Wound-induced triacylglycerol biosynthesis is jasmonoy-l-isoleucin and abscisic acid independent.

Triacylglycerol (TAG) plays a significant role during plant stress - it maintains lipid homeostasis. Upon wounding plants accumulate TAG, likely as a storage form of fatty acids (FAs) that originate from damaged membranes. This study asked if this process depends on the two phytohormones jasmonoyl-isoleucine (JA-Ile) and abscisic acid (ABA), which are involved in wound signalling. To analyse regulation of wound-induced TAG accumulation, we used mutants deficient in JA-Ile, with reduced ABA and the myb96 mutant, which is deficient in an ABA-dependent transcription factor. The expression of genes involved in TAG biosynthesis, and TAG content after wounding were analysed via LC-MS and GC-FID, plastidial lipid content in all mentioned mutant lines was also determined. The localization of newly synthesized TAG was investigated using lipid droplet staining. TAG accumulation upon wounding was confirmed as well as the fact that the newly synthesized TAG are mostly composed of polyunsaturated fatty acids. Nevertheless, all tested mutant lines were able to accumulate TAG similar to the WT. We observed differences in reduction of plastidial lipids - in WT plants this was higher than in mutant lines. Newly synthesized TAGs were stored in lipid droplets at and around the wounded area. Our results show that TAG accumulation upon wounding is not dependent on JA-Ile or ABA. The newly synthesized TAG species are composed of unsaturated fatty acids of membrane origin, and most likely serves as a transient energy store.

[Transgastric-, transhepatic endosonography-guided biliary drainage (EUCD) in a patient with locally advanced cholangiocarcinoma]. / Endosonografisch gesteuerte transgastrisch-, transhepatische Cholangiodrainage (EUCD) bei einer Patientin mit lokal fortgeschrittenem Gallengangskarzinom.

We report on a 63-year-old female patient with locally advanced cholangiocarcinoma of the extrahepatic biliary tract. She was admitted with progressive obstructive jaundice, initiating cholangitis and distinctive itching. The biliodigestive anastomosis was secondarily barred by tumour infiltration and not accessible via an endoscopic route. Because the patient asked expressively for internal drainage, we successfully performed an endosonography-guided transgastric, transhepatic internal biliary drainage (EUCD). The jaundice and itching were regredient and the patient was discharged in a stable condition.

Cloning and expression of a new cDNA from monocotyledonous plants coding for a diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from barley (Hordeum vulgare).

From a cDNA library generated from mRNA of white leaf tissues of the ribosome-deficient mutant 'albostrians' of barley (Hordeum vulgare cv. Haisa) a cDNA was isolated carrying 54.2% identity to a recently published cDNA which codes for the diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) hydrolase of Lupinus angustifolius (Maksel et al. (1998) Biochem. J. 329, 313-319), and 69% identity to four partial peptide sequences of Ap4A hydrolase of tomato. Overexpression in Escherichia coli revealed a protein of about 19 kDa, which exhibited Ap4A hydrolase activity and cross-reactivity with an antibody raised against a purified tomato Ap4A hydrolase (Feussner et al. (1996) Z. Naturforsch. 51c, 477-486). Expression studies showed an mRNA accumulation in all organs of a barley seedling. Possible functions of Ap4A hydrolase in plants will be discussed.

Isolation of a cDNA coding for an ubiquitin-conjugating enzyme UBC1 of tomato--the first stress-induced UBC of higher plants.

A clone of an ubiquitin-conjugating enzyme (UBC) was isolated from a lambda-ZAP-cDNA library, generated from mRNA of tomato (Lycopersicon esculentum) cells grown in suspension for 3 days. The open reading frame called LeUBC1, encodes for a polypeptide with a predicted molecular mass of 21.37 kDa, which was confirmed by bacterial overexpression and SDS-PAGE. Database searches with LeUBC1 showed highest sequence similarities to UBC1 of bovine and yeast. By Southern blot analysis LeUBC1 was identified as a member of a small E2 subfamily of tomato, presumably consisting of at least two members. As revealed by Northern blot analysis LeUBC1 is constitutively expressed in an exponentially growing tomato cell culture. In response to heat shock an increase in LeUBC1-mRNA was detectable. A strong accumulation of the LeUBC1-transcript was observed by exposure to heavy metal stress which was performed by treatment with cadmium chloride (CdCl2). The cellular uptake of cadmium was controlled via ICP-MS measurements. The data suggest that like in yeast, in plants a certain subfamily of UBC is specifically involved in the proteolytic degradation of abnormal proteins as result of stress.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase from tomato (Lycopersicon esculentum cv. Lukullus)--purification, biochemical properties and behaviour during stress.

Dinucleoside 5',5"'-P1,P4-tetraphosphate hydrolase (EC 3.6.1.17) has been purified to homogeneity from tomato (Lycopersicon esculentum) cells grown in suspension. The purification procedure comprised ammonium sulphate fractionation following five standard chromatography steps and a final chromatography on Ap4A-Sepharose. The homogeneous hydrolase has a molecular mass of 20 kDa and an isoelectric point of 4.5. The enzyme hydrolyses diadenosine tetraphosphate (Ap4A) asymmetrically to AMP and ATP. Among other naturally occurring dinucleoside oligophosphates, Ap5A and Ap6A are substrates whereas Ap3A is not. Of various phosphonate analogues tested, the Ap5A analogue, AppCH2pCH2ppA, was not cleaved and the Ap3A analogue, ApCH2CH2ppA, was a very poor substrate. Enzyme activity is stimulated by 5 mM Mg2+ and inhibited by fluoride anion; I50 = 6.25 microM. The K(m) value for Ap4A is 0.8 microM. The enzyme exhibits a broad pH optimum from pH 6.5 to 9.0. In order to analyze the protein at the molecular level an internal peptide sequence from the homogeneous enzyme was identified. Within the sequence of 17 amino acids a kinase II motif as a general part of a conserved sequence of nucleotide binding sites was found. Against the internal peptide sequence a polyclonal antiserum was raised. By investigating the intracellular level of Ap4A hydrolase under different kinds of environmental stress, no changes occurred in response to heat shock. But, heavy metal stress and phosphate deprivation lead to a decrease in Ap4A hydrolase.

Pharmacokinetics and dosage adjustment of antibiotics during continuous extracorporeal lung assistance and hemofiltration.

Fifty-eight patients undergoing continuous volume-controlled hemofiltration (CVHF) and extracorporeal lung assistance (ECLA) received drug therapy determined by use of a new predictive algorithm in a validation study. Amikacin, netilmicin, tobramycin, ceftriaxone, vancomycin, and teicoplanin doses were given as predicted from clinical parameters. Corrections were necessary in 15 cases (e.g., CVHF 68% and ECLA 82% precision). There were no statistical differences between predicted and monitored drug doses. The correlation coefficient gave r2 = 0.921. Preliminary results from not yet analyzed drugs (ceftriaxone, teicoplanin) fit well into the curve. Because of rapid intraindividual changes, toxic drugs should continue to be monitored.

Distribution and properties of human intestinal diamine oxidase and its relevance for the histamine catabolism.

High activities of diamine oxidase (EC 1.4.3.6) were measured in the intestinal tract of human subjects and of several mammalian species. The enzyme was localized in the mucosa and was distributed primarily in the cytoplasm; the only exception being the guinea-pig where it was located in the particulate fraction. Despite its instability the enzyme from human colonic mucosa was purified 80-fold. During the purification a soluble monoamine oxidase (EC 1.4.3.4) was separated from diamine oxidase. The pH optima of diamine oxidase for putrescine and histamine were 6.6-7.0 and 6.4-6.6, respectively. Short-chain aliphatic diamines were deaminated with the highest reaction velocity, but histamine and N tau-methylhistamine were also excellent substrates. The Km for putrescine was 8.3 x 10(-5) M, for histamine 1.9 x 10(-5) M and for N tau-methylhistamine 9.7 x 10(-5) M. Typical substrates of monoamine oxidase were not deaminated by the enzyme. Aminoguanidine strongly inhibited human intestinal diamine oxidase (IC50 = 1.1 x 10(-8) M). Because of its properties the intestinal diamine oxidase is considered to play a protective role against histamine in diseases such as ischaemic bowel syndrome, mesenteric infarction and ulcerative colitis.

