Involvement of hydrophobic amino acid residues in C7–C8 loop of Aspergillus oryzae hydrophobin RolA in hydrophobic interaction between RolA and a polyester

Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. The Cys3–Cys4 and Cys7–Cys8 loops of hydrophobins are thought to form hydrophobic segments involved in adsorption of hydrophobins on hydrophobic surfaces. When the fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate-co-adipate (PBSA), A. oryzae produces hydrophobin RolA, which attaches to PBSA. Here, we analyzed the kinetics of RolA adsorption on PBSA by using a PBSA pull-down assay and a quartz crystal microbalance (QCM) with PBSA-coated electrodes. We constructed RolA mutants in which hydrophobic amino acids in the two loops were replaced with serine, and we examined the kinetics of mutant adsorption on PBSA. QCM analysis revealed that mutants with replacements in the Cys7–Cys8 loop had lower affinity than wild-type RolA for PBSA, suggesting that this loop is involved in RolA adsorption on PBSA.     

Alcohol dehydrogenase (ADH) isozymes in the AdhN/AdhN strain ofPeromyscus maniculatus (ADH− deermouse) and a possible role of class III ADH in alcohol metabolism

Although the Adh^N/Adh^N strain ofPeromyscus maniculatus (so-called ADH^? deermouse) has been previously considered to be deficient in ADH, we found ADH isozymes of Classes II and III but not Class I in the liver of this strain. On the other hand, the Adh^F/Adh^F strain (so-called ADH⁺ deermouse), which has liver ADH activity, had Class I and III but not Class II ADH in the liver. In the stomach, Class III and IV ADHs were detected in both deermouse strains, as well as in the ddY mouse, which has the normal mammalian ADH system with four classes of ADH. These ADH isozymes were identified as electrophoretic phenotypes on the basis of their substrate specificity, pyrazole sensitivity, and immunoreactivity. Liver ADH activity of the ADH^? strain was barely detectable in a conventional ADH assay using 15 mM ethanol as substrate; however, it increased markedly with high concentrations of ethanol (up to 3M) or hexenol (7 mM). Furthermore, in a hydrophobic reaction medium containing 1.0M t-butanol, liver ADH activity of this strain at low concentrations of ethanol (<100 mM) greatly increased (about sevenfold), to more than 50% that of ADH⁺ deermouse. These results were attributable to the presence of Class III ADH and the absence of Class I ADH in the liver of ADH^? deermouse. It was also found that even the ADH⁺ strain has low liver ADH activity (<40% that of the ddY mouse) with 15 mM ethanol as substrate, probably due to low activity in Class I ADH. Consequently, liver ADH activity of this strain was lower than its stomach ADH activity, in contrast with the ddY mouse, whose ADH activity was much higher in the liver than in the stomach, as well as other mammals. Thus, the ADH systems in both ADH^? and ADH⁺ deermouse were different not only from each other but also from that in the ddY mouse; the ADH^? strain was deficient in only Class I ADH, and the ADH⁺ strain was deficient in Class II ADH and down-regulated in Class I ADH activity. Therefore, Class III ADH, which was found in both strains and activated allosterically, may participate in alcohol metabolism in deermouse, especially in the ADH^? strain.     

Anti-diabetic effects of vanadium(III, IV, V)–chlorodipicolinate complexes in streptozotocin-induced diabetic rats

