Physicochemical characterization of α-, β-, and γ-cyclodextrins bioesterified with decanoate chains used as building blocks of colloidal nanoparticles

Nanoparticles of amphiphilic α-, β-, and γ-cyclodextrins were obtained by formulation of cyclodextrins enzymatically transesterified with vinyl decanoate. The product of this synthesis is a mixture of bioesterified cyclodextrins with various degrees of substitution (DS) presenting for a same DS different regio-isomers. In a first step, the efficiency of a MALDI-TOF procedure to characterize the average molecular weight of the derivative bulk mixture was demonstrated by comparing the results with those obtained from complementary NMR and HPLC techniques. In a second step, the ultrastructure of nanoparticles prepared from three different batches of synthesis was investigated and correlated with the average molecular weight and DS of the parent derivative.     

Investigation of Intramolecular Dynamics and Conformations of α-, β- and γ-Synuclein

The synucleins are a family of natively unstructured proteins consisting of α-, β-, and γ-synuclein which are primarily expressed in neurons. They have been linked to a wide variety of pathologies, including neurological disorders, such as Parkinson’s disease (α-synuclein) and dementia with Lewy bodies (α- and β-synuclein), as well as various types of cancers (γ-synuclein). Self-association is a key pathological feature of many of these disorders, with α-synuclein having the highest propensity to form aggregates, while β-synuclein is the least prone. Here, we used a combination of fluorescence correlation spectroscopy and single molecule Förster resonance energy transfer to compare the intrinsic dynamics of different regions of all three synuclein proteins to investigate any correlation with putative functional or dysfunctional interactions. Despite a relatively high degree of sequence homology, we find that individual regions sample a broad range of diffusion coefficients, differing by almost a factor of four. At low pH, a condition that accelerates aggregation of α-synuclein, on average smaller diffusion coefficients are measured, supporting a hypothesis that slower intrachain dynamics may be correlated with self-association. Moreover, there is a surprising inverse correlation between dynamics and bulkiness of the segments. Aside from this observation, we could not discern any clear relationship between the physico-chemical properties of the constructs and their intrinsic dynamics. This work suggests that while protein dynamics may play a role in modulating self-association or interactions with other binding partners, other factors, particularly the local cellular environment, may be more important.     

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.     

Molecular weights of wheat γ2-, β6-, α7-, α8- and α9-gliadins

The molecular weights of wheat γ₂-, β6-, α7-, α8- and α9-gliadins were calculated with the aid of a computer technique from sedimentation equilibrium data obtained in an ultracentrifuge equipped with photoelectric scanner. The dissociative solvents, all at pH 3.1 by addition of HCl, included 3 M urea, 0.15 M KCl; 8 M urea, 0.15 M KCl and 6 M guanidine-HCl. The minimum molecular weights for γ₂-, α7- and α9-gliadins, obtained in 6 M guanidine-HCl, were 34 600, 30 400 and 30 900, respectively. The β6- and α8-gliadins gave minimum molecular weights of 33 000 and 36 900, respectively, in 3 M urea, 0.15 M KCl.     

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.     

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.     

Association of Renal Na,K-ATPase α-Subunit with the β- and γ-Subunits Based on Cryoelectron Microscopy

Na,K-ATPase transports Na(+) and K(+) across cell membranes and consists of alpha- and beta-subunits. Na,K-ATPase also associates with small FXYD proteins that regulate the activity of the pump. We have used cryoelectron microscopy of two-dimensional crystals including data to 8 A resolution to determine the three-dimensional (3-D) structure of renal Na,K-ATPase containing FXYD2, the gamma-subunit. A homology model for the alpha-subunit was calculated from a Ca(2+)-ATPase structure and used to locate the additional beta- and gamma-subunits present in the 3-D map of Na,K-ATPase. Based on the 3-D map, the beta-subunit is located close to transmembrane helices M8 and M10 and the gamma-subunit is adjacent to helices M2 and M9 of the alpha-subunit.     

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.     

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.     

