Activity of plasma membrane β-galactosidase and β-glucosidase

Human fibroblasts produce ceramide from sialyllactosylceramide on the plasma membranes. Sialidase Neu3 is known to be plasma membrane associated, while only indirect data suggest the plasma membrane association of β-galactosidase and β-glucosidase. To determine the presence of β-galactosidase and β-glucosidase on plasma membrane, cells were submitted to cell surface biotinylation. Biotinylated proteins were purified by affinity column and analyzed for enzymatic activities on artificial substrates. Both enzyme activities were found associated with the cell surface and were up-regulated in Neu3 overexpressing cells. These enzymes were capable to act on both artificial and natural substrates without any addition of activator proteins or detergents and displayed a trans activity in living cells.     

Accumulation of Phosphorylated β-Catenin Enhances ROS-Induced Cell Death in Presenilin-Deficient Cells

Presenilin (PS) is involved in many cellular events under physiological and pathological conditions. Previous reports have revealed that PS deficiency results in hyperproliferation and resistance to apoptotic cell death. In the present study, we investigated the effects of PS on β-catenin and cell mortality during serum deprivation. Under these conditions, PS1/PS2 double-knockout MEFs showed aberrant accumulation of phospho-β-catenin, higher ROS generation, and notable cell death. Inhibition of β-catenin phosphorylation by LiCl reversed ROS generation and cell death in PS deficient cells. In addition, the K19/49R mutant form of β-catenin, which undergoes normal phosphorylation but not ubiquitination, induced cytotoxicity, while the phosphorylation deficient S37A β-catenin mutant failed to induce cytotoxicity. These results indicate that aberrant accumulation of phospho-β-catenin underlies ROS-mediated cell death in the absence of PS. We propose that the regulation of β-catenin is useful for identifying therapeutic targets of hyperproliferative diseases and other degenerative conditions.     

The Development of Activity of Cell Wall Bound β-Fructofuranosidase with Ripening and Senescence of Tomato Fruit

Optically active trisubstituted and tetrasubstituted cyclopentanes, with proper functional groups of proper stereochemistry as precursors for chiral synthesis of brefeldin A and prostaglandins, were prepared stereoselectively from carbohydrate.     

Mycelial β-N-Acetylglucosaminidase of Streptomyces sp. Having β-N-Acetylgalactosaminidase Activity

Formation of transfer products from soybean arabinogalactan and glycerol by endo-1,4-β-d-galactanase from Penicillium citrinum was described. The amount of transfer products depended on the glycerol concentration. About 50% of the galactose residues which could be liberated from the polysaccharide by the enzyme were transferred to glycerol at an acceptor concentration of 2.5% (w/v). Transfer products with various polymerization degrees were accumulated at the beginning of the reaction and then those with higher polymerization degrees were degraded gradually. At a final stage of the reaction, two transfer products in addition to two hydrolysis products (galactose and galactobiose) were mainly accumulated. The two transfer products were isolated and their structures were examined. They were 2-O-β-d-galactosyl glycerol and O-β-d-galactosyl-(1 → 4)-O-β-d-galactosyl-(1 → 2)glycerol.     

Isolation and Characterization of Mutations in the β-Tubulin Gene of SACCHAROMYCES CEREVISIAE

Of 173 mutants of Saccharomyces cerevisiae resistant to the antimitotic drug benomyl (BenR), six also conferred cold-sensitivity for growth and three others conferred temperature-sensitivity for growth in the absence of benomyl. All of the benR mutations tested, including the nine conditional-lethal mutations, were shown to be in the same gene. This gene, TUB2, has previously been molecularly cloned and identified as the yeast structural gene encoding beta-tubulin. Four of the conditional-lethal alleles of TUB2 were mapped to particular restriction fragments within the gene. One of these mutations was cloned and sequenced, revealing a single amino acid change, from arginine to histidine at amino acid position 241, which is responsible for both the BenR and the cold-sensitive lethal phenotypes. The terminal arrest morphology of conditional-lethal alleles of TUB2 at their restrictive temperature showed a characteristic cell-division-cycle defect, suggesting a requirement for tubulin function primarily in mitosis during the vegetative growth cycle. The TUB2 gene was genetically mapped to the distal left arm of chromosome VI, very near the actin gene, ACT1; no CDC (cell-division-cycle) loci have been mapped previously to this location. TUB2 is thus the first cell-division-cycle gene known to encode a cytoskeletal protein that has been identified in S. cerevisiae.     

Immunocytochemical localization of conglutin γ and legumin-like globulin in developing and mature seeds ofLupinus albus L.

