In vitro demetalation of central magnesium in various chlorophyll derivatives using Mg-dechelatase homolog from the chloroflexi Anaerolineae.

In the metabolic pathway of chlorophylls (Chls), an enzyme called STAY-GREEN or SGR catalyzes the removal of the central magnesium ion of Chls and their derivatives to their corresponding free bases, including pheophytins. The substrate specificity of SGR has been investigated through in vitro reactions using Chl-related molecules. However, information about the biochemical properties and reaction mechanisms of SGR and its substrate specificity remains elusive. In this study, we synthesized various Chl derivatives and investigated their in vitro dechelations using an SGR enzyme. Chl-a derivatives with the C3-vinyl group on the A-ring, which is commonly found as a substituent in natural substrates, and their analogs with ethyl, hydroxymethyl, formyl, and styryl groups at the C3-position were prepared as substrates. In vitro dechelatase reactions of these substrates were performed using an SGR enzyme derived from an Anaerolineae bacterium, allowing us to investigate their specificity. Reactivity was reduced for substrates with an electron-withdrawing formyl or sterically demanding styryl group at the C3-position. Furthermore, the Chl derivative with the C8-styryl group on the B-ring was less reactive for SGR dechelation than the C3-styryl substrate. These results indicate that the SGR enzyme recognizes substituents on the B-ring of substrates more than those on the A-ring.

Predicted Structure of the BciC Enzyme Catalyzing the Removal of the C132-Methoxycarbonyl Group for Biosynthesis of Chlorosomal Chlorophylls: A Mechanism for Dual Catalytic Functions of Hydrolysis and Decarboxylation inside Its Active Site.

Green photosynthetic bacteria, one of the phototrophs, have the largest and most efficient light-harvesting antenna systems, called chlorosomes. The core part of chlorosomes consists of unique bacteriochlorophyll c/d/e molecules. In the biosynthetic pathway of these molecules, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. Two sequential reactions have been proposed for the BciC enzymatic demethoxycarbonylation: the BciC enzyme would catalyze the hydrolysis of the C132-methoxycarbonyl group, and the resulting carboxylic acid would be rapidly decarboxylated to generate pyrochlorophyllide a. In this study, we computationally predicted the three-dimensional structure of the BciC protein. Its active site was proposed based on structural analysis using docking simulation. In vitro enzymatic reaction assays of mutated BciC supported the prediction. The BciC enzymatic hydrolysis would be an aspartic/glutamic acid hydrolase, which involves the amino residues E85 and D180. Furthermore, Y58 and H126 might depend on stabilization and/or recognition with the substrate. Most importantly, H137 would protonate 13-C═O or deprotonate C132-COOH in the hydrolyzed product to promote decarboxylation. In conclusion, the BciC enzyme has the dual functions of hydrolysis and decarboxylation.

Characterization of regioisomeric diterpenoid tails in bacteriochlorophylls produced by geranylgeranyl reductase from Halorhodospira halochloris and Blastochloris viridis.

Geranylgeranyl reductase (GGR) encoded by the bchP gene catalyzes the reductions of three unsaturated C = C double bonds (C6 = C7, C10 = C11, and C14 = C15) in a geranylgeranyl (GG) group of the esterifying moiety in 17-propionate residue of bacteriochlorophyll (BChl) molecules. It was recently reported that GGR in Halorhodospira halochloris potentially catalyzes two hydrogenations, yielding BChl with a tetrahydrogeranylgeranyl (THGG) tail. Furthermore, its engineered GGR, in which N-terminal insertion peptides characteristic for H. halochloris were deleted, performed single hydrogenation, producing BChl with a dihydrogeranylgeranyl (DHGG) tail. In some of these enzymatic reactions, it remained unclear in which order the C = C double bond in a GG group was first reduced. In this study, we demonstrated that the (variant) GGR from H. halochloris catalyzed an initial reduction of the C6 = C7 double bond to yield a 6,7-DHGG tail. The intact GGR of H. halochloris catalyzed the further hydrogenation of the C14 = C15 double bonds to give a 6,7,14,15-THGG group, whereas deleting the characteristic peptide region from the GGR suppressed the C14 = C15 reduction. We also verified that in a model bacterium, Blastochloris viridis producing standard BChl-b, the reduction of a GG to phytyl group occurred via 10,11-DHGG and 6,7,10,11-THGG. The high-performance liquid chromatographic elution profiles of BChls-a/b employed in this study are essential for identifying the regioisomeric diterpenoid tails in the BChls of phototrophic bacteria distributed in nature and elucidating GGR enzymatic reactions.

