Isolation,culture, and characterization of primordial germ cells in Mongolian sheep

This study was performed to culture and preliminarily identify the primordial germ cells (PGCs) isolated from the genital ridge of the Mongolian sheep fetus. The growth characteristics of the sheep PGCs were detected in different culture systems such as culture media, resources, and state and passages of feeder cells. The obtained embryonic germ (EG) cells were identified by morphology, enzymology, and immunofluorescence. The results showed that the sheep EG cell colonies were ridgy, typically nest like, and compact, and had regular edges. Alkaline phosphatase staining reaction was weakly positive. EG cells expressed Kit, Rex-1, Nanog, and Oct-4. Immunofluorescence detection was weakly positive for Oct3/4, whereas positive for SSEA-1, SSEA-3, SSEA-4, TRA-1-61, and TRA-1-80.     

Current limitations of SNP data from the public domain for studies of complex disorders: a test for ten candidate genes for obesity and osteoporosis

Background

Public SNP databases are frequently used to choose SNPs for candidate genes in the association and linkage studies of complex disorders. However, their utility for such studies of diseases with ethnic-dependent background has never been evaluated.

Results

To estimate the accuracy and completeness of SNP public databases, we analyzed the allele frequencies of 41 SNPs in 10 candidate genes for obesity and/or osteoporosis in a large American-Caucasian sample (1,873 individuals from 405 nuclear families) by PCR-invader assay. We compared our results with those from the databases and other published studies. Of the 41 SNPs, 8 were monomorphic in our sample. Twelve were reported for the first time for Caucasians and the other 29 SNPs in our sample essentially confirmed the respective allele frequencies for Caucasians in the databases and previous studies. The comparison of our data with other ethnic groups showed significant differentiation between the three major world ethnic groups at some SNPs (Caucasians and Africans differed at 3 of the 18 shared SNPs, and Caucasians and Asians differed at 13 of the 22 shared SNPs). This genetic differentiation may have an important implication for studying the well-known ethnic differences in the prevalence of obesity and osteoporosis, and complex disorders in general.

Conclusion

A comparative analysis of the SNP data of the candidate genes obtained in the present study, as well as those retrieved from the public domain, suggests that the databases may currently have serious limitations for studying complex disorders with an ethnic-dependent background due to the incomplete and uneven representation of the candidate SNPs in the databases for the major ethnic groups. This conclusion attests to the imperative necessity of large-scale and accurate characterization of these SNPs in different ethnic groups.     

三乙醇胺基强碱性聚苯乙烯树脂共价固定胰酶的研究

用多孔强碱型三乙醇胺基聚苯乙烯阴离子交换树脂做为载体,用CNBr与载体上的多羟基作用共价偶联了胰酶。红外光谱表明:其共价偶联反应机理与用CNBr活化多糖类载体并接酶的机理相类似。最适偶联条件研究表明:CNBr用量增多,酶蛋白载量增加。但比活下降。偶联pH为10时,固定化酶有适宜的载量和较高的比活。由于胰酶水解蛋白反应释放出H~+质子,这些质子在载体内积累,使微环境内H~+质子浓度增加,进而使得固定化胰酶的pH—活性曲线在pH9～11范围内未出现下降。在变温和60℃恒温下对固定化酶的热稳定性测试表明:固相酶的热稳定性比天然酶的热稳定性有所提高。     

矮嵩草草甸植物蒸腾强度的初步研究

 此项研究工作于1986年5—9月植物生长季节内,在海北高寒草甸生态系统定位站进行。用钴纸法测定了矮嵩草等10种植物的蒸腾强度、垂穗披碱草等3种植物的蒸腾日进程和不同植被覆盖地段的蒸腾—蒸发量,在测定时记录了气温、湿度等有关气象资料,以便分析。研究结果表明：1．矮嵩草等10种植物的蒸腾强度随植物种和所处的物候期而变化,植物生长早期蒸腾强度较低,进入生殖阶段,蒸腾强度明显提高。2．垂穗披碱草等3种植物蒸腾强度日进程呈明显的单峰型曲线,在中午或午后出现峰值,没有午休现象。这同气孔一直开着有关,是矮嵩草草甸植物蒸腾的特征之一。 3．不同植被覆盖地段的蒸腾—蒸发表明,有植被覆盖的地段的蒸腾—蒸发量较裸地的蒸发量为高。     

Design,synthesis and biological evaluation of novel dual inhibitors of acetylcholinesterase and β-secretase

To explore novel effective drugs for the treatment of Alzheimer’s disease (AD), a series of dual inhibitors of acetylcholineterase (AChE) and β-secretase (BACE-1) were designed based on the multi-target-directed ligands strategy. Among them, inhibitor 28 exhibited good dual potency in enzyme inhibitory potency assay (BACE-1: IC₅₀ = 0.567 μM; AChE: IC₅₀ = 1.83 μM), and also showed excellent inhibitory effects on Aβ production of APP transfected HEK293 cells (IC₅₀ = 98.7 nM) and mild protective effect against hydrogen peroxide (H₂O₂)-induced PC12 cell injury. Encouragingly, intracerebroventricular injection of 28 into amyloid precursor protein (APP) transgenic mice caused a 29% reduction of Aβ_1–40 production. Therefore, 28 was demonstrated as a good lead compound for the further study and more importantly, the strategy of AChE and BACE-1 dual inhibitors might be a promising direction for developing novel drugs for AD patients.     

Missense Mutations in the MEFV Gene Are Associated with Fibromyalgia Syndrome and Correlate with Elevated IL-1β Plasma Levels

Background

Fibromyalgia syndrome (FMS), a common, chronic, widespread musculoskeletal pain disorder found in 2% of the general population and with a preponderance of 85% in females, has both genetic and environmental contributions. Patients and their parents have high plasma levels of the chemokines MCP-1 and eotaxin, providing evidence for both a genetic and an immunological/inflammatory origin for the syndrome (Zhang et al., 2008, Exp. Biol. Med. 233: 1171–1180).

Methods and Findings

In a search for a candidate gene affecting inflammatory pathways, among five screened in our patient samples (100 probands with FMS and their parents), we found 10 rare and one common alleles for MEFV, a gene in which various compound heterozygous mutations lead to Familial Mediterranean Fever (FMF). A total of 2.63 megabases of genomic sequence of the MEFV gene were scanned by direct sequencing. The collection of rare missense mutations (all heterozygotes and tested in the aggregate) had a significant elevated frequency of transmission to affecteds (p = 0.0085, one-sided, exact binomial test). Our data provide evidence that rare missense variants of the MEFV gene are, collectively, associated with risk of FMS and are present in a subset of 15% of FMS patients. This subset had, on average, high levels of plasma IL-1β (p = 0.019) compared to FMS patients without rare variants, unaffected family members with or without rare variants, and unrelated controls of unknown genotype. IL-1β is a cytokine associated with the function of the MEFV gene and thought to be responsible for its symptoms of fever and muscle aches.

Conclusions

Since misregulation of IL-1β expression has been predicted for patients with mutations in the MEFV gene, we conclude that patients heterozygous for rare missense variants of this gene may be predisposed to FMS, possibly triggered by environmental factors.     

Genotype-dependent expression of endothelial nitric oxide synthase (eNOS) and its regulatory proteins in cultured endothelial cells

