Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K_m, V_max, and K_cat values of 11.58 μM, 1.5 μmol/min, and 102.18 s⁻¹, respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.     

CXCR4 and PTEN are involved in the anti-metastatic regulation of anethole in DU145 prostate cancer cells

Anethole has been known to have chemopreventive activities as a suppressor of the incidence and multiplicity of both invasive and noninvasive carcinomas. The goal of this study was to understand the anti-metastatic effect of anethole through C-X-C chemokine receptor type 4 (CXCR4)/tumor suppressor phosphatase and tensin homologue (PTEN) axis in DU145 prostate cancer cells. Anethole reduced both of the RNA level and the protein level of CXCR4 in a dose-dependent manner without cytotoxicity. Anethole also reduced the expression of CXCR4 and prolonged the expression of PTEN in DU145 prostate cancers. The phosphorylation of AKT and phosphatidylinositol-3kinase (PI3K) were decreased with anethole. The inhibition metastatic effect of anethole was arisen from down-regulating CXCR4 and up-regulating PTEN. Morphologically, anethole significantly inhibited the invasion of DU145 cell and down-regulated the activities of matrix-metalloproteinase (MMPs) in a dose-dependent manner. However, anethole didnot decrease the phosphorylation of PI3K and AKT while PTEN was silenced. Furthermore, the CXCR4 inhibition of anethole was not caused to proteasomal or lysosomal of CXCR4.     

Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms,Actively Released via Membrane Vesicles,and Influenced by Components of the Protein Secretion Machinery

Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.     

Fenofibrate but not fenofibric acid inhibits 11beta-hydroxysteroid dehydrogenase 1 in C2C12 myotubes

Microtubules (MTs) play an important role in cell division, and their functions are regulated by a set of microtubule-associated proteins (MAPs). Tubulin polymerization promoting protein family member 3 (TPPP3), also known as p20, is a new member of the tubulin polymerization promoting protein (TPPP) family. Previous studies have demonstrated that TPPP3 specifically binds to MTs and positively regulates MTs assembly, which leads to significant ultrastructural alterations of the MTs network. However, the physiological function of TPPP3 is still largely unknown. In the present study, we showed that knockdown of endogenous TPPP3 by RNA interference (RNAi) suppressed cell proliferation and induced cell cycle arrest in HeLa cells. Furthermore, we showed that the depletion of TPPP3 caused mitotic abnormalities, such as the formation of multipolar spindles and chromosome segregation errors, which lead to apoptosis in HeLa cells. Our study suggested that TPPP3 played a crucial role in cell mitosis by regulating centrosomes amplification and/or spindles translocation processes.     

Matrix metalloproteinase sensitive gold nanorod for simultaneous bioimaging and photothermal therapy of cancer

Herein, we developed matrix metalloprotease (MMP) sensitive gold nanorods (MMP-AuNR) for cancer imaging and therapy. It was feasible to absorb NIR laser and convert into heat as well as visualize MMP activity. We showed the possibility of gold nanorods as a hyperthermal therapeutic agent and MMP sensitive imaging agent both in vitro and in vivo condition. The results suggested potential application of MMP-AuNR for simultaneous cancer diagnosis and therapy.     

Polymorphisms in the selenoprotein S and 15-kDa selenoprotein genes are associated with altered susceptibility to colorectal cancer

Selenium (Se), a dietary trace metal essential for human health, is incorporated into ~25 selenoproteins including selenoprotein S (SelS) and the 15-kDa selenoprotein (Sep15) both of which have functions in the endoplasmic reticulum protein unfolding response. The aim of this study was to investigate whether genetic variants in such selenoprotein genes are associated with altered risk of colorectal cancer (CRC). A Korean population of 827 patients with CRC and 733 healthy controls was genotyped for 7 SNPs in selenoprotein genes and one SNP in the gene encoding manganese superoxide dismutase using Sequenom technology. Multivariate logistic regression analysis showed that after adjustment for lifestyle factors three SNP variants were associated with altered disease risk. There was a mean odds ratio of 2.25 [95% CI 1.13,4.48] in females homozygous TT for rs34713741 in SELS with the T variant being associated with higher risk of rectal cancer, and odds ratios of 2.47 and 2.51, respectively, for rs5845 and rs5859 in SEP15 with the minor A and T alleles being associated with increased risk of male rectal cancer. The data indicate that the minor alleles for rs5845, rs5859 and rs34713741 are associated with increased rectal cancer risk and that the effects of the three SNPs are dependent on gender. The results highlight potential links between Se, the function of two selenoproteins involved in the protein unfolding response and CRC risk. Further studies are required to investigate whether the effects of the variants on CRC risk are also modulated by dietary Se intake.     

Metabolomic screening and star pattern recognition by urinary amino acid profile analysis from bladder cancer patients

Metabolomic analysis of urinary amino acids (AAs) from patients with bladder cancer was performed by gas chromatography–mass spectrometry. The β-aminoisobutyric acid (P < 0.05) and pyroglutamic acid (P < 0.03) levels among 21 AAs had relatively low P-values in the cancer group. The distorted star pattern of the cancer group was different from the heneicosagonal shape of the control mean. The present metabolomic screening combined with star pattern recognition method as clinical monitoring tool might be useful for the visual discrimination of bladder cancer group from normal group.     

Effect of pore architecture on oxygen diffusion in 3D scaffolds for tissue engineering

The aim of this study was to maximize oxygen diffusion within a three-dimensional scaffold in order to improve cell viability and proliferation. To evaluate the effect of pore architecture on oxygen diffusion, we designed a regular channel shape with uniform diameter, referred to as cylinder shaped, and a new channel shape with a channel diameter gradient, referred to as cone shaped. A numerical analysis predicted higher oxygen concentration in the cone-shaped channels than in the cylinder-shaped channels, throughout the scaffold. To confirm these numerical results, we examined cell proliferation and viability in 2D constructs and 3D scaffolds. Cell culture experiments revealed that cell proliferation and viability were superior in the constructs and scaffolds with cone-shaped channels.     

Classification of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>l. japonica nipponbare) immunophilins (FKBPs,CYPs) and expression patterns under water stress

Background  

FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses.