Application of rapid biochemical methods for detecting avocado sunblotch disease

The method of Palukaitis & Symons (1980) for extracting low molecular weight ribonucleic acids from plant tissue was improved by CF-11-cellulose chromatography and further simplified for use in routine biochemical indexing for avocado sunblotch viroid (ASBV). Extracts were prepared routinely at 10 g dry weight equivalents (DWE) of tissue per ml; more concentrated than previously possible with many avocado cultivars. Conditions for assaying extracts of ASBV were standardised and the lower limits for detection determined as 80–230 ng ASBV/g DWE of tissue for polyacrylamide gel electrophoresis (PAGE) and 1 ng/g DWE of tissue for complementary DNA (cDNA) probe assays. The concentration of ASBV in a single infected tree varied from 5 to 5000 ng/g DWE between branches, but only to a minor degree between mature leaves and young blossoms within branches. Six independent sources of sunblotch disease were examined and all proved positive for ASBV by PAGE. The ASBV extracted from five of these sources hybridised with cDNA prepared from the sixth or standard source (Hass/SB-1), with hybridisation values ranging from 43% to 89%. In a survey of 76 trees intended for propagation in Australia, all of 17 trees previously accepted as healthy on the basis of graft transmission tests were negative for ASBV by PAGE and had cDNA hybridisation values ranging from 1.8% to 12.1%. Amongst 59 trees apparently free of sunblotch symptoms but not previously indexed, only one tree was positive for ASBV by both PAGE and cDNA probe assay. The other 58 trees were negative by PAGE but had hybridisation values ranging from 1.0% to 42.8%. Forty-nine trees had values consistent with known healthy trees (<12% hybridisation), while the results of the remaining nine trees will require confirmation by additional tests before a conclusion about ASBV is made. The cDNA probe assay successfully detected ASBV in avocado seedlings graft inoculated with Hass/SB-1, 1–2 months before symptoms were displayed but not until 6 months after inoculation. Methods for improving the cDNA probe assay still further are discussed.     

Analytical methods in environmental effects-directed investigations of effluents

Effluent discharges are released into aquatic environments as complex mixtures for which there is commonly either no knowledge of the toxic components or a lack of understanding of how known toxicants interact with other effluent components. Effects-directed investigations consist of chemical extraction and iterative fractionation steps directed by a biological endpoint that is designed to permit the identification or characterization of the chemical classes or compounds in a complex mixture responsible for the observed biological activity. Our review of the literature on effects-directed analyses of effluents for non-mutagenic as well as mutagenic endpoints showed that common extraction and concentration methods have been used. Since the mid-1980s, the methods have evolved from the use of XAD resins to C18 solid-phase extraction (SPE). Blue cotton, blue rayon, and blue chitin have been used specifically for investigations of mutagenic activity where polycyclic compounds were involved or suspected. After isolation, subsequent fractionations have been accomplished using SPE or a high-pressure liquid chromatography (HPLC) system commonly fitted with a C18 reverse-phase column. Substances in active fractions are characterized by gas chromatography/mass spectrometry (GC-MS) and/or other spectrometric techniques for identification. LC-MS methods have been developed for difficult-to-analyze polar substances identified from effects-directed studies, but the potential for LC-MS to identify unknown polar compounds has yet to be fully realized. Salmonella-based assays (some miniaturized) have been coupled with fractionation methods for most studies aimed at identifying mutagenic fractions and chemical classes in mixtures. Effects-directed investigations of mutagens have focused mostly on drinking water and sewage, whereas extensive investigations of non-mutagenic effects have also included runoff, pesticides, and pulp mill effluents. The success of effects-directed investigations should be based on a realistic initial objective of each project. Identification of chemical classes associated with the measured biological endpoint is frequently achievable; however, confirmation of individual compounds is much more difficult and not always a necessary goal of effects-directed chemical analysis.     

