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.     

Findings of preserved implicit attention in methamphetamine dependent subjects

Long-term methamphetamine (MA) abuse is associated with a wide range of deficits on explicit tasks of selective attention. Less is known however about the effects of MA abuse on implicit measures of attention. Accordingly, we used a computerized spatial priming task to assess implicit attentional processes in 54 MA dependent subjects (mean age=37.04+/-8.9 years) and 32 healthy controls without history of any form of substance abuse (mean age=33.63+/-7.05 years). The MA dependent subjects had been drug-abstinent a minimum of 3 weeks with a mean duration of MA use of 13.27+/-7.75 years. The MA dependent subjects did not differ significantly from controls on either inhibitory priming [p=.37] or facilitory priming) [p=.69]. This result comports with our earlier findings of intact object-based priming in MA dependent individuals and suggests that intact priming effects extend across spatial domains. Further, this pattern of sparing suggests that cortical brain systems typically supporting implicit attentional functioning are relatively intact in long-term MA dependent individuals whereas brain systems supporting explicit attentional processes are affected.     

MicroRNA-301b-3p accelerates the growth of gastric cancer cells by targeting zinc finger and BTB domain containing 4

MicroRNAs (miRNAs) have been found to be aberrantly expressed and exert essential roles in the tumorigenesis and progression of gastric cancer (GC). miR-301b-3p has been recognized as a cancer-related miRNA in lung cancer, bladder cancer and hepatocellular carcinoma. However, the function of miR-301b-3p in GC progression and its underlying mechanism have not been studied yet. In this study, we found that miR-301b-3p expression was up-regulated in GC tissues compared to adjacent noncancerous tissues. Furthermore, the elevated levels of miR-301b-3p were detected in GC cell lines (SGC-7901, AGS, MKN-45 and MGC-803) as compared with GES-1 cells. Interestingly, GC tissues from patients with tumor size ≥ 5 cm and advanced tumor stages showed obvious higher levels of miR-301b-3p compared to matched controls. Functionally, miR-301b-3p knockdown prominently inhibited cell proliferation, and induced cell cycle arrest at G1 phase and apoptosis in MGC-803 cells. Meanwhile, ectopic expression of miR-301b-3p conversely regulated these biological behaviors of MKN-45 cells. Next, we found that miR-301b-3p knockdown increased, whereas miR-301b-3p overexpression reduced the expression of zinc finger and BTB domain containing 4 (ZBTB4) in GC cells. Accordingly, luciferase reporter assay identified ZBTB4 as a direct target of miR-301b-3p. ZBTB4 overexpression markedly restrained the growth of MGC-803 cells. More importantly, ZBTB4 silencing partially reversed miR-301b-3p knockdown-induced tumor suppressive effects on MGC-803 cells. In conclusion, we firstly revealed that miR-301-3p was highly expressed in GC and contributed to tumor progression via attenuating ZBTB4, which might provide a novel molecular-targeted strategy for GC treatment.     

c—myc和c—neu癌基因在外耳和中耳鳞状细胞癌组织中的表达及其相关性

目的：探讨在上皮癌变过程中c-myc及c-neu的表达及二者与外耳道和中耳鳞癌发生的相关性,以进一步阐明该类肿瘤的病因和发生机理。方法：收集患者外耳道和中耳黏膜上皮,包括正常上皮（N）7例,慢性非特异性炎症（IF）5例,癌旁正常组织（EAC）8例,鳞状细胞癌（SCC）30例（外耳癌12例,中耳癌18例）。一抗分别为抗c-myc及抗c-neu单抗,采用免疫组化SP法检测c-myc及c-neu基因蛋白。结果：（1）SCC组阳性率与其它组阳性率差异有显著性（P＜0．05）;（2）两种癌基因在高分化组中的表达阳性率均高。结论：c-myc和c-neu与SCC关系密切,二者的表达具有高度相关性。     

眼底樱桃红斑(唾液酸沉积症Ⅰ型1例报道及文献复习)

目的探讨晚发型唾液酸沉积症Ⅰ型的临床特点。方法收集1例明确诊断的唾液酸沉积症Ⅰ型患者的临床资料,就病史、体征、常规实验室检查、眼底摄片、脑电图、头颅MRI和NEU1基因检测结果结合文献复习进行分析。结果患者为17岁女性;视力减退6年余,反复癫发作、行走不稳1年余。查体存在小脑体征。眼科检查示双侧樱桃红斑伴视神经萎缩。脑电图见尖慢波、棘慢波、多棘慢波。基因检测示NEU1基因存在杂合突变c.544AG(S182G)及c.239CT(P80L)、分别来自其父母,且均为已知突变,结合表型可诊断为唾液酸沉积症Ⅰ型。结论唾液酸沉积症Ⅰ型以晚发的视力损害、肌阵挛、樱桃红斑、共济失调、癫为特征,酶活性分析、电镜检查及基因检测为可靠的确诊手段。     

Five novel mutations in the lysosomal sialidase gene (NEU1) in type II sialidosis patients and assessment of their impact on enzyme activity and intracellular targeting using adenovirus-mediated expression

Sialidosis is an autosomal recessive disease resulting from a deficiency of lysosomal sialidase. Type II sialidosis is a rare disease characterized clinically by hydrops fetalis, hepatosplenomegaly, and severe psychomotor retardation. Genomic DNA from four unrelated sialidosis patients was screened for mutations within the sialidase gene NEU1. Five novel mutations were identified. Four are missense and one is nonsense: c.674G>C (p.R225P), c.893C>T (p.A298V), c.3G>A (p.M1?), c.941C>G (p.R341G), and c.69G>A (p.W23X). We have used our findings and diagnostic tools to confirm the presence of a homozygous null allele in a neonate sibling. Recombinant adenoviruses expressing the mutant sialidase alleles in primary cell cultures were utilized to assess the impact of each mutation on enzyme activity and intracellular localization. None of the mutant alleles expressed significant enzymatic activity. The p.R341G mutation exerts its pathological effect by perturbing substrate binding, while the p.A298V and p.R225P mutations appear to impair the folding of the sialidase enzyme. Our findings point to mutation‐sensitive amino acids involved in catalytic function or structural stability and indicate the potential utility of these mutations for molecular diagnosis of this rare disease. Hum Mutat 23:32–39, 2004. © 2003 Wiley‐Liss, Inc.     

Autophagy is decreased in triple-negative breast carcinoma involving likely the MUC1-EGFR-NEU1 signalling pathway

Triple-negative breast carcinoma (TN) is a heterogeneous cancer type expressing EGFR in 75% of cases. MUC1 is a large type I sialylated glycoprotein comprising two subunits (α and β chains, also called respectively MUC1-VNTR and MUC1-CT), which was found to regulate EGFR activity through endocytic internalisation. Endocytosis and autophagy use the lysosome pathway involving NEU1. Recently, a molecular EGFR-MUC1-NEU1 complex was suggested to play a role in EGFR pathway. In the aim to understand the relationship between EGFR-MUC1-NEU1 complex and autophagy in breast carcinoma, we compared triple negative (TN) showing a high-EGFR expression with luminal (LUM) presenting low-EGFR level. We studied the expression of MUC1-VNTR, MUC1-CT and NEU1 in comparison with those of two molecular actors of autophagy, PI3K (p110β) and Beclin1. A total of 87 breast cancers were split in two groups following the immunohistochemical classification of breast carcinoma: 48 TN and 39 LUM. Our results showed that TN presented a high expression of EGFR and a low expression of MUC1-VNTR, MUC1-CT, NEU1, Beclin-1 and PI3Kp110β. Moreover, in TN, a positive statistical correlation was observed between Beclin-1 or PI3Kp110β and MUC1-VNTR or NEU1, but not with EGFR. In conclusion, our data suggest that autophagy is reduced in TN leading likely to the deregulation of EGFR-MUC1-NEU1 complex and its associated cellular pathways.     

Age- and dose-dependent transplacental carcinogenesis byN-nitrosoethylurea in Syrian golden hamsters

Syrian golden hamsters have a very short period (15 days) of gestation. The implantation of the balstocyst occurs on day 5, embryogenesis proceeds very rapidly thereafter and neural tube closure is completed by day 9. In the present study the effects of two different doses ofN-nitrosoethylurea (NEU) administered at various stages of gestation were quantitatively evaluated in Syrian golden hamsters. NEU at either 0.2 or 0.5 mmol/kg was administered transplacentally as a single i.p. injection to pregnant hamsters on gestation days 7, 8, 9, 10, 11, 12, 13, or 14. The incidence, latency period and multiplicity of tumors varied with the dose of NEU and the stage of development at the time of NEU administration. Although tumors of the peripheral nervous system predominated, a variety of other tumors, including melanomas and visceral tumors of epithelial and mesenchymal origin, were also observed in hamster offspring exposed transplacentally to NEU. Sensitivity to transplacental carcinogenesis was maximal during late gestation and very low before day 9.Abbreviations NEU N-nitrosoethylurea - PNS peripheral nervous system - CNS central nervous system     

The imbalanced profile and clinical significance of T helper associated cytokines in bone marrow microenvironment of the patients with acute myeloid leukemia

Background

Immunological disorder has shown to be related to the pathogenesis of acute myeloid leukemia (AML). The microenvironment of AML is immunosuppressive, favoring the survival of malignant hematopoietic cells. However, the systematic research on AML abnormal immune microenvironment, especially the T helper (Th) cells imbalance, remains unsettled.

Design and methods

The levels of cytokines in bone marrow plasma including Th1-associated cytokine (IFN-γ), Th2-associated cytokine (IL-4), Th17-associated cytokines (IL-17, IL-6, TGF-β, and IL-21), regulatory T cell (Treg)-associated cytokines (IL-35 and IL-10) and Th22-associated cytokine (IL-22) were examined by enzyme-linked immunosorbent assay (ELISA) in AML patients and controls. The relative expression levels of IL-4, IL-10, and IL-21 mRNA were analyzed by real time polymerase chain reaction (PCR).

Results

Significant differences on cytokine levels tested were observed among the AML newly-diagnosed (ND) patients, AML patients in complete remission (CR) and controls except IL-21 and IL-35. In AML-ND group IFN-γ level was positively correlated with IL-21 or IL-22 level. Additionally, significant associations were observed between IL-17, IL-21 and some clinical characteristics.

Conclusion

Our results showed that many cytokines were abnormal in AML bone marrow microenvironment. The dysregulation of Th subsets cytokines is thought to contribute to the pathogenesis of AML.     

Subchronic toxicity study of Coptidis Rhizoma in rats

Ethnopharmacological relevance

Coptidis Rhizoma (CR) is a medical herb from the family Ranunculacease that has been used to treat gastroenteritis, dysentery, diabetes mellitus, and severe skin diseases.

Aim of the study

To evaluate the no-observed-adverse-effect level (NOAEL) and the toxicity of CR, following repeat oral administration to rats for 13 weeks.

Materials and methods

CR was administered by oral gavage to groups of rats (n=10/group, each sex) at dose levels of 0 (control), 25, 74, 222, 667 or 2000 mg/kg/day 5 times per week for 13 weeks. Mortality, clinical signs, body weights, food consumption, hematology, serum chemistry, urinalysis, vaginal cytology and sperm morphology, organ weights, gross and histopathological findings were compared between control and CR groups.

Results

Urinalysis showed a significant increase in N-acety1-β-glucosaminidase in males in the 2000 mg/kg/day group (P<0.01). However, no mortality or remarkable clinical signs were observed during this 13-week study. No adverse effects on body weight, food consumption, hematology, serum chemistry, organ weights, gross lesion, histopathology, vaginal cytology, sperm motility, or deformity were observed in the males or female rats treated with CR.

Conclusions

On the basis of these results, the NOAEL of CR is determined to be 667 mg/kg/day for males and 2000 mg/kg/day for females.