Summary The distribution of two seed proteins, namely conglutin and a legumin-like globulin, in developing and mature seeds ofLupinus albus L. has been examined by immunocytochemistry and the concomitant modifications of their constituent polypeptides followed by SDS-PAGE. Both proteins were found within vacuolar protein bodies in various tissues of the cotyledons, although with some differences in the distribution patterns. The legumin-like protein was found to be deposited within the large storage parenchyma cells of the cotyledons in a manner similar to that reported for other storage proteins; little or no immunolabelling was associated with the cotyledonary epidermal and vascular parenchyma cells. In contrast conglutin was present in all cell types.A precursor of the legumin-like protein accumulated transiently in the developing cotyledon, but was subsequently modified by proteolytic cleavage. The onset of such modification was concomitant with a transition in the predominant vacuolar forms within the storage parenchyma cells. No precursor molecules of conglutin have been detected in this study, thus indicating that this protein is deposited in the protein bodies in its mature form.Abbreviations LM light microscopy - EM electron microscopy - DAF days after flowering - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - GAR goat antirabbit antiserum     

Multiple molecular forms of β-amylase in seeds and vegetative tissues of barley

The molecular forms of -amylase present in developing, mature, germinating and malted grains of barley (Hordeum vulgare L.), and in vegetative tissues, have been studied using Western-blot analyses and isoelectric focusing of isoenzymes. Five isoforms with different relative molecular masses (M_rs) could be recognised. The major isoform present in the mature grain, called isoform B, had an M_r of about 60 000. This was converted on malting or germination to two lower-M_r forms called C and D. Previous work (R. Lundgard and B. Svensson, 1986, Carlsberg Res. Commun. 51, 487–491) has shown that these result from partial proteolysis of isoform B. Isoenzyme analyses showed complex patterns of bands, with pIs between about 5.0 and 6.0. Two allelic types were present in the eight lines. A number of new bands with a range of pIs appeared during germination and malting.An isoform with the same M_r as D and a minor low-M_r isoform (E) were present in young developing whole caryopses (8–12 d after anthesis), but not in older developing endosperms (14–21 d after anthesis). Isoenzyme analyses also showed different patterns of bands in these two tissues, while hybrid-dot analyses indicated the presence of separate populations of mRNAs. It is suggested that the early endosperm isoforms (D and E) are green -amylases present in the pericarp and-or testa of the young caryopses.Roots but not shoots or leaves also contained an isoform with the same M_r as D, although the pattern of isoenzymes differed from that present in the seed tissues.The fifth isoform, A, was a diffuse high-M_r form present in small amounts in all seed and vegetative tissues, and may correspond to a constitutively expressed form.These multiple molecular forms of -amylase are discussed in relation to the recent report that -amylase is encoded by two structural loci, with a total copy number of two to three per haploid genome (Kreis et al, 1988, Genet. Res. Camb. 51, 13–16).Abbreviations M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

β-Tubulin accumulation and DNA synthesis are sequentially resumed in embryo organs of cucumber (Cucumis sativus L.) seeds during germination

Summary Resumption of DNA synthetic activities and -tubulin accumulation was studied in embryo organs of germinating cucumber (Cucumis sativus L.) seeds. Flow-cytometric analysis indicated the existence of 2C, 4C, and 8C nuclei in the radicle of mature embryos, whereas in cotyledons most of the cells contained nuclei with 2C DNA content. Upon imbibition of water, nuclear DNA replication was initiated in the radicle within 15 h, subsequently spreading towards the cotyledons. Bromodeoxyuridine incorporation preceded detectable changes in the relative amounts of DNA, implying the occurrence of putative DNA repair. Organellar DNA synthesis occurred independently of the nuclear DNA synthetic cycle. Western blotting and immunohistochemical localization demonstrated that the constitutive level of -tubulin originated from preserved -tubulin granules. During imbibition, disappearance of fluorescent tubulin granules, accumulation of -tubulin, and formation of microtubular cytoskeleton were found in the radicle, but not in the cotyledon areas. Mitosis only occurred after radicle protrusion at 21 h of imbibition. It is concluded that the differences in the initiation and progress of these cellular and molecular events are associated with the discrete behaviors of the radicle and the cotyledons upon imbibition. The formation of cortical microtubular cytoskeleton and the accumulation of tubulins are important features in preparation of radicle protrusion, whereas DNA synthesis may contribute to postgerminative growth.     

