Models for glycosidic juvenogens: Enzymic formation of selected alkyl-β-D-glucopyranosides and alkyl-β-D-galactopyranosides under microwave irradiation

2-(4-Methoxybenzyl)-1-cyclohexyl--d-glucopyra nosides (1b and 2b) and 2-(4-methoxybenzyl)-1-cyclohexyl--d-galactopy ranosides (1c and 2c), models for glycosidic juvenogens, were synthesized using either D-glucose or D-galactose [in their natural form (3 and 5) or activated form (4 and 6)], and the respective racemic cis or trans isomers of 2-(4-methoxybenzyl)-1-cyclohexanol (1a and 2a) by either enzymic reverse hydrolysis or transglycosylation under both standard heating and microwave irradiation. Commercially available almond -glucosidase (EC 3.2.1.21) or -galactosidase (EC 3.2.1.23) from Escherichia coli were employed using different acetonitrile/water mixtures [9/1 (v/v) for the reverse hydrolysis, and 4/1 (v/v) for the transglycosylation].     

Binding of kynurenine to catecholamine

Summary Papiliochrome II is a pale yellow pigment of butterflies and consists of one molecule each ofL-kynurenine and N--alanyldopamine (NBAD). The aromatic amino nitrogen of kynurenine is bonded to the-carbon of NBAD. There are isomers IIa and IIb which show opposite circular dichroism. The-alanine contents of IIa and IIb were determined and the molar ratio of IIa to IIb has proved to be 1.17. The IIa and IIb were decomposed toL-kynurenine and N--alanylnorepinephrine (NBANE) by being heated in water at 80°C for 30 min. In both IIa and IIb, circular dichroism of the NBANE showed the same positive peak at 280 nm. The NBANE were further decomposed to-alanine and norepinephrine (NE) by being heated in 1 N HC1 at 100°C for 2 hr. The NE was submitted to enantioseparation and has proved to be a racemic mixture in both cases of IIa and IIb. These results are discussed in the light of the enzymic synthesis of IIa and IIb.     

Effects of Lovastatin and Pravastatin on Amyloid Processing and Inflammatory Response in TgCRND8 Brain

Previous studies suggest that treatment with statins reduce beta amyloid (Abeta) deposition in brains of mouse models of Alzheimer's disease (AD) and may reduce the prevalence of AD in humans. Since lipophilicity influences the biological efficacy of statins, we compared the effects of lovastatin, a lipophilic statin, to effects of the hydrophilic pravastatin on amyloid processing and inflammatory responses in brain. Three-month old TgCRND8 mice expressing mutant human amyloid precursor protein (mHuAPP) were treated daily with various doses of either statin. After 1 month, levels of cerebral soluble and fibrillar Abeta peptides, soluble sAPPalpha, and inflammatory cytokines were measured. Both statins caused dose-dependent reductions in total Abeta peptides with parallel increases in total sAPPalpha. At all doses, slightly greater effects were observed with lovastatin than with pravastatin. In contrast, only lovastatin significantly increased levels of IL-1beta and of TNFalpha in a dose-dependent manner. Lovastatin, but not pravastatin, decreased succinic dehydrogenase and increased lactate dehydrogenase activities in skeletal muscle and increased TUNEL staining in liver. Our data demonstrate that both statins shift the balance of APP processing from excessive beta-toward the normal alpha-cleavage while reducing the total amyloid burden in TgCRND8 brain and that lovastatin, but not pravastatin, potentiates cerebral inflammation and is associated with liver and muscle histotoxicity in these animals. These data show that pravastatin can reduce amyloid burden without potentiating inflammatory responses in brain and, therefore, may have a wider dose-range of safety than have lipophilic statins in the treatment or prevention of AD.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

APP Processing Enzymes (Secretases) as Therapeutic Targets: Insights from the Use of Transgenics (Tgs) and Transfected Cells

