Enzymatic and non-enzymatic lipid peroxidation in leaf development.

Enzymatic and non-enzymatic lipid peroxidation has been implicated in programmed cell death, which is a major process of leaf senescence. To test this hypothesis we developed a high-performance liquid chromatography (HPLC) method for a simultaneous analysis of the major hydro(pero)xy polyenoic fatty acids. Quantities of lipid peroxidation products in leaves of different stages of development including natural senescence indicated a strong increase in the level of oxygenated polyenoic fatty acids (PUFAs) during the late stages of leaf senescence. Comprehensive structural elucidation of the oxygenation products by means of HPLC, gas chromatography/mass spectrometry and (1)H nuclear magnetic resonance suggested a non-enzymatic origin. However, in some cases a small share of specifically oxidized PUFAs was identified suggesting involvement of lipid peroxidizing enzymes. To inspect the possible role of enzymatic lipid peroxidation in leaf senescence, we analyzed the abundance of lipoxygenases (LOXs) in rosette leaves of Arabidopsis. LOXs and their product (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid were exclusively detected in young green leaves. In contrast, in senescing leaves the specific LOX products were overlaid by large amounts of stereo-random lipid peroxidation products originating from non-enzymatic oxidation. These data indicate a limited contribution of LOXs to total lipid peroxidation, and a dominant role of non-enzymatic lipid peroxidation in late stages of leaf development.

Metabolic profiling of oxylipins upon salicylate treatment in barley leaves--preferential induction of the reductase pathway by salicylate(1).

In barley leaves, 13-lipoxygenases (13-LOXs) are induced by salicylate (SA) and jasmonate. Here, we show by metabolic profiling that upon SA treatment, free linolenic acid and linoleic acid accumulate in a 10:1 ratio reflecting their relative occurrence in leaf tissues. Furthermore, 13-LOX-derived products are formed and specifically directed into the reductase branch of the LOX pathway leading mainly to the accumulation of (13S,9Z,11E,15Z)-13-hydroxy-9, 11,15-octadecatrienoic acid (13-HOT). Under these conditions, no accumulation of other products of the LOX pathway has been found. Moreover, exogenously applied 13-HOT led to PR1b expression suggesting for the time a role of hydroxy polyenoic fatty acid derivatives in plant defense reactions.

In vitro production of monoclonal antibodies in high concentration in a new and easy to handle modular minifermenter.

This paper describes a new and easy to handle reusable minifermenter for high-density culture of hybridoma and other cells. The culture apparatus is composed of two modules: a 40 ml disposable cell culture and antibody production chamber (the 'production module') and a 550 ml medium reservoir (the 'supply module'). The two modules are separated from each other by a dialysis membrane allowing passage of low molecular mass nutrients and metabolites. The monoclonal antibodies are produced and enriched in the production module. The outer part of this module is made from a thin gas-permeable silicone rubber membrane allowing exchange of gases (oxygen and carbon dioxide). To start the culture, the cells are injected into the production module through ports in the silicone rubber which are equipped with Luer Lock connectors. Samples can be removed in the same way. For culturing, the minifermenter is rolled on a roller apparatus in a carbon dioxide-supplied incubator. Depending on the individual properties of the hybridoma cells cultured, cell densities of more than 10 x 10(6) (in some cases up to 35 x 10(6)) cells per ml and monoclonal antibody concentrations of several mg per ml can be obtained in the new minifermenter. On average, 61 mg (range: 9-159 mg) could be produced within 1-4 weeks. In terms of their properties the monoclonal antibodies produced in the new modular minifermenter were indistinguishable from antibodies prepared from ascitic fluid or from the supernatant of conventional stationary culture. The culture method is a useful alternative to the in vivo production method in mice. In addition, it represents a completely new, inexpensive and easy to handle general solution to the problem of culturing cells in high density and obtaining cellular products in high concentrations.

The glutathione level of retinal Müller glial cells is dependent on the high-affinity sodium-dependent uptake of glutamate.

