Effects of Yariv dyes, arabinogalactan-protein binding reagents, on the growth and viability of Brazilian pine suspension culture cells

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins implicated in several aspects of plant growth and development. (β-d-glucosyl)₃ Yariv phenylglycoside (β-GlcY), commonly known as Yariv reagent, selectively binds AGPs. We treated cell suspension cultures of Araucaria angustifolia, the Brazilian pine, with β-GlcY and observed inhibition of biomass increase in a culture medium with 50 μM β-GlcY. However, the growth was not inhibited by (α-d-galactosyl)₃ Yariv phenylglycoside (α-GalY) which does not bind AGPs. Fluorescein diacetate staining of cells indicated that β-GlcY severely affected cell viability. However, cell swelling, bursting and release of cellular contents, all characteristics of necrotic cell death, were not observed in β-GlcY-treated cells. Instead, programmed cell death (PCD) structural changes such as cytoplasmic shrinkage and condensation were observed in β-GlcY-treated cells. In addition, callose accumulation, which is another marker of PCD, was also observed in β-GlcY-treated cells. The use of both, Ac-VEID-CHO, an inhibitor of caspase-like proteolytic activity related to PCD, and phenyl methyl sulphonyl fluoride (PMSF), a protease inhibitor known to suppress PCD, in the culture medium did not reverse the growth inhibition caused by β-GlcY. These data indicate that the β-GlcY-induced inhibition of Araucaria cell’s growth is related to AGP perturbation, and also that this growth inhibition is due to increased cell death not driven by necrosis.     

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's Iymphoma and germinal centre B cells

A new monoclonal antibody (TU-1) directed against the Galα1-4Galβ1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer (Galα1-4Galβ1-4Glcβ1-1′Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Galβ1-4Glcβ1-1′Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer), galactosylparagloboside (Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer), sulfatide (HSO3-3Galβ1-1′Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes. Abbreviations: ATL, adult T-cell leukaemia; BSA, bovine serum albumin; Cer, ceramide; DPPC, L-α-dipalmitoylphosphatidylcholine; EBV, Epstein-Barr virus; FCS, fetal calf serum; GalCer, Galβ1-1′Cer; GlcCer, Glcβ1-1′Cer; LacCer, Galβ1-4Glcβ1-1′Cer; Gb3Cer, Galα1-4Galβ1-4Glcβ1-1′Cer; Iyso-Gb3Cer, Galα1-4Galβ1-4Glc1-1′Sph; Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glc1-1′Cer; galactosylparagloboside, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; Gg3Cer, GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; Gg4Cer, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; GM3, Neu5Acα2-3Galβ1-4Glcβ1-1′Cer; GM2, GalNAcβ1-4(Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; GM1a, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1a, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1b, Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GT1b, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MAb, monoclonal antibody; MPL, monophosphoryl lipid A; P1 antigen, Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; PVP, polyvinylpyrolidone; Sph, sphingosine; sulfatide, HSO3-Galβ1-1′Cer; TDM, trehalose dimycolate; TLC, thin-layer chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Effects of Yariv phenylglycosides onRosa cell suspensions: Evidence for the involvement of arabinogalactan-proteins in cell proliferation

Treatment of ‘Paul's Scarlet rose (Rosa sp.) cell suspensions with β-D-glucosyl Yariv phenylglycoside (β-D-Glc)₃, a chromophoric molecule that selectively binds arabinogalactan-proteins (AGPs), caused inhibition of cell growth in a concentration-dependent manner, with complete inhibition of growth occurring at 50 μM (β-D-Glc)₃ in the culture medium. Growth was not inhibited by either α-D-galactosyl or β-D-mannosyl Yariv phenylglycosides which do not bind AGPs. Staining of cells with fluorescein diacetate indicated that (β-D-Glc)₃ did not affect cell viability. Upon transfer of 50 μM (β-D-Glc)₃-treated cells to control conditions, cell growth recovered with a time-course similar to that of control cells. Cell sizes in control and (β-D-Glc)₃-treated cultures were similar, indicating that the mechanism of growth inhibition by (β-D-Glc)₃ involved suppression of cell division. Two different analyses of (β-D-Glc)₃-treated cells both showed that approximately 95% of the bound (β-D-Glc)₃ was in the cell wall. Molecules that bound (β-D-Glc)₃ were extracted from the cell wall and were identified as AGPs, as judged by their carbohydrate and amino acid compositions.     

