Production of Higher-quality Plastein from a Crude Single-cell Protein

From defatted n-paraffin-assimilating yeast cells, a crude protein was obtained by alkaliextraction followed by acid-precipitation. Then the protein was treated with ether until extractable substances were removed exhaustively at this stage. However, at the next stage where the ether-treated protein had been partially hydrolyzed with pepsin, when the hydrolysate was retreated with ether, it was found that ether-extractable substances totalling 270 mg/100 g were obtainable additionally. Chromatographic investigations demonstrated that the substances included significant amounts of aliphatic and aromatic hydrocarbons, some indoles, and a ubiquinone (n = 8).

From the protein hydrolysate (substrate) after the above ether-treatment, a plastein was synthesized with Bioprase under the specific conditions. The plastein was obtained as a precipitate when the whole reaction mixture was treated with aqueous ethanol or acetone. The quantity and quality (nitrogen content) of the plastein depended on the ethanol or acetone concentration. Roughly speaking, the higher the concentration, the more the plastein quantity. The converse relation held for the quality; a plastein precipitated by treatment solely with water showed a higher quality than any other case.     

Hindlimb muscle fiber types in two frogs (Rana catesbeiana and Litoria caerulea) with different locomotor behaviors: histochemical and enzymatic comparison

To test how differences in locomotor behaviors may be reflected in muscle fiber-type diversity within anurans, a comparison of hindlimb muscles between the powerful terrestrial hopper, Rana catesbeiana, and the tree frog, Litoria caerulea, was done. One postural muscle (tibialis posticus, TP) and one primary hopping muscle (plantaris longus, PL), were characterized to identify muscle fiber types using standard histochemical methods. In addition, spectophotometric analysis of activity levels of the oxidative enzyme citrate synthase (CS) and the glycolytic enzyme lactate dehydrogenase (LDH) were done in each muscle. In spite of presumed differences in behavior between the species, we found no significant differences in the proportions of the identified fiber types when the muscles were compared across species. In addition, there were no significant differences in the proportions of the different fiber types between the postural versus phasic muscles within species. Within Rana, the postural muscle (TP) had greater oxidative capacity (as measured by CS activity) than did the phasic muscle (PL). Both muscles had equivalent LDH activities. Within Litoria, PL and TP did not differ in either LDH or CS activities. Both PL and TP of Litoria had less LDH activity and greater CS activity than their homologs in Rana. Thus, in spite of the uniform populations of fiber types between muscles and species, the metabolic diversity based on enzyme activity is consistent with behavioral differences between the species. These results suggest that the range of functional diversity within fiber types may be very broad in anurans, and histochemical fiber typing alone is not a clear indicator of their metabolic or functional properties.     

SSA/Ro52 autoantigen interacts with Dcp2 to enhance its decapping activity

We identified human decapping enzyme 2 (hDCP2) as a binding protein with Ro52, being colocalized in processing bodies (p-bodies). We also showed that the N-terminus and C-terminus of Ro52 bound to hDCP2. Moreover, Ro52 enhanced decapping activity of hDCP2 in a dose-dependent manner. Our data support the novel notion of the association between Ro52 with hDCP2 protein in cytoplasmic p-bodies, playing a role in mRNA metabolism in response to cellular stimulation.     

