Establishment of a human ovarian clear cell carcinoma cell line (RMG-I) and its single cell cloning--with special reference to the stem cell of the tumor

A cell line, designated RMG-I, has been established from ascites of a patient with ovarian clear cell carcinoma. RMG-I cells have grown well for more than 6 years with mirror ball formation. Chromosome analysis revealed aneuploidy with a modal number of 47 and 3 marker chromosomes. The histological characteristics of the transplanted tumor were similar to those observed in the original tumor which was composed of dark cells, clear cells, and hobnail cells. Thus, the RMG-I cell line was identified to be an ovarian clear cell carcinoma. Immunohistochemically, basic fetoprotein (BFP) and ferritin were demonstrated in the original tumor, the cultured cells, and the transplanted tumor. Furthermore, in order to clarify whether these 3 kinds of cells originate from one stem cell or not, monoclonal cell population were transplanted into nude mice, and its histological investigation revealed that 3 types of cells existed in the transplanted tumor, proving that they originate from one stem cell.     

Phosphorylation of neurofilament proteins by protein kinase C

The low molecular mass (70 kDa) subunit of neurofilaments (NF-L) contains at least three phosphorylation sites in vivo and is phosphorylated by multiple kinases in a site-specific manner [(1987) J. Neurochem. 48, S101; Sihag, R.K. and Nixon, R.A. submitted]. In this study, we observed that the three subunits of neurofilament proteins from retinal ganglion cell neurons are substrates for purified mouse brain protein kinase C. Two-dimensional alpha-chymotryptic phosphopeptide map analyses of the NF-L subunit demonstrated that protein kinase C phosphorylates four polypeptide sites, two of which incorporate phosphate when retinal ganglion cells are pulse-radiolabeled with [32P]orthophosphate in vivo.     

Intracerebroventricular administration of calcitonin enhances glucose-stimulated release of insulin

Intracerebroventricular (i.c.v.) administration of salmon calcitonin (500 ng) augmented glucose-stimulated release of insulin in rats. Vagotomy increased this enhancement effect of i.c.v. calcitonin significantly, whereas peripheral atropine treatment did not change it. Adrenal catecholamines did not participate in the centrally mediated insulinotropic effect of calcitonin since acute adrenalectomy did not modify the enhancement effect of i.c.v. calcitonin. Destruction of the sympathetic ganglia by neonatal treatment with 6-hydroxydopamine abolished the enhancement effect of i.c.v. calcitonin, which suggests that the sympathetic nervous system participates in the central action of calcitonin to enhance glucose-stimulated release of insulin.     

Meiosis in Coprinus VII. The prekaryogamy S-phase and the postkaryogamy DNA replication in C. lagopus.

The kinetics of incorporation of 32P into DNA have unequivocally shown that the premeiotic S-phase in Coprinus lagopus occurs before the onset of karyogamy. It takes 8 h under the control conditions (25 degrees C with a 16 h light-8 h dark regime) but only 6 h under the arrest-release conditions. An important discovery in this study is that the initiation of premeiotic DNA replication is subject to an arrest by restrictive conditions (35 degrees C under a continuous light regime) whereas that of the mitotic replication is not. Once initiated, meiotic DNA replication can continue even under the restrictive conditions. Incorporation of 32P into DNA at pachytene is quite extensive. These replications are considered to be repair replications.     

