Lipocalin-interacting-membrane-receptor (LIMR) mediates cellular internalization of β-lactoglobulin

β-Lactoglobulin (BLG) is a member of the lipocalin protein family and a major food-borne allergen in humans. Numerous in vitro studies have suggested a role for BLG in molecular transport processes; however, its physiological role remains enigmatic. A cellular receptor for BLG has been proposed, but has not yet been identified. Here we show that human LIMR, known to act as an endocytic receptor for lipocalin-1, also binds bovine BLG and mediates its cellular uptake. The specificity of this interaction is corroborated by a complete block of cellular uptake of BLG in the presence of LIMR antibodies or LIMR downregulation by antisense RNA. Furthermore, heterologous expression of human LIMR in insect cells mediates cellular internalization of FITC-BLG. Since LIMR is highly expressed in the human intestine, it might also function in the uptake of food-borne BLG.     

Theoretical investigation of direct amination of β-ketoesters catalyzed by copper(II)-bisoxazoline(BOX)

The mechanism of the direct amination of β-keto esters catalyzed by copper(II)-bisoxazoline has been studied by means of density functional theory of B3LYP method. The computational results support the present mechanism, which involves (i) the generation of the enol from β-keto esters, which coordinates to copper(II)-bisoxazoline. The coordination step appears to be fast, exothermic, and irreversible. (ii) The formation of the σ(N-C) bond via a six-membered ring transition state after azo dicarboxylate coordination with the chiral catalyst. This step is chirality-control step. (iii) Intramolecular hydrogen migration generates a catalyst-product complex, which can finally yield product. The hydrogen shift is the rate-determining step, which affords the experimentally observed (R)-product. The stereochemical predictions have been rationalized in terms of steric repulsions, showing good agreement with experimental data. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12–28)

Abstract

NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12–28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H₂0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH_α resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12–28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1–28) and the parent β-amyloid peptide.     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12-28)

Abstract NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12-28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H(2)0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH(α) resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12-28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1-28) and the parent β-amyloid peptide.     

Effect of β-Aminobutyrie Acid on Anthocyanin of Leaves of Arabidopsis thaliana (Cruciferae)

To analyze the effect of β aminobutyrie acid (BABA) on anthocyanin of leaves of Arabidopsis, 30 old plants were sprayed with BABA while the control were sprayed with water. After treated with BABA, the content of anthocyanin was significantly lower than that of control. Furthermore, the results from RT PCR showed that CHS, LDOX, UF3GT were down regulated compared with contro1, while PAL showed an opposite trend. At the same time, the activity of PPO, which played an important role in the degradation of anthocyanin, showed higher level than control. In addition, the antioxidant capacity, the death rate of cells and electrical conductivity of leaves were also decreased with BABA treatment. All results suggested that BABA might inhibit the accumulation of anthocyanin in leaves of Arabidopsis in vitro.     

Synthesis of mixed sphere β-diketonate nickel(II) complexes

Complexes of the type [Ni(β-diketonate)(L L)_1,2] (LL = diphosphine, (2), en or dipy, (3)) have been synthesized from [Ni(β-diketonate)₂] (1) and the free ligand under stoichiometric conditions in ethanol. Complexes 2 are square-planar species, while compounds 3 are hexacoordinate complexes, all being quite stable toward ligand disproportionation. The ligand-set {P₂O₂^-} appears to be particularly stable in the nickel(II) coordination sphere and turns out to exert a fairly strong average ligand field.     

Volatile compounds of β-diketonates of dioxouranium(VI) with tetrahydropyran and tetrahydrothiophene

Complexes of the type [UO₂(β-diket)₂·nB] where β-diket=CF₃COCHCOR (R=CF₃, CH₃, C₄H₃S or C₆H₅ and denoted by HFAA, TFAA, TTA or BTA, respectively), B=tetrahydropyran (THP) and tetrahydrothiophene (THT) and n= 0.5–2.0, have been synthesized and characterised by infrared, ¹H NMR and mass spectral techniques. Vapour pressure of UO₂(HFAA)₂·THP was measured by the transpiration method between the temperature range of 323–391 K. The enthalpy of vaporisation of UO₂(HFAA)₂·THP was estimated to be 18.4 kcal/mol.     