Effect of clenbuterol on apoptosis,adipogenesis, and lipolysis in adipocytes

Clenbuterol, a beta₂-adrenergic receptor (β₂-AR) selective agonist, has been shown to decrease body fat in animals and can induce apoptosis in adipose tissue in mice. We hypothesized that direct actions of a β-adrenergic receptor agonist on adipocytes could trigger the observed apoptotic effect. The hypothesis was inspected by investigating the direct effect of clenbuterol on apoptosis, adipogenesis, and lipolysis in vitro using the 3T3-L1 cell line and rat primary adipocytes. Cells were treated with 10⁻⁹ to 10⁻⁵ M clenbuterol depending on the experiments. There was no apoptotic effect of clenbuterol both in 3T3-L1 cells and rat primary adipocytes. Adipogenesis monitored by Oil Red O staining and AdipoRed™ assay was modestly decreased by clenbuterol treatment (p < 0.05). In fully differentiated primary adipocytes, clenbuterol increased basal lipolysis compared with the control (p < 0.01). In summary, direct stimulation of β₂-AR by clenbuterol does not cause apoptosis in adipocytes, despite a direct lipolytic stimulation and attenuation of adipogenesis.     

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

Background  

Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS).     

Exposure to airborne microorganisms,hyphal fragments,and pollen in a field of organically grown strawberries

Many working environments are predisposed for larger than average amounts of fungi and other microorganisms often due to organic material being handled. From 2003 to 2007, the area used for strawberry production in Denmark increased by 62%. The purpose of this study was to determine the levels of exposure to microorganisms, endotoxin, (1→3)-β-d-glucan (β-glucan), and pollen in a field of strawberries. The study was carried out in eastern Denmark from the middle of June to the beginning of August 2008. The strawberries were grown organically, and microbiological pest control agents (MPCAs) were applied during this and former growth seasons. In order to measure exposure to inhalable bioaerosol components, we used stationary filter samplers. Bioaerosol sampling was performed during 4 working days, and a total of 57 samplings were performed. The filters were analysed for contents of fungi, MPCAs, endotoxin, β-glucan, and pollen. The mean exposure was 6,154 CFU Cladosporium sp. m⁻³, 1.0 × 10⁵ fungal spores m⁻³, 4.1 × 10⁴ hyphal fragments m⁻³, 5.8 × 10³ pollen m⁻³, 57.3 ng β-glucan m⁻³, and 8.9 endotoxin units (EU) m⁻³. A significant and positive correlation was found between β-glucan and fungal spores and between CFU of Cladosporium sp. and CFU of fungi. We selected specifically for Metarhizium anisopliae, Beauveria bassiana, and the applied MPCAs Trichoderma harzianum, T. polysporum, and Bacillus thuringiensis but found none of these species. In conclusion, our study shows that berry pickers in this organic strawberry field were potentially subjected to higher levels of fungal spores, Cladosporium sp., hyphal fragments, pollen, and thus also β-glucan than is usually seen in outdoor air. Exposure to MPCAs was not seen. The exposure to endotoxin was only slightly higher than e.g. in a town.     

Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes

In human intestinal epithelial crypt (HIEC) cells, the PI3-K/Akt-1 pathway is crucial for the promotion of cell survival and suppression of anoikis. Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit. Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known. Herein, we analyzed the expression of PI3-K isoforms in HIEC cells and determined their roles in cell survival, as well as in the β1 integrin/Fak/Src-mediated suppression of anoikis. We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent). Hence, HIEC cells selectively express PI3-K isoform complexes, translating into distinct roles in Akt-1 activation and cell survival, as well as in a selective engagement by Fak and/or Src within the context of β1 integrin/Fak/Src-mediated suppression of anoikis.     