Detection of 132-carboxy-chlorin produced by the in vitro BciC enzymatic hydrolysis of zinc chlorophyllide.

Green photosynthetic bacteria with an efficient light-harvesting system contain special chlorophyll molecules, called bacteriochlorophylls c, d, e, in their main antennae. In the biosynthetic pathway, a BciC enzyme is proposed to catalyze the hydrolysis of the C132-methoxycarbonyl group of chlorophyllide a, but the resulting C132-carboxy group has not been detected yet because it is spontaneously removed due to the instability of the ß-keto-carboxylic acid. In this study, the in vitro BciC enzymatic reactions of zinc methyl (131R/S)-hydroxy-mesochlorophyllides a were examined and a carboxylic acid possessing the C132S-OH was first observed as the hydrolyzed product of the C132-COOCH3.

In Vitro Hydrolysis of Zinc Chlorophyllide a Homologues by a BciC Enzyme.

Chlorosomes in green photosynthetic bacteria are the largest and most efficient light-harvesting antenna systems of all phototrophs. The core part of chlorosomes consists of bacteriochlorophyll c, d, or e molecules. In their biosynthetic pathway, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. In this study, the in vitro enzymatic reactions of chlorophyllide a analogues, C132-methylene- and ethylene-inserted zinc complexes, were examined using a BciC protein from Chlorobaculum tepidum. As the products, their hydrolyzed free carboxylic acids were observed without the corresponding demethoxycarbonylated compounds. The results showed that the in vivo demethoxycarbonylation of chlorophyllide a by an action of the BciC enzyme would occur via two steps: (1) an enzymatic hydrolysis of a methyl ester at the C132-position, followed by (2) a spontaneous (nonenzymatic) decarboxylation in the resulting carboxylic acid.

BciC-Catalyzed C132 -Demethoxycarbonylation of Metal Pheophorbide†a Alkyl Esters.

Bacteriochlorophyll c molecules self-aggregate to form large oligomers in the core part of chlorosomes, which are the main light-harvesting antenna systems of green photosynthetic bacteria. In the biosynthetic pathway of bacteriochlorophyll c, a BciC enzyme catalyzes the removal of the C132 -methoxycarbonyl group of chlorophyllide a, which possesses a free propionate residue at the C17-position and a magnesium ion as the central metal. The in vitro C132 -demethoxycarbonylations of chlorophyll a derivatives with various alkyl propionate residues and central metals were examined by using the BciC enzyme derived from one green sulfur bacteria species, Chlorobaculum tepidum. The BciC enzymatic reactions of zinc pheophorbide a alkyl esters were gradually suppressed with an increase of the alkyl chain length in the C17-propionate residue (from methyl to pentyl esters) and finally the hexyl ester became inactive for the BciC reaction. Although not only the zinc but also nickel and copper complexes were demethoxycarbonylated by the BciC enzyme, the reactions were largely dependent on the coordination ability of the central metals: Zn>Ni>Cu. The above substrate specificity indicates that the BciC enzyme would not bind directly to the carboxy group of chlorophyllide a, but would bind to its central magnesium to form the stereospecific complex of BciC with chlorophyllide a, giving pyrochlorophyllide a, which lacks the (132 R)-methoxycarbonyl group.

Synthesis of chlorophyll-a homologs by C132-substitutions and their physico- and biochemical properties.

