and the posture of the surgeon have any influence on the use of the handles. METHOD: A ring handle positioned in-line (the Microsurge/Vygon 01-1007), an axial handle (the Aesculap PM-953), a shank handle (the Wilo 25.00), and a functional model of a new ergonomic multifunctional handle (the MFEHG Schafreuter) were tested and evaluated by 15 volunteers during an objective test using a transparent pelvitrainer with the left and right hand in a frontal and left and right lateral positions. Our analysis was based on their subjective answers to a questionnaire developed from ergonomic checklists and the semiquantitative observations of the test leader about their posture during testing. RESULTS: There were no significant differences in the results of the objective tests. Subjectively, the shank handle was preferred by most test persons, followed by the functional model for the right hand and the axial handle for the left hand; the ring handle positioned in-line scored poorly for both right and left hands. Arm movements were greater in the latter than in the frontal position. CONCLUSIONS: A clear recommendation for any one of the three currently available handles cannot be given. The results obtained with a simple model of a multifunctional handle were highly promising, and it may be possible to extend it to a real multifunctional instrument.

Equipment Design↗

PubMed1999

Crystal structure of brefeldin A esterase, a bacterial homolog of the mammalian hormone-sensitive lipase.

Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and inactivates BFA, a potent fungal inhibitor of intracellular vesicle-dependent secretory transport and poliovirus RNA replication. We have solved the crystal structure of BFAE and we discovered that the previously reported amino acid sequence was in serious error due to frame shifts in the cDNA sequence. The correct sequence, inferred from the experimentally phased electron density map, revealed that BFAE is a homolog of the mammalian hormone sensitive lipase (HSL). It is a canonical alpha/beta hydrolase with two insertions forming the substrate binding pocket. The enzyme contains a lipase-like catalytic triad, Ser 202, Asp 308 and His 338, consistent with mutational studies that implicate the homologous Ser 424, Asp 693 and His 723 in the catalytic triad in human HSL.

Amino Acid Sequence↗

PubMed1998

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↗

PubMed1998

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↗

PubMed1997

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↗

PubMed1997

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↗

PubMed1997

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↗

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

Crystallization of a novel esterase which inactivates the macrolide toxin brefeldin A.

A novel esterase obtained from Bacillus subtilis and capable of hydrolyzing the phytotoxin brefeldin A was crystallized using the hanging-drop technique. The crystals have two forms and both are monoclinic: form I, space group P2(1) with a = 101.7, b = 64.1, c = 55.4 A and beta = 102.5 degrees, and form II, space group C2 with a = 140.7, b = 82.6, c = 81.5 A and beta = 112.5 degrees. There are two molecules related by a pronounced non-crystallographic dyad per asymmetric unit in both crystal forms. The crystals diffract to 2.3 A using a rotating-anode X-ray source.

Journal Article↗

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↗