Differential storage of hydroxyethyl starch (HES) in the skin: an immunoelectron-microscopical long-term study

Hydroxyethyl starch (HES) is widely used as a plasma substitute. Serious side effects occur only rarely, whereas a high incidence of severe pruritus has been reported. Moreover, tissue storage of HES has been demonstrated in various organs. The aim of the current study has been to examine precisely the intracellular uptake and long-term storage of HES in the skin. Skin biopsies from 119 patients who received HES of various preparations and cumulative dosage were obtained 30 min to 130 months after infusion therapy. The samples were analysed by ultrastructural and immunoelectron microscopy with HES-specific monoclonal and polyclonal antibodies. A characteristic vacuolisation of perivascular histiocytes was a regular finding in all skin biopsies as early as 1 day after a single infusion of 30 g. Immunoreactivity for HES was demonstrable within the vacuoles. Generally, the size and number of vacuoles in the histiocytes increased concomitantly with the cumulative dosage. Following administration of higher HES dosages, vacuoles were demonstrable in endothelial cells of blood and lymphatic vessels, basal keratinocytes, epithelia of sweat glands and in small peripheral nerves, the last mentioned being associated with pruritus. A subsequent reduction of the vacuoles in size and number could be demonstrated within 52 months. In nerves, HES deposits persisted no longer than 17 months paralleling the cessation of pruritus. Biopsies taken after 94 months exhibited no HES deposits in the skin. The condensation and final dissolution of the vacuoles may either indicate the release and subsequent redistribution of HES into the circulation or lysosomal degradation.     

Molecular methods for the analysis of Clostridium perfringens relevant to food hygiene

Clostridium perfringens continues to be a common cause of food-borne disease. It produces an enterotoxin (CPE) which is released upon lysis of the vegetative cell during sporulation in the intestinal tract. Catering premises with insufficient cooling and reheating devices often seem to be the cause of outbreaks of C. perfringens food poisoning. Typing of C. perfringens is of great importance for investigating sources of food poisoning cases and for studying the epidemiology of this microorganism. This report describes the examination of 155 C. perfringens isolates by molecular methods. Isolates were taken from 10 food poisoning outbreaks and cases (n = 34, food and fecal isolates) and from meat and fish pastes (n = 121). Isolates were characterized by plasmid profiling, ribotyping, and/or macrorestriction analysis by pulsed-field gel electrophoresis (PFGE). Results show that all three methods are suitable for classifying C. perfringens isolates below the species level. Ribopatterns and PFGE patterns can be interpreted more easily than plasmid profiling results and can be recommended for contamination studies and epidemiologic investigation of food poisonings associated with C. perfringens.     

Tyrosinase involved in betalain biosynthesis of higher plants

A tyrosine-hydroxylating enzyme was partially purified from betacyanin-producing callus cultures of Portulaca grandiflora Hook. by using hydroxyapatite chromatography and gel filtration. It was characterized as a tyrosinase (EC 1.14.18.1 and EC 1.10.3.1) by inhibition experiments with copper-chelating agents and detection of concomitant o-diphenol oxidase activity. The tyrosinase catalysed both the formation of L-(3,4-dihydroxyphenyl)-alanine (Dopa) and cyclo-Dopa which are the pivotal precursors in betalain biosynthesis. The hydroxylating activity with a pH optimum of 5.7 was specific for L-tyrosine and exhibited reaction velocities with L-tyrosine and D-tyrosine in a ratio of 1:0.2. Other monophenolic substrates tested were not accepted. The enzyme appeared to be a monomer with an apparent molecular mass of ca. 53 kDa as estimated by gel filtration and SDS-PAGE. Some other betalain-producing plants and cell cultures were screened for tyrosinase activity; however, activities could only be detected in red callus cultures and plants of P. grandiflora as well as in plants, hairy roots and cell cultures of Beta vulgaris L. subsp. vulgaris (Garden Beet Group), showing a clear correlation between enzyme activity and betacyanin content in young B. vulgaris plants. We propose that this tyrosinase is specifically involved in the betalain biosynthesis of higher plants. Received: 14 July 1998 / Accepted: 23 October 1998     

Phenolic dimers and an indole alkaloid from Campylospermum flavum (Ochnaceae)

From the leaves and stem bark of Campylospermum flavum (Ochnaceae), three compounds, namely 4?-O-methylagathisflavone, flavumchalcone, and flavumindole have been isolated together with 10 known compounds, including three flavonoids, two biflavonoids, two alkaloids, two nitrile glucosides, and glucopyranosyl-β-sistosterol. The structures of these compounds and their relative configurations were established by 1D and 2D NMR experiments. The methanolic crude extracts of leaves and stem bark of C. flavum and compounds displayed a significant cytotoxicity towards Artemia salina larvae.     