Enhanced Solubility,Stability, and Transcorneal Permeability of Delta-8-Tetrahydrocannabinol in the Presence of Cyclodextrins

The purpose of this study was to investigate the effect of cyclodextrins (CDs) on aqueous solubility, stability, and in vitro corneal permeability of delta-8-tetrahydrocannabinol (Δ⁸-THC). Phase solubility of Δ⁸-THC was studied in the presence of 2-hydroxypropyl-β-cyclodextrin (HPβCD), randomly methylated-β-cyclodextrin (RMβCD) and sulfobutyl ether-β-cyclodextrin sodium salt (SβCD). Stability of Δ⁸-THC in 5% w/v aqueous CD solutions, as a function of pH, was studied following standard protocols. In vitro corneal permeation of Δ⁸-THC (with and without CDs) across excised rabbit cornea was also determined. Phase-solubility profile of Δ⁸-THC in the presence of both HPβCD and RMβCD was of the A_P type, whereas, with SβCD an A_L type was apparent. Aqueous solubility of Δ⁸-THC increased to 1.65, 2.4, and 0.64 mg/mL in the presence of 25% w/v HPβCD, RMβCD, and SβCD, respectively. Significant degradation of Δ⁸-THC was not observed within the study period at the pH values studied, except for at pH 1.2. Transcorneal permeation of Δ⁸-THC was dramatically improved in the presence of CDs. The results demonstrate that CDs significantly increase aqueous solubility, stability, and transcorneal permeation of Δ⁸-THC. Thus, topical ophthalmic formulations containing Δ⁸-THC and modified beta CDs may show markedly improved ocular bioavailability.     

Origin and evolution of the sulawesi macaques 2. complete amino acid sequences of seven β chains of three molecular types

Seven β chains were identified as the typical molecular types carried by the seven species of Sulawesi macaques based on isoelectric focusing and urea starch gel electrophoresis. These β chains include the β3 chains ofmaura, tonkeana, nigra, andbrunnescens, β1 chains ofhecki andochreata and β5 chain ofnigra. The results of chromatography on cation-exchange and reversed phase columns and the amino acid compositions of the tryptic peptides suggested substitutions at the 9th and 13th amino acids from the N-terminal. Sequence analyses of these seven β chains from the N-terminal to the 18th amino acid and those of purified tryptic peptides from βT3 to βT15 by Edman degradation revealed the following facts: (1) the amino acid sequences of the β3 chains carried by the four species coincided with each other and as did those of the β1 chains of the two named species; and (2) the 9th and 13th amino acids were Lys and Thr in β3, Asn and Asn in β1, and Asp and Thr in the β5 chain, respectively. These three β chains are related with each other by at least two-base changes. The evolution of the β chains of the Sulawesi macaques was inferred to be as follows. (1) The β3 chain might have been dominant β chain in the past among Sulawesi macaques, since peripheral species separately carried this chain; (2) the β1 and β5 chains might have derived from a “missing link” because of more than two-base substitutions between β3 and β1 and between β3 and β5; (3) eight other macaque species, including the lion-tailed macaque (M. silenus), bear Asn and Thr at these two positions, while the Barbary macaque (M. sylvanus) has Thr and Thr; and (4) thus, if the parsimonious rule is followed, the type with Asn-Thr is the most plausible “missing link,” since only the Asn-Thr type can combine these five β chains by minimum one-base change. Two genetic events are postulated in the evolutionary process of the Sulawesi β chains: the first Lys-Thr type (β3) was distributed over the whole island, and next Asn-Thr, the common type in other macaques, produced Asn-Asn (β1) and Asp-Thr (β5).     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Construction of antisense RNA expression vectors and correction of splicing defect in human β-globin gene (IVS-2-654 C→T mutant) in HeLa cells

