MicroRNA expression and clinical outcome of small cell lung cancer

The role of microRNAs in small-cell lung carcinoma (SCLC) is largely unknown. miR-34a is known as a p53 regulated tumor suppressor microRNA in many cancer types. However, its therapeutic implication has never been studied in SCLC, a cancer type with frequent dysfunction of p53. We investigated the expression of a panel of 7 microRNAs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in 31 SCLC tumors, 14 SCLC cell lines, and 26 NSCLC cell lines. We observed significantly lower miR-21, miR-29b, and miR-34a expression in SCLC cell lines than in NSCLC cell lines. The expression of the 7 microRNAs was unrelated to SCLC patients' clinical characteristics and was neither prognostic in term of overall survival or progression-free survival nor predictive of treatment response. Overexpression or downregulation of miR-34a did not influence SCLC cell viability. The expression of these 7 microRNAs also did not predict in vitro sensitivity to cisplatin or etoposide in SCLC cell lines. Overexpression or downregulation of miR-34a did not influence sensitivity to cisplatin or etoposide in SCLC cell lines. In contrast to downregulation of the miR-34a target genes cMET and Axl by overexpression of miR-34a in NSCLC cell lines, the intrinsic expression of cMET and Axl was low in SCLC cell lines and was not influenced by overexpression of miR-34a. Our results suggest that the expression of the 7 selected microRNAs are not prognostic in SCLC patients, and miR-34a is unrelated to the malignant behavior of SCLC cells and is unlikely to be a therapeutic target.     

Purification and characterization of enzymes exhibiting beta-D-xylosidase activities in stem tissues of Arabidopsis

This work describes the purification and characterization of enzymes that exhibit beta-d-xylosidase activity in stem tissues of Arabidopsis. This is the first detailed investigation that concerns the characterization of catalytic properties and sequence identity of enzymes with beta-D-xylosidase activities in a dicotyledonous plant. Three different enzymes, ARAf, XYL4, and XYL1 with apparent molecular masses of 75, 67, and 64 kD, respectively, were purified to homogeneity. ARAf was identified as a putative alpha-L-arabinofuranosidase, and XYL4 and XYL1 as putative beta-D-xylosidases using matrix-assisted laser-desorption ionization time of flight. ARAf belongs to family 51 and XYL4 and XYL1 to family 3 of glycoside hydrolases. ARAf and XYL1 have highest specificity for p-nitrophenyl-alpha-L-arabinofuranoside and XYL4 for p-nitrophenyl-beta-D-xylopyranoside and natural substrates such as xylobiose and xylotetraose. XYL4 was shown to release mainly D-Xyl from oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, and oligoarabinoxylans. ARAf and XYL1 can also release D-Xyl from these substrates but less efficiently than XYL4. Moreover, they can also release L-Ara from arabinoxylans and arabinan. Overall, the results indicate that XYL4 possesses enzymatic specificity characteristic for a beta-D-xylosidase, while ARAf and XYL1 act as bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidases. Analysis of the activity of these three enzymes in stem tissues at different stages of development has shown that young stems possess the highest activities for all three enzymes in comparison to the activities of the enzymes present in stems at older stages of development. High enzyme activities are most likely related to the necessary modifications of cell wall structure occurring during plant growth.     

An affinity-based method for the purification of fluorescently-labeled biomolecules

Due to the difficulty of separating mixtures of labeled and unlabeled biomolecules, a general new method for the affinity purification of modified proteins has been developed. A Sepharose-based solid support bearing beta-cyclodextrin groups was used to capture chromophore-modified proteins selectively, while unmodified proteins remained in solution. After isolation of the resin, the modified proteins were released by treating the sample with a competitive cyclodextrin binder, such as adamantane carboxylic acid. This procedure was demonstrated for several dyes displaying a wide range of spectral characteristics and diverse chemical structures. Preliminary studies have shown that this method can also be used to enrich modified peptide fragments present in proteolytic digests. This technique is anticipated to accelerate the development of new protein modification reactions and could provide a useful tool for proteomics applications.     

Division site placement in E.coli: mutations that prevent formation of the MinE ring lead to loss of the normal midcell arrest of growth of polar MinD membrane domains

The MinE protein functions as a topological specificity factor in determining the site of septal placement in Escherichia coli. MinE assembles into a membrane-associated ring structure near midcell and directs the localization of MinD and MinC into a membrane- associated polar zone that undergoes a characteristic pole-to-pole oscillation cycle. Single (green fluorescent protein) and double label (yellow fluorescent protein/cyan fluorescent protein) fluorescence labeling experiments showed that mutational alteration of a site on the alpha-face of MinE led to a failure to assemble the MinE ring, associated with loss of the ability to support a normal pattern of division site placement. The absence of the MinE ring did not prevent the assembly and disassembly of the MinD polar zone. Mutant cells lacking the MinE ring were characterized by the growth of MinD polar zones past their normal arrest point near midcell. The results suggested that the MinE ring acts as a stop-growth mechanism to prevent the MinCD polar zone from extending beyond the midcell division site.     

Bioethanol production from Gracilaria verrucosa using Saccharomyces cerevisiae adapted to NaCl or galactose

This study examined the pretreatment, enzymatic saccharification, and fermentation of the red macroalgae Gracilaria verrucosa using adapted saccharomyces cerevisiae to galactose or NaCl for the increase of bioethanol yield. Pretreatment with thermal acid hydrolysis to obtain galactose was carried out with 11.7% (w/v) seaweed slurry and 373 mM H₂SO₄ at 121 °C for 59 min. Glucose was obtained from enzymatic hydrolysis. Enzymatic saccharification was performed with a mixture of 16 U/mL Celluclast 1.5L and Viscozyme L at 45 °C for 48 h. Ethanol fermentation in 11.7% (w/v) seaweed hydrolysate was carried out using Saccharomyces cerevisiae KCTC 1126 adapted or non-adapted to high concentrations of galactose or NaCl. When non-adapted S. cerevisiae KCTC 1126 was used, the ethanol productivity was 0.09 g/(Lh) with an ethanol yield of 0.25. Ethanol productivity of 0.16 and 0.19 g/(Lh) with ethanol yields of 0.43 and 0.48 was obtained using S. cerevisiae KCTC 1126 adapted to high concentrations of galactose and NaCl, respectively. Adaptation of S. cerevisiae KCTC 1126 to galactose or NaCl increased the ethanol yield via adaptive evolution of the yeast.

     

Regulation of the formation of proteinases inBacillus megaterium

Absrract The exocellular proteinases from the asporogenic and sporogenic strain ofBacillus megaterium KM were purified and characterized. They are both neutral metalloenzymes, having an optimum pH of 7.2. The bivalent metal cations, particularly calcium or magnesium, are essential for their activity. The curve of the relationship between the reaction velocity and the concentrations of Ca²⁺ resembles the Michaelis curve for substrate concentration. The enzymes also require metal cations for their stability. Both proteinases are inactivated byo-phenanthroline (lmm) and are resistant against diisopropyl fluorophosphate (lmm) and sodium-p-chloromercuribenzoate (lmm) treatment. In spite of the difference in biochemical regulation of their synthesis, these exocellular proteinases seem to be similar. The terms, megaterioproteinase A and megaterioproteinase S have been proposed for these enzymes.