The dependence of intracellular glutathione, an important radical scavenger, on the extracellular glutamate and cystine concentration and the velocity of the high affinity sodium/glutamate transporter was studied in freshly-isolated Müller glial cells of the guinea-pig, kept in vitro for up to 11 h. To this end the relative Müller cell glutathione levels were measured using the fluorescent dye monochlorobimane, using different concentrations of glutamate and cystine in Ringer solution. In some experiments L-buthionine-[S,R]-sulfoximine, a blocker of glutathione synthesis, or L-trans-pyrrolidine-2,4-dicarboxylic acid and L-alpha-aminoadipic acid, inhibitors of glutamate uptake, were added. The Müller cells maintained about 80% of the normal glutathione level when maintained in Ringer solution containing 100 microM glutamate for 11 h. When under these conditions 100 microM cystine was added, the glutathione level increased to values, which were even higher than those at the beginning of the incubation period. Addition of cystine without glutamate caused a run down of the glutathione level to about 45% of the normal level, which is comparable to the run down in pure Ringer solution. Likewise, application of L-buthionine-[S,R]-sulfoximine (5 mM) lead to a strong run down of the glutathione level even in glutamate/cystine (100 microM)-containing solution. A similar suppressing effect was observed using L-trans-pyrrolidine-2,4-dicarboxylic acid and L-alpha-aminoadipic acid in the presence of 100 microM cystine and glutamate. We conclude that the intracellular glutamate concentration of the Müller cells is determined by the extracellular glutamate concentration and the velocity of the sodium/glutamate uptake. Consequently, cystine uptake into Müller cells, which is performed by the cystine/glutamate antiporter, is fueled by the sodium/glutamate transporter with intracellular glutamate. Both glutamate and cystine are also substrates for glutathione synthesis. The glutathione level is logically limited by the capacity of the sodium/glutamate transporter to provide glutamate intracellularly for, first, cystine uptake and, second, direct insertion into glutathione. Accordingly, the glutathione level is reduced when the sodium/glutamate transporter is blocked. Thus, a diminution of the glutathione level should be taken into consideration when the effects of sodium/glutamate uptake failure and reduced intracellular glutamate concentrations are discussed.

[Ultrasound progress control of retroperitoneal tumours during chemotherapy (author's transl)]. / Sonographische Verlaufskontrollen bei retroperitonealen Tumoren unter Chemotherapie

In a retrospective study the chemotherapeutic response of retroperitoneal tumours was controlled by means of ultrasound and lymphangiography 34 patients. It could be demonstrated that ultrasound is a reliable tool for the evaluation of bulky retroperitoneal tumours, frequently undetectable by lymphangiography. The reliability was better than 90%.

Hydration behaviour of POPC/C(12)-Bet mixtures investigated by sorption gravimetry, (31)P NMR spectroscopy and X-ray diffraction.

The hydration behaviour of mixtures of the zwitterionic phospholipid 1-palmitoyl-2-oleolyl-sn-glycero-3-phosphocholine (POPC) and the zwitterionic surfactant N,N-dimethyl-N-dodecyl-betain (C(12)-Bet) was investigated by sorption gravimetry, solid-state (31)P NMR-spectroscopy and small angle X-ray diffraction (SAXD). Negative excess hydration (dehydration) was found for almost all hydration degrees investigated. This behaviour is explained by the formation of an inner salt between the dipoles of phospholipid and surfactant headgroups that show a reverse sequence of partial charges with respect to the hydrocarbon backbone. The formation of an inner-salt most probably reduces potential water binding sites. Moreover, NMR data suggest that the incorporation of the zwitterionic surfactant into the phospholipid membrane is correlated with reorientation of the phosphate axis towards the membrane director as well as with reduced lateral and wobbling diffusion.

Culture and stimulation of human T- and B-lymphocytes.

The cultivation and stimulation of human peripheral blood cells by different mitogens were studied using a serum-free culture technique. It shows the possibility to culture human PBL in a commercially available serum-free medium over a period of at least 7 days. In a serum-free medium with high protein content the viability of the cells was higher than in one with low protein content or in a serum containing medium. It is possible to stimulate human T cells by phytohaemagglutinin in a serum-free medium. After measurement of [3H]thymidine incorporation, a significant difference between control and test cultures was found. There is also a difference between serum-free medium with high and low protein content. Human peripheral B lymphocytes stimulated by pokeweed mitogen did not show any measurable IgG-secretion if they were cultured in serum-free medium.

[Surgical therapy of diffuse peritonitis--staged lavage]. / Chirurgische Therapie der diffusen Peritonitis--Etappenlavage.

Better understanding of pathophysiology and improved techniques of intensive medical care resulted in new concepts of aggressive treatment for diffuse peritonitis. All consider the importance of surgical removal of the infectious focus but also the necessity of further treatment following first surgical intervention in cases of severe peritonitis. No randomised clinical study yet exists. Indication and application of selected therapeutic technique result from clinical criteria and experience. Presently used concepts are described and evaluated from a clinical viewpoint.

[Computer tomographic and neurologic observations in the acute phase of closed craniocerebral injuries]. / Computertomographische und neurologische Verlaufsbeobachtungen in der Akutphase gedeckter Schädelhirnverletzungen.

Repeated CT-scans and neurological examinations in 59 patients with severe closed head injuries showed that in the acute post-traumatic phase the CT gives no reliable evidence of the degree of brain damage and that during the following period an increase of pathological CT-findings is to be expected, some of which have to be operated on. Prognosis about the course of illness can hardly be given on the basis of the CT in the early stage, but worsening of the findings in controls later on indicates a bad prognosis especially in patients with multifocal or bilateral lesions in the initial CT.