DNA polymorphisms in endothelial nitric oxide synthase (eNOS) gene have been shown to be associated with constitutive eNOS expression and coronary artery disease (CAD). In the present study we explored the hypothesis whether genotype-dependent effects can be maintained in vitro during replication, or the effect is conditional on in vivo biological environments. Human umbilical vein endothelial cells (HUVEC) were collected and cultured from 89 normal deliveries of Mexican Americans. The cells were treated with or without cigarette smoking extracts (CSE) and genotypes of eNOS polymorphisms were determined by PCR. We measured the levels of eNOS by ELISA and its binding proteins including heat-shock protein 90 (Hsp-90) and caveolin-1 by Western blotting. The rare C allele for the promoter T786C polymorphism (0.2), and the rare 4 x 27-bp repeat allele in the intron 4 (0.30) were different from those reported in other populations. Yet, the rare T allele in the exon 7 (G894T polymorphism) was similar as others. After four passages in vitro, both the intron 4 and promoter polymorphisms maintained significant effects on eNOS mRNA levels in HUVECs (P < 0.05). However, the effects on eNOS protein and enzyme activity were less consistent. Although primary smokers had significantly lower eNOS protein levels (P < 0.05), the in vitro CSE treatment on cultured HUVECs only resulted in a significant reduction in NO levels as measured by the stable metabolites of nitrite/nitrate (P < 0.001). Neither Hsp-90 nor caveolin-1--important eNOS regulators--appears to mediate the genotypesmoking effects on eNOS expression although HUVECs did produce more Hsp-90 when exposed to CSE. Our study demonstrates that endothelial cells maintain genotype-dependent expression even after the deprivation of in vivo environment. However, the cigarette smoking-genotype interaction may require such in vivo conditions to be manifested.     

Oleuropein induces apoptosis via abrogating NF-κB activation cascade in estrogen receptor–negative breast cancer cells

Oleuropein is one of the most abundant phenolic compounds found in olives. Epidemiological studies have indicated that an increasing intake of olive oil can significantly reduce the risk of breast cancer. However, the potential effect(s) of oleuropein on estrogen receptor (ER)-negative breast cancer is not fully understood. This study aims to understand the anticancer effects and underlying mechanism(s) of oleuropein on ER-negative breast cancer cells in vitro. The effect of oleuropein on the viability of breast cancer cell lines was examined by mitochondrial dye-uptake assay, apoptosis by flow cytometric analysis, nuclear factor-κB (NF-κB) activation by DNA binding/reporter assays and protein expression by Western blot analysis. In the present report, thiazolyl blue tetrazolium bromide assay results indicated that oleuropein inhibited the viability of breast cancer cells, and its effects were more pronounced on MDA-MB-231 as compared with MCF-7 cells. It was further found that oleuropein increased the level of reactive oxygen species and also significantly inhibited cellular migration and invasion. In addition, the activation of NF-κB was abrogated as demonstrated by Western blot analysis, NF-κB-DNA binding, and luciferase assays. Overall, the data indicates that oleuropein can induce substantial apoptosis via modulating NF-κB activation cascade in breast cancer cells.     

The rs738409 (I148M) variant of the PNPLA3 gene and cirrhosis: a meta-analysis

The human patatin-like phospholipase domain-containing-3 (PNPLA3) gene rs738409 C>G polymorphism is associated with several types of liver disease. The aim of this meta-analysis was to assess the risk of cirrhosis on the basis of rs738409 allele frequency and genotype. Medline, the Cochrane Library, EMBASE, and Google Scholar were searched for prospective and retrospective studies assessing the effect of the rs738409 polymorphism on liver cirrhosis. Seven studies, involving 2,023 patients with cirrhosis, were included. The G allele was associated with a significantly increased risk of cirrhosis versus the C allele [pooled odds ratio (OR) = 1.86, 95% confidence interval (CI) = 1.64–2.12, Z = 9.55, P < 0.001]. Both the GC and GG genotypes were associated with a significantly increased risk of cirrhosis versus the CC genotype (GC vs. CC: pooled OR = 1.73, 95% CI = 1.51–1.98, Z = 7.86, P < 0.001; GG vs. CC: pooled OR = 3.41, 95% CI = 2.77–4.18, Z = 11.65, P < 0.001). There was no evidence of publication bias. Our findings suggest that patients at risk for liver cirrhosis may benefit from PNPLA3 genotyping and thus more intensive monitoring if the rs738409 C>G polymorphism is identified.