Klotho is a substrate for α-, β- and γ-secretase

Klotho is an anti-aging protein with different functions of the full-length membrane protein and the secreted hormone-like form. Using overexpression and knock-down approaches as well as embryonic fibroblasts of knock-out mice we present evidence that Klotho is shedded by the α-secretases ADAM10 and 17 as well as by the β-secretase β-APP cleaving enzyme 1. The remaining membrane-bound fragment is a substrate for regulated intramembrane proteolysis by γ-secretase. Our data suggest that therapeutic approaches targeting these proteases should be carefully analyzed for potential side effects on Klotho-mediated physiological processes.     

Characterization of the mRNAs for α-, β- and γ-actin

The mRNAs for α-, β- and γ-actin have been characterized with respect to molecular weight and poly(A) content. Polyacrylamide gel electrophoresis under denaturing conditions shows that the mRNA for α-actin (muscle-specific actin) is approximately 4.6 × 10⁵ daltons in size, and that the mRNAs for β- and γ-actin (nonmuscle actins) are much larger, approximately 6.6 × 10⁵ daltons in size. We therefore calculate that the noncoding regions of the β- and γ-actin mRNAs contain about 800 nucleotides. This is in marked contrast to the noncoding regions of α-actin mRNA which contain only about 180 nucleotides. During electrophoresis in high-resolution nondenaturing gels, the β-actin mRNA migrates slightly slower than the γ-actin mRNA. This indicates either that β-actin mRNA is about 100 nucleotides longer than γ-actin mRNA, or that these mRNAs differ in secondary structure. Fractionation of actin mRNA on the basis of poly(A) content shows that a substantial portion of the β-actin mRNA, but very little of the α- or γ-actin mRNAs, fails to bind to oligo(dT)-cellulose. Much of this poly(A)-deficient β-actin mRNA, however, does bind to poly(U)-Sepharose, a substrate with higher affinity for short poly(A) sequences. This indicates that many of these β-actin mRNA molecules are polyadenylated, but that they have unusually short poly(A) tails. The finding that β- and γ-actins are translated from mRNAs of different electrophoretic mobility and different poly(A) content strongly suggests that these two closely related proteins are products of different genes.     

Fluorescence Polarization of αs1, κ-Caseins and αs1-κ-Casein Complex

Conjugates of α^s1-,κ-caseins and α^s1-,κ-casein complex were prepared with dimethylaminonaphthalenesulfonate and pyrenebutyrate. Their fluorescence lifetimes and the rotational relaxation times were measured by single photon counting technique and fluorescence depolarization technique, respectively. Both dimethylaminonaphthalenesulfonate and pyrenebutyrate conjugates had more than two lifetimes and the longer lifetime of pyrenebutyrate conjugates was near 140 nsec.

The rotational relaxation time of pyrenebutyrate α^s1-,κ-casein complex was smaller than that of pyrenebutyrate κ-casein polymer, which suggested that the complex formation of α^s1- and κ-casein polymers led to dissociation of the κ-casein polymer.

Changes of the rotational relaxation time as a function of weight ratio of α^s1- and κ-casein polymers (α^s1/κ) showed the specific variation and it was suggested that 4 moles of α^s1-κ-casein complex were formed from one mole of κ-casein polymer.     

Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent α-glucosidase

The gene for the α-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hy-drolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritimaα-glucosidase has the unusual property of requiring NAD⁺ and Mn²⁺ for activity. Co²⁺ and Ni²⁺ also activated AglA, albeit less efficiently than Mn²⁺. T. maritima AglA represents the first example of a maltodextrin-degrading α-glucosidase with NAD⁺ and Mn²⁺ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or β-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD⁺ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of d-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90°C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50°C and 70°C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h. Received: October 6, 1999 / Accepted: February 9, 2000     

Inhibition of α-, β-, γ-, and δ-carbonic anhydrases from bacteria and diatoms with N′-aryl-N-hydroxy-ureas

The inhibition of α-, β-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N’-aryl-N-hydroxy-ureas is reported. The α-/β-CAs from V. cholerae (VchCAα and VchCAβ) were effectively inhibited by some of these derivatives, with K_Is in the range of 97.5?nM – 7.26?µM and 52.5?nM – 1.81?µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (K_Is of 4.75 – 8.87?µM). The β-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAβ) was not inhibited by these compounds (K_Is?>?10?µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (K_Is of 59.8?nM – 6.42?µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (K_Is of 33.3?nM – 8.74?µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (K_Is?>?10?µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.     

