Increase of lycopene production by supplementing auxiliary carbon sources in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

In the fed-batch culture of glycerol using a metabolically engineered strain of Escherichia coli, supplementation with glucose as an auxiliary carbon source increased lycopene production due to a significant increase in cell mass, despite a reduction in specific lycopene content. l-Arabinose supplementation increased lycopene production due to increases in cell mass and specific lycopene content. Supplementation with both glucose and l-arabinose increased lycopene production significantly due to the synergistic effect of the two sugars. Cell growth by the consumption of carbon sources was related to endogenous metabolism in the host E. coli. Supplementation with l-arabinose stimulated only the mevalonate pathway for lycopene biosynthesis and supplementation with both glucose and l-arabinose stimulated synergistically only the mevalonate pathway. In the fed-batch culture of glycerol with 10 g l⁻¹ glucose and 7.5 g l⁻¹ l-arabinose, the cell mass, lycopene concentration, specific lycopene content, and lycopene productivity after 34 h were 42 g l⁻¹, 1,350 mg l⁻¹, 32 mg g cells⁻¹, and 40 mg l⁻¹ h⁻¹, respectively. These values were 3.9-, 7.1-, 1.9-, and 11.7-fold higher than those without the auxiliary carbon sources, respectively. This is the highest reported concentration and productivity of lycopene.     

Cystatin SN neutralizes the inhibitory effect of cystatin C on cathepsin B activity

Cystatin SN (CST1) is one of the several salivary cystatins that form tight equimolar complexes with cysteine proteases, such as the cathepsins. High expression of CST1 is correlated with advanced pTNM stage in gastric cancer. However, the functional role of CST1 in tumorigenesis has not been elucidated. In this study, we showed that CST1 was highly expressed in colon tumor tissues, compared with nontumor regions. Increased cell proliferation and invasiveness were observed in HCT116 cell lines stably transfected with CST1 cDNA (HCT116-CST1) but not in CST3-transfected cells. We also demonstrated that CST1-overexpressing cell lines exhibited increased tumor growth as well as metastasis in a xenograft nude mouse model. Interestingly, CST1 interacted with cystatin C (CST3), a potent cathepsin B (CTSB) inhibitor, with a higher affinity than the interaction between CST3 and CTSB in the extracellular space of HCT116 cells. CTSB-mediated cellular invasiveness and proteolytic activities were strongly inhibited by CST3, but in the presence of CST1 CTSB activities recovered significantly. Furthermore, domain mapping of CST1 showed that the disulfide-bonded conformation, or conserved folding, of CST1 is important for its secretion and for the neutralization of CST3 activity. These results suggest that CST1 upregulation might be involved in colorectal tumorigenesis and acts by neutralizing the inhibition of CTSB proteolytic activity by CST3.     

Synergistic production of L-arabinose from arabinan by the combined use of thermostable endo- and exo-arabinanases from Caldicellulosiruptor saccharolyticus

The optimum conditions for the production of l-arabinose from debranched arabinan were determined to be pH 6.5, 75 °C, 20 g l⁻¹ debranched arabinan, 42 U ml⁻¹ endo-1,5-α-l-arabinanase, and 14 U ml⁻¹ α-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus and the conditions for sugar beet arabinan were pH 6.0, 75 °C, 20 g l⁻¹ sugar beet arabinan, 3 U ml⁻¹ endo-1,5-α-l-arabinanase, and 24 U ml⁻¹ α-l-arabinofuranosidase. Under the optimum conditions, 16 g l⁻¹l-arabinose was obtained from 20 g l⁻¹ debranched arabinan or sugar beet arabinan after 120 min, with a hydrolysis yield of 80% and a productivity of 8 g l⁻¹ h⁻¹. This is the first reported trial for the production of l-arabinose from the hemicellulose arabinan by the combined use of endo- and exo-arabinanases.     

Gene delivery platforms

One of the key challenges in the experimental and therapeutic use of gene delivery agents is the development of methods that can efficiently deliver nucleic acids into living systems. During the past decade, the development of effective and safe gene delivery systems has been intensively investigated. This review summarizes the current state of gene delivery methods based on viral and non-viral agents.     

Optimizing of metadata management in large-scale file systems

As modern computer systems face the challenge of large data, filesystems have to deal with a large number of files. This leads to amplified concerns of metadata operations as well as data operations. Most filesystems manage metadata of files by constructing in-memory data structures, such as directory entry (dentry) and inode. We found inefficiencies on management of metadata in existing filesystems, such as path traversal mechanism. In this article, we optimize the metadata operations by (1) looking up dentry cache (dcache) hash table in backward manner. To adopt the backward finding mechanism, we devise the rename and permission-granted mechanism. We also propose (2) compacting the metadata into dentry structures for in-memory space efficiency. We evaluate our optimized metadata managing mechanisms with several benchmarks, including a real-world workload. These optimizations significantly reduce dcache lookup latency by up to 40% and improve overall throughput by up to 72% in a real-world benchmark.     