The antisense fragments, which were available inin vitro system, were cloned into the mammalian expression vector pcDNA3, and were transfected into H654 cells, a mammalian cell line stably expressing the thalassaemic (IVS-2-654 C→T) human β-globin gene. In these transfected cells, the level of correctly spliced β-globin mRNA in total β-globin mRNA (β/(β + β*)) was improved from 0.07 (0 d) to 0.22 (3 d), and this effect persisted for up to 15 d post transfection. All the results demonstrated that antisense RNAs were able to be transcribed from the antisense fragment expression vectors stably and effectively suppressed aberrant splicing pattern of the mutated β-globin gene (IVS-2-654 C→T) and restored correct splicing pathway. This work provided a novel approach with potential clinical significance to gene therapy of this kind of splicing mutants including β-thalassaemia (IVS-2-654 C→T) by antisense RNAs. Project supported in part by the National Natural Science Foundation of China (Grant Nos. 39780019, 39392903) and the Shanghai Life Sciences Research Centre.     

Ontogeny of respiratory area of a marine teleost, porgy,pagrus major

Relationships of respiratory areas (gill, body surface and fin areas) (A) to body mass (W) were determined with a marine teleost, the porgyPagrus major of 0.0002–1230 g (just after hatch to 3+ years old), based on the allometric formula A=αW^β. (1) Early larvae (0.0002–0.0003 g) did not have the secondary lamellae that were responsible for gas exchange at the gills. After this stage, a tetraphasic relationship was observed between lamellar area (total area of secondary lamellae, often called gill area) (GA_L) and boby mass. During the late larval and early juvenile stages, the GA_L-W relationship showed a triphasic positive allometry with β-values of 3.773, 1.561 and 1.111 corresponding to the first half of the late larval stage (0.00034–0.001g), the second half of the stage (0.001–0.01 g) and the early juvenile stage (0.02–0.1 g), respectively, During the squamated juvenile and later stages (0.1–1080g), there was a negative allometry with a β-value of 0.813. (2) A triphasic relationship was observed between the total cutaneous surface area (body surface area and fin area) (CA_b+f) and body mass. During the early larval stage, in which an increase of body mass was very small. from 0.0002 to 0.00025 g, CA_b+p/W increased with growth with a β-value of 3.986. After this stage, the CA_b+t W relationship showed a diphasic negative allometry with β-values of 0.562, during the late larval stage (0.00028–0.0045 g) and 0.652 during the early juvenile and later stages (0.0045–1230 g). (3) Based on these results, factors controlling the metabolism-size relationship are discussed.     

Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

Background  

Endonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG.     

Effect of α-domain substitution on the structure, property and function of human neuronal growth inhibitory factor

Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), is attractive due to its distinct neuronal growth inhibitory activity, which is not shown by other human MT isoforms. It has been reported that the neuronal growth inhibitory activity arises from the N-terminal β-domain rather than its C-terminal α-domain. However, previous bioassay results have shown that the single β-domain is less effective at inhibiting the neuron growth than that in intact hMT3 on a molar basis, which suggests that the α-domain is indispensable to the neuronal growth inhibitory activity of hMT3. In order to confirm this assumption, we constructed two domain-hybrid mutants, the β(MT3)–β(MT3) mutant and the β(MT3)–α(MT1) mutant, and investigated their structural and metal binding properties by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, etc. The results showed that stability of the Cd₃S₉ cluster of the β(MT3)–β(MT3) mutant decreased significantly while the Cd₃S₉ cluster of the β(MT3)–α(MT1) mutant had a similar stability and solvent accessibility to that of hMT3. Interestingly, the bioassay results showed that the neuronal growth inhibitory activity of the β(MT3)–β(MT3) mutant decreased significantly, while the β(MT3)–α(MT1) mutant showed similar inhibitory activity to hMT3. Based on these results, we conclude that the α-domain is indispensable and plays an important role in modulating the stability of the metal cluster in the β-domain by domain–domain interactions, thus influencing the bioactivity of hMT3. Z.-C. Ding and Q. Zheng contributed equally to this work.     