Secretases degrade amyloid precursor protein (APP) releasing fragments (-peptides A, A_x) that assemble to form hallmark extracellular deposits in Alzheimer's disease (AD) correlating with disease severity. As such, secretases supply targets for therapeutic intervention and form the focus of this overview. Progress in elucidating secretases and their modes of catalysis come from exploiting the use of transgenics or transfected cells. In addition to Ax, secretases also release C-terminal fragments with putative signaling properties (amyloid intracellular domain, AICD) similar in concept to those available for conversion of the Notch-r to release the nuclear transactivator NICD. The review considers lingering questions on APP fragmentation by secretase action, ancillary proteins such as presenilins (PS1/2), nicastrin, XII, or proteases (caspases), and the influence of familial mutations (mAPP, mPS) in terms of fibrillogenesis.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Expression of one of the members of the Arabidopsis chaperonin 60β gene family is developmentally regulated and wound-repressible

To study the pattern of gene regulation of the plastid chaperonin 60 gene family a chimaeric gene was constructed fusing the 5-flanking region of the chaperonin 60 B3 gene to the -glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

Amyloid Beta Peptide Membrane Perturbation Is the Basis for its Biological Effects

Experimental studies have indicated that the mechanisms offered for explaining the neurotoxicity of amyloid beta peptide (AP) are diverse, and include altered enzyme activities, disrupted calcium homeostasis, and increased free radical formation. AP appears to interact at the cell membrane with a multitude of receptor sites and also inserts physically into the membrane matrix. This membrane insertion affects the membrane fluidity and potentially influences the function of resident membrane proteins. We propose a unifying hypothesis to explain the experimental observations of the diverse cellular responses to AP. The indiscriminate physical insertion of AP into the cell membrane unspecifically activates a host of membrane processes by perturbation of the membrane proteins. This recurrent activation of membrane processes eventually culminates in neuronal cell death. We recommend that successful therapeutic interventions should be directed at reducing or preventing the interaction of AP with neuronal cell membranes.     

Novel plasmid marker rescue transformation system for molecular cloning in Bacillus subtilis enabling direct selection of recombinants

Summary A versatile plasmid marker rescue transformation system was developed for homology-facilitated cloning in Bacillus subtilis. It is based on the highly efficient host-vector system 6GM15-pHPS9, which allows the direct selection of recombinants by means of -galactosidase -complementation. The system offers several advantages over previously described cloning systems: (1) the convenient direct selection of recombinants; (2) the ability to effectively transform B. subtilis competent cells with plasmid monomers, which allows the forced cloning of DNA fragments with high efficiency; (3) the availability of 6 unique target sites, which can be used for direct clone selection, SphI, NdeI, NheI, BamHI, SmaI and EcoRI; and (4) the rapid segregational loss of the helper plasmid from the transformed cells.     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .     

Light and drug induced changes of epiphysial synaptic ribbons

Summary In the present investigation experiments were carried out to determine whether the functionally obscure synaptic ribbons of mammalian pinealocytes can be affected by acute changes in environmental lighting and which chemical processes may be involved in their regulation. Experiments carried out in male guinea-pigs have shown that the amounts of synaptic ribbons are immediately affected by changes in the lighting pattern. Extension of the light period reduced the normally occurring increase, whereas extension of the dark period inhibited the normally occurring decrease in the amount of synaptic ribbons. Results following injections of a number of drugs known to influence pineal function (noradrenaline, L-DOPA, propranolol, reserpine and p-chlorophenylalanine, respectively) suggest that synaptic ribbons may be directly or indirectly regulated by -adrenergic mechanisms.Dedicated to Professor Wolfgang Bargmann on the occasion of his 70th birthday.     