Biogeographic,climatic and spatial drivers differentially affect α-, β- and γ-diversities on oceanic archipelagos

Island biogeographic studies traditionally treat single islands as units of analysis. This ignores the fact that most islands are spatially nested within archipelagos. Here, we took a fundamentally different approach and focused on entire archipelagos using species richness of vascular plants on 23 archipelagos worldwide and their 174 constituent islands. We assessed differential effects of biogeographic factors (area, isolation, age, elevation), current and past climate (temperature, precipitation, seasonality, climate change velocity) and intra-archipelagic spatial structure (archipelago area, number of islands, area range, connectivity, environmental volume, inter-island distance) on plant diversity. Species diversity of each archipelago (γ) was additively partitioned into α, β, nestedness and replacement β-components to investigate the relative importance of environmental and spatial drivers. Multiple regressions revealed strong effects of biogeography and climate on α and γ, whereas spatial factors, particularly number of islands, inter-island distance and area range, were key to explain β. Structural equation models additionally suggested that γ is predominantly determined by indirect abiotic effects via its components, particularly β. This highlights that β and the spatial arrangement of islands are essential to understand insular ecology and evolution. Our methodological framework can be applied more widely to other taxa and archipelago-like systems, allowing new insights into biodiversity origin and maintenance.     

The effect of α-tocopherol, α- and γ-tocotrienols on amyloid-β aggregation and disaggregation in vitro

One of the neuropathological hallmarks of Alzheimer's disease (AD)—causing neurodegeneration and consequent memory deterioration, and eventually, cognitive decline—is amyloid-β (Aβ) aggregation forming amyloid plaques. Our previous study showed the potential of a tocotrienol-rich fraction—a mixture of naturally occurring of vitamin E analogs—to inhibit Aβ aggregation and restore cognitive function in an AD mouse model. The current study examined the effect of three vitamin E analogs—α-tocopherol (α-TOC), α-tocotrienol (α-T3), and γ-tocotrienol (γ-T3)—on Aβ aggregation, disaggregation, and oligomerization in vitro. Thioflavin T (ThT) assay showed α-T3 reduced Aβ aggregation at 10 μM concentration. Furthermore, both α-T3 and γ-T3 demonstrated Aβ disaggregation, as shown by the reduction of ThT fluorescence. However, α-TOC showed no significant effect. We confirmed the results for ThT assays with scanning electron microscopy imaging. Further investigation in photo-induced cross-linking of unmodified protein assay indicated a reduction in Aβ oligomerization by γ-T3. The present study thus revealed the individual effect of each tocotrienol analog in reducing Aβ aggregation and oligomerization as well as disaggregating preformed fibrils.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Helix induction potential of N-terminal α-methyl, α-amino acids

Summary A series of longer analogues of the C-peptide of RNAse A has been synthesized with the aim of assessing the helix induction potential in water of α-methyl, α-amino acids at the N-terminus of the chain. The circular dichroism data indicate that one isovaline residue is effective in increasing the helix content of the 13-residue peptide by about 7%.     

Bromine oxidation of methyl α- and β-pyranosides of D-galactose,D-glucose,and D-mannose

Methyl α- and β-pyranosides of D-galactose, D-glucose, and D-mannose have been oxidized with bromine in aqueous solution at various pH values. The resulting keto glycosides were converted into their more-stable O-methyloxime derivatives which were characterized by spectroscopy and chromatography. Oxidation at a ring carbon atom where the hydrogen is axial is hindered by bulky substituents in syn (i.e., a 1,3) diaxial relationship. Thus, the aglycon group in the α anomers protects position 3, the axial HO-4 in galactopyranosides protects position 2, and the axial HO-2 in mannopyranosides protects position 4 from oxidation.     

α1,3 Fucosyltransferase, α-L-fucosidase, α-D-galactosidase, β-D-galactosidase, and Lex glycoconjugates in developing rat brain

Fucosyltransferases (FTs) and various glycosidases that are involved in the biosynthesis or degradation of SSEA-1 (Lex) antigens and their precursors in the CNS are developmentally regulated. In forebrain and cerebellum with lactosamine (LacNAc) as acceptor the FT activity was maximal at P15–P22, but with the glycolipid substrate paragloboside (nLc4) the maximal activity in cerebellum was obtained at P10–P15. The FT activity, with these substrates, was insensitive to N-ethylmaleimide (NEM) and the glycolipid product had an α1,3 linkage (Fuc to GlcNAc) suggesting similarities of the investigated enzyme to the cloned human and rat FT IV. However, the observation of different patterns of FT activity in isoelectrofocused fractions (pH 3.5–10) with different types of acceptors, and the differential expression of Lex containing glycolipids and glycoproteins during development strongly suggest the presence of more than one type of FT during development. Data on developmental expression of the hydrolytic enzymes, α-L-fucosidase, β-D-galactosidase and α-D-galactosidase, which can potentially hydrolyse SSEA-1 or its precursors, support the notion that SSEA-1 expression is the result of a dynamic balance between the activity of transferases and hydrolases. © 1998 Rapid Science Ltd     

Subunit isoform selectivity in assembly of Na,K-ATPase α-β heterodimers

To catalyze ion transport, the Na,K-ATPase must contain one α and one β subunit. When expressed by transfection in various expression systems, each of the four α subunit isoforms can assemble with each of the three β subunit isoforms and form an active enzyme, suggesting the absence of selective α-β isoform assembly. However, it is unknown whether in vivo conditions the α-β assembly is random or isoform-specific. The α(2)-β(2) complex was selectively immunoprecipitated by both anti-α(2) and anti-β(2) antibodies from extracts of mouse brain, which contains cells co-expressing multiple Na,K-ATPase isoforms. Neither α(1)-β(2) nor α(2)-β(1) complexes were detected in the immunoprecipitates. Furthermore, in MDCK cells co-expressing α(1), β(1), and β(2) isoforms, a greater fraction of the β(2) subunits was unassembled with α(1) as compared with that of the β(1) subunits, indicating preferential association of the α(1) isoform with the β(1) isoform. In addition, the α(1)-β(2) complex was less resistant to various detergents than the α(1)-β(1) complex isolated from MDCK cells or the α(2)-β(2) complex isolated from mouse brain. Therefore, the diversity of the α-β Na,K-ATPase heterodimers in vivo is determined not only by cell-specific co-expression of particular isoforms, but also by selective association of the α and β subunit isoforms.     