Early developmental changes of the lactate dehydrogenase isoenzyme pattern in mouse,rat, guinea-pig,Syrian hamster and rabbit

Summary The LDH-isoenzyme pattern was studied by microdisc electrophoresis in pre-implantation and early post-implantation embryos of the mouse, rat, guinea-pig and Syrian hamster. Prior to implantation only LDH1 (β subunits) is present. After implantation only LDH5 (α subunits) is demonstrable; additional isoenzymes appear subsequently. This indicates that in rodent species in general the total pre-implantation LDH activity is based on the maternally transmitted β subunits while the activation of the embryonal LDH genes starts only with implantation. Moreover, the gene for α-subunits is activated first and the gene for β subunits follows later on. As in rodents, LDH1 is also the only LDH isoenzyme present in the ovarian oocyte in the rabbit. However, in contrast to rodents, the embryonal LDH genes start functioning long before implantation in the rabbit: in the 86-hour-old rabbit embryo (middle blastocyst) isoenzymes formed of α and β subunits are already present.

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Zusammenfassung Bei Maus, Ratte, Meerschwein und Goldhamster wurde das LDH-Isoenzymmuster w?hrend der preimplantativen und frühen postimplantativen Entwicklung mit Hilfe der Mikro-Disk-Elektrophorese untersucht. Dabei fand sich vor der Implantation nur LDH1 (β-Untereinheiten), nach der Implantation zun?chst nur LDH5 (α-Untereinheiten); weitere Isoenzyme kamen in Abh?ngigkeit vom Alter der Embryonen sp?ter hinzu. Daraus kann geschlossen werden, da? bei Rodentiern allgemein w?hrend der preimplantativen Entwicklung ausschlie?lich der aus β-Ketten bestehende mütterlich übertragen LDH-Vorrat vorhanden ist und die Aktivierung der embryonalen LDH-Gene erst nach der Implantation erfolgt. Dabei wird zuerst das Gen für α-Ketten und sp?ter das Gen für β-Ketten der LDH aktiviert. Im Gegensatz dazu werden beim Kaninchen offensichtlich die embryonalen LDH-Gene bereits l?ngere Zeit vor der Implantation aktiviert: w?hrend in Oocyten aus dem Ovar wie bei Rodentiern ebenfalls nur β-Ketten nachweisbar sind, k?nnen bei 86 Std alten Embryonen (mittlere Blastocyste) mehrere LDH-Isoenzyme nachgewiesen werden, an deren Bildung α- und β-Untereinheiten beteiligt sind.

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Supported by the Deutsche Forschungsgemeinschaft     

Association between apolipoprotein ε4 allele,factor V Leiden,and plasma lipid and lipoprotein levels with sickle cell disease in southern Iran

To investigate whether there is any association between various APOE alleles and factor V Leiden (FVL) with lipid profiles and sickle cell disease (SCD) in Southern Iran. 65 SCD patients consisting of 35 sickle cell anemia homozygous (SS), 15 sickle cell heterozygous (AS) and 15 sickle cell/βThalassemia (S/βthal) patients and 68 healthy individuals with normal hematological indices were studied. APOE and FVL polymorphisms were detected by PCR–RFLP and serum lipid level was measured enzymatically. The frequencies of FVL and APOE-ε4 allele were significantly higher in SCD patients than in control (15.4 vs. 4.4 and 13.7% vs. 3.3%, respectively). The distributions of APOE ε3ε3, ε2ε3 and ε2ε4 + ε3ε4 alleles in SCD patients were significantly different from those in the control group. The SCD subjects particularly SS/S βthal (SS + S/βthal) and SS patients have significantly lower frequency of APOE ε3ε3 allele (P < 0.05) whereas SCD, SS patients and AS individuals have a significantly higher frequency of APOE ε4 allele (ε2ε4 + ε3ε4; P = 0.003, P = 0.011 and P = 0.035, respectively) compared to the control group. The LDL-C (P = 0.006) and total cholesterol (P < 0.001) levels in SCD subjects were found to be significantly lower than those in the control group. In addition, the presence of non-APOE ε4 allele (ε2ε3 + ε3ε3) resulted in a significant decrease in the level of LDL-C and total cholesterol in SCD subjects in general and in SS and SS/S βthal patients in particular compared to controls. Furthermore, the presence of APOE ε4 allele (ε2ε4 + ε3ε4) was found to be associated with the risk of sickle cell anemia [OR = 4.1, P = 0.04]. The presence of either FVL mutation (OR = 4.6; CI: 0.91–24, P = 0.07) or APOE-ε4 allele (OR = 4.07; CI: 1.01–16.4, P = 0.048) is associated with the risk of sickle cell disease in Southern Iran. This data suggest that the activation of coagulation system enhances thrombus generation and decreases antioxidant activity in SCD patients from Southern Iran.     