Quantitative assessment of primary cerium reaction product formed by glucose-6-phosphatase activity in a male rat liver: an image analysis study

 Glucose-6-phosphatase (G6Pase) activity has been determined in periportal and pericentral areas of the liver of normal male rats. Measurements were performed on unfixed cryostat sections mounted on semipermeable membranes. In the present study, the oxidized primary reaction product of a cerium-based histochemical method [Ce(IV)perhydroxyphosphate] instead of the final reaction product after a second-step incubation was measured. For quantification of the amount of Ce(IV)perhydroxyphosphate formed the digital image analyzing system Quantimet 500+ was used. Estimated values of optical densities of Ce(IV)perhydroxyphosphate over test areas were employed for calculation of kinetic parameters of (G6Pase). Highest activities of G6Pase (higher K _m and V _max levels) were found in periportal areas of the rat liver, indicating a higher amount of active enzyme molecules and a lower affinity for the substrate. Differences in values for both K _m and V _max between periportal and pericentral zones were highly significant and closely comparable to those for male fed rats. Correlations between K _m and V _max were significant for periportal as well for pericentral liver areas. The results of the present study thus allow the same biological implications as histochemical methods employing a final reaction for quantification of enzyme activities. The present method avoids the drawbacks of enhancement reactions and demonstrates the feasibility of in situ analysis of enzyme kinetic parameters by quantification of oxidized primary cerium reaction products. Accepted: 8 January 1996     

Developmental studies on two ecdysone deficient mutants ofDrosophila melanogaster

Summary This paper describes two ecdysone-deficient, recessive-lethal mutants,lethal(1)giant ring gland (grg) andlethal(1)suppressor of forked ^mad-ts (mad-ts: Jürgens and Gateff 1979) and compares their ecdysteroid titers with that of the wild-type. Mutant larvae show a much reduced ecdysteroid content, amounting to 1/10 to 1/30 of the wild-type values, but never a true titer peak. They fail to pupate and die after 1–3 weeks. Ecdysteroid feeding elicits different responses in the larvae of the two mutants.mad-ts larvae pupate within 24 h, thus showing that their low ecdysteroid titer is directly connected to their inability to pupate.mad-ts resembles the mutantlethal (3)ecdysone-1 ^ts (Garen et al. 1977). Thegrg mutant larvae, on the other hand, fail to pupate after 20-hydroxyecdysone feeding as well as injection. The primary defect of thegrg mutant is not entirely clear. Thegrg larval salivary gland cells appear to possess normal ecdysteroid receptors. Furthermore, the low ecdysteroid titer ingrg is not the result of an increased ecdysteroid catabolism. The primary defect in the mutant may lie in the malfunctioning neurosecretory cells which do not show neurosecretion in histological preparations. Further support for this notion comes from electronmicrographs of the enlargedgrg ring glands which, in contrast to the wild-type, do not possess nerve endings.In the wild-type three ecdysteroid peaks were found: one shortly before puparium formation, the second at approximately 12 h and the third at about 30 h after pupation. The ecdysteroid titer peak in late third instar, wild-type larvae is mainly due to the presence of 20-dydroxyecdysone as shown by radioimmunoassays after thin layer chromatography and derivatization followed by gas liquid chromatography and mass spectroscopy. In addition, a number of unidentified polar and apolar metabolites were also present.     

New cytotoxic alkylbenzoquinone derivatives from leaves and stem of Ardisia kivuensis (Myrsinaceae)

Five new alkylbenzoquinone derivatives, ardisiaquinones L–P (1–5) along with the known ardisiaquinone K were isolated from the MeOH extracts of leaves and stems of Ardisia kivuensis Taton (Myrsinaceae). Ardisiaquinones L, M and N were isolated from the leaves while ardisiaquinones K, O and P were obtained from the stem. Ardisiaquinone O was obtained in mixture with ardisiaquinone N, and P together with K, respectively. Their structures were elucidated on the basis of spectroscopic data. All the compounds showed cytotoxicity against Artemia salina and moderate antimicrobial activity.