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.     

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.     

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.     

α-, γ- and δ-tocopherols reduce inflammatory angiogenesis in human microvascular endothelial cells

Vitamin E, a micronutrient (comprising α-, β-, γ- and δ-tocopherols, α-, β-, γ- and δ-tocotrienols), has documented antioxidant and non-antioxidant effects, some of which inhibit inflammation and angiogenesis. We compared the abilities of α-, γ- and δ-tocopherols to regulate human blood cytotoxicity (BEC) and lymphatic endothelial cytotoxicity (LEC), proliferation, invasiveness, permeability, capillary formation and suppression of TNF-α-induced VCAM-1 as in vitro models of inflammatory angiogenesis. α-, γ- and δ-tocopherols were not toxic to either cell type up to 40 μM. In BEC, confluent cell density was decreased by all concentrations of δ- and γ-tocopherol (10–40 μM) but not by α-tocopherol. LEC showed no change in cell density in response to tocopherols. δ-Tocopherol (40 μM), but not other isomers, decreased BEC invasiveness. In LEC, all doses of γ-tocopherol, as well as the highest dose of α-tocopherol (40 μM), decreased cell invasiveness. δ-Tocopherol had no effect on LEC invasiveness at any molarity. δ-Tocopherol dose dependently increased cell permeability at 48 h in BEC and LEC; α- and γ-tocopherols showed slight effects. Capillary tube formation was decreased by high dose (40 μM) concentrations of α-, γ- and δ-tocopherol, but showed no effects with smaller doses (10–20 μM) in BEC. γ-Tocopherol (10–20 μM) and α-tocopherol (10 μM), but not δ-tocopherol, increased LEC capillary tube formation. Lastly, in BEC, α-, γ- and δ-tocopherol each dose-dependently reduced TNF-α-induced expression of VCAM-1. In LEC, there was no significant change to TNF-α-induced VCAM-1 expression with any concentration of α-, γ- or δ-tocopherol. These data demonstrate that physiological levels (0–40 μM) of α-, γ- and δ-tocopherols are nontoxic and dietary tocopherols, especially δ-tocopherol, can limit several BEC and LEC endothelial behaviors associated with angiogenesis. Tocopherols may therefore represent important nutrient-signals that limit cell behaviors related to inflammation/angiogenesis, which when deficient, may predispose individuals to risks associated with elevated angiogenesis such as inflammation and cancer; further differences seen from the tocopherols may be due to their blood or lymphatic cell origin.     

O-Succinyl-l-homoserine-based C4-chemical production: succinic acid,homoserine lactone, γ-butyrolactone, γ-butyrolactone derivatives,and 1,4-butanediol

There has been a significant global interest to produce bulk chemicals from renewable resources using engineered microorganisms. Large research programs have been launched by academia and industry towards this goal. Particularly, C4 chemicals such as succinic acid (SA) and 1,4-butanediol have been leading the path towards the commercialization of biobased technology with the effort of replacing chemical production. Here we present O-Succinyl-l-homoserine (SH) as a new, potentially important platform biochemical and demonstrate its central role as an intermediate in the production of SA, homoserine lactone (HSL), γ-butyrolactone (GBL) and its derivatives, and 1,4-butanediol (BDO). This technology encompasses (1) the genetic manipulation of Escherichia coli to produce SH with high productivity, (2) hydrolysis into SA and homoserine (HS) or homoserine lactone hydrochloride, and (3) chemical conversion of either HS or homoserine lactone HCL (HSL·HCl) into drop-in chemicals in polymer industry. This production strategy with environmental benefits is discussed in the perspective of targeting of fermented product and a process direction compared to petroleum-based chemical conversion, which may reduce the overall manufacturing cost.     

Partially Folded Aggregation Intermediates of Human γD-, γC-, and γS-Crystallin Are Recognized and Bound by Human αB-Crystallin Chaperone

Human γ-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone α-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human γ-crystallin substrate species recognized by human α-crystallin. The three major human lens monomeric γ-crystallins, γD, γC, and γS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human αB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human γ-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The αB-crystallin oligomers formed long-lived stable complexes with their γD-crystallin substrates. Using α-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate γ-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with α-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.     

The metabolism of γ-2,3,4,5,6-pentachlorocyclohex-1-ene and γ-hexachlorocyclohexane in rats

1. After intraperitoneal administration, gamma-hexachlorocyclohexane (Gammexane) and gamma-2,3,4,5,6-pentachlorocyclohex-1-ene were converted by rats into 2,3,5- and 2,4,5-trichlorophenol, which were excreted as free phenols and as sulphuric acid and glucuronic acid conjugates. 2. Derivatives of 2,4,5-trichlorophenol and 2,4,5-trichlorophenyl glucosiduronic acid and 2,4-dichlorophenylmercapturic acid were isolated from the urine as metabolites of gamma-2,3,4,5,6-pentachlorocyclohex-1-ene. 3. The phenolic metabolites of gamma-hexachlorocyclohexane and gamma-2,3,4,5,6-pentachlorocyclohex-1-ene isolated from urine were similar to those of 1,2,4-trichlorobenzene, which indicates that the two latter compounds are intermediates in gamma-hexachlorocyclohexane metabolism in rats.     

Kinetic study of the quenching reaction of singlet oxygen by α-, β-, γ-, δ-tocotrienols,and palm oil and soybean extracts in solution

Measurements of the singlet oxygen (¹O₂) quenching rates (k_Q (S)) and the relative singlet oxygen absorption capacity (SOAC) values were performed for 11 antioxidants (AOs) (eight vitamin E homologues (α-, β-, γ-, and δ-tocopherols and -tocotrienols (-Tocs and -Toc-3s)), two vitamin E metabolites (α- and γ-carboxyethyl-6-hydroxychroman), and trolox) in ethanol/chloroform/D₂O (50:50:1, v/v/v) and ethanol solutions at 35?°C. Similar measurements were performed for five palm oil extracts 1–5 and one soybean extract 6, which included different concentrations of Tocs, Toc-3s, and carotenoids. Furthermore, the concentrations (wt%) of Tocs, Toc-3s, and carotenoids included in extracts 1–6 were determined. From the results, it has been clarified that the ¹O₂-quenching rates (k_Q (S)) (that is, the relative SOAC value) obtained for extracts 1–6 may be explained as the sum of the product {Σ k_Q^AO-i (S) [AO-i]/100} of the rate constant (k_Q^AO-i (S)) and the concentration ([AO-i]/100) of AO-i (Tocs, Toc-3s, and carotenoid) included.     

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.     

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.     

The Construction of γ-TIP Prokaryotic Expression Vector and Preparation of the γ- TIP Antibody

A prokaryotic expression vector, pGEX-TIP, was constructed from Arabidopsis thaliana (L.) Heynh. Employing PCR, 205 bp fragment near 3' end of γ-TIP cDNA, which has specific aquaporin activity, was amplified and cloned into pGEX-KG. Restriction endonuclease analysis and sequencing confirmed the correct construction, and 0.4 mmoL/L IPTC can induce high expression of GST-TIP fused protein which was about 50% in total of E. coli proteins. The IPTG induced E. coli was collected and ]ysed by supersonic treatment. The fusion protein was mainly recovered as an inclusion body. The expressed GST-TIP was purified by SDS-PAGE according to their molecular weight, which was about 32 kD. The purified protein was used to immune rabbits directly or was electrophoretically eluted before it was used for immunization. The highly qualified antibody for GST-TIP was obtained, which provides a very useful protein probe for the research on localization and function of aquaporins.     

Modulation of the conformation,fibrillation, and fibril morphologies of human brain α-, β-, and γ-synuclein proteins by the disaccharide chemical chaperone trehalose

Human α-, β-, and γ-synuclein (syn) are natively unfolded proteins present in the brain. Deposition of aggregated α-syn in Lewy bodies is associated with Parkinson's disease (PD) and γ-syn is known to be involved in both neurodegeneration and breast cancer. At physiological pH, while α-syn has the highest propensity for fibrillation followed by γ-syn, β-syn does not form any fibrils. Fibril formation in these proteins could be modulated by protein structure stabilizing osmolytes such as trehalose which has an exceptional stabilizing effect for globular proteins. We present a comprehensive study of the effect of trehalose on the conformation, aggregation, and fibril morphology of α-, β-, and γ-syn proteins. Rather than stabilizing the intrinsically disordered state of the synucleins, trehalose accelerates the rate of fibril formation by forming aggregation-competent partially folded intermediate structures. Fibril morphologies are also strongly dependent on the concentration of trehalose with ≤ 0.4M favoring the formation of mature fibrils in α-, and γ-syn with no effect on the fibrillation of β-syn. At ≥ 0.8M, trehalose promotes the formation of smaller aggregates that are more cytotoxic. Live cell imaging of preformed aggregates of a labeled A90C α-syn shows their rapid internalization into neural cells which could be useful in reducing the load of aggregated species of α-syn. The findings throw light on the differential effect of trehalose on the conformation and aggregation of disordered synuclein proteins with respect to globular proteins and could help in understanding the effect of osmolytes on intrinsically disordered proteins under cellular stress conditions.     

Partial sequence homologies between cytoskeletal proteins,c-myc,Rous sarcoma virus and adenovirus proteins,transducin, and β- and γ-crystallins

Computer based sequence comparisons indicate partial sequence homology between human c-myc, Rous sarcoma virus, adenovirus 7, and simian sarcoma virus proteins and the cytoskeletal proteins desmin, keratin and vimentin. In addition, sections of the oncogene proteins showed partial but significant homology to and subunits of transducin, -II and -BP crystallins showed partial but significant homology to the cytoskeletal proteins keratin, vimentin, desmin, and -tubulin, and to adenovirus 7 and simian sarcoma virus transforming gene proteins. -BP crystallin showed partial but significant homology to Rous sarcoma virus protein, and to and y subunits of transducin. Both crystallins showed partial sequence homology to the GTP-binding protein elongation factor TU fromEscherichia coli . These sequence homologies suggest a link between the mechanisms of normal lens cell differentiation, involving modifications to the cytoskeleton and subsequent changes to the pattern of protein synthesis, and mechanisms of neoplastic transformation. Furthermore the transducin-like region on -crystallin may be important for its interaction with lens membranes and the maintenance of short-range order for lens transparency.     

Structure-function relationships in human α- and γ-thrombins

Summary Human pro-coagulant -thrombin may be proteolyzed under controlled conditions to the non-coagulant - and -thrombin forms. These derivative forms nonetheless retain esterase and amidase activities with small substrates as well as several other thrombin functions. Structurally, human -thrombin consists of three non-covalently associated fragments which retain structural integrity as measured by several spectroscopic criteria as well as enzymatic function. The protein folding characteristics of three-chain -thrombin indicate that each fragment (domain) contains sufficient information to result in a correct renaturation of protein conformation. Those subtle structural differences which distinguish - from -thrombin are most likely the obstructions to fibrinogen binding which account for the loss of clotting activity.     

Occurrence of γ-(Guanylureido)butyric Acid in a Red Alga,Gymnogongrus flabelliformis

From a red alga, Gymnogongrus flabelliformis, a new guanidino compound of an empirical formula, C₆H₁₂O₃N₄ was isolated. The decomposition products identified were carbon dioxide, guanidine and γ-aminobutyric acid on hydrolysis at 120°C, succinic acid and guanylurea on oxidation with potassium permanganate, and γ-aminobutyric and γ-ureidobutyric acids on hydrolysis with barium hydroxide, respectively. These results led the authors to postulate that the substance is 1-amidino-3-(3-carboxypropyl)urea or γ-(guanylureido)butyric acid.     

Fragmentation of a linoleate-derived γ-hydroperoxy-α,β-unsaturated epoxide to γ-hydroxy- and γ-oxo-alkenals involves a unique pseudo-symmetrical diepoxycarbinyl radical

Many of the pathological effects of lipid peroxidation are mediated by aldehydes generated through fragmentation of lipid peroxides. Among these aldehydes, the γ-hydroxy- and γ-oxo-α,β-alkenals, e.g., 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), are especially prone to modifying proteins and DNA through covalent adduction. In addition the "mirror image" γ-hydroxy- and γ-oxo-α,β-alkenal phospholipids can serve as high-affinity ligands for biological receptors triggering pathology. Therefore, the mechanisms by which these aldehydes are generated in vivo are under intense scrutiny. We now report observations supporting the intermediacy of a unique pseudo-symmetrical diepoxycarbinyl radical that accounts for the coproduction of HNE, ONE, and their mirror image analogues 9-hydroxy-12-oxo-10(E)-dodecenoic acid and 9-keto-12-oxo-10-dodecenoic acid upon fragmentation of 13-hydroperoxy-cis-9,10-epoxyoctadeca-11-enoic acid.     

Binding of indium,used in the perturbed γ-γ angular correlation studies,on DNA and DNA moieties

The two successive gamma rays emitted from bound indium (¹¹¹In) are utilized in the Perturbed γ-γ Angular Correlation (PAC) method for the study of the molecular dynamics of cellular macromolecules such as DNA. In this paper, evidence is presented indicating that indium binds on both phosphate and the base moieties of DNA and of the nucleotides and that, the binding of indium, under the conditions used in the PAC method, does not alter the hydrodynamic properties of the DNA macromolecule or its UV absorbance spectrum.