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.     

5′-Nitro-indirubinoxime induces G1 cell cycle arrest and apoptosis in salivary gland adenocarcinoma cells through the inhibition of Notch-1 signaling

Background

5′-Nitro-indirubinoxime (5′-NIO) is a new derivative of indirubin that exhibits anti-cancer activity in a variety of human cancer cells. However, its mechanism has not been fully clarified.

Methods

Human salivary gland adenocarcinoma (SGT) cells were used in this study. Western blot and RT-PCR analyses were performed to determine cellular Notch levels. The cell cycle stage and level of apoptosis were analyzed using flow cytometry analysis.

Results

5′-NIO significantly inhibited the mRNA levels of Notch-1 and Notch-3 and their ligands (Delta1, 2, 3, and Jagged-2) in SGT cells. Immunocytochemistry analysis showed that 5′-NIO specifically decreased the level of Notch-1 in the nucleus. In addition, 5′-NIO induced G1 cell cycle arrest by reducing levels of CDK4 and CDK6 in SGT cells. Using flow cytometry and immunoblotting analysis, we found that 5′-NIO induces apoptosis following the secretion of cytochrome c and the activation of caspase-3 and caspase-7. Intracellular Notch-1 overexpression led to a decrease in G1 phase arrest and an inhibition of 5′-NIO-induced apoptosis.

Conclusion

These observations suggest that 5′-NIO induces cell cycle arrest and apoptosis by down-regulating Notch-1 signaling.

General significance

This study identifies a new mechanism of 5′-NIO-mediated anti-tumor properties. Thus, 5′-NIO could be used as a candidate for salivary gland adenocarcinoma therapeutics.     

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.     

Expression and identification of a minor extracellular fibrinolytic enzyme (Vpr) from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KCTC 3014

Previously, three extracellular proteases, Vpr, PepT, and subtilisin were identified from Bacillus subtilis KCTC 3014. To confirm the activity of Vpr, two recombinant Vpr proteins, full Vpr with TTG (pGST-fTTG-Vpr) and full Vpr with ATG (pGST-fATG-Vpr) as an initiation codon were expressed using a pGEX-2T vector encoding glutathione S-transferase (GST) in Escherichia coli. Vpr was produced in two forms, occurring as four spots on a 2-DE gel, 68 and 75 kDa proteins with similar pI values (4.0 ∼ 4.5). Activity was detected in a fibrin zymography at the expected molecular size of 68 kDa (mature form) processed from full Vpr. However, the recombinant 75 kDa of GST-fVpr did not exhibit activity. Replacement of the TTG codon with ATG led to 1.9-fold increased enzyme activity in 68 kDa. Interestingly, the expression of GSTVpr resulted in the proteolytic degradation of the protein and no GST fusion Vpr protein was detected.     

Discovery of a vorapaxar analog with increased aqueous solubility

An analog of the thrombin receptor antagonist vorapaxar (SCH 530348) with increased aqueous solubility, compound 9c (SCH 602539), was discovered through incorporation of polar substituents on the pyridine ring of the himbacine-derived lead series. This analog retained the excellent potency, pharmacokinetic and safety properties of vorapaxar while increasing the aqueous solubility by 20-fold. Also presented are in vivo evaluations of this compound in a cynomolgus monkey platelet aggregation assay and in a Folts model of thrombosis in anesthetized monkeys.     

BROTHER OF FT AND TFL1 (BFT), a member of the FT/TFL1 family,shows distinct pattern of expression during the vegetative growth of Arabidopsis

Transition to the flowering stage is precisely controlled by a few classes of regulatory molecules. BROTHER OF FT AND TFL1 (BFT) is a member of FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family, an important class of flower development regulators with unidentified biochemical function. BFT has a TFL1-like activity and plays a role in axillary inflorescence development. To elucidate the expression pattern of BFT, we analyzed the subcellular localization and conditional expression of BFT in this study. We generated 35S::BFT:GFP plants to investigate the subcellular localization of BFT protein. 35S::BFT:GFP plants showed late flowering, similarly as did 35S::BFT plants. BFT:GFP fusion protein was localized in the nucleus and the plasma membrane, which was different from the localization pattern of FT and TFL1. BFT expression was induced by abiotic stress conditions. ABA, drought, and osmotic stress treatments induced BFT expression, whereas cold, salt, and heat stress conditions did not, suggesting that BFT plays a role in regulating flowering time and inflorescence structure under drought conditions. The induction pattern of BFT was different from those of other FT/TFL1 family genes. Our studies indicated that BFT showed a distinct expression pattern from its homologous genes during the vegetative growth in Arabidopsis.Key words: flowering time, flowering locus T, terminal flower 1, brother of FT and TFL1, abiotic stress, subcellullar localizationThe FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family is a small gene family whose members play a pivotal role in flower development in Arabidopsis. The family includes FT, TFL1, TWIN SISTER OF FT (TSF), Arabidopsis thaliana CENTRORADIALIS homologue (ATC), MOTHER OF FT AND TFL1 (MFT) and BROTHER OF FT AND TFL1 (BFT).^{3,5,6,9,15,17} FT is a floral promoter that integrates signal inputs from various pathways that regulate flowering time in Arabidopsis.^5,6 TFL1 plays an antagonistic role to that of FT, functioning as a floral inhibitor. Unlike FT, TFL1 also plays an important role in controlling plant architecture by regulating the expression of LEAFY (LFY) and APETALA1 (AP1), two important floral meristem identity genes in the shoot apical meristem (SAM).^3,7 TSF regulates flowering by a mechanism similar to that of FT, although a lesion in TSF does not have an apparent effect on the determination of flowering time. MFT has a weak FT-like activity.¹⁷ ATC acts as a floral repressor, and its role is similar to that of TFL1.⁹ Finally, BFT has a TFL1-like activity, in spite of its amino acid homology to FT,^2,4,16 and functions redundantly with TFL1 in inflorescence meristem development in Arabidopsis.¹⁶ Although genetic studies elucidated an intricate role of the FT/TFL1 family genes, not much is known about the expression pattern of the remaining members except FT and TFL1. Here, we report that BFT expression showed a distinct pattern from its homologous genes during the vegetative phase. BFT:GFP fusion protein was detected in the nucleus and the plasma membrane. BFT expression was induced by abiotic stress conditions, distinct from other FT/TFL1 family genes, raising the possibility that BFT plays a role in regulating flowering time and inflorescence structure under drought conditions.     

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.     

Molecular characterization of flavonol synthase from poplar and its application to the synthesis of 3-<Emphasis Type="Italic">O</Emphasis>-methylkaempferol

Biosynthesis of flavonoid derivatives requires enzyme(s) having high reactivity as well as regioselectivity. We have synthesized 3-O-kaempferol from naringenin using two enzymes. The first reaction, in which naringenin is converted to kaempferol, is mediated by flavonol synthase (FLS). An FLS (PFLS) with strong catalytic activity was cloned and characterized from the genome sequence of the poplar (Populus deltoides). PFLS consists of a 1,008 bp ORF encoding a 38 kDa protein. PFLS was expressed in Escherichia coli with a glutathione-S-transferase (GST) tagging. The purified recombinant PFLS was characterized. Catalytically, it was more efficient than the previously characterized FLSs. A mixture of two E. coli transformants harboring either PFLS or ROMT9 (a kaempferol 3-O-methyltransferase) converted naringenin into 3-O-methylkaempferol.