Comparison of two β-glucosidases for the enzymatic synthesis of β-(1-6)-β-(1-3)-gluco-oligosaccharides

A domain of epiglucan was synthesized by beta-glucosidases. Two beta-glucosidases, an extracellular beta-glucosidase derived from Sclerotinia sclerotiorum grown on xylose, and a commercial lyophilized preparation of beta-glucosidase from Aspergillus niger, were used to synthesize gluco-oligosaccharides from cellobiose and, specially, beta-(1-6) branched beta-(1-3) gluco-oligosaccharides, corresponding to the structure of epiglucan. Gentiobiose, cellotriose, cellotetraose, beta-Glc-(1-3)-beta-Glc-(1-4)-Glc, beta-Glc-(1-6)-beta-Glc-(1-4)-Glc and beta-Glc-(1-6)-beta-Glc-(1-3)-Glc were synthesized from cellobiose by both enzymes. The latter compound was preferentially synthesized by the beta-glycosidase from Sclerotinia sclerotiorum. Under the best conditions, only 7 g l(-1) of beta-Glc-(1-6)-beta-Glc-(1-3)-Glc was synthesized by the beta-glycosidase from Aspergillus niger compared to 20 g l(-1) synthesized with beta-glycosidase from Sclerotinia sclerotiorum.     

CutA divalent cation tolerance homolog (Escherichia coli) (CUTA) regulates β-cleavage of β-amyloid precursor protein (APP) through interacting with β-site APP cleaving protein 1 (BACE1)

Accumulation of the neurotoxic β-amyloid (Aβ) peptide in the brain is central to the pathogenesis of Alzheimer disease. Aβ is derived from the β-amyloid precursor protein (APP) through sequential cleavages by β- and γ-secretases, and the production of Aβ is greatly affected by the subcellular localization of these factors. CUTA, the mammalian CutA divalent cation tolerance homolog (E. coli), has been proposed to mediate acetylcholinesterase activity and copper homeostasis, which are important in Alzheimer disease pathology. However, the exact function of CUTA remains largely unclear. Here we show that human CUTA has several variants that differ in their N-terminal length and are separated as heavy (H) and light (L) components. The H component has the longest N terminus and is membrane-associated, whereas the L component is N-terminally truncated at various sites and localized in the cytosol. Importantly, we demonstrate that the H component of CUTA interacts through its N terminus with the transmembrane domain of β-site APP cleaving enzyme 1 (BACE1), the putative β-secretase, mainly in the Golgi/trans-Golgi network. Overexpression and RNA interference knockdown of CUTA can reduce and increase BACE1-mediated APP processing/Aβ secretion, respectively. RNA interference of CUTA decelerates intracellular trafficking of BACE1 from the Golgi/trans-Golgi network to the cell surface and reduces the steady-state level of cell surface BACE1. Our results identify the H component of CUTA as a novel BACE1-interacting protein that mediates the intracellular trafficking of BACE1 and the processing of APP to Aβ.     

Antiradical and NO-Inhibiting activities of β-hydroxy(ethoxy) derivatives of nitrous heterocycles

The antiradical and NO-inhibiting activities of β-hydroxy(ethoxy) derivatives of nitrous heterocycles (3-hydroxypyridine, 5-hydroxybenzimodazole, and 6-hydroxy(alkoxy)-benzothiazole) have been studied. The antiradical activity has been studied using a homogeneous hydrophilic chemiluminescent system, and the quenching constants (K _i ) have been determined. For the most reactive compound, 4-methylthiobenzimidazolyl-3-hydroxypyridine, K _i = 4.5 × 10⁵ M⁻¹. The NO-inhibiting activity was estimated on a model of the endotoxin shock of experimental animals using a spin trap of nitric oxide radicals based on complexes of iron with sodium diethyldithiocarbamate. It was found that the compounds at doses of 0.25–1 mmol/kg have both the inhibitory and stimulating action on the production of nitric oxide in the liver of animals. The results obtained suggest that some derivatives of nitrous heterocycles can be used as effective antioxidant preparations.     