Cloning and characterization of a human cDNA ACAD10 mapped to chromosome 12q24.1

Mitochondrial fatty acid -oxidation is an important energy resource for many mammal tissues. Acyl-CoA dehydrogenases (ACADs) are a family of flavoproteins that are involved in the -oxidation of the fatty acyl-CoA derivatives. Deficiency of these ACADs can cause metabolic disorders including muscle fatigue, hypoglycaemia, hepatic lipidosis and so on. By large scale sequencing, we identified a cDNA sequence of 3960 base pairs with a typical acyl-CoA dehydrogenase function domain. RT-PCR result shows that it is widely expressed in human tissues, especially high in liver, kidney, pancreas and spleen. It is hypothesized that this is a novel member of ACADs family. Abbreviations: ACADs – acyl-CoA dehydrogenases, FAD – flavinadenine dinucleotide, SCAD – short-chain acyl-CoA dehydrogenase,MCAD – medium-chain acyl-CoA dehydrogenase, LCAD – long-chain acyl-CoAdehydrogenase, VLCAD – very long- chain acyl-CoA dehydrogenase, IVD –isocalery-CoA dehydrogenase, SBCAD – short/branched chain acyl-CoAdehydrogenase, GCD – glutaryl- CoA dehydrogenase, ETF – electron transferflavoprotein, ACAD8 – acyl-CoA dehydrogenase 8, ACAD9 – acyl-CoAdehydrogenase 9, ACAD10 – acyl-CoA dehydrogenase 10.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

Involvement of β 1,4 galactosyltransferase 1 and Galβ 1→4GlcNAc groups in human hepatocarcinoma cell apoptosis

1,4 galactosyltransferase 1 ( 1,4GT1) synthesizes Gal 14GlcNAc groups in N-linked sugar chains of animal glycoproteins, which have been demonstrated to play an important role in many biological events, including sperm-egg interaction, cell migration and mammalian embryonic development. In this study, the mRNA level of 1,4GT1 was found to increase greatly during the 7721 hepatocarcinoma cells apoptosis induced by cycloheximide. Ricinus Communis Agglutinin-I staining indicated generous increase of Gal 14GlcNAc groups during apoptosis. Further study showed that the 7721 hepatocarcinoma cells transiently transfected with 1,4GT1 were more susceptible to the apoptosis induced by cycloheximide. The increased susceptibility was in accordance to the transfection concentration of 1,4GT1, which also led to the increased Gal 14GlcNAc groups on the transfected cell surface. All the observations suggested that 1,4GT1 and Gal 14GlcNAc groups might be associated with the apoptosis of human hepatocarcinoma cells.     

Activities ofβ-glucanases andβ-glucosidases during blastospore formation inSaccharomycopsis fibuligera

Summary The activities of three glycosidases, -glucosidase and (1,3)- and (1,6)-glucanases have been monitored during growth and blastospore formation inSaccharomycopsis fibuligera. The assays were carried out on the cell-free culture and in a cell-free extract and a wall autolysate preparation from the growing cells. In complex medium containing 1% glucose an increase in the level of all three enzymes was associated with the transition from mycelium to blastospores. When the level of glucose was increased to 5% blastospore formation was repressed and the level of -glucanases only increased at the end of the fermentation. The -glucosidase activity increased during the growth phase. In a defined medium in which slow growth in a wholly yeast-like form was observed, growth was not associated with a high level of -glucanase activity.     

Effect of murine interferon α/β on tumor-induced suppressor function

T-lymphocyte-mediated immunosuppression has been described in several animal models and in man. In animal models, T-cell-mediated immunosuppression can hasten the development of cancers, permit the growth of tumors in immunocompetent hosts, and inhibit otherwise effective antitumor immunotherapy. Cyclophosphamide can abrogate the T-cell-mediated immunosuppression. However, inappropriately administered cyclophosphamide can adversely affect antitumor immunity. On the basis of data showing that interferon / (IFN/) and IFN selectively abrogate the T-cell-mediated dinitrofluorobenzene-specific suppressor function, we investigated the efficacy of purified murine IFN/ in manipulating tumorinduced T-cell-mediated immunosuppression in the wellcharacterized P815 mastocytoma model. In this model, generation of cytotoxicity in vitro and its inhibition by T cells correlates with antitumor immunity in vivo. We report that IFN/ selectively diminishes the generation of tumor-induced suppressor activity.     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.