Physiological and biochemical investigations on egg stickiness in common carp

The properties and behaviour of common carp, Cyprinus carpio, eggs in water and in ovarian fluids were studied at different temperature, pH, and with divalent cation concentrations. The biochemical composition of zona radiata externa (ZRE) was analyzed qualitatively and quantitatively on amino acids, carbohydrates, uronic acid and sialic acids using chemical assays; on proteins using electrophoresis. Comparative biochemical studies were performed on the chub, Leuciscus cephalus, the vimba, Vimba vimba and the bleak, Chalcalburnus chalcoides. Eggs of common carp became sticky within seconds after mixing with water. Egg stickiness was not affected by water pH in a range of 6-9, by water temperatures between 4 and 30 degrees C, by divalent cations in concentrations < or =20 mmol/l, and by sodium chloride concentrations < or =50 mmol/l. Our investigations indicated that specific proteins of the cyprinid ovarian fluid are controlling (inhibiting) the initiation of egg stickiness: egg stickiness did not develop as long as the eggs were incubated in ovarian fluid. When however the ovarian fluid proteins were removed from the ovarian fluid by heat treatment, eggs developed stickiness within seconds, like they do in water. Biochemically, the ZRE consisted of nine types of proteins whereby four of them were glycoproteins. Glucose, fructose, galactose, and uronic acids were the major carbohydrates. Treatment of the egg membrane with invertase or amyloglucosidase did not affect the egg stickiness. Treatment with protease prevented stickiness. From these results and from additional histochemical results, we conclude that glycoproteins are likely to be the molecules responsible for stickiness.     

Application of multivariate resolution methods to the study of biochemical and biophysical processes

Multivariate resolution methods make up a set of mathematical tools that may be applied to the analysis and interpretation of spectroscopic data recorded when monitoring a physical or chemical process with multichannel detectors. The goal of resolution methods is the recovery of chemical and/or physical information from the experimental data. Such data include, for example, the number of intermediates present in a reaction, the rate or equilibrium constants, and the spectra for each one of those intermediates. Multivariate resolution methods have been shown to be useful for the study of biophysical and biochemical processes such as folding/unfolding of proteins or nucleic acids. The present article reviews the most frequently used resolution methods, the limitations on their use, and their latest applications in protein and nucleic acid research.     

A glycogen storage disease in rats. Morphological and biochemical investigations

Liver and heart from a substrain of the NZR/Gd rat in which there is an inherited deficiency of liver phosphorylase b kinase was examined by light and electron microscopy and compared to material from a related, but normal substrain. Hepatic tissue differed markedly from that of control animals. Hepatocytes contained more than twice as much free glycogen and visible lipid. Glycogen particles had an abnormal appearance and some glycogen was sequestered within large, membrane-bound vesicles. Hepatocyte lysosomes were increased by a third and mean cell volume by more than half. Lobular architecture was distorted by the presence of enlarged, irregularly-shaped hepatocytes. Free glycogen was present in the space of Disse and sinusoids and within lysosomes in Kupffer cells. There were increased amounts of collagen in the space of Disse. The changes resemble those described in human glycogen storage disease IXa. A study of hepatic tissue from fasted rats showed that affected animals have an impaired ability to mobilise their liver glycogen stores. An increase in visible lipid also occurred in affected, fasted animals. Cardiac tissue appeared to be normal.     

Disaccharidase activities in camel small intestine: biochemical investigations of maltase-glucoamylase activity