Paraoxon, 4-nitrophenyl phosphate and acetate are substrates of α- but not of β-, γ- and ζ-carbonic anhydrases

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to α-, β-, γ- and ζ-classes and from various organisms, ranging from the bacteria, archaea to eukarya domains, were investigated for their esterase/phosphatase activity with 4-nitrophenyl acetate, 4-nitrophenyl phosphate and paraoxon as substrates. Only α-CAs showed esterase/phosphatase activity, whereas enzymes belonging to the β-, γ- and ζ-classes were completely devoid of such activity. Paraoxon, the metabolite of the organophosphorus insecticide parathione, was a much better substrate for several human/murine α-CA isoforms (CA I, II and XIII), with k_cat/K_M in the range of 2681.6–4474.9 M^?1 s^?1, compared to 4-nitrophenyl phosphate (k_cat/K_M of 14.9–1374.4 M^?1 s^?1).     

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.     

Crystallographic Studies on α- and β-D-glucopyranosyl Formamide Analogues,Inhibitors of Glycogen Phosphorylase

Abstract

The catalytic site of glycogen phosphorylase (GP) is currently under investigation as a target for inhibition of hepatic glycogenolysis under high glucose conditions. Three D-glucopyranosyl analogues, C-(1-azido-α-D-glucopyranosyl) formamide, C-(1-acetamido-α-D-glucopyranosyl) formamide, and C-(1-hydroxy-β-D-glucopyranosyl) formamide, were recognised as moderate competitive inhibitors of muscle glycogen phosphorylase b (GPb) [with respect to α-D-glucose 1-phosphate (Glc-1-P)] with K_i values of 1.80 (±0.2) mM, 0.31 (±0.01) mM, and 0.88 (±0.04) mM, respectively. In order to elucidate the structural basis of inhibition, we determined the structure of muscle GPb complexed with the three compounds at 2.1, 2.06 and 2.0 Å resolution, respectively. The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provide a rationalisation for understanding potency of the inhibitors. The glucopyranose moiety makes the standard hydrogen bonds and van der Waals contacts as observed in the GPb-glucose complex, while the substituent groups in the α- and β-position of the C1 atom make additional hydrogen bonding and van der Walls interactions to the protein.     

Anion inhibition profiles of α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae

Among the numerous metalloenzymes known to date, carbonic anhydrase (CA, EC 4.2.1.1) was the first zinc containing one, being discovered decades ago. CA is a hydro-lyase, which catalyzes the following hydration–dehydration reaction: CO₂ + H₂O ⇋ HCO₃⁻ + H⁺. Several CA classes are presently known, including the α-, β-, γ-, δ-, ζ- and η-CAs. In prokaryotes, the existence of genes encoding CAs from at least three classes (α-, β- and γ-class) suggests that these enzymes play a key role in the physiology of these organisms. In many bacteria CAs are essential for the life cycle of microbes and their inhibition leads to growth impairment or growth defects of the pathogen. CAs thus started to be investigated in detail in bacteria, fungi and protozoa with the aim to identify antiinfectives with a novel mechanism of action. Here, we investigated the catalytic activity, biochemical properties and anion inhibition profiles of the three CAs from the bacterial pathogen Vibrio cholera, VchCA, VchCAβ and VchCAγ. The three enzymes are efficient catalysts for CO₂ hydration, with k_cat values ranging between (3.4 − 8.23) × 10⁵ s⁻¹ and k_cat/K_M of (4.1 − 7.0) × 10⁷ M⁻¹ s⁻¹. A set of inorganic anions and small molecules was investigated for inhibition of these enzymes. The most potent VchCAγ inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_I values ranging between 44 and 91 μM.     

Interplay between α-, β-, and γ-Secretases Determines Biphasic Amyloid-β Protein Level in the Presence of a γ-Secretase Inhibitor