Synthesis and characterization of β-diketiminate germanium(II) and tin(II) bromides

The equimolar reaction of a β-diketiminate lithium salt LLi(OEt₂) [L = HC(CMeNAr)₂; Ar = 2,6-iPr₂C₆H₃] with either GeBr₂ or SnBr₂ in diethyl ether affords the synthetically useful monomeric β-diketiminate-element halides LGeBr (1) and LSnBr (2), respectively. Both are soluble in hydrocarbon solvents, stable in inert atmosphere, and have been characterized by elemental analysis, NMR spectroscopy, and single-crystal X-ray diffraction analysis.     

Investigation of poly ( β-L-malic acid) production by strains of Aureobasidium pullulans

 Eight strains of the genus Aureobasidium obtained from culture collections were tested for their capability to produce poly(β-L-malic acid) (PMA). Four of the tested strains showed positive results. The most productive strain, A. pullulans CBS 591.75, was used to study the production of PMA in stirred-tank reactors. It was found that PMA was mainly produced in the late exponential phase, and the production related positively to glucose consumption. At the beginning of the fermentation the pH increased from 4.0 to about 7.0; subsequently the pH decreased and remained stable at around 3.0–3.5 for several days. Temperatures higher than 25^°C were detrimental to PMA production and cell growth. PMA production and cell growth at 20^°C and 25^°C exhibited no significant differences. PMA production and cell growth were studied under pH-controlled fermentation (at pH 2.0, 4.0, 5.5). The highest PMA production occurred at pH 4.0. PMA production was reduced at pH 2.0 although quite reasonable cell growth occurred at this pH value. Under optimized conditions 9.8 g PMA/l was produced during 9 days of fermentation in the stirred-tank reactors with an overall yield of 0.11 g PMA/g glucose. A procedure for the isolation of PMA and its separation from the other components of the fermentation broth was developed. The isolated PMA was characterized by ¹H and ¹³C-NMR spectroscopy as well as by infrared absorption spectroscopy. Gel-permeation chromatography revealed a relative molecular mass of approximately 3000–5000 by comparison with polyethylene glycol standards. Received: 13 February 1996/Received revision: 25 April 1996/Accepted: 1 May 1996     

Effects of adeno-associated virus (AAV) of transforming growth factors β1 and β3 (TGFβ1,3) on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type Ⅱ were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type Ⅱ, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Kinetics and mechanism of the oxidative addition of iodomethane to β-diketonatobis(triphenylphosphite)rhodium(I) complexes

The kinetics and mechanism of the oxidative addition of CH₃I to [Rh(β-diketone)(P(OPh)₃)₂] complexes was studied in acetone medium at various temperatures. The experimental rate law is R = k[Rh(β-diketone)(P(OPh₃)₂][CH₃I]. The order of the effect of the β-diketone on the reactivity of the complexes is acac >; BA >; DBM >; TFAA >; TFBA >; HFAA indicating that electronegative substituents of the β-diketone decrease the reactivity of the complexes towards oxidative addition reactions. The volume of activation for some of the reactions was determined in various solvents. The large negative values of the volume and entropy of the activation indicated a mechanism which occurs via a polar transition state.     

Differential Regulation of the Ten-Eleven Translocation (TET) Family of Dioxygenases by O-Linked β-N-Acetylglucosamine Transferase (OGT)

The ten-eleven translocation (TET) family of dioxygenases (TET1/2/3) converts 5-methylcytosine to 5-hydroxymethylcytosine and provides a vital mechanism for DNA demethylation. However, how TET proteins are regulated is largely unknown. Here we report that the O-linked β-GlcNAc (O-GlcNAc) transferase (OGT) is not only a major TET3-interacting protein but also regulates TET3 subcellular localization and enzymatic activity. OGT catalyzes the O-GlcNAcylation of TET3, promotes TET3 nuclear export, and, consequently, inhibits the formation of 5-hydroxymethylcytosine catalyzed by TET3. Although TET1 and TET2 also interact with and can be O-GlcNAcylated by OGT, neither their subcellular localization nor their enzymatic activity are affected by OGT. Furthermore, we show that the nuclear localization and O-GlcNAcylation of TET3 are regulated by glucose metabolism. Our study reveals the differential regulation of TET family proteins by OGT and a novel link between glucose metabolism and DNA epigenetic modification.     

