Determination of free and conjugated indole-3-acetic acid, tryptophan, and tryptophan metabolites in grape must and wine.

Tryptophan (Trp) and its metabolites, especially indole-3-acetic acid (IAA), are considered to be potential precursors of 2-aminoacetophenone (AAP), an aroma compound that causes an "untypical aging off-flavor" (UTA) in Vitis vinifera wines. In this study, RP-HPLC with fluorescence detection was used for the qualitative and quantitative analysis of Trp and Trp metabolites in grape musts and wines to which different viticultural measures had been applied (time of harvest, soil treatment, leaf plucking, vine prune). An alkaline hydrolysis was developed to release bound IAA and Trp. A sensitive and selective determination of different Trp metabolites was achieved after solid phase extraction using a strong anion exchange material. In the examined grape musts, more than 95% of the total IAA was bound either as ester conjugate or as amide conjugate. Free IAA and other Trp metabolites were below the detection limit (<3 microg/L) or could be determined only in traces. Their amounts increased significantly during fermentation, whereas the amount of Trp decreased. It could be shown that the different viticultural measures applied (except the vine prune) as well as the climatic conditions of the vintage exhibited significant influences on the amounts of Trp and Trp metabolites in grape musts or wines.

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.

CD44 in normal human pancreas and pancreatic carcinoma cell lines.

CD44 is an integral cell-surface glycoprotein. Overexpression of the CD44 standard (CD44st) and its variants (CD44v) has been implicated in transformation and progression of many cancer types. Here, we investigated expression of CD44st, CD44v3-7, CD44v7/8, and v10 in five human pancreatic tumor cell lines and normal human pancreatic duct cells transfected with the SV40 large T antigen. CD44st and its variant proteins were quantified using immunocytochemistry and flow cytometry. CD44v7 was expressed at low levels, whereas CD44st, CD44v3, CD44 v4, CD44v, and CD44v6 were expressed at moderate levels in all pancreatic tumor cell lines. In contrast, CD44v7/8 and CD44v10 were expressed at very low levels in two out of the five pancreatic tumor cell lines. Overall, staining of CD44st and CD44 variants was significantly weaker compared to another surface molecule, ICAM-1, reported to be overexpressed in pancreatic cancer cells. Furthermore, the SV40 large T transfected duct cells showed only a weak staining for CD44st, CD44v5, and CD44v6. To determine a possible mechanism for the regulation of surface expression of CD44st, v5 and v6, we incubated Panc-1 cells with bFGF, TGF-beta1, EGF, TNFalpha, and IFNgamma. Only IFNgamma affected the CD44 expression by down-regulation of CD44v6. The constitutive expression of CD44 variants seems to be associated with the malignant state of invasive carcinoma.

Determination of tryptophan and tryptophan metabolites in grape must and wine.

Tryptophan (Trp) and its metabolites, especially indole-3-acetic acid (IAA), are considered as potential precursors of 2-aminoacetophenone (AAP), an aroma compound which causes the "untypical aging off-flavor" (UTA) in Vitis vinifera white wines. In this study RP-HPLC with fluorescence detection was used for the qualitative and quantitative analysis of Trp and Trp-metabolites in 39 grapes, 22 grape musts and 16 wines, to which different viticultural conditions (ripeness, pruning, strip of leaves, soil condition) have been applied. A sensitive and selective determination was achieved after solid phase extraction using an anion exchange material. Only traces of Trp-metabolites could be determined in the examined grapes and grape musts, but their amounts increased significantly during fermentation, whereas the amount of Trp decreased. Different viticultural measures, besides the time of grape harvest, showed no significant influences on the amount of Trp and Trp-metabolites.

Cessation of insulin infusion at night-time during CSII-therapy: comparison of regular human insulin and insulin lispro.

Development of hyperglycemia with subsequent ketoacidosis is one of the potential risks of a sudden cessation of insulin delivery during continuous insulin infusion therapy with insulin pumps in patients with IDDM. To evaluate differences in the development of ketoacidosis after a sudden pump stoppage between regular human insulin and insulin lispro, we performed an open label randomized crossover investigation with 7 patients (6 male/1 female, mean age (SD: 40.9 +/- 12.9 years). At 10 p.m., 4 hours after a light dinner with a preprandial injection of the corresponding insulin, the catheter was pulled out of the skin. During the observation period, blood glucose (every hour), pH-values and base excess values (every two hours) were measured until 7 a.m. One patient, in the insulin lispro treatment arm, discontinued because early interruption criteria were met after 7 hours. With insulin lispro, the metabolic changes developed 1.5 to 2 hours earlier than with regular human insulin (after 3 hours: difference in base excess (BE) mean +/- SD: regular human insulin: -0.41 +/- 1.04 mmol/l; insulin lispro: -1.69 +/- 0.83 mmol/l, p < 0.05; blood glucose: regular human insulin: 4.93 +/- 2.87 mmol/l, insulin lispro: 8.97 +/- 3.48, p < 0.05; pH values: regular human insulin: 7.38 +/- 0.02, insulin lispro: 7.36 +/- 0.02, n.s.). In general, metabolic deterioration tended to be more pronounced with insulin lispro than with regular human insulin (deltaBE after 7 h: regular human insulin: -2.39 +/- 1.30 mmol/l; insulin lispro: -3.27 +/- 2.43 mmol/l, n.s.). In conclusion, if patients want to be treated with insulin lispro in an insulin pump, they have to be well-educated about the pharmacokinetic properties of the insulin analogue and about the possibility that ketoacidotic deterioration after an interruption of the insulin delivery may occur earlier in comparison to regular human insulin. It is anyway recommendable to perform a pump stop test when starting CSII-treatment in patients with diabetes mellitus.

[Hot air coagulator for the treatment of bleeding of parenchymal organs]. / Heissluftkoagulator zur Behandlung von Blutungen parenchymatöser Organe.

Experiences are described about the hemostasis in parenchymatous organs by means of a self-constructed hot air coagulator. Bleedings in liver, spleen, and kidney were caused experimentally in 15 dogs. A certain control of hemorrhage were carried out by hot air. The own hot air coagulator was superior to other coagulators by the little weight and the fast and noiseless working.

The use of protein beads as immunoadsorbent for the column fractionation of lymphocytes.

Beads of calf serum proteins (CSB) with antigen-antibody complexes or antigen by activation with glutaraldehyde were used as immunoadsorbents for the fractionation of mouse lymphocytes. T-lymphocytes could be separated by this method with high purity. The precursors of antibody-forming cells were completely eliminated from spleen and bone marrow cell suspensions. The unspecific binding of lymphocytes to CBS or Sepharose was similar and not selective for T or B lymphocytes.

[Oxygen pressure values in the ischemic muscle tissue of patients with occlusive arterial diseases (author's transl)]. / Sauerstoffdruckwerte im ischämischen Muskelgewebe von Patienten mit chronischen peripheren arteriellen Verschusskrankheiten

The local oxygen pressure of muscle tissue was measured in 10 patients with occlusive atherosclerosis and in 9 healthy volunteers using a micro-Pt-electrode. In 3 cases, the muscular pO2 values of an ischemic leg were compared to the non-ischemic leg of the same patient. In both groups, there was a statistically significant difference in the oxygen pressure of the muscle tissue in normal legs of volunteers as compared to the ischemic legs. Measurements of the local oxygen pressure in the muscle enables us to have a quantitative measure for the ischemic situtation. It is a useful diagnostic method in patients suffering from ischemic claudication and seems to be an objective criterium of therapeutical methods.