Vanadium(III, IV, V)–chlorodipicolinate (dipic-Cl) complexes, including H[V^III(dipic-Cl)₂] · 5H₂O (V₃dipic-Cl), V^IVO(dipic-Cl)(H₂O)₂ (V₄dipic-Cl) and K[V^VO₂(dipic-Cl)] (V₅dipic-Cl), were prepared with the indicated oxidation states. Our aim was to evaluate the anti-diabetic effects of V₃dipic-Cl, V₄dipic-Cl and V₅dipic-Cl in streptozotocin-induced diabetic rats. Vanadium complexes were orally administered to diabetic rats at concentrations of 0.1–0.3 mg/ml in the drinking water. We found that vanadium–chlorodipicolinate (V–dipic-Cl) complexes at the concentration of 0.1 mg/ml did not exhibit blood glucose-lowering effects when administered to diabetic rats for 20 days. However, the levels of fasting blood glucose in diabetic rats were decreased after treatment with 0.3 mg/ml of V₄dipic-Cl and V₅dipic-Cl complexes for the following 20 days. Although administration of both V₄dipic-Cl and V₅dipic-Cl significantly lowered diabetic hyperglycemia, the vanadium intake from administration of V₄dipic-Cl is nearly 1.5-fold greater compared to that of V₅dipic-Cl. Treatment with the H₂dipic-Cl ligand and all three V–dipic-Cl complexes significantly lowered serum cholesterol, while administration of the V₅dipic-Cl complex lowered serum cholesterol significantly more than administration of the ligand alone. Treatment with ligand alone did not have an effect on serum triglyceride, while administration of the V₄dipic-Cl and V₅dipic-Cl significantly lowered the elevated serum triglyceride associated with diabetes. Oral administration of the ligand and all V–dipic-Cl complexes did significantly lower diabetes elevated serum alkaline phosphatase. Treatment with H₂dipic-Cl ligand and V₄dipic-Cl and V₅dipicCl significantly lowered diabetes elevated aspartate amino transferase. These results indicate that the health of the treated animals did not seem to be further compromised compared to that of diabetic animals. In addition, oral administration of H₂dipic-Cl, V₃dipic-Cl, V₄dipic-Cl and V₅dipic-Cl did not alter diabetic serum creatinine and blood urea nitrogen levels, suggesting no significant side effects of vanadium treatment on renal functions at the dose of 0.3 mg/ml in diabetic rats. The results presented here suggest that the anti-diabetic effects of treatment with V–dipic-Cl complexes were likely associated in part with the oxidation state of vanadium.     

Synthesis and synergetic effects of chrysin–organogermanium (IV) complex as potential anti-oxidant

Organogermanium(IV) (Ge) is considered to play an important role in the anti-oxidative activities of some Chinese medicines. Here, a new chrysin–organogermanium (Chry–Ge) complex was synthesized and investigated for its potential biological activities. The radicals-sensitive Ge–O bond was introduced to Chry–Ge complex to enhance bioactivities of organic Ge or Chry. Results showed that Chry–Ge complex possessed great anti-oxidative activities, showing stronger hydroxyl scavenging effects than their corresponding ligands. We also demonstrated Chry–Ge complex inhibited ROS-dependent oxidative damage in cells. Moreover, the morphological and biophysical recoveries in oxidation-damaged cells induced by Chry–Ge complex were characterized by atomic force microscopy. All these results collectively suggested that Chry–Ge complex has synergetic effect for radicals scavenging and could be served as promising pharmacologically active agent against anti-oxidative treatment.     

Discovery of a novel class of 2-mercaptohexanoic acid derivatives as highly active PPARα agonists

A novel and robust scaffold for highly active PPARα agonists based on the 2-mercaptohexanoic acid substructure is presented. Systematic structural variation of the substitution pattern of the phenolic backbone yielded detailed SAR especially of ortho and meta substituents. We corroborated the importance of the sulfur atom as well as of the n-butyl chain for PPARα activity in the 2-mercaptohexanoic acid head group by preparation of carbon analogs and α-unsubstituted derivatives. Compound 10 represents a low nano molar active PPARα activator with excellent selectivity towards PPARγ.     

Identification of a four-base deletion (delTCAT296–299) in the dihydropyrimidine dehydrogenase gene with variable clinical expression

Dihydropyrimidine dehydrogenase catalyzes the first and rate-limiting step in the breakdown of thymine, uracil, and the widely used antineoplastic drug, 5-fluorouracil. Sequence analysis of the dihydropyrimidine dehydrogenase cDNA in a Dutch consanguineous family identified a novel four-base deletion (delTCAT_296–299) leading to premature termination of translation. The deletion is located in a TCAT tandem-repeat sequence and most likely results from unequal crossing-over or slipped mispairing. In this family we identified three homozygous individuals for this mutation. Two of these showed convulsive disorders but one was clinically normal. This observation suggests that, at least in this family, there is no clear correlation between the dihydropyrimidine dehydrogenase genotype and phenotype. Received: 20 January 1997 / Accepted: 10 March 1997     

Effect of tranexamic acid and δ-aminovaleric acid on lipoprotein(a) metabolism in transgenic mice

The assembly of lipoprotein(a) (Lp(a)) is a two-step process which involves the interaction of kringle-4 (K-IV) domains in apolipoprotein(a) (apo(a)) with Lys groups in apoB-100. Lys analogues such as tranexamic acid (TXA) or δ-aminovaleric acid (δ-AVA) proved to prevent the Lp(a) assembly in vitro. In order to study the in vivo effect of Lys analogues, transgenic apo(a) or Lp(a) mice were treated with TXA or δ-AVA and plasma levels of free and low density lipoprotein bound apo(a) were measured. In parallel experiments, McA-RH 7777 cells, stably transfected with apo(a), were also treated with these substances and apo(a) secretion was followed. Treatment of transgenic mice with Lys analogues caused a doubling of plasma Lp(a) levels, while the ratio of free:apoB-100 bound apo(a) remained unchanged. In transgenic apo(a) mice a 1.5-fold increase in plasma apo(a) levels was noticed. TXA significantly increased Lp(a) half-life from 6 h to 8 h. Incubation of McA-RH 7777 cells with Lys analogues resulted in an up to 1.4-fold increase in apo(a) in the medium. The amount of intracellular low molecular weight apo(a) precursor remained unchanged. We hypothesize that Lys analogues increase plasma Lp(a) levels by increasing the dissociation of cell bound apo(a) in combination with reducing Lp(a) catabolism.     

The covalent structure of cartilage collagen. Amino acid sequence of residues 552–661 of bovine α1(II) chains

The covalent structure of the first 111 residues from the N-terminus of peptide α1(II)-CB10 from bovine nasal-cartilage collagen is presented. This region comprises residues 552–661 of the α1(II) chain. The sequence was determined by automated Edman degradation of peptide α1(II)-CB10 and of peptides produced by cleavage with trypsin and hydroxylamine. Comparison of this region of the α1(II) chain with the homologous segment of the α1(I) chain indicated a homology level of 85%, slightly higher than that of 81% reported for the N-terminal region of the α1(II) chain (Butler, Miller & Finch (1976) Biochemistry 15, 3000–3006). The occurrence of two residues of glycosylated hydroxylysine was established at positions 564 and 603, the first present exclusively as galactosylhydroxylysine and the latter as a mixture of galactosylhydroxylysine and glucosylgalactosylhydroxylysine. Also, two residues at positions 648 and 657 were tentatively identified as glycosylated hydroxylysines. The amino acid sequences adjacent to the hydroxylysine residues so far identified in the α1(II) chain were compared with the homologous regions of the α1(I) and α2 chains, but no obvious prerequisite for hydroxylation could be seen. From comparison with the homologous sequence of the α1(I) chain, it appears that the α1(II)-chain sequence presented here contains three more amino acids than that reported for the α1(I) chain. This triplet would be interposed between residues 63 and 64 of the reported sequence of peptide α1(I)-CB7 from calf skin collagen. Data on the purification of the subpeptides and their amino acid compositions have been deposited as Supplementary Publication SUP 50087 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Diverse effects of residues 74–78 in ribosomal protein S12 on decoding and antibiotic sensitivity

Ribosomal protein S12 plays key roles in the ribosome’s response to the error-promoting antibiotic streptomycin and in modulating the accuracy of translation. The discovery that substitutions at His76 in S12, distant from the streptomycin binding site, conferred streptomycin resistance in the thermophilic bacterium Thermus thermophilus prompted us to make similar alterations in the S12 protein of Escherichia coli. While, none of the E. coli S12 mutations confers streptomycin resistance, they all have distinct effects on the accuracy of translation. In addition, a subset of the S12 alterations renders the cells hypersensitive to fusidic acid, an inhibitor of the translocation step of translation. These results indicate that the His 76 region of ribosomal protein S12 plays key roles in tRNA selection and translocation steps of protein synthesis, consistent with its interaction with elongation factors EF-Tu and EF-G, as deduced from structural studies of ribosomal complexes.     

A new missense mutation in FGF23 gene in a male with hyperostosis–hyperphosphatemia syndrome (HHS)

Hyperostosis–hyperphosphataemia syndrome (HHS) is a rare autosomal recessive metabolic disorder, characterized by recurrent painful swelling of long bones, periosteal new bone formation and cortical hyperostosis or intramedullary sclerosis, hyperphosphatemia and low intact fibroblast growth factor 23 (FGF23) protein levels. It is caused by mutations in 2 genes, N-acetylgalactosaminyltransferase 3 (GalNAc-transferase; GALNT3) and FGF23. We have performed mutation analysis of the GALNT3 and FGF23 genes in a patient with HHS and detected a homozygous mutation in exon 3 of FGF23 gene (NM_020638.2: c.471C>A) which results in amino acid change from phenylalanine 157 to leucin (p.F157L) in receptor interaction site.     

N-terminal truncation of a maleate cis–trans isomerase from Rhodococcus jostii RHA1 results in a highly active enzyme for the biocatalytic production of fumaric acid

As part of the project to develop an efficient biocatalytic process for the production of fumaric acid, a full-length putative maleate cis–trans isomerase gene from Rhodococcus jostii RHA1 was synthesized and expressed in Escherichia coli Rosetta2 (DE3) pLysS, but the protein was not soluble and showed no catalytic activity. Bioinformatics analysis of the protein sequence indicated that there were two hydrophilic and two hydrophobic amino acid clusters in an alternate arrangement at the N-terminus, and 50 extra amino acid residues at the N-terminus were not present in the known maleate cis–trans isomerases. The alternate hydrophilic and hydrophobic clusters at the N-terminus were thus truncated one by one to evaluate their effect on the gene expression and enzyme activity. Three mutants (MaiR-D41/42-304AA, MaiR-D48/49-304AA and MaiR-D52/53-304AA) without the hydrophilic and hydrophobic clusters were expressed as soluble protein with maleate cis–trans isomerase activity. Among them, MaiR-D48 was purified and its properties were studied. The purified enzyme had a temperature optimum of 40 °C and a wide pH range (5.0–9.0) with the optimum pH being 8.0. The whole cells of E. coli expressing MaiR-D48 catalyzed the isomerization of maleic acid to fumaric acid at 1 M substrate concentration, showing its potential for industrial use.     

Effect of diets containing γ-linolenic acid on n-6 highly unsaturated fatty acid content of rotifer (Brachionus plicatilis)

The effect of various diets containing linoleic and/or -linolenic acids was studied on n-6 fatty acid composition of the rotifer Brachionus plicatilis. The rotifer's abilities for transformations of n-6 fatty acids were evaluated. Diets containing only linolenic acid as n-6 fatty acid induced low levels of other n-6 fatty acids in rotifers while a diet containing also -linolenic acid led to substantial amounts of di homo -linolenic acid in the rotifers through elongation. Desaturation of -linoleic acid to gamma linolenic appears to be the limiting factor of n-6 highly unsaturated fatty acid biosynthesis by the rotifer. Two sets of experiments were compared using different techniques and different sources of -linolenic acid: Spirulina in inert food or borage oil in emulsion with baker's yeast. Rotifers fed with inert diet with Spirulina contained arachidonic acid while those fed with borage oil had very low arachidonic content. High level of n-3 fatty acids incorporated into the diets seemed to exert inhibitory effects on n-6 transformation rate.     

Thermotropic and structural effects of poly(malic acid) on fully hydrated multilamellar DPPC–water systems

The thermotropic and structural effects of low molecular weight poly(malic acid) (PMLA) on fully hydrated multilamellar dipalmitoylphosphatidylcholine (DPPC)–water systems were investigated using differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), and freeze-fracture transmission electron microscopy (FFTEM). Systems of 20 wt% DPPC concentration and 1 and 5 wt% PMLA to lipid ratios were studied. The PMLA derivatives changed the thermal behavior of DPPC significantly and caused a drastic loss in correlation between lamellae in the three characteristic thermotropic states (i.e., in the gel, rippled gel and liquid crystalline phases). In the presence of PBS or NaCl, the perturbation was more moderate. The structural behavior on the atomic level was revealed by FTIR spectroscopy. The molecular interactions between DPPC and PMLA were simulated via modeling its measured infrared spectra, and their peculiar spectral features were interpreted. Through this interpretation, the poly(malic acid) is inferred to attach to the headgroups of the phospholipids through hydrogen bonds between the free hydroxil groups of PMLA and the phosphodiester groups of DPPC.     

Conserved residues of the Pro103–Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase

The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteric signal in the Escherichia coli ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize that a Pro103–Arg115 loop is part of an activation path. It had strongly correlated movements with regions of the enzyme associated with regulation and ATP binding, and a network analysis showed that the optimal network pathways linking ATP and the activator binding Lys39 mainly involved residues of this loop. This hypothesis was biochemically tested by mutagenesis. We found that several alanine mutants of the Pro103–Arg115 loop had altered activation profiles for fructose-1,6-bisphosphate. Mutants P103A, Q106A, R107A, W113A, Y114A, and R115A had the most altered kinetic profiles, primarily characterized by a lack of response to fructose-1,6-bisphosphate. This loop is a distinct insertional element present only in allosterically regulated sugar nucleotide pyrophosphorylases that could have been acquired to build a triggering mechanism to link proto-allosteric and catalytic sites.     

Anti-tumor activity evaluation of novel chrysin–organogermanium(IV) complex in MCF-7 cells

Chrysin (5,7-dihydroxylflavone, Chry) is a natural product extracted from plants, honey, and propolis. In this work, a novel chrysin–organogermanium(IV) complex (Chry–Ge) with enhanced anticancer activities was synthesized, and its potential anticancer effects against cancer cells were measured using various methods. MTT results showed that Chry–Ge had significant inhibition effects on the proliferation of MCF-7, HepG2 and Colo205 human cancer cell lines in a dose-dependent manner while had little cytotoxic effects on MCF-10A human normal cells (MCF-10A cells) with the same treatment of Chry–Ge. These results suggested that Chry–Ge possessed enhanced anticancer effects and high selectivity between cancer cells and normal cells. The immuno-staining results showed that the nuclei of MCF-7 cells represented a total fragmented morphology and a disorganized cytoskeletal network in MCF-7 cells after Chry–Ge treatment. Besides, atomic force microscopy (AFM) was applied to detect the changes of ultrastructural and biomechanical properties of MCF-7 cellular membrane induced by Chry–Ge. The AFM data indicated that Chry–Ge treatment directly caused the decrease of cell rigidity and adhesion force of MCF-7 cells, suggesting that membrane toxicity might be one of the targets for Chry–Ge in MCF-7 cells. Moreover, the fluorescence-based flow cytometric analysis demonstrated that Chry–Ge could induce apoptosis in MCF-7 cells in ROS-dependent mitochondrial pathway. All results collectively showed that Chry–Ge could be as a promising anticancer drug for cancer therapy.     

The effects of structural variations of thiophene-containing Ru(II) complexes on the acid–base and DNA binding properties

A phenylthiophenyl-bearing Ru(II) complex of [Ru(bpy)₂(Hbptip)](PF₆)₂ {bpy?=?2,2′-bipyridine, Hbptip?=?2-(4-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline} was synthesized and characterized by elemental analysis, ¹H NMR spectroscopy, and electrospray ionization mass spectrometry. The ground- and excited-state acid–base properties of the complex were studied by UV–visible absorption and photoluminescence spectrophotometric pH titrations and the negative logarithm values of the ground-state acid ionization constants were derived to be pK _a1?=?1.31?±?0.09 and pK _a2?=?5.71?±?0.11 with the pK _a2 associated deprotonation/protonation process occurring over 3 pK _a units more acidic than thiophenyl-free parent complex of [Ru(bpy)₂(Hpip)]²⁺ {Hpip?=?2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline}. The calf thymus DNA-binding properties of [Ru(bpy)₂(Hbptip)]²⁺ in Tris–HCl buffer (pH 7.1 and 50?mM NaCl) were investigated by DNA viscosities and density functional theoretical calculations as well as UV–visible and emission spectroscopy techniques of UV–visible and luminescence titrations, steady-state emission quenching by [Fe(CN)₆]^4?, DNA competitive binding with ethidium bromide, DNA melting experiments, and reverse salt effects. The complex was evidenced to bind to the DNA intercalatively with binding affinity being greater than those for previously reported analogs of [Ru(bpy)₂(Hip)]²⁺, [Ru(bpy)₂(Htip)]²⁺, and [Ru(bpy)₂(Haptip)]²⁺ {Hip?=?1H-imidazo[4,5-f][1,10]phenanthroline, Htip?=?2-thiophenimidazo[4,5-f][1,10]phenanthroline, Haptip?=?2-(5-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline}.     

A causal C–A mutation in the second exon of GS3 highly associated with rice grain length and validated as a functional marker

Comparative sequencing of GS3, the most important grain length (GL) QTL, has shown that differentiation of rice GL might be principally due to a single nucleotide polymorphism (SNP) between C and A in the second exon. A total of 180 varieties representing a wide range of rice germplasm were used for association analysis between C–A mutation and GL in order to confirm the potential causal mutation. A cleaved amplified polymorphic sequence (CAPS) marker, SF28, was developed based on the C–A polymorphism in the GS3 gene. A total of 142 varieties carried allele C with GL from 6.4 to 8.8 mm, while the remaining 38 varieties carried allele A with GL from 8.8 to 10.7 mm. Twenty-four unlinked SSR markers were selected to genotype 180 varieties for population structure analysis. Population structure was observed when the population was classified to three subpopulations. Average GL of either genotype A or genotype C within japonica among the three subpopulations had no significant difference from that in indica, respectively, although indica rice had longer grains on average than japonica in the 180 varieties. However, genotype C always had longer grain length on average than genotype A among three subpopulations. The mutation could explain 79.1, 66.4 and 34.7% of GL variation in the three subpopulations, respectively. These results clearly confirmed the mutation between C and A was highly associated with GL. The SF28 could be a functional marker for improvement of rice grain length.