A novel mutation in the β-protein coding region of the amyloid β-protein precursor (APP) gene

Summary A novel mutation, a C to T transition at base pair 2124 in exon 17 of the amyloid -protein precursor (APP) gene, has been identified by direct sequencing of amplified DNA from two Alzheimer's disease (AD) patients. A simple oligonucleotide-hybridization procedure was developed to allow population studies of this DNA variation. The mutation, which is silent at the protein level, was present in 2 out of 12 investigated AD patients, in 1 out of 60 non-AD patients and in 1 out of 30 healthy individuals. The mutation can be used as a new marker for linkage studies involving the APP gene, although more comprehensive population studies are required to determine the status of the mutation as a possible risk factor for the development of AD.     

Improvement in the catalytic activity of β-agarase AgaA from Zobellia galactanivorans by site-directed mutagenesis

In this study, site-directed mutagenesis was performed on the β-agarase AgaA gene from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutant enzymes, S63K, C253I, and S63K-C253I, were 126% (1,757.78 U/mg), 2.4% (33.47 U/mg), and 0.57% (8.01 U/mg), respectively, relative to the wildtype beta-agarase AgaA (1,392.61 U/mg) at 40°C. The stability of the mutant S63K enzyme was 125% of the wild-type up to 45°C, where agar is in a sol state. The mutant S63K enzyme produced 166%, 257%, and 220% more neoagarohexaose, and 230%, 427%, and 350% more neoagarotetraose than the wild-type in sol, gel, and nonmelted powder agar, respectively, at 45°C over 24 h. The mutant S63K enzyme produced 50% more neoagarooligosaccharides from agar than the wild-type beta-agarase AgaA from agarose under the same conditions. Thus, mutant S63K β-agarase AgaA may be useful for the production of functional neoagarooligosaccharides.     

Role of the N-terminal region of the a chain in β1-bungarotoxin from the venom ofBungarus multicinctus (Taiwan-banded krait)

The N-terminal α-amino groups of β₁-bungarotoxin (β₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the α-amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between β₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that β₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the α-amino group of the A chain was in the vicinity of substrate binding site and that the TNP α-amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for β₁-Bgt.     

Changes of the Level of G Protein α-Subunit mRNA by Tolerance to and Withdrawal from Pentobarbital in Rats

Pentobarbital was continuously infused intracerebroventricularly (i.c.v.) at the rate of 300 g/10 l/h for 7 days, and withdrawal from pentobarbital was rendered 24 h after the stopping of the infusion. To eliminate the induction of hepatic metabolism by systemic administration of pentobarbital, an i.c.v. infusion model of tolerance to and withdrawal from pentobarbital was used. Little is known about the functional modulation of the G protein -subunits at the molecular level. The effects of continuous infusion of pentobarbital on the modulation of G protein -subunits mRNA were investigated by using in situ hybridization study. In situ hybridization showed that the level of Gs mRNA was increased in the septum and brainstem, and the level of Go mRNA was elevated in the cortex during the pentobarbital withdrawal. The level of Gi mRNA was significantly elevated in almost all area of brain during the pentobarbital withdrawal. These results suggest that region-specific changes of G protein -subunit mRNA were involved in the withdrawal from pentobarbital, whereas -subunit is not so highly involved in the pentobarbital tolerance.     

A polymorphic region defined by pCN2 (the 3′ nontranslated region of N-ras) maps to chromosome 9cen-p12

Summary Abnormalities of chromosome 9p have been reported in human leukemias and lymphomas, and in cell lines lacking the enzyme methylthioadenosine phosphorylase. It has been shown pCN2, the 3 nontranslated region of the N-ras oncogene, crosshybridizes with unknown DNA segments on chromosome 6, 9p, and 22, in addition to the N-ras oncogene itself on chromosome 1p. To use pCN2 to study chromosome 9p abnormalities in malignancies, we undertook to localize the pCN2 crosshybridizing region in chromosome 9p. By analyzing the copy numbers of the pCN2 crosshybridizing bands associated with chromosome 9p among various chromosomally aberrant human cell lines, we mapped the pCN2 hybridizing region to 9cen-p12. Since there is no other available probe in this region, pCN2 should prove very useful in studying abnormalities of chromosome 9p in human malignancies.This is publication number 5969-MEM from the Research Institute of Scripps Clinic, La Jolla, California     