Identification of Plasma Membrane Glycoproteins Specific to Human Glioblastoma Multiforme Cells Using Lectin Arrays and LC‐MS/MS

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most malignant type of brain cancer and has poor prognosis with a median survival of less than one year. While the structural changes of tumor cell surface carbohydrates are known to be associated with invasive behavior of tumor cells, the cell surface glycoproteins to differentiate the low‐ and high‐grade glioma cells can be potential diagnostic markers and therapeutic targets for GBMs. In the present study, lectin arrays consisting of eight lectins were employed to explore cell surface carbohydrate expression patterns on low‐grade oligodendroglioma cells (Hs683) and GBM cells (T98G). Griffonia simplicifolia I (GS I) was found to selectively bind to T98G cells and not to Hs683 cells. For identification of the glioblastoma‐specific cell surface markers, the glycoproteins from each cell type were captured by a GS I lectin column and analyzed by LC‐MS/MS. The identified proteins from the two cell types were quantified using label‐free quantitative analysis based on spectral counting. Of cell surface glycoproteins showing significant increases in T98G cells, five proteins were selected for verification of both protein and glycosylation level changes using Western blot and GS I lectin‐based immunosorbent assay.     

Effect of high hydrostatic pressure treatment on isoquercetin production from rutin by commercial α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase

Objectives

To optimize conversion of rutin to isoquercetin by commercial α-l-rhamnosidase using high hydrostatic pressure (HHP).

Results

The de-rhamnosylation activity of α-l-rhamnosidase for isoquercetin production was maximal at pH 6.0 and 50 °C using HHP (150 MPa). The enzyme showed high specificity for rutin. The specific activity for rutin at HHP was 1.5-fold higher than that at atmospheric pressure. The enzyme completely hydrolysed 20 mM rutin in tartary buckwheat extract after 2 h at HHP, with a productivity of 10 mM h^?1. The productivity and conversion were 2.2- and 1.5-fold higher at HHP than at atmospheric pressure, respectively.

Conclusions

This is the first report concerning the enzymatic hydrolysis of isoquercetin in tartary buckwheat at HHP.

     

Glucooligosaccharide production by <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> fermentation with efficient pH control,using a calcium hydroxide-sucrose solution

95.3% of the sucrose in a feed batch fermentation (300 g/L) was hydrolyzed by Leuconostoc mesenteroides subp. mesenteroides NRRL B-23188 glucansucrase. Further, the glucose of sucrose formed glucooligosaccharides (GOS) of degree of polymerization (DP) over 2, together with 91.6% of the maltose (200 g/L). Lime saccharate (lime sucrate) was used to control the pH during fermentation. The GOS products had DP between 2 and 7. When Streptococcus mutans mutansucrase (0.1 U/mL) reacted with 0.1% sucrose, addition of 0.1 ~ 10% GOS to the mutansucrase reaction digest resulted in a 56 ~ 90% reduction of mutan formation. GOS also reduced E. coli (72.2%) and Salmonella sp. (over 40.0%) growth, when 2.5% GOS was used as a single carbon source, compared to growth using glucose. The calculated glycemic index and glycemic load of GOS was 8 and 1, respectively, based on a 10 g carbohydrate serving. GOS was calculated to have 2.43 kcal/g. After a glucose tolerance test was performed using C57BL/6 mice, we found that mice treated with GOS showed a 59.4% lower increase in plasma glucose than those treated with maltose.     

RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity

RraA is a protein inhibitor of RNase E (Rne), which catalyzes the endoribonucleolytic cleavage of a large proportion of RNAs in Escherichia coli. The antibiotic-producing bacterium Streptomyces coelicolor also contains homologs of RNase E and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2, respectively. Here, we report that RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity. Analyses of the steady-state level of RNase E substrates indicated that coexpression of RraAS2 in E. coli cells overproducing Rns effectively inhibits the ribonucleolytic activity of full-length RNase ES, but its inhibitory effects were moderate or undetectable on other truncated forms of Rns, in which the N- or/and C-terminal scaffold domain was deleted. In addition, RraAS2 more efficiently inhibited the in vitro ribonucleolytic activity of RNase ES than that of a truncated form containing the catalytic domain only. Coimmunoprecipitation and in vivo cross-linking experiments further showed necessity of both scaffold domains of RNase ES for high-affinity binding of RraAS2 to the enzyme, resulting in decreased RNA-binding capacity of RNase ES. Our results indicate that RraAS2 is a protein inhibitor of RNase ES and provide clues to how this inhibitor affects the ribonucleolytic activity of RNase ES.