Disaccharidases (maltase, cellobiase, lactase, and sucrase), alpha-amylase, and glucoamylase in the camel small intestine were investigated to integrate the enzymatic digestion profile in camel. High activities were detected for maltase and glucoamylase, followed by moderate levels of sucrase and alpha-amylase. Very low activity levels were detected for lactase and cellobiase. Camel intestinal maltase-glucoamylase (MG) was purified by DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of camel small intestinal MG4 and MG6 were estimated to be 140,000 and 180,000 using Sephacryl S-200. These values were confirmed by SDS-PAGE, where the two enzymes migrated as single subunits. This study encompassed characterization of MGs from camel intestine. The Km values of MG4 and MG6 were estimated to be 13.3 mM and 20 mM maltose, respectively. Substrate specificity for MG4 and MG6 indicated that the two enzymes are maltase-glucoamylases because they catalysed the hydrolysis of maltose and starch with alpha-1,4 and alpha-1,6 glycosidic bonds, but not sucrose with alpha-1,2 glycosidic bond which was hydrolyzed by sucrase-isomaltase. Camel intestinal MG4 and MG6 had the same optimum pH at 7.0 and temperature optimum at 50 degrees C and 40 degrees C, respectively. The two enzymes were stable up to 50 degrees C and 40 degrees C, followed by strong decrease in activity at 60 degrees C and 50 degrees C, respectively. The effect of divalent cations on the activity of camel intestinal MG4 and MG6 was studied. All the examined divalent cations Ca(2+), Mn(2+), Mg(2+), Co(2+) and Fe(3+) had slight effects on the two enzymes except Hg(2+) which had a strong inhibitory effect. The effect of different inhibitors on MG4 and MG6 indicated that the two enzymes had a cysteine residue.     

Computational methods for diffusion-influenced biochemical reactions

MOTIVATION: We compare stochastic computational methods accounting for space and discrete nature of reactants in biochemical systems. Implementations based on Brownian dynamics (BD) and the reaction-diffusion master equation are applied to a simplified gene expression model and to a signal transduction pathway in Escherichia coli. RESULTS: In the regime where the number of molecules is small and reactions are diffusion-limited predicted fluctuations in the product number vary between the methods, while the average is the same. Computational approaches at the level of the reaction-diffusion master equation compute the same fluctuations as the reference result obtained from the particle-based method if the size of the sub-volumes is comparable to the diameter of reactants. Using numerical simulations of reversible binding of a pair of molecules we argue that the disagreement in predicted fluctuations is due to different modeling of inter-arrival times between reaction events. Simulations for a more complex biological study show that the different approaches lead to different results due to modeling issues. Finally, we present the physical assumptions behind the mesoscopic models for the reaction-diffusion systems. AVAILABILITY: Input files for the simulations and the source code of GMP can be found under the following address: http://www.cwi.nl/projects/sic/bioinformatics2007/     

Comperative investigations of non chemical weed management methods in Hungary

Organic farming has an increasing tendency in Hungary because of growing consumers' demands according to organic products not only in inland but also in the countries of the European Union. Developments of weed control methods in organically cropped field plants have become conspicuous next to developing chemical weed management methods of convencionally cropped cultural plants. The aim of our investigations was to make comperative investigations of non chemical weed control methods in wide rowed plants.     

Further biochemical investigations on glycosaminoglycans extracted from Euglena gracilis]

Polyanionic glycans extracted from Euglena gracilis have been studied by biochemical, chromatographic and electrophoretic analysis. Our results show the presence of a fraction which precipitate with CPC and another one which not precipitate with CPC. The CPC precipitable material fractionated on CPC-Cellulose column shows the presence of 5 Glycosaminoglycans; the not CPC precipitable material contains uronic acid, galactose, sulfate, galactosamine and cannot be related to Keratan sulfate.     

Computational investigations into the origins of short-term biochemical memory in T cell activation

Recent studies have reported that T cells can integrate signals between interrupted encounters with Antigen Presenting Cells (APCs) in such a way that the process of signal integration exhibits a form of memory. Here, we carry out a computational study using a simple mathematical model of T cell activation to investigate the ramifications of interrupted T cell-APC contacts on signal integration. We consider several mechanisms of how signal integration at these time scales may be achieved and conclude that feedback control of immediate early gene products (IEGs) appears to be a highly plausible mechanism that allows for effective signal integration and cytokine production from multiple exposures to APCs. Analysis of these computer simulations provides an experimental roadmap involving several testable predictions.