Effect of temperature on locomotor activity in the goldfish (Carassius auratus) and the bluegill (Lepomis macrochirus): Presence of an ‘activity well’ in the region of the final preferendum

Goldfish (Carassius auratus) and bluegill sunfish (Lepomis macrochirus) were placed in aquaria where their locomotor activity was monitored by photocells, and tested at various acclimation temperatures over a range encompassing their final thermal preferenda. Activity was pooled over 24-hour periods to eliminate any circadian rhythm effects. Both species exhibited an activity well of reduced locomotor activity in the region of the final preferendum. Goldfish, tested either singly or in groups of 2–5 individuals, exhibited a social-interaction effect which became more pronounced at higher temperatures. These results are discussed in relation to a thermokinetic interpretation of thermo-regulatory behavior in fishes, and to the correspondence between thermal preferenda and thermal optima.     

Dissection of events in the resistance to β-lactam antibiotics mediated by the protein BlaR1 from Staphylococcus aureus

A heterologous expression system was used to evaluate activation of BlaR1, a sensor/signal transducer protein of Staphylococcus aureus with a central role in resistance to β-lactam antibiotics. In the absence of other S. aureus proteins that might respond to antibiotics and participate in signal transduction events, we documented that BlaR1 fragmentation is autolytic, that it occurs in the absence of antibiotics, and that BlaR1 directly degrades BlaI, the gene repressor of the system. Furthermore, we disclosed that this proteolytic activity is metal ion-dependent and that it is not modulated directly by acylation of the sensor domain by β-lactam antibiotics.     

Back to translation: removal of aIF2 from the 5′-end of mRNAs by translation recovery factor in the crenarchaeon Sulfolobus solfataricus

The translation initiation factor aIF2 of the crenarchaeon Sulfolobus solfataricus (Sso) recruits initiator tRNA to the ribosome and stabilizes mRNAs by binding via the γ-subunit to their 5′-triphosphate end. It has been hypothesized that the latter occurs predominantly during unfavorable growth conditions, and that aIF2 or aIF2-γ is released on relief of nutrient stress to enable in particular anew translation of leaderless mRNAs. As leaderless mRNAs are prevalent in Sso and aIF2-γ bound to the 5′-end of a leaderless RNA inhibited ribosome binding in vitro, we aimed at elucidating the mechanism underlying aIF2/aIF2-γ recycling from mRNAs. We have identified a protein termed Trf (translation recovery factor) that co-purified with trimeric aIF2 during outgrowth of cells from prolonged stationary phase. Subsequent in vitro studies revealed that Trf triggers the release of trimeric aIF2 from RNA, and that Trf directly interacts with the aIF2-γ subunit. The importance of Trf is further underscored by an impaired protein synthesis during outgrowth from stationary phase in a Sso trf deletion mutant.     

Membrane-microdomain localization of amyloid β-precursor protein (APP) C-terminal fragments is regulated by phosphorylation of the cytoplasmic Thr668 residue

Amyloid β-precursor protein (APP) is primarily cleaved by α- or β-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr(668) and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648-695)) with Thr(P)(668) did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aβ generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aβ in Alzheimer disease.     

Decreasing the sialidase activity of multifunctional Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) by site-directed mutagenesis

Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2-6-sialyltransferase, α2-3-sialidase, and α2-3-trans-sialidase activities in addition to its major α2-3-sialyltransferase activity. The presence of the α2-3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2-3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2-3-sialidase activity of PmST1 can be significantly decreased by protein crystal structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2-3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2-3-linked sialosides.     

