Synthesis of Polyoxamic Acid (2-Amino-2-deoxy-l-xylonic Acid

The rate of precipitation of the retrograded amylose product from a dil. amylose solution was determined by the centrifugal method. The results showed that the relation of the quantity of precipitate vs. time did not fit the typical second order reaction for the coalescence of colloidal particles but fitted the crystallization formula, in appearance.

The rate of precipitation was in proportion to (c-c_a)^1.5, where c is the amylose concentration and c_a the concentration of the dil. solution phase in the phase-separated solution. When the temperature dependence of the rate was treated according to the crystallization of polymers, it was found that the rate was in proportion to T_m²/T(ΔT)², where T_m is the melting point of the polymer in solution and ΔT is (T_m?T). The T_m thus obtained was 120°C for an amylose solution. These results suggested a certain correlation between the amylose retrogradation and the crystallization.     

CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation

Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.     

Which one of the two common reporter systems is more suitable for chemiluminescent enzyme immunoassay: alkaline phosphatase or horseradish peroxidase?

Alkaline phosphatase and horseradish peroxidase are the most commonly used reporter systems in chemiluminescent enzyme immunoassay (CLEIA). Which one, therefore, would be better when establishing a CLEIA method for a new target substance? There was no standard answer. In this study, both reporters were compared systematically including luminescence kinetics, conjugation methods, optimal condition and detection performance, using two common drugs, SD‐methoxy‐pyrimidine and enrofloxacin, as determination objects. The results revealed that there was much difference between the luminescence kinetics of the two systems. However, there was little difference between these systems when detecting the same substance, including in optimal conditions and determination of performance. Both reporters were suitable for establishing chemiluminescent enzyme immunoassays. Therefore, the choice of alkaline phosphatase or horseradish peroxidase as the reporter system in chemiluminescent enzyme immunoassays depends on availability. Conversely, these two report systems could be applied in simultaneous analysis of multicomponents due to their different optical behaviors and similar performances. But attention should be paid to conjugation method and coating buffer, which affected the luminescent intensity of different determination targets. Copyright © 2015 John Wiley & Sons, Ltd.     

The Combined Effect of Cu,Zn and Pb on Enzyme Activities in Soil from the Vicinity of a Wellhead Protection Area

Heavy metals are known to have adverse effects on soil ecosystems, while soil enzyme activities are sensitive to soil pollution. This study investigated the combined effects of Cu, Zn and Pb on the activities of invertase (IN), urease (U) and alkaline phosphatase (ALP) in soil obtained from the vicinity of a wellhead protection area via an orthogonal array (OA) design method. The experimental results showed the following: (1) Cu showed higher inhibition on the activities of all three enzymes than Zn and Pb when three metals were all present in the soil sample. IN activity, U activity and ALP activity decreased as the levels of Cu increased, and ranged from 15.9% to 55.7%, 3.57% to 78.6%, and 3.23% to 75.3%, respectively. Their lowest values were found in samples at 35 days with 400 mg/kg Cu. (2) Zn and Pb had different influences on the activities of the three enzymes. The lowest IN activity (the highest reduction 58.0%) and U activity (76.8%) were observed when Zn was at the concentration of 100 mg/kg after 35 days, whereas the highest inhibitory function of Zn on ALP activity (75.3%) was at 300 mg/kg after 7 days. When the concentration of Pb increased from 35 to 350 mg/kg, the activities of IN (62.5%) and U (69.6%) were most inhibited at 35 days and 14 days, respectively. However, when Pb was at the concentration of 500 mg/kg after 14 days, ALP activity (72.0%) showed the lowest value. (3) With respect to the three hydrolases in this study, ALP was the most sensitive to the two-variable interactive effects of Cu, Zn and Pb, especially Cu?×?Pb. It is concluded that the soil ALP activity may be a sensitive tool for assessing additive toxic effect on soil biochemical parameters. To provide more information about the potential ecological risk of chemicals on soil ecosystems, much more should be done to clearly determine the mechanisms of the combined effects of heavy metals in soil.     

Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.     

Cyanobacterial bioactive metabolites—A review of their chemistry and biology

Cyanobacterial blooms occur when algal densities exceed baseline population concentrations. Cyanobacteria can produce a large number of secondary metabolites. Odorous metabolites affect the smell and flavor of aquatic animals, whereas bioactive metabolites cause a range of lethal and sub-lethal effects in plants, invertebrates, and vertebrates, including humans. Herein, the bioactivity, chemistry, origin, and biosynthesis of these cyanobacterial secondary metabolites were reviewed. With recent revision of cyanobacterial taxonomy by Anagnostidis and Komárek as part of the Süβwasserflora von Mitteleuropa volumes 19(1–3), names of many cyanobacteria that produce bioactive compounds have changed, thereby confusing readers. The original and new nomenclature are included in this review to clarify the origins of cyanobacterial bioactive compounds.Due to structural similarity, the 157 known bioactive classes produced by cyanobacteria have been condensed to 55 classes. This review will provide a basis for more formal procedures to adopt a logical naming system. This review is needed for efficient management of water resources to understand, identify, and manage cyanobacterial harmful algal bloom impacts.     

透明质酸钠联合硫酸氨基酸葡萄糖钾胶囊对踝关节损伤患者血清骨性标志物水平及关节功能的影响

目的:探讨透明质酸钠联合硫酸氨基酸葡萄糖钾胶囊对踝关节损伤患者血清骨性标志物水平及关节功能的影响。方法:选择2015年1月至2017年1月在我院接受治疗的120例踝关节损伤患者,将其随机分为观察组和对照组,每组各60例。对照组患者采用透明质酸钠联治疗,观察组则在对照组治疗方案的基础上联合硫酸氨基酸葡萄糖钾胶囊进行治疗。检测和比较两组患者治疗前后血清骨钙素(BGP)、I型前胶原羧基端肽(PICP)、骨源性碱性磷酸酶(BALP)水平及Baird-Jackson踝关节评分的变化情况及治疗后的临床疗效。结果:治疗后,观察组的总有效率(91.67%)明显高于对照组(78.33%,P0.05)。两组患者治疗后的血清BGP、PICP、BALP水平及Baird-Jackson踝关节评分均较治疗前显著升高,且观察组明显高于对照组(P0.05)。两组治疗过程中均无明显不良反应发生。结论:透明质酸钠联合硫酸氨基酸葡萄糖钾胶囊治疗踝关节损伤患者的临床疗效显著优于单用透明质酸钠联治疗,其可有效改善血清骨性标志物水平,且利于踝关节功能的恢复。     

с-Met receptor can be activated by extracellular alkaline medium

Receptor tyrosine kinase (RTK) Met or c-Met is a target of hepatocyte growth factor (HGF) and it plays an important role under normal and pathological conditions. Activation of Met signaling pathway is associated with several cellular processes, such as proliferation, survival, motility, angiogenesis, invasion, and metastasis. In this article, we describe the ability of Met to activate upon a mild alkali treatment. To identify potential alkali-regulated proteins, CAKI-1 cells were treated with alkaline media and further tested for protein phosphorylation changes. By anti-phosphotyrosine antibody precipitation and lectin chromatography, we identified Met as a major cytoplasmic membrane protein that responded to pH changes by its phosphorylation. The activation of Met by alkali occurred at pH >8.0 and was dose-dependent. Specificity of the Met response to alkali was confirmed by the treatment with Met kinase inhibitor SU11274 and also by Met receptor knockout using CRISPR/CAS9 genome editing system. Both approaches completely blocked the Met phosphorylation response in CAKI-1 cells. Similar pH-dependent Met activation was observed in the HeLa cell line. Our data suggest existence of ligand-independent mechanism of Met receptor activation.     

枯草芽孢杆菌中一种新型双向启动子的功能鉴定

本研究旨在通过转录组分析预测的方法,由地衣芽孢杆菌中筛选获得一种新型双向启动子,鉴定其启动强度。以已知强组成型启动子pShuttle-09为对照,检测其对克劳氏芽孢杆菌碱性蛋白酶基因的表达活性。成功构建了3种重组碱性蛋白酶表达载体及对应的工程菌株。在新型启动子pLA和其反向启动子pLB调控转录下,克劳氏芽孢杆菌碱性蛋白酶表达活性达到164 U/mL和111 U/mL。结果表明,pLA的启动强度明显高于pShuttle-09和pLB,pLA启动子与pLB启动子均可表达碱性蛋白酶。从而为枯草芽孢杆菌表达系统中异源基因的表达提供一个新的方向,也为原核生物中共同表达两种基因提供了新的思路。