Characterization of a β-xylosidase from Aspergillus terreus (IJIRA 6.2)

Aspergillus terreus (IJIRA 6.2), a common soil microorganism, produces an extracellular β-xylosidase during its growth on wheat bran. The enzyme has been purified 328 fold (with a sp act of 4233 units/mg protein) by chromatography on DEAE-Sephadex A-25, hydroxyapatite, ConA-Sepharose and gel filtration on Sephacryl-S-300. Molecular mass of β-xylosidase by gel filtration was estimated to be about 95,000 and sedimentation coefficient of 5.6S was determined by glycerol density gradient centrifugation. The enzyme displayed maximum activity at pH 5.0 and 40°C; and in the absence of substrate, the β-xylosidase was stable up to 50°C and between pH 4.5 to 6.5. The purified enzyme hydrolysed p-nitrophenyl-β-Dxylopyranoside (PNPX) and xylooligosaccharides but not xylan, carboxymethyl cellulose or cellobiose. With PNPX as the substrate, the purified β-xylosidase exhibited a Km of 1.0 mM and D(+) xylose served as a competitive inhibitor with a K₁ of 10.5 mM.     

Interaction of human β-defensin 2 (HBD2) with glycosaminoglycans

Human β-defensin 2 (HBD2) is a member of the defensin family of antimicrobial peptides that plays important roles in the innate and adaptive immune system of both vertebrates and invertebrates. In addition to their direct bactericidal action, defensins are also involved in chemotaxis and Toll-like receptor activation. In analogy to chemokine/glycosaminoglycan (GAG) interactions, GAG-defensin complexes are likely to play an important role in chemotaxis and in presenting defensins to their receptors. Using a gel mobility shift assay, we found that HBD2 bound to a range of GAGs including heparin/heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate. We used NMR spectroscopy of (15)N-labeled HBD2 to map the binding sites for two GAG model compounds, a heparin/HS pentasaccharide (fondaparinux sodium; FX) and enzymatically prepared DS hexasaccharide (DSdp6). We identified a number of basic amino acids that form a common ligand binding site, which indicated that these interactions are predominantly electrostatic. The dissociation constant of the [DSdp6-HBD2] complex was determined by NMR spectroscopy to be 5 ± 5 μM. Binding of FX could not be quantified because of slow exchange on the NMR chemical shift time scale. FX was found to induce HBD2 dimerization as evidenced by the analysis of diffusion coefficients, (15)N relaxation, and nESI-MS measurements. The formation of FX-bridged HBD2 dimers exhibited features of a cooperative binding mechanism. In contrast, the complex with DSdp6 was found to be mostly monomeric.     

Synthesis and characterization of alkynes β-diketonate copper(I) complexes

The reactions of 1,3,5-tris-(trimethylsilylethynyl)benzene (1) with Cu₂O and 1,1,1,5,5,5,-hexafluoroacetylacetone in alkyne to Cu ratios 1:0.5, 1:1 and 1:3 in CH₂Cl₂ at room temperature give copper complexes (η²-1,3,5-tris(trimethylsilylethynyl)benzene)(Cu(hfac)) (2), (η²:η²-1,3,5-tris(trimethylsilylethynyl)benzene)(Cu(hfac))₂ (3) and (η²:η²:η²-(1,3,5- tris(trimethylsilylethynyl)benzene))₂(Cu(hfac))₃(4), respectively. In the same conditions, 2,5-bis-(trimethylsilylethynyl)thiophene (5) reacts with 0.5 or 1 equiv. of Cu₂O to give (η²:η²-2,5-bis(trimethylsilylethynyl)thiophene)(Cu(hfac)) (6) and (η²:η²-2,5-bis(trimethylsilylethynyl)thiophene)(Cu(hfac))₂ (7), respectively, and 1,4-bis(trimethylsilyl)-1,3-butadiyne (8) with 0.5 equiv. of Cu₂O give (η²:η²-1,4-bis(trimethylsilyl)-1,3-butadiyne)(Cu(hfac))₂(9). All the new compounds have been characterized by analytical and spectroscopic methods and their thermal properties were examined by thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC).