Desulfurization of model and diesel oils by resting cells of Gordona sp.

The desulfurization activity of the resting cells of Gordona sp. CYKS1 was strongly depended on harvest time and the highest value when the cells had been harvested in the early growth phase (0.12 mg sulfur g^–1 cell^–1 h^–1). For the model oil, hexadecane containing dibenzothiophene, the specific desulfurization rate decreased as the reaction proceeded. Both the specific and the volumetric desulfurization rates were not significantly affected by the aqueous-to-oil phase ratio. The diesel oils, light gas oil and a middle distillate unit feed were desulfurized at higher rates (ca. 0.34 mg sulfur g^–1 cell^–1 h^–1) than the model oil (0.12 mg sulfur g^–1 cell^–1 h^–1).     

Single-molecule fluorescence measurements reveal the reaction mechanisms of the core-RISC,composed of human Argonaute 2 and a guide RNA

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection. [BMB Reports 2015; 48(12): 643-644]     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

Microstructures in the Organ of Corti Help Outer Hair Cells Form Traveling Waves along the Cochlear Coil

According to the generally accepted theory of mammalian cochlear mechanics, the fluid in the cochlear scalae interacts with the elastic cochlear partition to generate transversely oscillating displacement waves that propagate along the cochlear coil. Using a computational model of cochlear segments, a different type of propagating wave is reported, an elastic propagating wave that is independent of the fluid-structure interaction. The characteristics of the propagating wave observed in the model, such as the wavelength, speed, and phase lag, are similar to those observed in the living cochlea. Three conditions are required for the existence of the elastic propagating wave in the cochlear partition without fluid-interaction: 1), the stiffness gradient of the cochlear partition; 2), the elastic longitudinal coupling; and 3), the Y-shaped structure in the organ of Corti formed by the outer hair cell, the Deiters cell, and the Deiters cell phalangeal process. The elastic propagating waves in the cochlear partition disappeared without the push-pull action provided by the outer hair cell and Deiters cell phalangeal process. The results suggest that the mechanical feedback of outer hair cells, facilitated by the organ of Corti microstructure, can control the tuning and amplification by modulating the cochlear traveling wave.     

Rapid fractionation of bovine transferrin using immobilized gangliosides.

Bovine transferrin (BTF) was fractionated from bovine whey using ganglioside affinity chromatography. After loading the immobilized matrix with a 2% whey solution, the matrix was washed with sodium acetate buffer at pH 4 containing 1 M NaCl before elution of BTF with sodium phosphate buffers at pH 7. Concanavalin-A affinity and ion exchange chromatography were used for further purification. The ganglioside column showed a 74.2% BTF recovery from whey and BTF was enriched to 61% purity with ion exchange chromatography. Bovine transferrin was identified by SDS-PAGE and western analysis. The Concanavalin-A affinity and ion exchange chromatography steps enriched BTF in the samples and removed other whey proteins from ganglioside purified fractions. These results indicate that immobilized ganglioside can be used to fractionate BTF from bovine whey. Our novel ganglioside affinity chromatography is rapid and efficient for the fractionation of BTF from whey.     

Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.     

On two tests of fit for HLA data with no double blanks.

Stevens suggests a test of fit, based on Bernstein's estimators, of the Hardy-Weinberg law for the ABO system. Nam and Gart extend this test to the generalized ABO-like system and apply it to HLA data. When the recessive gene is rare, Huether and Murphy recall Haldane's point that its Bernstein's estimator is negatively biased and go on to suggest novel corrected versions of it. With the identification of more HLA antigens, it is not uncommon to find, in certain populations, that the sample data contain no double blanks; that is, every individual reacts to at least one antigen for a given locus. Gart and Nam give a simple score test of a zero true recessive-gene frequency for such situations. Here we examine the extended test of Stevens as a test of this hypothesis. We find that it is fully efficient for two codominant alleles but that when the number exceeds two its efficiency may be 50% or lower or as high as 100%, depending on the number of alleles and the pattern of gene frequencies. The tests are applied to a set of HLA data.     

Urinary gonadotropin fragment (UGF) measurements in the diagnosis and management of ovarian cancer

UGF is a small peptide present in the urines and tissues of patients with gynecologic cancers. Published research (which, at present, mainly comes from our laboratory) on the general application of UGF as a tumor marker, and on its use in the diagnosis of ovarian cancer, is reviewed, and new studies on its use, alone and with CA125, in the management of patients with ovarian cancer, are presented. In 234 healthy women, 89 with benign disease, and 79 with ovarian cancer, UGF levels were above 3 fmol/ml (low cut-off) in 12 percent, 7 percent, and 82 percent, respectively, and above 8 fmol/ml (high cut-off) in 1.7 percent, less than 1.1 percent, and 59 percent, respectively. Similarly, 11 percent, 14 percent, and 70 percent, respectively, had CA125 levels above 35 U/ml (low cut-off), and less than 1.9 percent, 1.2 percent, and 49 percent had levels above a 200 U/ml (high cut-off). Ideally, the higher UGF and CA125 cut-offs should be used for diagnostic applications, like differentiation of a benign from a malignant pelvic mass (false-positive rate: UGF, less than 1.1 percent; CA125, 1.2 percent), but raising the cut-offs diminishes sensitivities for malignancy (UGF, 59 percent; CA125, 49 percent). The populations detected by the two markers only partially overlap, however, so that, together, UGF or CA125 can identify 75 percent of malignant pelvic masses. Levels of UGF (cut-off, greater than 3 fmol/ml) and CA125 (35 U/ml) were also monitored in 30 women undergoing therapy for ovarian cancer. Clinical observations were reflected at each clinic visit by UGF alone in 67 percent, by CA125 alone in 57 percent, and by UGF and CA125 together in 87 percent of cases. While separately UGF and CA125 levels predicted 71 percent and 57 percent, together they forecast 86 percent of recurrent cancers prior to clinical manifestations. UGF and CA125 should be used together in the detection and management of ovarian cancers.