Molecular pathology of NEU1 gene in sialidosis

Lysosomal sialidase (EC 3.2.1.18) has a dual physiological function; it participates in intralysosomal catabolism of sialylated glycoconjugates and is involved in cellular immune response. Mutations in the sialidase gene NEU1, located on chromosome 6p21.3, result in autosomal recessive disorder, sialidosis, which is characterized by the progressive lysosomal storage of sialylated glycopeptides and oligosaccharides. Sialidosis type I is a milder, late-onset, normosomatic form of the disorder. Type I patients develop visual defects, myoclonus syndrome, cherry-red macular spots, ataxia, hyperreflexia, and seizures. The severe early-onset form, sialidosis type II, is also associated with dysostosis multiplex, Hurler-like phenotype, mental retardation, and hepatosplenomegaly. We summarize information on the 34 unique mutations determined so far in the sialidase gene, including four novel missense and one novel nonsense mutations found in two Czech and two French sialidosis patients. The analysis of sialidase mutations in sialidosis revealed considerable molecular heterogeneity, reflecting the diversity of clinical phenotypes that make molecular diagnosis difficult. The majority of sialidosis patients have had missense mutations, many of which have been expressed; their effects on activity, stability, intracellular localization, and supramolecular organization of sialidase were studied. A structural model of sialidase allowed us to localize mutations in the sialidase molecule and to predict their impact on the tertiary structure and biochemical properties of the enzyme.     

Novel missense mutations in the human lysosomal sialidase gene in sialidosis patients and prediction of structural alterations of mutant enzymes

Three novel missense mutations in the human lysosomal sialidase gene causing amino acid substitutions (P80L, W240R, and P316S) in the coding region were identified in two Japanese sialidosis patients. One patient with a severe, congenital form of type 2 sialidosis was a compound heterozygote for 239C-to-T (P80L) and 718T-to-C (W240R). The other patient with a mild juvenile-onset phenotype (type 1) was a homozygote for the base substitution of 946C-to-T (P316S). None of these mutant cDNA products showed enzymatic activity toward an artificial substrate when coexpressed in galactosialidosis fibroblastic cells together with protective protein/cathepsin A (PPCA). All mutants showed a reticular immunofluorescence distribution when coexpressed with the PPCA gene in COS-1 cells, suggesting that the gene products were retained in the endoplasmic reticulum/Golgi area or rapidly degraded in the lysosomes. Homology modeling of the structural changes introduced by the mutations predicted that the P80L and P316S transversions cause large conformational changes including the active site residues responsible for binding the sialic acid carboxylate group. The W240R substitution was deduced to influence the molecular surface structure of a limited region of the constructed models, which was also influenced by previously identified V217M and G243R transversions. Received: Stptember 21, 2001 / Accepted: November 2, 2001     

唾液酸酶法校正和区分细菌性阴道病病原菌形态研究

目的 探讨唾液酸酶法校正和区分细菌性阴道病（BV）的病原菌形态和特性差异，以提高BV的确诊率。方法 选取细菌性评分≥6分的阴道分泌物标本进行涂片染色计分，并做唾液酸酶试验、细菌培养及药敏试验。结果 染色法≥7分及唾液酸酶法阳性，病原菌镜检以小菌大量增长为主，占86．61％；染色法≥7分而唾液酸酶法阴性，病原菌镜检则以小杆菌大量增长为主，占13．39％。经统计检验，上述两种方法对BV的检测结果差异无显著性（X^2=2．23，P=0．1356）。病原菌以在高倍镜下呈小菌样大量增长及混合弯曲菌时，唾液酸酶法阳性率达100％；病原菌镜检以小杆菌大量增长为主时，其唾液酸酶活性检测均为阴性。结论 唾液酸酶法有助于染色计分法区分阴道菌群临界过渡态和BV态，可用以校正涂片染色法的评分，提高计分的准确性、客观性。     

In the Shadow of Hemagglutinin: A Growing Interest in Influenza Viral Neuraminidase and Its Role as a Vaccine Antigen

Despite the availability of vaccine prophylaxis and antiviral therapeutics, the influenza virus continues to have a significant, annual impact on the morbidity and mortality of human beings, highlighting the continued need for research in the field. Current vaccine strategies predominantly focus on raising a humoral response against hemagglutinin (HA)—the more abundant, immunodominant glycoprotein on the surface of the influenza virus. In fact, anti-HA antibodies are often neutralizing, and are used routinely to assess vaccine immunogenicity. Neuraminidase (NA), the other major glycoprotein on the surface of the influenza virus, has historically served as the target for antiviral drug therapy and is much less studied in the context of humoral immunity. Yet, the quest to discern the exact importance of NA-based protection is decades old. Also, while antibodies against the NA glycoprotein fail to prevent infection of the influenza virus, anti-NA immunity has been shown to lessen the severity of disease, decrease viral lung titers in animal models, and reduce viral shedding. Growing evidence is intimating the possible gains of including the NA antigen in vaccine design, such as expanded strain coverage and increased overall immunogenicity of the vaccine. After giving a tour of general influenza virology, this review aims to discuss the influenza A virus neuraminidase while focusing on both the historical and present literature on the use of NA as a possible vaccine antigen.     

大黄素-8-O-β-D-吡喃葡萄糖苷对牙龈卟啉单胞菌唾液酸酶活性及其毒力基因表达影响研究

目的    探讨大黄素-8-O-β-D-吡喃葡萄糖苷对牙龈卟啉单胞菌（P. gingivalis）唾液酸酶活性及其毒力基因表达的影响。方法    使用不同质量浓度的大黄素-8-O-β-D-吡喃葡萄糖苷（0.2、0.5、2、5、10 mg/mL）处理P. gingivalis W83（实验组）,用未加药物的P. gingivalis W83作对照（对照组）,采用荧光法检测大黄素-8-O-β-D-吡喃葡萄糖苷对P. gingivalis唾液酸酶活性的作用。5 mg/mL大黄素-8-O-β-D-吡喃葡萄糖苷作用于P. gingivalis W83,Real-time PCR法检测毒力基因fimA、fimR、fimS、kgp、rgpA和rgpB的表达情况。结果    大黄素-8-O-β-D-吡喃葡萄糖苷对P. gingivalis唾液酸酶活性产生了抑制作用,当其质量浓度为0.2、0.5、2、5、10 mg/mL时,对唾液酸酶活性的抑制率分别为11.4%、32.23%、40.21%、73.54%、84.31%。与对照组比较,实验组（5 mg/mL大黄素-8-O-β-D-吡喃葡萄糖苷处理）的fimA、fimR、fimS、kgp、rgpA和rgpB基因表达均下降,差异均有统计学意义（均P < 0.05）。结论    大黄素-8-O-β-D-吡喃葡萄糖苷可有效抑制P. gingivalis唾液酸酶活性,其抑制作用会降低细菌毒力基因表达,有望成为预防及治疗牙周炎的新型药物。     

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??Objective    To compare the biofilm formation of P. gingivalis  W83 and its sialidase-deficient mutant strain??ΔPG0352?? under stressful conditions. Methods    P. gingivalis  W83 and ΔPG0352 were cultured in trypticase soy broth??TSB??and supplemented with 5 μg/mL hemin and 1 μg/mL menadione under stressful conditions??including temperature??oxidatie stress and pH values. Biofilms were formed on the 96-well plates for four days??and stained with crystal violet. Detect the biofilm formation with spectrophotometer. Results    Both P. gingivalis  W83 and ΔPG0352 strains could form biofilms in normal culture conditions. P. gingivalis  W83 formed more biofilm??compared to ?PG0352 ??P < 0.05??. The biofilm formations of P. gingivalis  W83 and ΔPG0352 decreased in 34?? and 41?? ??P < 0.05??. As the concentration of H2O2 increased??biofilm formations of both strains decreased more and more seriously. The biofilm formations of ΔPG0352 were less than those of P. gingivalis  W83??when the ultimate concentration of hydrogen peroxide was 0.1??0.25 and 0.5 mmol/L. Both P. gingivalis  W83 and ?PG0352 formed less biofilm in alkaline condition??pH = 9????ΔPG0352 even less??and were unable to form biofilm in acidic condition??pH = 5??. Conclusion    Sialidase gene deletion can affect P. gingivalis  biofilm formation. Biofilms formations of both P. gingivalis  W83 and ΔPG0352 will decrease under stressful conditions.      

快速检测试剂盒在细菌性阴道病诊断中的应用

目的：探讨唾液酸酶法快速检测试剂盒在细菌性阴道病诊断中的应用价值。方法：应用BV快速诊断试剂盒对女性阴道分泌物进行检测。以Amsel法作金标准。结果：以Amsel法作标准，快速法敏感性为93．81％，特异性为98．29％，假阳性为1．14％，假阴性为6．19％。结论：该方法可以作为临床独立诊断指标，适合作常规检测，可广泛使用。     

前列腺癌PC-3M细胞株膜结合型唾液酸酶的活性测定与基因表达

目的:研究前列腺癌PC-3M细胞株膜结合型唾液酸酶(hmSD)的活性和基因表达.方法:收集指数生长期的PC-3M细胞,匀浆后离心取上清,与底物共同孵育,以TBA显色反应检测所生成的唾液酸,计算唾液酸酶活性;应用RT-PCR方法检测hmSD的mRNA表达.结果:在PC-3M细胞株可检测到hmSD活性及其RNA的表达.结论:PC-3M细胞在基因和蛋白水平有hmSD的表达.     

Sialidase NEU3 contributes neoplastic potential on colon cancer cells as a key modulator of gangliosides by regulating Wnt signaling

The plasma membrane‐associated sialidase NEU3 is a key enzyme for ganglioside degradation. We previously demonstrated remarkable up‐regulation of NEU3 in various human cancers, with augmented malignant properties. Here, we provide evidence of a close link between NEU3 expression and Wnt/β‐catenin signaling in colon cancer cells by analyzing tumorigenic potential and cancer stem‐like characteristics. NEU3 silencing in HT‐29 and HCT116 colon cancer cells resulted in significant decrease in clonogenicity on soft agar and in vivo tumor growth, along with down‐regulation of stemness and Wnt‐related genes. Analyses further revealed that NEU3 enhanced phosphorylation of the Wnt receptor LRP6 and consequently β‐catenin activation by accelerating complex formation with LRP6 and recruitment of GSK3β and Axin, whereas its silencing exerted the opposite effects. NEU3 activity‐null mutants failed to demonstrate the activation, indicating the requirement of ganglioside modulation by the sialidase for the effects. Under sphere‐forming conditions, when stemness genes are up‐regulated, endogenous NEU3 expression was found to be significantly increased, whereas NEU3 silencing suppressed sphere‐formation and in vivo tumor incidence in NOD‐SCID mice. Increased ability of clonogenicity on soft agar and sphere formation by Wnt stimulation was abrogated by NEU3 silencing. Furthermore, NEU3 was found to regulate phosphorylation of ERK and Akt via EGF receptor and Ras cascades, thought to be additionally required for tumor progression. The results indicate an essential contribution of NEU3 to tumorigenic potential through maintenance of stem‐like characteristics of colon cancer cells by regulating Wnt signaling at the receptor level, in addition to tumor progression via Ras/MAPK signaling.