The leaves of the common box,Buxus sempervirens (Buxaceae), become red as the level of a red carotenoid,anhydroeschscholtzxanthin, increases

Carotenoids from the leaves of the common box,Buxus sempervirens (Buxaceae), which turn red in late autumn to winter, were analyzed by reversed-phase HPLC. A novel carotenoid, monoanhydroeschscholtzxanthin (3), was isolated from the red-colored leaves. UV-VIS, MS,¹H-NMR and CD spectral data showed that the structure of 3 was (3S)-2′, 3′, 4′, 5′-tetradehydro-4, 5′-retro-β, β-caroten-3-ol. As well as anhydroeschscholtzxanthin (2), the major red carotenoid in the leaves, eschscholtzxanthin (4) was identified. Very small amounts of yellow carotenoids (neoxanthin, violaxanthin, lutein and β-carotene), which are major components of green leaves, were present in the red-colored leaves. The amounts of chlorophylla andb in the leaves decreased markedly during coloration, even at the early stages, whereas those of the yellow carotenoids decreased gradually. In contrast, the content of 2, a red carotenoid, increased steadily during coloration. The biosynthetic pathway of 2 inB. sempervirens was deduced tentatively on the basis of the individual carotenoid contents during autumnal coloration.     

The Phosphatidyl Inositol 3 Kinase-Glycogen Synthase Kinase 3β Pathway Mediates Bilobalide-Induced Reduction in Amyloid β-Peptide

Bilobalide (BB), a sesquiterpenoid extract of Ginkgo biloba leaves, has been demonstrated to have neuroprotective effects. The neuroprotective mechanisms were suggested to be associated with modulation of intracellular signaling cascades such as the phosphatidyl inositol 3-kinase (PI3K) pathway. Since some members of intracellular signalling pathways such as PI3K have been demonstrated to be involved in amyloid precursor protein (APP) processing, the present study investigated whether BB has an influence on the β-secretase-mediated APP cleavage via PI3K-dependent pathway. Using HT22 cells and SAMP8 mice (a senescence-accelerated strain of mice), this study showed that BB treatment reduced generation of two β-secretase cleavage products of APP, the amyloid β-peptide (Aβ) and soluble APPβ (sAPPβ), via PI3K-dependent pathway. Additionally, glycogen synthase kinase 3β (GSK3β) signaling might be involved in BB-induced Aβ reduction as a downstream target of the activated PI3K pathway. BB showed no significant effects on β-site APP cleaving enzyme 1 (BACE-1) or γ-secretase but inhibited the β-secretase activity of another protease cathepsin B, suggesting that BB-induced Aβ reduction was probably mediated through modulation of cathepsin B rather than BACE-1. Similarly, inhibition of GSK3β did not affect BACE-1 activity but decreased cathepsin B activity, suggesting that the PI3K-GSK3β pathway was probably involved in BB-induced Aβ reduction. Increasing evidence suggests that decreasing Aβ production in the brain via modulation of APP metabolism should be beneficial for the prevention and treatment of Alzheimer’s disease (AD). BB may offer such an approach to combat AD.