镉胁迫对家蚕脂肪体脂质过氧化物含量及抗氧化酶活性和mRNA表达的影响

【目的】探讨鳞翅目模式昆虫家蚕Bombyx mori作为重金属污染的监测指示生物在镉胁迫下的酶反应及相关的基因表达。【方法】给家蚕幼虫期全龄添食镉(Cd²⁺), 调查不同性别家蚕5龄幼虫脂肪体中脂质过氧化物丙二醛（MDA）的含量, 超氧化物歧化酶（SOD）、 过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）的活性及其基因表达水平的变化。【结果】Cd²⁺胁迫对雌雄家蚕MDA 含量均具有浓度效应关系, MDA含量随Cd²⁺胁迫浓度的升高而增加。Cd²⁺胁迫下, SOD和CAT活性表现为先升后降的变化趋势, Pearson相关性分析显示SOD和CAT活性变化有显著相关性(雄: R=0.770, P=0.001; 雌: R=0.854, P=0.000）。雌性家蚕脂肪体中CAT活性变化和Cat mRNA水平的表达具有正相关性（R=0.712, P=0.003）。雄性家蚕脂肪体中GSH-Px活性随Cd²⁺胁迫浓度的升高而增加, 显示浓度 效应关系, 12.5~50 mg/kg Cd²⁺胁迫组GSH-Px活性与对照相比有显著差异（P<0.05）, 其活性和GSH-Px mRNA水平的表达具有正相关性（R=0.834, P=0.000）; 雌性家蚕脂肪体中GSH-Px活性表现为先升后降的变化趋势, 12.5 mg/kg Cd²⁺胁迫组GSH-Px活性与对照相比有显著增加（P<0.01）。【结论】结果表明, 急性镉胁迫对家蚕脂肪体有明显的毒性作用, 其作用机制与脂质过氧化加剧和抗氧化酶活性变化有关。家蚕对重金属镉的解毒机制有性别相关性。     

紫茎泽兰入侵对土壤酶活性和理化因子的影响

紫茎泽兰是我国危害最严重的外来入侵植物之一,为探明其入侵对土壤肥力的影响,比较研究了紫茎泽兰、云南菅、狗尾草群落和撂荒地下0~30 cm的4层土壤中6种酶活性和12种理化因子。结果表明群落类型和土壤深度对测定的各参数均有显著影响。随土壤深度的增加,多酚氧化酶、碱性磷酸酶、脲酶活性,以及有机质、全氮、全磷、全钙、水解氮、有效磷、速效钾含量和pH值均降低。总的看来,紫茎泽兰群落下碱性磷酸酶和脲酶活性,有机质、全氮、全磷、全钙、水解氮和有效磷含量,以及pH值均较高,全钾含量较低,但速效钾含量并不低,表明紫茎泽兰入侵多年后土壤肥力水平提高,形成了对其生长有利的土壤环境。     

Thermostable Alkaline Phosphatase of Bacterium Meiothermus ruber: Gene Cloning,Expression in Escherichia coli,and Biochemical Characterization of the Recombinant Protein

The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60–65°C, pH 11.0, K _M = 0.055 mM with p-nitrophenyl phosphate. The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60°C and being completely inactivated in 2 h at 80°C. In substrate specificity assays, the highest activity was observed with p-nitrophenyl phosphate and dATP. Vanadate, inorganic phosphate, and SDS were inhibitory, while thiol-reducing agents had virtually no effect. The enzyme activity strongly depended on exogenous Mg²⁺ and declined in the presence of EDTA.     

The inhibitory effect of boric acid on hypoxia-regulated tumour-associated carbonic anhydrase IX

Carbonic anhydrases (EC 4.2.1.1) catalyse the reversible hydration of CO₂ into bicarbonate and protons. As a hypoxia-sensitive and tumour-associated isoform, isoform CA IX, is significantly overexpressed in various malignancies, being a validated target for new anticancer/antimetastatic drugs. A multitude of studies has shown that CA IX inhibition decreases cancer cell proliferation and metastasis through pHe/pHi modulation and enhancement of ferroptosis among others. Numerous studies demonstrated increased efficacy of cytotoxic drugs combined with CA inhibitors (CAIs) in various cancer types. We tested the inhibitory effect of boric acid (BA), an inorganic Lewis acid, on CA IX as well as other isoforms (CA I, II, and XII). BA acted as a millimolar in vitro CAI, decreased proliferation of two cancer cell lines, although not strong correlations between the in vitro inhibition and in vivo effects were observed. The mechanism of antiproliferative action of BA should be investigated in more detail.     

Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.