Structure of l-rhamnose isomerase in complex with l-rhamnopyranose demonstrates the sugar-ring opening mechanism and the role of a substrate sub-binding site

Abstractl-Rhamnose isomerase (l-RhI) catalyzes the reversible isomerization of l-rhamnose to l-rhamnulose. Previously determined X-ray structures of l-RhI showed a hydride-shift mechanism for the isomerization of substrates in a linear form, but the mechanism for opening of the sugar-ring is still unclear. To elucidate this mechanism, we determined X-ray structures of a mutant l-RhI in complex with l-rhamnopyranose and d-allopyranose. Results suggest that a catalytic water molecule, which acts as an acid/base catalyst in the isomerization reaction, is likely to be involved in pyranose-ring opening, and that a newly found substrate sub-binding site in the vicinity of the catalytic site may recognize different anomers of substrates

β-d-Altrose

The mol­ecule of the title compound, C6H12O6, [systematic name: (2R,3S,4R,5R,6R)-6-(hydroxy­meth­yl)oxane-2,3,4,5-tetrol] adopts a 4 C 1 chair conformation with the anomeric hydroxyl group in the equatorial position. All hydroxyl groups act as donors and acceptors in hydrogen bonding and the mol­ecule is involved in ten inter­molecular O—H⋯O inter­actions [O⋯O = 2.672 (5)–2.776 (4) Å] with eight neighbouring mol­ecules. Two independent O—H⋯O—H⋯ helices extending along the z axis are found in this structure

FOXO/TXNIP pathway is involved in the suppression of hepatocellular carcinoma growth by glutamate antagonist MK-801

BACKGROUND: Accumulating evidence has suggested the importance of glutamate signaling in cancer growth, yet the signaling pathway has not been fully elucidated. N-methyl-D-aspartic acid (NMDA) receptor activates intracellular signaling pathways such as the extracellular-signal-regulated kinase (ERK) and forkhead box, class O (FOXO). Suppression of lung carcinoma growth by NMDA receptor antagonists via the ERK pathway has been reported. However, series of evidences suggested the importance of FOXO pathways for the regulation of normal and cancer cell growth. In the liver, FOXO1 play important roles for the cell proliferation such as hepatic stellate cells as well as liver metabolism. Our aim was to investigate the involvement of the FOXO pathway and the target genes in the growth inhibitory effects of NMDA receptor antagonist MK-801 in human hepatocellular carcinoma. METHODS: Expression of NMDAR1 in cancer cell lines from different tissues was examined by Western blot. NMDA receptor subunits in HepG2, HuH-7, and HLF were examined by reverse transcriptase polymerase chain reaction (RT-PCR), and growth inhibition by MK-801 and NBQX was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of MK-801 on the cell cycle were examined by flow cytometry and Western blot analysis. Expression of thioredoxin-interacting protein (TXNIP) and p27 was determined by real-time PCR and Western blotting. Activation of the FOXO pathway and TXNIP induction were examined by Western blotting, fluorescence microscopy, Chromatin immunoprecipitation (ChIP) assay, and reporter gene assay. The effects of TXNIP on growth inhibition were examined using the gene silencing technique. RESULTS: NMDA receptor subunits were expressed in all cell lines examined, and MK-801, but not NBQX, inhibited cell growth of hepatocellular carcinomas. Cell cycle analysis showed that MK-801 induced G1 cell cycle arrest by down-regulating cyclin D1 and up-regulating p27. MK-801 dephosphorylated Thr24 in FOXO1 and induced its nuclear translocation, thus increasing transcription of TXNIP, a tumor suppressor gene. Knock-down of TXNIP ameliorated the growth inhibitory effects of MK-801. CONCLUSIONS: Our results indicate that functional NMDA receptors are expressed in hepatocellular carcinomas and that the FOXO pathway is involved in the growth inhibitory effects of MK-801. This mechanism could be common in hepatocellular carcinomas examined, but other mechanisms such as ERK pathway could exist in other cancer cells as reported in lung carcinoma cells. Altered expression levels of FOXO target genes including cyclin D1 and p27 may contribute to the inhibition of G1/S cell cycle transition. Induction of the tumor suppressor gene TXNIP plays an important role in the growth inhibition by MK-801. Our report provides new evidence that FOXO-TXNIP pathway play a role in the inhibition of the hepatocellular carcinoma growth by MK-801

D-Tagatose Effectively Reduces the Number of Streptococcus mutans and Oral Bacteria in Healthy Adult Subjects: A Chewing Gum Pilot Study and Randomized Clinical Trial