Amyloid-β (Aβ) is produced by the consecutive cleavage of amyloid precursor protein (APP) first by β-secretase, generating C99, and then by γ-secretase. APP is also cleaved by α-secretase. It is hypothesized that reducing the production of Aβ in the brain may slow the progression of Alzheimer disease. Therefore, different γ-secretase inhibitors have been developed to reduce Aβ production. Paradoxically, it has been shown that low to moderate inhibitor concentrations cause a rise in Aβ production in different cell lines, in different animal models, and also in humans. A mechanistic understanding of the Aβ rise remains elusive. Here, a minimal mathematical model has been developed that quantitatively describes the Aβ dynamics in cell lines that exhibit the rise as well as in cell lines that do not. The model includes steps of APP processing through both the so-called amyloidogenic pathway and the so-called non-amyloidogenic pathway. It is shown that the cross-talk between these two pathways accounts for the increase in Aβ production in response to inhibitor, i.e. an increase in C99 will inhibit the non-amyloidogenic pathway, redirecting APP to be cleaved by β-secretase, leading to an additional increase in C99 that overcomes the loss in γ-secretase activity. With a minor extension, the model also describes plasma Aβ profiles observed in humans upon dosing with a γ-secretase inhibitor. In conclusion, this mechanistic model rationalizes a series of experimental results that spans from in vitro to in vivo and to humans. This has important implications for the development of drugs targeting Aβ production in Alzheimer disease.     

Synthesis of methyl α- and β-maltotriosides and aryl β-maltotriosides

Reaction of β-maltotriose hendecaacetate with phosphorus pentachloride gave 2′,2″,3,3′,3″,4″,6,6′,6″,-nona-O-acetyl-(2)-O-trichloroacetyl-β-maltotriosyl chloride (2) which was isomerized into the corresponding α anomer (8). Selective ammonolysis of 2 and 8 afforded the 2-hydroxy derivatives 3 and 9, respectively; 3 was isomerized into the α anomer 9. Methanolysis of 2 and 3 in the presence of pyridine and silver nitrate and subsequent deacetylation gave methyl α-maltotrioside. Likewise, methanolysis and O-deacetylation of 9 gave methyl β-maltotrioside which was identical with the compound prepared by the Koenigs—Knorr reaction of 2,2′,2″,3,3′,3″,4″,6,6′,6″-deca-O-acetyl-α-maltotriosyl bromide (12) with methanol followed by O-deacetylation. Several substituted phenyl β-glycosides of maltotriose were also obtained by condensation of phenols with 12 in an alkaline medium. Alkaline degradation of the o-chlorophenyl β-glycoside decaacetate readily gave a high yield of 1,6-anhydro-β-maltotriose.     

Combining α - and β -diversity models to fill gaps in our knowledge of biodiversity

For many taxonomic groups, sparse information on the spatial distribution of biodiversity limits our capacity to answer a variety of theoretical and applied ecological questions. Modelling community-level attributes (α- and β-diversity) over space can help overcome this shortfall in our knowledge, yet individually, predictions of α- or β-diversity have their limitations. In this study, we present a novel approach to combining models of α- and β-diversity, with sparse survey data, to predict the community composition for all sites in a region. We applied our new approach to predict land snail community composition across New Zealand. As we demonstrate, these predictions of metacommunity composition have diverse potential applications, including predicting γ-diversity for any set of sites, identifying target areas for conservation reserves, locating priority areas for future ecological surveys, generating realistic compositional data for metacommunity models and simultaneously predicting the distribution of all species in a taxon consistent with known community diversity patterns.     

Effect of 1-Anilino-8-naphthalenesulfonate on Properties and Associations of αs-, β- and κ-Caseins

It was indicated from fluorescence spectra and fluorescence titration that a hydrophobic probe, 1-anilino-8-naphthalenesulfonate (ANS), binds to casein components (α_s-, β- and κ-caseins). Fluorescence intensity and affinity of ANS-κ-casein complex were larger than that of ANS-α_s- and ANS-β-casein complexes. Enhancements of fluorescence intensity of complexes of casein components were observed by the addition of KCI or CaCl₂. Reason for the enhancement was postulated to be the increase of the quantum yield of the ANS fluorescence caused by the environmental change of ANS binding region of the casein components.

Marked increase of sedimentation coefficient of β-casein in the presence of KCl or CaCl₂ at 10°C was caused by the addition of ANS. This may be responsible for the stimulation of the Ca-dependent precipitation of β-casein by the addition of ANS.

It was found that α_s · κ-association was prevented by ANS and that hydrophobic interaction have an important role for α_s · κ-association.     

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.