Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Single mid-chain GlcNAcβ1-6Galβ1-4R sequences of linear oligosaccharides are resistant to endo-β-galactosidase ofBacteroides fragilis

Endo--galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml^–1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc1-6Gal1-4GlcNAc, or those of the tetrasaccharides Gal1-4GlcNAc1-6Gal1-4GlcNAc and Gal1-4GlcNAc1-6Gal1-4Glc. The isomeric glycans which contained the GlcNAc1-3Gal1-4GlcNAc/Glc sequence were readily cleaved.Abbreviations GlcNAc 2-acetamido-2-deoxy-d-glucose - Lact lactose - MT maltotriose - MTet maltotetraose - R _MTet chromatographic migration rate in relation to that of maltotetraose     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.     

Characterization of two beta-tubulin genes from Geotrichum candidum.

Summary The -tubulin genes G1 and G2 from the phytopathogenic hemiascomycete Geotrichum candidum were found to be highly diverged in amino acid sequence from those of other filamentous fungi. G1 and G2 were also divergent from each other, with the coding regions sharing only 66% nucleotide sequence homology and 64% amino acid identity. However, the proteins shared 82% similarity and only 25 of the 161 non-identical amino acid substitutions were non-conservative. The organization of G1 is similar to other fungal -tubulin genes, but G2 has several unusual features; it has 2 amino acid additions in the N-terminal 40 residues and must employ an uncommon 5 splice junction sequence in preference to an overlapping perfect consensus. The amino acid change found to confer benomyl resistance in Neurospora crassa was also present in G2. G1 has four introns which are located similarly to those of -tubulin genes in other fungi. G2, however, has a single intron in a unique location. Translational fusions employing the 5 non-coding regions of the two Geotrichum -tubulin genes were made with the hygromycin phosphotransferase gene and shown to function in Schizosaccharomyces pombe and Trichoderma hamatum. However, G. candidum could not be transformed with these or other tested plasmids commonly used for fungal transformation.     

β-Ether cleavage of the lignin model compound 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether and derivatives by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 4-ethoxy-3-methoxyphenyl-glycerol--guaiacyl ether (V) in low nitrogen, stationary cultures under which conditions the ligninolytic enzyme system is expressed. 4-Ethoxy-3-methoxyphenylglycerol XIII, guaicol and 4-ethoxy-3-methoxybenzyl alcohol (II) were isolated as metabolic products. Exogenously added XIII was rapidly converted to 4-ethoxy-3-methoxybenzyl alcohol indicating that it is an intermediate in the metabolism of V. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-3-hydroxypropane VI. The degradation pathway for this dimer also included initial -ether cleavage and -hydroxylation of the diol product 1-(4-ethoxy-3-methoxyphenyl) 2,3 dihydroxypropane (XI) to yield the triol XIII which was cleaved at the , bond to yield 4-ethoxy-3-methoxybenzyl alcohol. Finally P. chrysosporium also cleaved the dimer 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1-hydroxypropane (VIII) at the -ether linkage yielding 1-(4-ethoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IX) which was subsequently cleaved at the , bond to yield II. All of the results indicate that oxidative -ether cleavage is an important initial reaction in the metabolism of -aryl ether lignin substructure dimeric compounds. Metabolities were identified after comparison with chemically synthesized standards by gas liquid chromatography-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography     

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.     

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.     

Ability of a generalist insect,Schistocerca gregaria,to overcome thioglucoside defense in desert plants: tolerance or adaptation?

Desert locusts, Schistocerca gregaria (Forskål) (Orthoptera, Acrididae), are generalist herbivores that, in the Sahara desert, may at times feed only on Schouwia purpurea (Forskål) (Brassicaceae), that is 10 times richer in thioglucosides than currently observed in other crucifers (>100 moles/g d.w.). Thioglucosides, when ingested, release products that are usually toxic to generalist insects.We studied the short-term (8 days) and long-term (21–26 days) consequences of a Schouwia-only diet on the digestion of these insects. The response was compared to the effects of a diet of Brassica oleracea, a crucifer well consumed in laboratory rearing conditions (7 moles/g glucosinolates d.w.).We found that the production of a myrosinase was induced in the midgut as early as 8 days following exposure to glucosinolates. No negative short-term effects were observed on the growth of the insect, but the activity of -glucosidases decreased in the midgut. The long-term exposure to the Schouwia diet affected activities of -glucosidases and -galactosidases, growth and assimilation efficiency. The limited adaptation of the desert locust to plant glucosides is compensated by an ability to tolerate high concentrations of allelochemicals for a short period.     

Interleukin-1β Stimulates Mitogen-Activated Protein Kinase in U373 Astrocytoma Cells without the Production of Lipid Second Messengers

IL-1 is one of the cytokines known to affect astroglial cells in normal brain development, brain injury and neurodegenerative diseases. IL-1 causes astrocytes to become more reactive, alter the expression and release of molecules and in some cases to proliferate. We have investigated the mitogenic effect and signal transduction pathway induced by IL-1 in U373 cells, a human astrocytoma cell-line. Recombinant human IL-1 induced mitogenesis of U373 cells in a dose-dependent fashion as assessed by tritiated thymidine incorporation. The following signal transduction mechanisms, reported to be induced in other systems by IL-1, were investigated in U373 cells: (1) activation of phosphatidylcholine-specific phospholipase C as assayed by incorporation of tritiated choline into cellular phospholipids, (2) production of diacylglycerol, a lipid second messenger, (3) activation of sphingomyelinase, and (4) activation of mitogen-activated protein kinase (MAPK). Of these, IL-1 activated only MAPK. In cultured rat astrocytes, IL-1 caused activation of MAPK without inducing proliferation.     

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].     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

Interactions of Aβ(1–40) with Glycerophosphocholine and Intact Erythrocyte Membranes: Fluorescence and Circular Dichroism Studies

Deposition of amyloid peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimers disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid (1–40) peptide (A) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of A and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled A studies indicate GPC enhances A aggregation. CD spectroscopy reveals that A in the presence of GPC adopts 14% more -sheet structure than does A alone. Fluorescamine anisotropy measurements show that GPC and A interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble A and GPC insert into the phospholipid head-group region of the membrane where they interact leading to -sheet formation in soluble A which enhances A aggregation.     

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.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Molecular cloning and sequencing analysis of a β-tubulin gene from Lupinus albus

Genomic -Dash library constructed from Lupinus albus nuclear DNA was screened using a fragment of the -tubulin cDNA ( 8–31) clone of Chlamydomonas reinhardtii as probe. One of the positive recombinant phages was isolated, subcloned and analysed by sequencing. We present here nucleotide and derived amino acid sequences of the -tubulin gene, designated as L1 and identified by similarity with other -tubulins. The L1-encoded protein reveals a very high degree of similarity with other plant tubulins and contains consensus sequences for binding guanine base, phosphate and Mg²⁺. Northern analysis of total RNA isolated from roots, leaves, flowers and pools revealed that Lupinus albus -tubulin genes are constitutively expressed in all studied plant tissues.     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.     

Neurotoxic Potential of Three Structural Analogs of β-N-oxalyl-α,β-Diaminopropanoic Acid (β-ODAP)

Lathyrism is a non-progressive motor neuron disease produced by consumption of the excitatory amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (-ODAP). To learn more about the mechanisms underlying Lathyrism three structural analogs of -ODAP were synthesized. Carboxymethyl-,-diaminopropanoic acid (CMDAP) evoked inward currents which were antagonized by APV (30 M), but not by CNQX (10 M). N-acetyl-,-diaminopropanoic acid (ADAP) evoked no detectable ionic currents but potentiated N-methyl-D-aspartate (NMDA)-activated currents. The potentiation of NMDA currents by ADAP was blocked by 7-chlorokynurenic acid. Carboxymethylcysteine (CMC) did not activate any detectable ionic currents. None of the three -ODAP analogs produced visible symptoms of toxicity in day old chicks when administered for 2–3 consecutive days. Ligand binding studies demonstrated that all the three compounds were effective to in displacing [³H]glutamate. The maximum inhibition was 92% for CMDAP, 61% for ADAP, 65% for CMC and 99% for -ODAP. These data indicate that analogs of -ODAP may interact with glutamate receptors without producing neurotoxicity.     

Primary structure of two nonallelicβ-globin chains from DBA/2 mice

The amino acid sequences of the major and minor globin chains from DBA/2 mice have been determined. This information is of interest because DBA/2 mice are the strain of origin for most murine erythroleukemia lines. The primary structure of DBA/2 globins agrees completely with that predicted from the coding properties of BALB/c globin genes. This identity does not support a rapid evolutionary divergence in inbred mouse strains, at least at these loci in these strains.This work was supported in part by National Institutes of Health Grants AM26956, HL24908, and AM14923 and by the Department of Energy under Contract DE-AC05-840R21400 with Martin Marietta Systems. The SUNY Buffalo Sequencing Facility was supported by a grant from the James Cummings Foundation and National Institutes of Health Grants RR05400 and RR07066.     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Development and isoproterenol-induced regulation of adrenoceptor binding in cultured rat neocortical explants is seen only with the beta-1, not with the beta-2 subtype

The presence and time-course of -adrenoceptor density in cultured explants of neocortex obtained from 6-day-old rat pups were investigated using a [¹²⁵I]ICYP binding assay. A delayed, but more pronounced, increase in the receptor expression was observed as compared to the situation previously described in vivo. These changes only occurred for the ₁-subtype of the receptor, whereas the ₂-subtype binding remained constant up to 3 weeks in vitro. The delay of ₁-adrenoceptor expression may be due to the incomplete presence of the proper maturational input, and the late enhancement of receptor expression to upregulation related to the absence in vitro of noradrenergic input. Decreased -adrenoceptor levels could be induced by chronic treatment of the -agonist isoproterenol (1 M) introduced either for 3 or 13 days. Again, changes in density were found only for the ₁-adrenoceptor binding sites. There is no reduction of receptor density following return to control conditions for 10 days after a 3-day treatment with isoproterenol, demonstrating the ability of this model to attain its final receptor density notwithstanding the developmental insult.Special issue dedicated to Dr. Robert Balázs