Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.     

Release of Pollen Protoplasts in Large Quantities in Brassica napus and B. compestris var. purpurea

Protoplasts were released in large quantities from mature pollen of Brassica napus L. and B. campestris var. purpurea for the first time, with a yield up to 66.7% and 70.4% respectively. Most of the pollen protoplasts were viable as tested by fluorochromatic reaction with fluorescein diacetate. The success of isolation of pollen protoplasts in these two Brassica species relied on a technique modified from the previous method developed for several monocotyledonous flowers. The procedure included two steps: First, the pollen was hydrated in 1 mol/L of sucrose solution at 28–30℃ for ca. 9h. During this process, the exine of most pollen dehisced and was detached from the pollen grains which were then covered by intine alone. Second, the hydrated pollen was transferred into an enzyme solution containing 1% cellulase, 1% pectinase, I mol/L mannitol, 0.5% potassium dextran sulphate and Ks medium salts. After 4–6 of enzymatic maceration, the intine was degradated resulting in the release of protoplasts. Factors affecting the two steps have been investigated.     

Microbial desulfurization of gasoline by free whole-cells of Rhodococcus erythropolis XP

Rhodococcus erythropolis XP could grow well with condensed thiophenes, mono-thiophenic compounds and mercaptans present in gasoline. Rhodococcus erythropolis XP was also capable of efficiently degrading the condensed thiophenes in resting cell as well as biphasic reactions in which n-octane served as a model oil phase. Free whole cells of R. erythropolis XP were adopted to desulfurize fluid catalytic cracking (FCC) and straight-run (SR) gasoline oils. About 30% of the sulfur content of FCC gasoline and 85% of sulfur in SR gasoline were reduced, respectively. Gas chromatography analysis with atomic emission detection also showed depletion of sulfur compounds in SR gasoline. Rhodococcus erythropolis XP could partly resist the toxicity of gasoline and had an application potential to biodesulfurization of gasoline.     

滨海白首乌(耳叶牛皮消)药理活性研究进展

滨海白首乌(耳叶牛皮消)是一种传统中药材,为萝藦科鹅绒藤属植物耳叶牛皮消的块根。耳叶牛皮消具有抗肿瘤、调节免疫、抗氧化、抗衰老、调血脂、促进毛发生长、保护脏器等多种药理活性。本文分析了耳叶牛皮消药理活性,提出了今后耳叶牛皮消研究与开发利用方向。     

The Peutz-Jegher gene product LKB1 is a mediator of p53-dependent cell death.

Here, we investigate the mechanism and function of LKB1, a Ser/Thr kinase mutated in Peutz-Jegher syndrome (PJS). We demonstrate that LKB1 physically associates with p53 and regulates specific p53-dependent apoptosis pathways. LKB1 protein is present in both the cytoplasm and nucleus of living cells and translocates to mitochondria during apoptosis. In vivo, LKB1 is highly upregulated in pyknotic intestinal epithelial cells. In contrast, polyps arising in Peutz-Jegher patients are devoid of LKB1 staining and have reduced numbers of apoptotic cells. We propose that a deficiency in apoptosis is a key factor in the formation of multiple benign intestinal polyps in PJS patients, and possibly for the subsequent development of malignant tumors in these patients.     

Na transport proteins are expressed by rat alveolar epithelial type I cells

Despite a presumptive role for type I (AT1) cells in alveolar epithelial transport, specific Na transporters have not previously been localized to these cells. To evaluate expression of Na transporters in AT1 cells, double labeling immunofluorescence microscopy was utilized in whole lung and in cytocentrifuged preparations of partially purified alveolar epithelial cells (AEC). Expression of Na pump subunit isoforms and the alpha-subunit of the rat (r) epithelial Na channel (alpha-ENaC) was evaluated in isolated AT1 cells identified by their immunoreactivity with AT1 cell-specific antibody markers (VIIIB2 and/or anti-aquaporin-5) and lack of reactivity with antibodies specific for AT2 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte common antigen). Expression of the Na pump alpha(1)-subunit in AEC was assessed in situ. Na pump subunit isoform and alpha-rENaC expression was also evaluated by RT-PCR in highly purified (approximately 95%) AT1 cell preparations. Labeling of isolated AT1 cells with anti-alpha(1) and anti-beta(1) Na pump subunit and anti-alpha-rENaC antibodies was detected, while reactivity with anti-alpha(2) Na pump subunit antibody was absent. AT1 cells in situ were reactive with anti-alpha(1) Na pump subunit antibody. Na pump alpha(1)- and beta(1)- (but not alpha(2)-) subunits and alpha-rENaC were detected in highly purified AT1 cells by RT-PCR. These data demonstrate that AT1 cells express Na pump and Na channel proteins, supporting a role for AT1 cells in active transalveolar epithelial Na transport.     

Toward the physical basis of thermophilic proteins: linking of enriched polar interactions and reduced heat capacity of unfolding

The enrichment of salt bridges and hydrogen bonding in thermophilic proteins has long been recognized. Another tendency, featuring lower heat capacity of unfolding (DeltaC(p)) than found in mesophilic proteins, is emerging from the recent literature. Here we present a simple electrostatic model to illustrate that formation of a salt-bridge or hydrogen-bonding network around an ionized group in the folded state leads to increased folding stability and decreased DeltaC(p). We thus suggest that the reduced DeltaC(p) of thermophilic proteins could partly be attributed to enriched polar interactions. A reduced DeltaC(p) might serve as an indicator for the contribution of polar interactions to folding stability.