Genetic Variability of the β-Tubulin Genes in Benzimidazole-Susceptible and -Resistant Strains of Haemonchus Contortus

Benzimidazole anthelmintics are the most common chemotherapeutic agents used to remove intestinal helminths from farm animals. The development of drug resistance within helminth populations is wide-spread and can render these drugs essentially useless. The mechanism of benzimidazole resistance appears to be common to many species ranging from fungi to nematodes and involves alterations in the genes encoding β-tubulin. During the selection process resulting in resistance, there must be quantitative changes in the population gene pool. Knowledge of these changes would indicate the mechanisms underlying the spread of resistance in the population, which in turn could be used to design more effective drug administration strategies. To this end we have identified allelic variation at two β-tubulin genes in Haemonchus contortus using restriction map analysis of individual adults. Extremely high levels of variation were identified at both loci within a susceptible strain. In two independently derived benzimidazole resistant strains, allele frequencies at both loci were significantly different from the susceptible strain but not from each other. The same alleles at both loci, in both resistant strains, were favored by selection with benzimidazoles, suggesting that both loci are involved in determining benzimidazole resistance. These data confirm that changes in allele frequency, rather than novel genetic rearrangements induced by exposure to the drug, explain the changes associated with benzimidazole resistance. These results also show that any DNA based test for the development of benzimidazole resistance must take into account the frequency of alleles present in the population and not simply test for the presence or absence of specific allelic types.     

Identification and characterization of a rhizosphere β-galactosidase from Pisum sativum L.

Plant enzyme activities in the rhizosphere potentially are a resource for improved plant nutrition, soil fertility, bioremediation, and disease resistance. Here we report that a border cell specific β-galactosidase is secreted into the acidic extracellular environment surrounding root tips of pea, as well as bean, alfalfa, barrel medic, sorghum, and maize. No enzyme activity was detected in radish and Arabidopsis, species that do not produce viable border cells. The secreted enzyme activity was inhibited by galactose and 2-phenylethyl 1-thio-β-d-galactopyranoside (PETG) at concentrations that altered root growth without causing cell death. A tomato galactanase encoding gene was used as a probe to isolate a full length pea cDNA clone (BRDgal1) from a root cap-border cell cDNA library. Southern blot analysis using full length BRDgal1 as a probe revealed 1–2 related sequences within the pea genome. BRDgal1 mRNA expression was analysed by whole mount in situ hybridization (WISH) and found to occur in the outermost peripheral layer of the cap and in suspensions of detached border cells. No expression was detected within the body of the root cap. Repeated efforts to develop viable hairy root clones expressing BRDgal1 antisense mRNA under the control of the CaMV35S promoter, whose expression in the root cap is limited to cells at the root cap periphery only during root emergence, were unsuccessful. These data suggest that altered expression of this enzyme is deleterious to early root development. The first two authors contributed equally to the completion of this project.     

Accumulation of the β-subunit of polygalacturonase 1 in normal and mutant tomato fruit

Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B breaker - MG mature green - M_r relative molecular mass - nor non-ripening mutant - PAGE polyacrylamide gel electrophoresis - PG polygalacturonase - rin ripening inhibitor mutant - SDS sodium dodecyl sulphate     

Cell Cycle Dependent Regulation of Deoxycytidine Kinase,Deoxyguanosine Kinase,and Cytosolic 5′-Nucleotidase I Activity in MOLT-4 Cells

Activation of nucleoside analogues is dependent on kinases and 5′-nucleotidases and the balance between the activity of these enzymes. The purpose of this study was to analyze deoxycytidine kinase, deoxyguanosine kinase, and 4 different 5′-nucleotidases during cell cycle progression in MOLT-4 cells. The activity of both kinases was cell cycle dependent and increased during proliferation while the activity of cytosolic 5′-nucleotidase I decreased. We could show that the kinase activity was higher than the total nucleotidase activity, which was unchanged or decreased during cell cycle progression. These data may be important in designing modern combination therapy with nucleoside analogues.     

The Activity of Neutral α-Glucosidase and Selected Biochemical Parameters in the Annual Cycle of Breeding Carp (Cyprinus carpio L.)

The aim of the study was to demonstrate seasonal changes in the hydrolytic and transferase activity of neutral α-glucosidase, the level of glucose, cholesterol, triglycerides and total protein in the annual breeding cycle of the carp. The study was conducted on fish from a fish farm in Lower Silesia (Poland). Blood serum was collected from the heart in: June, September and December of two consecutive years. The results of the study show that the hydrolytic and transferase activity of neutral α-glucosidase, as well as the results of basic biochemical parameters are highest in summer, when the fish seek and intake food intensively. The lowest values were observed in spring, when carp have the lowest metabolism after the wintering period.