Aversion to nicotine is regulated by the balanced activity of β4 and α5 nicotinic receptor subunits in the medial habenula

Nicotine dependence is linked to single nucleotide polymorphisms in the CHRNB4-CHRNA3-CHRNA5 gene cluster encoding the α3β4α5 nicotinic acetylcholine receptor (nAChR). Here we show that the β4 subunit is rate limiting for receptor activity, and that current increase by β4 is maximally competed by one of the most frequent variants associated with tobacco usage (D398N in α5). We identify a β4-specific residue (S435), mapping to the intracellular vestibule of the α3β4α5 receptor in close proximity to α5 D398N, that is essential for its ability to increase currents. Transgenic mice with targeted overexpression of Chrnb4 to endogenous sites display a strong aversion to nicotine that can be reversed by viral-mediated expression of the α5 D398N variant in the?medial habenula (MHb). Thus, this study both provides insights into α3β4α5 receptor-mediated mechanisms contributing to nicotine consumption, and identifies the MHb as a critical element in the circuitry controlling nicotine-dependent phenotypes.     

Inter-ethnic polymorphism of the β-globin gene locus control region (LCR) in sickle-cell anemia patients

Sequence polymorphisms within the 5HS2 segment of human locus control region is described among sickle cell anemia patients. Distinct polymorphic patterns of a simple sequence repeat are observed in strong linkage disequilibrium with each of the five major ^S haplotypes. Potential functional relevance of this polymorphic region in globin gene expression is discussed.     

Hydrolysis of β-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-β-glucanase from the basidiomycete Phanerochaete chrysosporium

When Phanerochaete chrysosporium was grown with laminarin (a β-1,3/1,6-glucan) as the sole carbon source, a β-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear β-1,3-glucan, branched β-1,3/1,6-glucan, and β-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-β-glucanase (EC 3.2.1.6) with broad substrate specificity for β-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (β-d-Glcp-(1–>6)-β-d-Glcp-(1–>3)-β-d-Glcp-(1–>3)-d-Glc) and 4-O-glucosyl-laminaribiose (β-d-Glcp-(1–>4)-β-d-Glcp-(1–>3)-d-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes β-d-Glcp-(1–>3)-d-Glcp at subsites −2 and −1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a β-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched β-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular β-1,3-glucanases.     

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 β-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 β-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 β-glucosidase and as a member of the β-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 β-glucosidases, a β-xylosidase, β-1,4-glycanases of the enzyme family F (mostly xylanases), and other families of β-1,4-glycosyl hydrolases. This result indicates that BGA β-glucosidases may comprise one enzyme family within a large ‘enzyme order’ of retaining β-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.     

Structure and functional investigation of ligand binding by a family 35 carbohydrate binding module (CtCBM35) of β-mannanase of family 26 glycoside hydrolase from Clostridium thermocellum

Functional attributes of recombinant CtCBM35 (family 35 carbohydrate binding module) of β-mannanase of family 26 Glycoside Hydrolase from Clostridium thermocellum were deduced by biochemical and in silico approaches. Ligand-binding analysis of expressed CtCBM35 analyzed by affinity-gel electrophoresis and fluorescence spectroscopy exhibited association constants K _a ～ 1.2·10⁵ and 3.0·10⁵ M^?1 with locust bean galactomannan and mannotriose, respectively. However, CtCBM35 showed low ligand-binding affinity with insoluble ivory nut mannan with K _a of 5.0·10^?5 M^?1. Unfolding transition analysis by fluorescence spectroscopy explained the conformational changes of CtCBM35 in the presence of guanidine hydrochloride (5 M) and urea (6.25 M). This explained that CtCBM35 has good conformational stability and requires higher free energy of denaturation to invoke unfolding. The three-dimensional (3-D) model of CtCBM35 from C. thermocellum generated by Modeller9v8 displayed predominance of β-sheets arranged as β-jelly-roll fold. The secondary structure of CtCBM35 by PredictProtein showed the presence of two α-helices (3%), 12 β-sheets (45%), and 15 random coils (52%). Secondary structural element analysis of cloned, expressed, and purified recombinant CtCBM35 by circular dichroism also corroborated the in silico predicted secondary structure. Multiple sequence alignment of CtCBM35 showed conserved residues (Tyr123, Gly124, and Phe125), which are commonly observed in mannan specific CBMs. Docking analysis of CtCBM35 with manno-oligosaccharide displayed the involvement of Tyr26, Gln29, Asn43, Trp66, Tyr68, Leu69, Arg76, and Leu127 residues, making polar contact with the ligand molecules. Ligand docking analysis of CtCBM35 exhibiting higher binding affinity with mannotriose and galactomannan (Man-Gal-Man moiety) substantiated the affinity binding and fluorescence results, displaying similar values of K _a.     