The MTT-assay as a rapid test for cell proliferation and cell killing: application to human peripheral blood lymphocytes (PBL).

The possibility to use the colorimetric MTT assay for measuring proliferation and cell death of human peripheral blood lymphocytes (PBL) was studied. In a range from 100,000-800,000 cells/well a linear correlation between the optical signal (OD signal at 570 nm) and the cell number was found. It is necessary to incubate the cells with the MTT at least 2 hours. After stimulation by different PHA concentrations a very good correlation between [3H] thymidine incorporation and MTT assay was found. A comparison of daunomycin cytotoxicity, measurement by trypan blue exclusion and MTT assay, gave also a good correlation between both methods. It can be pronounced that the MTT assay is a suitable method to measure cell proliferation and cell death of human PBL. The assay is easy to handle, a large number of probes can be assayed in a relatively short time and no radioactivity is necessary. For the measurement of the colored product a common ELISA reader can be used.

Comparison of encephalotomograms and computerized tomograms in midline tumours in infants.

Even after the introduction of the computerized tomogram, midline tumours in infants may give diagnostic problems. In the present study 41 children with tumours in the midline were investigated by both computerized tomography and encephalotomography. The results could be divided into two groups, according to the method and according to the localization of the space-occupying lesion. The diagnostic procedure may be restricted to computerized tomography in cases with direct signs (density differences, contrast enhancement). In cases showing indirect signs on the computerized tomogram (dislocation of ventricles or cisterns or both), further investigation would appear to be valuable. Encephalotomography is the method of choice in patients with negative or uncertain CT findings and contradictory clinical symptomatology.

Formation of 4-hydroxy-2-alkenals in barley leaves.

In barley leaves 13-lipoxygenases are induced by jasmonates. This leads to induction of lipid peroxidation. Here we show by in vitro studies that these processes may further lead to autoxidative formation of (2E)-4-hydroxy-2-hexenal from (3Z)-hexenal.

Metabolic profiling of oxylipins upon sorbitol treatment in barley leaves.

In barley leaves 13-lipoxygenases (LOXs) are induced by salicylate and jasmonate. Here, we analyse by metabolic profiling the accumulation of oxylipins upon sorbitol treatment. Although 13-LOX-derived products are formed and specifically directed into the reductase branch of the LOX pathway, accumulation is much later than in the cases of salicylate and jasmonate treatment. In addition, under these conditions only the accumulation of jasmonates as additional products of the LOX pathway has been found.

[Comparison of the results from sonographic and lymphographic examination in retroperitoneal tumor formations (author's transl)]. / Sonographisch-lymphographische Vergleichsuntersuchungen be retroperitonealen Tumorbildungen.

Retroperitoneal sonographic (grey scale) and lymphographic examination of 51 patients was performed in a prospective study. With either method, an 1:1 correlation was detected in 47 patients (92%). In nine cases, sonographic examination directly revealed extended tumors, whereas lymphographically only lesions within the not-enlarged lymph nodes were represented at the same level. Defective lymph nodes with minimum tumorous affection, on the other hand, were not detectable by sonography. The indications resulting from these findings are defined more precisely with regard to the examinations.

[Variant of the subclavian steal syndrome associated with an anomalous origin of the left vertebral artery]. / Variante des Subclavian Steal Syndroms bei isoliert abgehender Arteria vertebralis sinistra.

A case with a variant of the subclavian steal syndrome associated with an anomalous origin of the left vertebral artery from the aortic arch is presented. Prior to the examination a carotid-subclavian bypass had been performed. The main angiographic finding is the blood supply of the distal subclavian artery through an ipsilateral cervical collateral system. Angiographic procedure and therapeutic consequences are discussed.

Classification of tumours in the sellar region using CT and encephalotomography.

Tumours in the sellar region may still present diagnostic problems. We therefore compared encephalotomographic and CT findings in sellar tumours and 91 patients were examined by both methods. For clinical purposes and operative treatment the degree of compression of the hypothalamus is of great importance. Encephalotomographic and CT findings are analysed and demonstrated. In cases of density differences between tumour and brain tissue shown by contrast enhancement, more information on the inner structure of a tumour (cystic or solid) can be obtained by computerized tomography. But if these are no density differences or/and contrast enhancement, tumour diagnosis is made easier by encephalotomography because of the great efficiency of this method.

Limitations of computerized tomography in diagnosis of subdural hematoma.

CT findings in 51 patients with subdural hematomas are studied. Direct signs (hyperdensity, hypodensity) and indirect signs (any form of mass lesion) are distinguished. CT is limited with regard to determining correlations between time and the attenuation values. The factors which influence density of the hematoma are discussed. For a rational approach with subdural hematomas, which present diagnostic problems and reveal the limitations of CT diagnosis, a diagnostic scheme is proposed.

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies.

A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid.