Isolation and partial characterization of basic proteinases from stem bromelain

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substratel-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosineserine) and position 20 (asparagine glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.02.01.02.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. ThepH optimum of F4 and F5 was betweenpH4.0 and 4.5 and for F9 close to neutralpH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (K _m 2.30 mM,k _cat 0.87 sec^–1 and F5 (K _m 2.42 mM,k _cat 0.68 sec^–1), and differed greatly from F9 (K _m 0.40 mM,k _cat 3.94 sec^–1).Dedicated to H. Tschesche, Bielefeld, Germany, on behalf of his 60th anniversary.     

Sterols and frost hardening of winter rape

Winter rape ( Brassica napus L., var. olifera cv. Górczanski) seedlings were exposed to hardening conditions and the content and composition of free sterols as well as the ratio of free sterols to total phospholipids were determined. There was a reduction in free sterol content in the leaves at the most advanced stage of hardening. The ratio of free sterol to total phospholipids was significantly reduced by hardening due to a decrease in the level of the former and an increase in that of the latter compounds. There was a negative correlation between this ratio and the temperature at which half of the seedlings died. Thus, adaptation of membranes to temperature takes place also at the level of sterol-phospholipid interactions. Exposing seedlings already hardened to freezing temperatures caused injury higher than 50%, and brought about a drastic increase in the level of free sterols and an elevation in the ratio of free sterols to phospholipids. The results are discussed in terms of a possible role of the molecular architecture of membranes in surviving at subzero temperatures.     

Frequency of large granular lymphocytes in peripheral blood of healthy persons and breast cancer patients

The frequency of large granular lymphocytes (LGL) in the peripheral blood of healthy persons (n = 56) and breast cancer patients (31 cases with stage-I and -II disease and 42 cases with stage-III and -IV disease) was studied. The frequency of LGL in peripheral blood was significantly depressed in cancer patients, and particularly in patients with advanced breast cancer.     

Distance measurements using paramagnetic ion-induced relaxation in the saturation transfer electron spin resonance of spin-labeled biomolecules: Application to phospholipid bilayers and interdigitated gel phases

The saturation transfer electron spin resonance (STESR) spectra of spin-labeled phosphatidylcholines in gel phase lipid bilayers are shown to be sensitive to dipolar spin-spin interactions with paramagnetic ions in the aqueous phase. The reciprocal integrated intensity of the STESR spectrum is linearly dependent on aqueous Ni²⁺ ion concentration, hence, confirming the expectation that the STESR intensity is directly proportional to the spin-lattice relaxation time of the spin label. The gradient of the relaxation rate with respect to Ni²⁺ ion concentration decreases strongly with the position of the nitroxide group down the sn-2 chain of the spin-labeled lipid and is consistent with a 1/R³ dependence on the distance, R, from the bilayer surface. The values derived for the dimensions of the bilayer and lipid molecules in the case of dipalmitoyl phosphatidylcholine (DPPC) are in good agreement with those available from x-ray diffraction studies. Allowance for the multibilayer nature of the DPPC dispersions gives an estimate of the water layer thickness that is also consistent with results from x-ray diffraction. The profile of the paramagnetic ion-induced relaxation is drastically changed with DPPC dispersions in glycerol for which the lipid chains are known to be interdigitated in the gel phase. The terminal methyl groups of the lipid chains are located approximately in register with the C-3 atoms of the sn-2 chain of the oppositely oriented lipid molecules in the interdigitated phase. The thickness of the lipid layer and the effective thickness of the lipid polar group are reduced by ~40% in the interdigitated phase as compared with the bilayer phase. The calibrations of the distance dependence established by use of spin labels at defined chain positions should be applicable to STESR measurements on other biological systems.     

Lectinomics I. Relevance of exogenous plant lectins in biomedical diagnostics

This review focuses on utilization of plant lectins as medical diagnostic reagents and tools. The lectin-related diagnostic is aimed at detection of several diseases connected to alteration of the glycosylation profiles of cells and at identification of microbial and viral agents in clinical microbiology. Certain lectins, proposed for or used as diagnostic tools could even recognize those cellular determinants, which are not detected by available antibodies. Broad information is presented on the lectinomics field, illustrating that lectin diagnostics might become practical alternative to antibody-based diagnostic products. In addition, the rising trend of lectin utilization in biomedical diagnostics might initiate a development of innovative methods based on better analytical technologies. Lectin microarray, a rapid and simple methodology, can be viewed as an example for such initiative. This technology could provide simple and efficient screening tools for analysis of glycosylation patterns in biological samples (cellular extracts, tissues and the whole cells), allowing thus personalized detection of changes associated with carbohydrate-related diseases.     