Fermentation of cellobiose to ethanol by industrial Saccharomyces strains carrying the β-glucosidase gene (BGL1) from Saccharomycopsis fibuligera

Constructs carrying the Saccharomycopsis fibuligera β-glucosidase gene (BGL1) under the control of a constitutive actin or a galactose-inducible promoter were introduced into eleven Saccharomyces strains. In ten of these recombinant strains, BGL1 expression driven by the actin promoter was between 1.6- and 18-fold higher than that obtained with the galactose-inducible promoter. Strains carrying the actin promoter yielded ethanol concentrations from cellobiose of between 0.5% and 14%, depending on their ability to accumulate Bgl1 (between 30 and 250 mU/mL) but also on their genetic background. Comparative analysis of a S. cerevisiae strain and its corresponding petite version showed similar ethanol yields, despite a 3-fold lower β-glucosidase production of the latter, suggesting that respiratory activity could be one of the factors influencing ethanol production when using carbon sources other than glucose. This study provides a selection of strains that may be good candidates as hosts for ethanol biosynthesis from cellulosic substrates.     

Unusual hydrophobic linker region of β-glucosidase (BGLII) from Thermoascus aurantiacus is required for hyper-activation by organic solvents

A gene encoding a putative β-glucosidase was isolated from Thermoascus aurantiacus IFO9748 and designated as bgl2. The recombinant enzyme showed β-glucosidase activity when p-nitrophenyl-β-glucose (pNP-Glc) was used as substrate. We also found that the enzyme activity was increased in the presence of organic solvents. An addition of 20 % (v/v) 1-octanol resulted in 54-fold higher activity of pNP-Glc hydrolysis, and transglycosylation activity was also found to be activated. The results of tryptophan fluorescence spectral analysis revealed the changes in the tertiary structure of the enzyme in the presence of 1-hexanol that may cause increased enzyme activity. BGLII has a distinctive hydrophobic linker region between N- and C-terminal domains. A chimeric enzyme in which the linker region was substituted by the corresponding region of another β-glucosidase failed to be activated by organic solvents, suggesting that the hydrophobic linker region may act as a molecular switch in BGLII.     

Generation of active oxygen species (AOS) and induction of β-Glucanase activity by fungal elicitor xylanase in the suspension cultured cells of Tobacco

It was investigated that active oxygen species (AOS) involved in the plant defense responses induced by fungal elicitor xylanase. When xylanase from the fungusTrichoderma viridae was treated to tobacco suspension cultured cells as an elicitor, β-glucanase activity was increased markedly. Lignin biosynthesis was also increased and peaked at 72 h after the treatment with xylanase. The treatment of H₂O₂ also dramatically increased β-glucanase activity at 24 h, which was much earlier than that of xylanase did. Using lucigenin-and luminol-dependent chemiluminescence, the effects of xylanase on oxidative burst were examined. Superoxide anion (O₂) production was peaked at 40 h and 52 h after xylanase treatment and hydrogen peroxide (H₂O₂) release was peaked at 44 h and 56 h, suggesting H₂O₂ burst was followed by O₂ generation. The scavengers of AOS, n-propyl gallate (PG) and mannitol, inhibited xylanase-induced β-glucanase activity by 85% and 50%, respectively. The activity of superoxide dismutase (SOD), which catalyzes the dismutation of O₂ to H₂O₂, began to increase from 24 h and reached to maximum at 48 h after xylanase treatment. Pretreatment of N,N,-diethyldithiocarbamate (DDC), known as a SOD inhibitor, caused the inhibition of H₂O₂ generation by 80% and reduced the β-glucanase activity by 60%. Treatment of 2,5-norbonadiene (NBD), a specific ethylene-action inhibitor, did not have any significant effect on xylanase-induced β-glucanase activity. This result suggested that ethylene did not involve in xylanase-induced response. Our results strongly suggest that the AOS generation is an essential component in plant defense response, in which cell wall degrading enzyme, glucanase, contributes to remove the necrotic tissue induced by pathogens.