A mixture of pheophytins-a/a', metal-free forms of photosynthetically active chlorophyll(Chl)s-a/a' bearing the 132-methoxycarbonyl group, was substituted at the C132-position by bimolecular nucleophilic substitution with methyl bromoacetate or Michael addition with methyl acrylate, followed by C132-demethoxycarbonylation and magnesium insertion at the central position, to afford Chl-a/a' homologs possessing a methoxycarbonylmethyl or 2-methoxycarbonylethyl group at the C132-position, respectively. These C132-methylene- and ethylene-inserted homologs were characterized by 1D/2D 1H NMR spectroscopy, and the optical properties of their C132-epimerically pure samples are investigated using visible absorption, fluorescence emission, and circular dichroism spectroscopies. The stereochemistry at the C132-chiral center of these Chl-a/a' homologs was not inverted in a basic solution, and the Chl-a homologs were effective for the substrates for the chlorophyllase reaction, hydrolysis of the phytyl ester.

In vitro C132-dealkoxycarbonylations of zinc chlorophyll a derivatives including C132-substitutes by a BciC enzyme.

Chlorosomes in the green photosynthetic bacteria are the largest and most efficient light-harvesting antenna systems of all phototrophs. The core part of chlorosomes consists of bacteriochlorophyll c, d, e, or f molecules. In their biosynthetic pathway, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. In this study, in vitro C132-dealkoxycarbonylations of zinc chlorophyll a derivatives bearing a methyl-, ethyl- or propyl-esterifying group and its methyl ester analogs with additional alkyl and hydroxy groups at the C132-position were examined using the BciC enzyme. The BciC-catalyzed reaction activity for the C132-methoxycarbonylated substrate was comparable to that for the ethoxycarbonylated compound; however, depropoxycarbonylation did not proceed. The BciC enzymatic demethoxycarbonylation of zinc methyl C132-alkylated pheophorbides a was gradually suppressed with the elongation of the alkyl chain and finally became inactive for the propyl substrate. The reaction of the C132-hydroxylated substrate (allomer) was accelerated compared to that of the C132-methyl analog possessing a similar steric size, and gave the corresponding C132-oxo product via further air-oxidation. All of the abovementioned enzymatic reactions occurred for one of the C132-epimers with the same configuration as in chlorophyllide a. The above substrate specificities and product distributions indicated the stereochemistry and size of the BciC enzymatic active site (pocket).

Influence of Living Environment during Childhood on Helicobacter pylori Infection in Japanese Young Adults.

INTRODUCTION: Helicobacter pylori infection is usually established during childhood, for which certain responsible environmental factors have been identified. However, the details of the infection routes remain unclear. OBJECTIVE: To determine the relation between H. pylori infection statuses and living environment of Japanese young adult. METHODS: The subjects were 449 healthy young adult medical students of Tsukuba University (299 men and 150 women, mean age: 22.8 years). The H. pylori infection statuses were investigated using the rapid urease test or urine antibody. Questionnaires regarding sanitary conditions including usage of pit toilet or well water and experience of living with one's grandparents during childhood were surveyed. Each item was compared between the H. pylori-positive and -negative groups. RESULTS: Among all participants, 33 (7.3%) were H. pylori-positive. The usage rates of pit toilets were 12.1 and 3.1% for the H. pylori-positive and -negative groups respectively (p = 0.03; OR 4.35, 95% CI 1.33-14.22). The usage rates of well water were 24.2 and 13.7% for the H. pylori-positive and -negative groups respectively (p = 0.07; OR 2.12, 95% CI 0.91-4.98). The proportion of participants with a history of living with their grandparents was significantly greater in the H. pylori-positive group (46.7%) than in the -negative group (20.9%; p = 0.03; OR 3.28, 95% CI 1.13-9.54). Only a history of living with one's grandparents during childhood showed statistical significance in the multivariate regression analysis (p = 0.04; OR 3.20, 95% CI 1.08-9.49). CONCLUSIONS: These results suggest that H. pylori infection is more strongly related to living with one's grandparents than living in a hygienic environment.

Augmented antitumor activity of 5-fluorouracil by double knockdown of MDM4 and MDM2 in colon and gastric cancer cells.

Inactivation of the TP53 tumor suppressor gene is essential during cancer development and progression. Mutations of TP53 are often missense and occur in various human cancers. In some fraction of wild-type (wt) TP53 tumors, p53 is inactivated by upregulated murine double minute homolog 2 (MDM2) and MDM4. We previously reported that simultaneous knockdown of MDM4 and MDM2 using synthetic DNA-modified siRNAs revived p53 activity and synergistically inhibited in vitro cell growth in cancer cells with wt TP53 and high MDM4 expression (wtTP53/highMDM4). In the present study, MDM4/MDM2 double knockdown with the siRNAs enhanced 5-fluorouracil (5-FU)-induced p53 activation, arrested the cell cycle at G1 phase, and potentiated the antitumor effect of 5-FU in wtTP53/highMDM4 human colon (HCT116 and LoVo) and gastric (SNU-1 and NUGC-4) cancer cells. Exposure to 5-FU alone induced MDM2 as well as p21 and PUMA by p53 activation. As p53-MDM2 forms a negative feedback loop, enhancement of the antitumor effect of 5-FU by MDM4/MDM2 double knockdown could be attributed to blocking of the feedback mechanism in addition to direct suppression of these p53 antagonists. Intratumor injection of the MDM4/MDM2 siRNAs suppressed in vivo tumor growth and boosted the antitumor effect of 5-FU in an athymic mouse xenograft model using HCT116 cells. These results suggest that a combination of MDM4/MDM2 knockdown and conventional cytotoxic drugs could be a promising treatment strategy for wtTP53/highMDM4 gastrointestinal cancers.

Newly developed endoscopic detachable snare ligation therapy for colonic diverticular hemorrhage: a multicenter phase II trial (with videos).

BACKGROUND AND AIMS: We previously reported preliminary safety results for a new method, endoscopic detachable snare ligation (EDSL), for diverticular hemorrhage. This method does not need endoscope removal to attach a ligation device after detection of the bleeding site. The aim of the present study was to evaluate the efficacy and safety of EDSL in a larger patient population. METHODS: This prospective study was conducted in 12 institutions. Patients suspected of having diverticular hemorrhage without serious systemic disease were enrolled. The primary endpoint was early (within 30 days) recurrent bleeding rate in patients treated with EDSL. The secondary endpoints were overall early recurrent bleeding rate in patients with definite diverticular bleeding and adverse events in patients treated with EDSL. RESULTS: From June 2015 to March 2017, bleeding diverticula were detected in 123 of 205 enrolled patients (60%), of whom 101 (82%) were treated with EDSL. Most patients (20/22) in whom EDSL was not successful were treated with clipping. The early recurrent bleeding rate was 7.9% (95% confidence interval, 2.6%-13.2%; 8/101) in patients who could be treated with EDSL. The median total endoscopic and EDSL procedure time was 40 minutes (interquartile range, 15-71) and 4 minutes (interquartile range, 1-7), respectively. Two mild adverse events, colonic diverticulitis and temporary abdominal pain, were observed. CONCLUSION: EDSL was confirmed to be useful and safe for treatment of colonic diverticular hemorrhage. (Clinical trial registration number: UMIN 000001858.).

A randomized phase II study comparing S-1 plus weekly split-dose cisplatin with S-1 plus standard-dose cisplatin as first-line chemotherapy for advanced gastric cancer.

BACKGROUND: S-1 plus weekly split-dose cisplatin demonstrated promising results in previous phase I and II studies for advanced gastric cancer (AGC) patients. METHODS: In this randomized phase II study, the efficacy and safety of S-1 plus weekly split-dose cisplatin (SWP, S-1 daily oral dose of 80-120 mg according to body surface area on days 1-14, and cisplatin 20 mg/m(2) i.v. on days 1 and 8 every 3 weeks) were compared with those of S-1 plus standard-dose cisplatin (SP) as first-line chemotherapy for AGC patients. The primary endpoint was 1-year survival rate. RESULTS: Patients were randomized into two groups: 18 in the SWP arm and 19 in the SP arm. This trial was terminated early because of low patient enrollment. The 1-year survival rate was 61 % [95 % confidence interval (CI), 36-86 %] and 53 % (95 % CI, 30-75 %) in the SWP and SP arms, respectively. However, the median survival time was 12.3 months (9.9-14.6 months) and 15.7 months (4.0-27.4 months), respectively (P = 0.064). Progression-free survival was significantly shorter in the SWP arm than in the SP arm (P = 0.047). Toxicity tended to be milder in the SWP arm than in the SP arm. For approximately 40 % of patients in the SWP arm, cisplatin was omitted on day 8 and treatment delayed because of prolonged myelosuppression. CONCLUSIONS: No clear benefits of adding cisplatin to S-1 in the SWP arm were demonstrated in this study. At this point, split-dose cisplatin combined with S-1 cannot be recommended for use in clinical practice.

Preparation of natural high-mannose-type oligosaccharides (Glc1Man9GlcNAc2) with the asparagine-glycine-threonine as consensus sequence from chicken egg yolk.

High-mannose-type glycan structure of N-glycoproteins plays important roles in the proper folding of proteins in sorting glycoprotein secretion and degradation of misfolded proteins in the endoplasmic reticulum (ER). The Glc1Man9GlcNAc2 (G1M9)-type N-glycan is one of the most important signaling molecules in the ER. However, current chemical synthesis strategies are laborious, warranting more practical approaches for G1M9-glycopeptide development. Wang et al. reported the procedure to give G1M9-Asn-Fmoc through chemical modifications and purifications from 40 chicken eggs, but only 3.3 mg of G1M9-glycopeptide was obtained. Therefore, better methods are needed to obtain more than 10 mg of G1M9-glycopeptide. In this study, we report the preparation of G1M9-glycopeptide (13.2 mg) linking Asn-Gly-Thr triad as consensus sequence from 40 chicken eggs. In this procedure, λ-carrageenan treatment followed by papain treatment was used to separate the Fc region of IgY antibody that harbors high-mannose glycans. Moreover, cotton hydrophilic interaction liquid chromatography was adapted for easy purification. The resulting G1M9-Asn(Fmoc)-Gly-Thr was identified by nuclear magnetic resonance and mass spectroscopy. G1M9-Asn(Fmoc)-Gly, G1M9-Asn(Fmoc), and G1M9-OH were also detected by mass spectroscopy. Here, our developed G1M9-tripeptide might be useful for the elucidation of glycoprotein functions as well as the specific roles of the consensus sequence.

In vitro reversible dehydration in C3-substituents of zinc chlorophyll analogs by BchF and BchV enzymes: Stereoselectivity and substrate specificity in the dehydration.

In the biosynthetic pathway of bacteriochlorophyll(BChl)-a/b/c/d/e molecules, BchF and BchV enzymes catalyze the hydration of a C3-vinyl to C3-1-hydroxyethyl group. In this study, the in vitro reactions catalyzed by BchF and BchV partially afforded a C31-epimeric mixture of the hydrated products (secondary alcohols), with the primary recovery of the C3-vinylated substrate. The stereoselectivity and substrate specificity for the in vitro reverse enzymatic dehydration were examined using zinc chlorophyll analogs as model substrates by BchF and BchV, which were obtained from extracts of Escherichia coli overexpressing the respective genes from Chlorobaculum tepidum and used without further purification. Both BchF and BchV preferred dehydration of the (31R)-epimers over the (31S)-epimers. The (31R)-epimer was directly dehydrated by BchF and BchV to give the C3-vinylated product. By contrast, two reaction pathways for BchF and BchV dehydrations of the (31S)-epimer were proposed: (1) the (31S)-epimer would be directly dehydrated to C3-vinyl group. (2) the (31S)-epimer would be epimerized to the (31R)-epimer, and the resulting epimer was dehydrated. The results indicated that both BchF and BchV did function as a hydratase/dehydratase and could play a role in the C31-epimerization. An increase in the alkyl size at the C8-position gradually suppressed the BchF and BchV-catalyzed dehydration in vitro, while the C121- and C20-methylation only slightly affected the reaction. Using the BchF dehydration, a large amount of 3-vinyl-bacteriochlorophyllide-a was successfully prepared, with the retention of the chemically labile, central magnesium atom.

Chiral-phase HPLC separation of (divinyl-)protochlorophyllide-a enantiomers as key precursors in chlorophyll biosynthesis from their 132-stereoisomeric prime forms.