Cell adhesion to anosmin via α5β1, α4β1, and α9β1 integrins

Anosmin is an extracellular matrix protein, and genetic defects in anosmin result in human Kallmann syndrome. It functions in neural crest formation, cell adhesion, and neuronal migration. Anosmin consists of multiple domains, and it has been reported to bind heparan sulfate, FGF receptor, and UPA. In this study, we establish cell adhesion/spreading assays for anosmin and use them for antibody inhibition analyses to search for an integrin adhesion receptor. We find that α5β1, α4β1, and α9β1 integrins are needed for effective adhesive receptor function in cell adhesion and cell spreading on anosmin; adhesion is inhibited by both RGD and α4β1 CS1-based peptides. This identification of anosmin-integrin adhesion receptors should facilitate studies of anosmin function in cell and developmental biology.     

Characterization of α2,3- and α2,6-sialyltransferases from Helicobacter acinonychis

Genome sequence data were used to clone and express two sialyltransferase enzymes of the GT-42 family from Helicobacter acinonychis ATCC 51104, a gastric disease isolate from Cheetahs. The deposited genome sequence for these genes contains a large number of tandem repeat sequences in each of them: HAC1267 (RQKELE)(15) and HAC1268 (EEKLLEFKNI)(13). We obtained two clones with different numbers of repeat sequences for the HAC1267 gene homolog and a single clone for the HAC1268 gene homolog. Both genes could be expressed in Escherichia coli and sialyltransferase activity was measured using synthetic acceptor substrates containing a variety of terminal sugars. Both enzymes were shown to have a preference for N-acetyllactosamine, and they each made a product with a different linkage to the terminal galactose. HAC1267 is a mono-functional α2,3-sialyltransferase, whereas HAC1268 is a mono-functional α2,6-sialyltransferase and is the first member of GT-42 to show α2,6-sialyltransferase activity.     

Crystal structures of rare disaccharides, α-D-glucopyranosyl β-D-psicofuranoside, and α-D-galactopyranosyl β-D-psicofuranoside

The crystal structures of α-D-glucopyranosyl β-D-psicofuranoside and α-D-galactopyranosyl β-D-psicofuranoside were determined by a single-crystal X-ray diffraction analysis, refined to R(1)=0.0307 and 0.0438, respectively. Both disaccharides have a similar molecular structure, in which psicofuranose rings adopt an intermediate form between (4)E and (4)T(3). Unique molecular packing of the disaccharides was found in crystals, with the molecules forming a layered structure stacked along the y-axis.     

Inhibitors of α-glucosidase, α-amylase and lipase from Chrysanthemum morifolium

A new endoperoxysesquiterpene lactone, 10α-hydroxy-1α,4α-endoperoxy-guaia-2-en-12,6α-olide (1), together with a flavanone, eriodictyol (2), and two flavone glycosides, acacetin-7-O-β-d-glucopyranoside (3) and acacetin-7-O-α-l-rhamopyranoside (4), were isolated from the methanol extract of Chrysanthemum morifolium flowers by a bioassay-guided fractionation. Compound 1 showed strong inhibitory effects against α-glucosidase and lipase activities, with IC₅₀ values of 229.3 and 161.0 μM, respectively. The flavone glycosides 3 and 4 inhibited both α-glucosidase and α-amylase, while flavanone 2 was only effective against α-amylase.     

Crystal structure of the γ-cyclodextrin n-propanol inclusion complex; Correlation of α-, β-, γ-cyclodextrin geometries

Crystals of the hydrated n-propanol inclusion complex of γ-cyclodextrin (γ-CD; cyclo-octaamylose) have space group P4, a = b = 23.759(7), c = 23.069(7)Å and six quarter γ-CD per asymmetric unit. The structure was solved by YZARC and refined to R = 14% using 6300 X-ray counter data. The γ-CD are stacked, n-propanol (not located) occupies the channel-type cavity and 27 water sites populate interstices between stacks. Within the stacks γ-CD are arranged head-to-head as well as head-to-tail and H-bonded with O(2), O(3), O(6) hydroxyls. In the series α-,β-,γ-CD, angles C(1′)-O(4)-C(4) reduce from 119°-117.7°-112.6°, virtual O(4′)?O(4) distances increase 4.23-4.39-4.48 Å. intramolecular H-bonding distances O(2)?O(3) between adjacent glucoses, 3.00 Å in α-CD are wider than ～2.83 Å in β- and γ-CD, indicating a greater flexibility of the former.