Kinetics and dynamics of annealing during sub-gel phase formation in phospholipid bilayers: A saturation transfer electron spin resonance study

The saturation transfer electron spin resonance (STESR) spectra of spin-labeled phosphatidylcholine have been used to follow the kinetics of conversion from the gel phase to the sub-gel phase in aqueous bilayers of dipalmitoyl phosphatidylcholine. This is a simple, well-defined model system for lipid domain formation in membranes. The integrated intensity of the STESR spectrum from the chain-labeled lipid first increases and then decreases with time of incubation in the gel phase at 0°C. The first, more rapid phase of the kinetics is attributed to the conversion of germ nuclei to growth nuclei of the sub-gel phase. The increase in STESR intensity corresponds to the reduction in chain mobility of spin labels located in the gel phase at the boundaries of the growth nuclei and correlates with the increase in the diagnostic STESR line height ratios over this time range. The second, slower phase of the kinetics is attributed to growth of the domains of the sub-gel phase. The decrease in STESR intensity over this time regime corresponds to exclusion of the spin-labeled lipids from the tightly packed sub-gel phase and correlates quantitatively with calibrations of the spin label concentration dependence of the STESR intensity in the gel phase. The kinetics of formation of the sub-gel phase are consistent with the classical model for domain formation and growth. At 0°C, the half-time for conversion of germ nuclei to growth nuclei is ∼7.7 h and domain growth of the sub-gel phase is characterized by a rate constant of 0.025 h^-1. The temperature dependence of the STESR spectra from samples annealed at 0°C suggests that the subtransition takes place via dissolution of sub-gel phase domains, possibly accompanied by domain fission.     

A new dermatophyte,Thallomicrosporon kuehnii Benedek, 1963

Summary A new species of a dermatophyte,Thallomicrosporon kuehnii Benedek, 1963, is described. In contradistinction to all other known dermatophytes, it reproduces exclusively by thallospores: 1) large, fusiform, macroconidia-like structures, 2) arthrospores and, 3) chlamydospores. Microconidia are present, rodlike or pyriform. The elicitation of a perfect form of fructification in hair-on-soil cultures was unsuccessful.

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Zusammenfassung Eine neue Art der Dermatophyten,Thallomicrosporon kuehnii Benedek, 1963, wird beschrieben. Sie unterscheidet sich von allen anderen Hauptilzen durch den Umstand, dass sie sich ausschliesslich durch Thallosporen reproduziert: 1) Formen, ähnlich fusiformen Makrokonidien; 2) Arthrosporen und 3) Chlamydosporen. Mikrokonidien werden gebildet. Das Hervorbringen der perfekten Phase der Fruktifikation in Kulturen von Haar an Humus ist nicht gelungen.

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Résumé Une espèce nouvelle des dermatophytes,Thallomicrosporon kuehnii Benedek, 1963, est présentée. Elle est distincte de tous les autres dermatophytes par le fait de sa reproduction exclusive par des thallospores: 1) des structures imitant des macroconidies fusiformes; 2) des arthrospores et 3) des chlamydospores. Des microconidies sont présentes. L'élicitation de la phase perfecte de fructification en culture des poils en sol n'a pas réussi.

     

Vergleichende Untersuchungen über die DDT-Empfindlichkeit von Kartoffelkäfer-Populationen (Leptinotarsa decemlineata Say) verschiedener Herkünfte

Für eine effiziente Resistenzzüchtung stellt eine Resistenzprüfmethode, die eine schnelle und zuverlässige Evaluierung einer möglichst großen Zahl von Genotypen erlaubt, eine wichtige Voraussetzung dar. Für das Freiland wurde eine Preßluftinjektionsmethode entwickelt, die eine nahezu 100 %ige Infektion von Maispflanzen mit dem Zuckerrohrmosaik‐Virus (sugarcane mosaic virus, SCMV) und dem Maisver‐zwergungsmosaik‐Virus (maize dwarf mosaic virus, MDMV) garantiert. Bei den empfindlicheren Pflanzen im Gewächshaus hatte eine mechanische Inokulation von Hand Vorteile.     

Detection of hydrogen peroxide by lactoperoxidase-mediated dityrosine formation

The aim of this work was to study the dityrosine-forming activity of lactoperoxidase (LPO) and its potential application for measuring hydrogen peroxide (H₂O₂). It was observed that LPO was able to form dityrosine at low H₂O₂ concentrations. Since dityrosine concentration could be measured in a simple fluorimetric reaction, this activity of the enzyme was utilized for the measurement of H₂O₂ production in different systems. These experiments successfully measured the activity of NADPH oxidase 4 (Nox4) by this method. It was concluded that LPO-mediated dityrosine formation offers a simple way for H₂O₂ measurement.