Functional neuroprotective effect of CGS 26303, a dual ECE inhibitor, on ischemic-reperfusion spinal cord injury in rats

Endothelin-1 (ET-1) has been implicated in many neurological diseases, including subarachnoid hemorrhage (SAH) and cerebral ischemia. ET-1 is also proved to deteriorate the ischemia-reperfusion injury in many organs. Our previous studies demonstrated that the endothelin-converting enzyme (ECE) inhibitor, CGS 26303, possessed beneficial effects for the treatment of SAH and transient middle cerebral artery occlusion. In this study, we investigated the neuroprotective effect of CGS 26303 on the locomotor function and mRNA expression of heme-oxygenase-1 (HO-1) in rats subjected to a 15-min spinal cord ischemia. The results showed that pretreatment with CGS 26303 significantly preserved the locomotor function and decreased the paraplegia rate at Days 1 and 3 as compared with a saline-treated group. Furthermore, rats pretreated with CGS 26303 had a significant increase in the levels of HO-1 mRNA expression at Day 3 when compared with animals pretreated with saline after spinal cord ischemia and the sham operation group. These results suggest that CGS 26303 may have a promising neuroprotective effect in the spinal cord after ischemia-reperfusion injury, and beneficial result may be due to an adaptive mechanism involved by HO-1 overexpression.     

Synthesis and evaluation of [18F]Fluorobutyl ethacrynic amide: a potential PET tracer for studying glutathione transferase

[(18)F]Flurobutyl ethacrynic amide ([(18)F]FBuEA) was prepared from the precursor tosylate N-Boc-N-[4-(toluenesulfonyloxy)butyl]ethacrynic amide with a radiochemical yield of 3%, a specific activity of 48 GBq/μmol and radiochemical purity of 98%. Chemical conjugation of [(18)F]FBuEA with glutathione (GSH) via a self-coupling reaction and enzymatic conjugation under catalysis of glutathiontransferase alpha (GST-α) and π provided about 41% yields of radiochemical conjugated product [(18)F]FBuEA-GSH, 85% and 5-16%, respectively. The catalytic selectivity of this tracer toward GST-alpha was addressed. Positron emission tomography (PET) imaging of [(18)F]FBuEA in normal rats showed that a homogeneous pattern of radioactivity was distributed in the liver, suggesting a catalytic role of GST. By contrast, PET images of [(18)F]FBuEA in rats with thioacetamide-induced cholangiocarcinoma displayed a heterogeneous pattern of radioactive accumulation with cold spots in tumor lesions. PET imaging with [(18)F]FBuEA could be used for early diagnosis of hepatic tumor with a low GST activity as well as liver function.     

蚯蚓在重金属污染土壤生物修复中的应用潜力

综述了不同生态类型的蚯蚓的特性及其土壤生态功能;总结了其对土壤重金属富集和有效性的作用及影响重金属活化的机理,并指出目前相关的生物化学机理研究的不足及其未来研究方向.同时,文章针对蚯蚓在农业和环境领域的应用现状,提出其应用于重金属污染土壤的植物提取技术的可能性,在未来工作中应加强真实污染土壤室内模拟和田间试验研究,进一步探索筛选和繁育本地蚯蚓品种以及添加有机物等相关技术.     

Whole blood-derived microRNA signatures in mice exposed to lipopolysaccharides

Background

Lipopolysaccharide (LPS) is recognized as the most potent microbial mediator presaging the threat of invasion of Gram-negative bacteria that implicated in the pathogenesis of sepsis and septic shock. This study was designed to examine the microRNA (miRNA) expression in whole blood from mice injected with intraperitoneal LPS.

Methods

C57BL/6 mice received intraperitoneal injections of varying concentrations (range, 10–1000 μg) of LPS from different bacteria, including Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enterica, and Serratia marcescens and were killed 2, 6, 24, and 72 h after LPS injection. Whole blood samples were obtained and tissues, including lung, brain, liver, and spleen, were harvested for miRNA expression analysis using an miRNA array (Phalanx miRNA OneArray® 1.0). Upregulated expression of miRNA targets in the whole blood of C57BL/6 and Tlr4^−/− mice injected with LPS was quantified using real-time RT-PCR and compared with that in the whole blood of C57BL/6 mice injected with lipoteichoic acid (LTA) from Staphylococcus aureus.

Results

Following LPS injection, a significant increase of 15 miRNAs was observed in the whole blood. Among them, only 3 miRNAs showed up-regulated expression in the lung, but no miRNAs showed a high expression level in the other examined tissues. Upregulated expression of the miRNA targets (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) following LPS injection on real-time RT-PCR was dose- and time-dependent. miRNA induction occurred after 2 h and persisted for at least 6 h. Exposure to LPS from different bacteria did not induce significantly different expression of these miRNA targets. Additionally, significantly lower expression levels of let-7d, miR-25, miR-92a, miR-103, and miR-107 were observed in whole blood of Tlr4^−/− mice. In contrast, LTA exposure induced moderate expression of miR-451 but not of the other 7 miRNA targets.

Conclusions

We identified a specific whole blood–derived miRNA signature in mice exposed to LPS, but not to LTA, from different gram-negative bacteria. These whole blood-derived miRNAs are promising as biomarkers for LPS exposure.     

Microglia recognize double-stranded RNA via TLR3

Microglia are CNS resident innate immune cells of myeloid origin that become activated and produce innate proinflammatory molecules upon encountering bacteria or viruses. TLRs are a phylogenetically conserved diverse family of sensors for pathogen-associated molecular patterns that drive innate immune responses. We have recently shown that mice deficient in TLR3 (TLR3(-/-) mice) are resistant to lethal encephalitis and have reduced microglial activation after infection with West Nile virus, a retrovirus that produces dsRNA. We wished to determine whether microglia recognize dsRNA through the TLR3 pathway. In vitro, murine wild-type primary cultured microglia responded to synthetic dsRNA polyinosinic-polycytidylic acid (poly(I:C)) by increasing TLR3 and IFN-beta mRNA and by morphologic activation. Furthermore, wild-type microglia dose dependently secreted TNF-alpha and IL-6 after poly(I:C) challenge, whereas TLR3(-/-) microglia produced diminished cytokines. Activation of MAPK occurred in a time-dependent fashion following poly(I:C) treatment of wild-type microglia, but happened with delayed kinetics in TLR3(-/-) microglia. As an in vivo model of encephalitis, wild-type or TLR3(-/-) mice were injected intracerebroventricularly with poly(I:C) or LPS, and microglial activation was assessed by cell surface marker or phospho-MAPK immunofluorescence. After intracerebroventricular injection of poly(I:C), microgliosis was clearly evident in wild-type mice but was nearly absent in TLR3(-/-) animals. When taken together, our results demonstrate that microglia recognize dsRNA through TLR3 and associated signaling molecules and suggest that these cells are key sensors of dsRNA-producing viruses that may invade the CNS.     

五株同源饲养层支持猕猴胚胎干细胞能力的比较

我们以前的研究建立了五株猕猴饲养层细胞系来支持猕猴胚胎干细胞(rESCs)的生长：一岁猴耳皮肤成纤维细胞(MESFs)、两岁猴输卵管成纤维细胞(MOFs)、成年猴卵泡颗粒成纤维样细胞(MFGs)、成年猴卵泡颗粒上皮样细胞(MFGEs),以及MESFs的克隆成纤维细胞(CMESFs)。我们发现MESFs、CMESFs、MOFs和MFGs,而不是MFGEs支持猕猴胚胎干细胞(rESCs, rhesus embryonic stem cells)的生长。通过半定量PCR的方法,我们在支持性的饲养层细胞中检测到了一些基因的高表达。在本研究中,我们运用Affymetrix公司的GeneChip® Rhesus Macaque Genome Array芯片来研究这五株同源饲养层的表达谱,希望发现哪些细胞因子和信号通路在维持rESCs中起到重要作用。结果表明,除MFGE外,包括GREM2、 bFGF,、KITLG,、DKK3、GREM1、AREG、SERPINF1 和 LTBP1等八个基因的mRNA在支持性的饲养层细胞中高表达。本研究结果提示,很多信号通路在支持rESCs的未分化生长和多潜能性方面可能起到了冗余的作用。