Intestinal monoamine oxidase: does it have a role in histamine catabolism?

The importance of intestinal diamine oxidase in histamine catabolism was proved in several series of experiments. However, intestinal monoamine oxidase might also be involved in histamine degradation either by direct deamination or by the deamination of methylated products. The soluble fraction of intestinal monoamine oxidase was purified and tested for the properties and substrate specificity by three different methods which are described in detail. Using 0.15 M phosphate buffer the optimum pH was 7.4--7.6. The Km values for serotonin and tyramine were 0.2 and 0.3 X 10(-3) M. The most favoured substrates of the enzyme were tyramine, tryptamine and serotonin, but it was not possible to classify the enzyme as a type A or B monoamine oxidase only by its substrate specificity. Histamine and ring methylated derivatives were not attacked by intestinal monoamine oxidase. This means that in the intestinal mucosa by the oxidative pathway of histamine is completely catalysed by diamine oxidase.

The start of a programme for measuring diamine oxidase activity in biopsy specimens of human renal mucosa.

In human subjects, apart from in the kidney, diamine oxidase occurs mainly in the gut. Therefore this enzyme can be used as an indicator of intestinal integrity. In biopsies of rectal mucosa the diamine oxidase activity was assayed in 55 patients, 41 having a histologically normal mucosa and 14 being diseased. The determinations of the enzymic activity were supervised by statistical quality control. In the unchanged rectal mucosa the diamine oxidase activity was 40 nmol/min X g on average. In 7 patients with rectal polyps the enzymic activity was significantly diminished in these benign tumors (mean = 7.7 nmol/min X g) apart from one, where it was elevated. A decrease in diamine oxidase activity was further observed in rectal carcinoma and ulcerative colitis. Whether the reduction of intestinal diamine oxidase activity accompanies premalignant or malignant states or whether it is a general sign of a disturbance of intestinal integrity remains questionable.

Human intestinal diamine oxidase: substrate specificity and comparative inhibitor study.

For an 80-fold purified preparation of human intestinal diamine oxidase the optimum conditions of incubation, the substrate and the inhibitor specificity were tested. Putrescine was the most favoured substrate but N tau-methylhistamine and 2-methylhistamine were metabolized at optimum conditions with nearly the same velocity. Histamine reached about 50% of the reaction velocity of putrescine. Aminoguanidine and semicarbazide inhibited the human intestinal enzyme like a classical diamine oxidase. However, a distinct inhibition of human intestinal and pea seedling diamine oxidase was observed in presence of beta-aminopropionitrile (weak inhibition of the human enzyme, strong inhibition of pea seedling diamine oxidase) and burimamide (strong inhibition of human intestinal enzyme, nearly no influence on pea seedling diamine oxidase). It is proposed to differentiate on the basis of functional considerations diamine oxidases with more histamine detoxicating activities from those being more involved in regulating polyamine levels in growing tissues.

The influence of carcinoma growth on diamine oxidase activity in human gastrointestinal tract.

The distribution of diamine oxidase (DAO) activity was studied in patients having no carcinoma disease. Besides in gut DAO occurred in high activity only in kidney and mesenteric lymph nodes. In patients with adenocarcinoma of the large bowel or of the stomach the enzymic activity was reduced in the tumour tissue itself as compared with the adjacent mucosa in which part the highest activities were observed. In stomach it seemed most important that the enzymic activity was enhanced in the whole mucosa, also in a 10 cm distance from tumour where no histological alterations were found. This fact should be checked for significance in early tumour diagnosis.

The importance of human intestinal diamine oxidase in the oxidation of histamine and/or putrescine.

Histamine, naturally methylated histamine and putrescine are good substrates for human intestinal diamine oxidase, N-Methyl-N-formylhydrazine, the constituent of the mashroom poison-gyromitrin is an inhibitor of human intestinal diamine oxidase. Burimamide inhibits more effectively mammalian intestinal diamine oxidases than pea seedling diamine oxidase, beta-aminopropionitrile is a better inhibitor of pea seedling enzyme than mammalian diamine oxidases. This inhibitory differences might be related to preference in histamine or putrescine oxidizing activity of these enzymes.