人源μ型阿片受体的cDNA克隆及转染CHO细胞的活性分析

μ型阿片受体是阿片类药物镇痛与成瘾的分子基础。从人脑组织总RNA通过RTPCR扩增获得μ型阿片受体的cDNA,将其克隆至pcDNA31(+)中,用酶切鉴定正确的重组质粒转染CHO细胞。筛选的单克隆细胞株,检测阳性的细胞克隆表达的μ型阿片受体介导胞内信号转导的能力。通过与激动剂和拮抗剂的信号转导分析证实,阳性的细胞克隆表达的μ型阿片受体与天然的μ型阿片受体具有基本一致的生物学特性,因此可以用来作为高效镇痛低成瘾药物筛选平台的候选细胞株。     

Cell-free DNA as a prognostic marker in stage I non-small-cell lung cancer patients undergoing stereotactic body radiotherapy

Abstract

Objective: To evaluate whether plasma cell-free DNA (cfDNA) was related to clinical outcome in inoperable stage I non-small cell lung cancer (NSCLC) patients undergoing stereotactic body radiotherapy (SBRT).

Materials and methods: Plasma cfDNA was assessed at baseline, before the last day and 45 days after the end of SBRT, in 22 NSCLC patients. Twenty-two healthy controls were also evaluated.

Results: Plasma cfDNA was higher in patients than in controls. An association with unfavourable disease-free survival was found for continuous baseline cfDNA increments (HR?=?5.9, 95%CI: 1.7–19.8, p?=?0.04).

Conclusion: Plasma cfDNA may be a promising prognostic biomarker in high-risk NSCLC patients.     

Cold shock proteins improve E. coli cell-free synthesis in terms of soluble yields of aggregation-prone proteins

Protein folding is usually slowed-down at low temperatures, and thus low-temperature expression is an effective strategy to improve the soluble yield of aggregation-prone proteins. In this study, we investigated the effects of a variety of cold shock proteins and domains (Csps) on an Escherichia coli cell extract-based cell-free protein synthesis system (CF). Most of the 12 Csps that were successfully prepared dramatically improved the protein yields, by factors of more than 5 at 16°C and 2 at 23°C, to levels comparable to those obtained at 30°C. Their stimulatory effects were complementary to each other, while CspD and CspH were inhibitory. The Csps’ effects correlated well with their Pfam CSD family scores (PF00313.22). All of the investigated Csps, except CspH, similarly possessed RNA binding and chaperon activities and increased the messenger RNA amount irrespective of their effect, suggesting that the proper balance between these activities was required for the enhancement. Unexpectedly, the 5′-untranslated region of cspA was less effective as the leader sequence. Our results demonstrated that the use of the Csps presented in this study will provide a simple and highly effective strategy for the CF, to improve the soluble yields of aggregation-prone proteins.     

Conversion from Tryptophan Precursor into Violacein Pigments by a Cell-free System from Chromobacterium violaceum

A cell-free system for converting tryptophan precursor into violacein pigments is reported. Crucial factors were the requirements of the reduced nicotinamides as a cofactor and Zn²⁺ as a metal ion. Optimal pHs were in the range of 8.5–9.5. The effectiveness of NADH was thirty times lower than that of NADPH at a low concentration of 2mm. The oxygenation mechanism(s) is discussed by using oxygenase inhibitors such as amethopterin, metyrapone, ancymidol, and prohexadione. Metal-chelating agents strongly inhibited the biosynthesis, suggesting that metallo-enzymes are involved in the biosynthesis.     

Assessment and Clinical Utility of a Non-Next-Generation Sequencing-Based Non-Invasive Prenatal Testing Technology

Background: Rolling-circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus, and 25 synthetic samples, were analyzed for the fetal risk of trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), by three laboratories on three continents. All the screen-positive pregnancies were provided post-test genetic counseling and confirmatory diagnostic invasive testing (e.g., amniocentesis). The screen-negative pregnancies were routinely evaluated at birth for fetal aneuploidies, using newborn examinations, and any suspected aneuploidies would have been offered diagnostic testing or confirmed with karyotyping. Results: The study found rolling-circle replication to be a highly viable technology for the clinical assessment of fetal aneuploidies, with 100% sensitivity for T21 (95% CI: 82.35–100.00%); 100.00% sensitivity for T18 (71.51–100.00%); and 100.00% sensitivity for T13 analyses (66.37–100.00%). The specificities were >99% for each trisomy (99.7% (99.01–99.97%) for T21; 99.5% (98.62–99.85%) for T18; 99.7% (99.03–99.97%) for T13), along with a first-pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling-circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening, and it would provide comparable results to other reported cfDNA methodologies.     

Nucleosomal Assembly in Vitro from Crypthecodinium cohnii Chromosomes in the Cell-free System

The typical eukaryote interphase nuclei were reconstructed in vitro following incubation of chromosomes of primitive eukaryote dinoflagellate Crypthecodinium cohnii E. in extracts of S phase eggs of Xenopus laevis L. Micrococcal nuclease digestion experiments indicated that the reconstructed nuclei contained typical nucleosomes. Since the chromosomes of C. cohnii do not contain histone and nucleosomes, these data suggest that nucleosome assembly is independent of specific DNA sequences, nuclear membranes and Lamin. Moreover, it has been demonstrated that Topoisomerase Ⅱ was partially involved in the process of nucleosome assembly. Taken together, these findings suggest that nuclear assembly is independent of formation of nucleosomes and that histone and non-histone may be the decisive factors in this process.     

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.     

Silurian carbonate platforms and extinction events—ecosystem changes exemplified from Gotland, Sweden

Recent and ancient carbonate platforms are major marine ecosystems, built by various carbonate-secreting organisms with different sensitivity for environmental change. For this reason, carbonate platforms are excellent sensors for changes in contemporaneous marine environments. A variety of ecosystem changes in carbonate platforms have previously been recognised in the aftermath of mass extinction events. This paper addresses how two Silurian extinction events among graptolites, conodonts, and pentamerid brachiopods can be related to changes in the style of carbonate production and general evolution of low latitude carbonate platforms in a similar way as previously reported from the major five mass extinctions of the Phanerozoic. Strata formed on Gotland during the Mulde and Lau events share remarkably many similarities but are strikingly different in composition compared to other strata on the island. The event-related strata is characterised by the sudden appearance of widespread oolites, deviating reef composition, flat-pebble conglomerates, abundant micro- and macro-oncoids, stromatolites, and other microbial facies suggesting decreased bioturbation levels in contemporaneous shelf seas. Importantly, these changes can be tied to high-resolution biostratigraphic frameworks and global stable isotope excursions. The anomalous intervals may therefore be searched for elsewhere in order to test their regional or global significance.     

Bidirectional Propagation of Signals and Nutrients in Fungal Networks via Specialized Hyphae

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