We examined the effect of D-Tagatose on the growth of oral bacteria including Streptococcus mutans (S. mutans). Saliva collected from 10 healthy volunteers was plated on BHI medium (to culture total oral bacteria) and MBS medium (to culture S. mutans, specifically). Agar plates of BHI or MBS containing xylitol or D-Tagatose were cultured under aerobic or anaerobic conditions. We then counted the number of colonies. In BHI plates containing D-Tagatose, a complete and significant reduction of bacteria occurred under both aerobic and anaerobic conditions. In MSB medium, significant reduction of S. mutans was also observed. We then performed a doubleblind parallel randomized trial with 19 healthy volunteers. They chewed gum containing xylitol, D-Tagatose, or both for 4 weeks, and their saliva was collected weekly and plated on BHI and MSB media. These plates were cultured under anaerobic conditions. Total bacteria and S. mutans were not effectively reduced in either the D-Tagatose or xylitol gum group. However, S. mutans was significantly reduced in volunteers chewing gum containing both D-Tagatose and xylitol. Thus, D-Tagatose inhibited the growth of S. mutans and many types of oral bacteria, indicating that D-Tagatose intake may help prevent dental caries, periodontitis, and many oral diseases

Differences and similarities in enzymes from the neopullulanase subfamily isolated from thermophilic species

Six glycoside hydrolase (GH) family 13 members, classified under the polyspecific neopullulanase subfamily GH13_20 (also termed cyclomaltodextrinase) were analysed. They originate from thermophilic bacterial strains (Anoxybacillus flavithermus, Laceyella sacchari, and Geobacillus thermoleovorans) or from environmental DNA, collected after in situ enrichments in Icelandic hot springs. The genes were isolated following the CODEHOP consensus primer strategy, utilizing the first two of the four conserved sequence regions in GH13. The typical domain structure of GH13_20, including an N-terminal domain (classified as CBM34), the catalytic module composed of the A-and B-domains, and a C-terminal domain, was found in five of the encoded enzymes (abbreviated Amy1, 89, 92, 98 and 132). These five enzymes degraded cyclomaltodextrins (CDs) and starch, while only three, Amy92 (L. sacchari), Amy98 (A. flavithermus) and Amy132 (environmental DNA), also harboured neopullulanase activity. The L. sacchari enzyme was monomeric, but with CD as the preferred substrate, which is an unusual combination. The sixth enzyme (Amy29 from environmental DNA), was composed of the ABC-domains only. Preferred substrate for Amy29 was pullulan, which was degraded to panose, and the enzyme had no detectable activity on CDs. In addition to its different activity profile and domain composition, Amy29 also displayed a different conservation (LPKF) in the fifth conserved region (MPKL) proposed to identify the subfamily. All enzymes had apparent temperature optima in the range 50–65°C, while thermostability varied, and was highest for Amy29 with a half-life of 480 min at 80°C. Calcium dependent activity or stability was monitored in four enzymes, but could not be detected for Amy29 or 98. Tightly bound calcium can, however, not be ruled out, and putative calcium ligands were conserved in Amy98

Functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain

Disruptions in ZIC3 cause heterotaxy, a congenital anomaly of the left–right axis. ZIC3 encodes a nuclear protein with a zinc finger (ZF) domain that contains five tandem C2H2 ZF motifs. Missense mutations in the first ZF motif (ZF1) result in defective nuclear localization, which may underlie the pathogenesis of heterotaxy. Here we revealed the structural and functional basis of the nuclear localization signal (NLS) of ZIC3 and investigated its relationship to the defect caused by ZF1 mutation. The ZIC3 NLS was located in the ZF2 and ZF3 regions, rather than ZF1. Several basic residues interspersed throughout these regions were responsible for the nuclear localization, but R320, K337 and R350 were particularly important. NMR structure analysis revealed that ZF1–4 had a similar structure to GLI ZF, and the basic side chains of the NLS clustered together in two regions on the protein surface, similar to classical bipartite NLSs. Among the residues for the ZF1 mutations, C253 and H286 were positioned for the metal chelation, whereas W255 was positioned in the hydrophobic core formed by ZF1 and ZF2. Tryptophan 255 was a highly conserved inter-finger connector and formed part of a structural motif (tandem CXW-C-H-H) that is shared with GLI, Glis and some fungal ZF proteins. Furthermore, we found that knockdown of Karyopherin α1/α6 impaired ZIC3 nuclear localization, and physical interactions between the NLS and the nuclear import adapter proteins were disturbed by mutations in the NLS but not by W255G. These results indicate that ZIC3 is imported into the cell nucleus by the Karyopherin (Importin) system and that the impaired nuclear localization by the ZF1 mutation is not due to a direct influence on the NLS

Conformational Changes in DNA upon Ligand Binding Monitored by Circular Dichroism

Circular dichroism (CD) spectroscopy is an optical technique that measures the difference in the absorption of left and right circularly polarized light. This technique has been widely employed in the studies of nucleic acids structures and the use of it to monitor conformational polymorphism of DNA has grown tremendously in the past few decades. DNA may undergo conformational changes to B-form, A-form, Z-form, quadruplexes, triplexes and other structures as a result of the binding process to different compounds. Here we review the recent CD spectroscopic studies of the induction of DNA conformational changes by different ligands, which includes metal derivative complex of aureolic family drugs, actinomycin D, neomycin, cisplatin, and polyamine. It is clear that CD spectroscopy is extremely sensitive and relatively inexpensive, as compared with other techniques. These studies show that CD spectroscopy is a powerful technique to monitor DNA conformational changes resulting from drug binding and also shows its potential to be a drug-screening platform in the future