Protochlorophyllide(PChlide)-a and its 8-vinylated analog, divinyl(DV)-PChlide-a, are common and essential intermediates in the biosynthesis of all naturally occurring chlorophyll (Chl) pigments. These porphyrinoid-type pigments have a single optically active (asymmetric) carbon atom at the 132-position, so their stereoisomers are (132R)- and (132S)-enantiomers. The former and latter are called (DV-)PChlide-a and (DV-)PChlide-a', respectively. In this study, chiral-phase HPLC separation of enantiomeric (DV-)PChlides-a/a' was demonstrated. The (132R)-enantiomeric PChlide-a was eluted more slowly than the corresponding (132S)-enantiomeric PChlide-a' under the present HPLC conditions. On the other hand, the elution order of (132R)-DV-PChlide-a and (132S)-DV-PChlide-a' was reverse to that of PChlides-a/a'. After the separation of each enantiomer by the chiral-phase HPLC, the stereoisomeric configuration at the 132-position was characterized by means of circular dichroism spectroscopy. The present chiral-phase HPLC method enables us to evaluate optical purities of (DV-)PChlide-a species. For example, PChlide-a and/or DV-PChlide-a extracted from the spent medium and harvested cells of cultured purple photosynthetic bacterial mutants, the former of which has been often used as the source of (DV-)PChlide-a substrates for enzymatic reactions, were revealed to be mostly racemized, giving enantiomeric mixtures of (DV-)PChlides-a/a'.

Effect of early nutritional initiation on post-cerebral infarction discharge destination: A propensity-matched analysis using machine learning.

AIM: Malnutrition is associated with poor outcomes in cerebral infarction patients, with research indicating that early nutritional initiation may improve the short-term prognosis of patients. However, evidence supported by big data is lacking. Here, to determine the effect of nutritional initiation during the first 3 days after hospital admission on home discharge rates, propensity score matching was conducted in patients with acute cerebral infarction. METHODS: This retrospective observational study, using the Diagnosis Procedure Combination anonymised database in Japan, included 41 477 ischaemic cerebral infarction patients hospitalised between 2016 and 2019. The patients were divided into two groups: those who received oral or enteral nutrition during the first 3 days of hospital admission (early nutrition group, n = 37 318) and those who did not (control group, n = 4159). One-to-one pair-matching was performed using propensity scores calculated via extreme gradient boosting to limit the confounding variables of the two groups. RESULTS: After propensity score matching, 3541 pairs of patients were selected. The dependence of home discharge rates on early nutrition was significant (p < 0.05), and the effectiveness of early nutrition for home discharge showed an odds ratio of 1.79 (95% confidence interval of 1.59-2.03 in Fisher's exact test). CONCLUSIONS: Our findings revealed that early nutritional initiation during the first 3 days of admission resulted in higher home discharge rates.

MDM4 as a Prognostic Factor for Patients With Gastric Cancer With Low Expression of p53.

BACKGROUND/AIM: The oncoproteins murine double minute (MDM) 2 and MDM4 inactivate tumor-suppressor protein p53. Their mutual relationship with the prognosis of gastric cancer (GC) remains unknown. PATIENTS AND METHODS: Expression of MDM2, MDM4, and p53 in tumors of 241 patients with GC were evaluated immunohistochemically. Effects of overexpression of MDM4 on tumor-growth properties and sensitivity to cytotoxic drugs were investigated using NUGC4 human GC cell line. RESULTS: High expression of p53 was associated with poor overall survival in the whole population. Among 173 patients with low expression of p53 (implying nonmutation), high expression of MDM4 was an independent factor of poor prognosis in both stage I-III and IV, but of MDM2 was not. MDM4-transduced NUGC4 cells formed twice as many colonies and had a higher 50% inhibitory concentration for 5-fluorouracil and oxaliplatin than did the control cells. CONCLUSION: MDM4 expression is a factor conferring poor prognosis in patients with GC with low expression of p53 and may confer drug resistance.

Enhanced G1 arrest and apoptosis via MDM4/MDM2 double knockdown and MEK inhibition in wild-type TP53 colon and gastric cancer cells with aberrant KRAS signaling.

Murine double minute homolog 2 (MDM2) is an oncoprotein that induces p53 degradation via ubiquitin-ligase activity. MDM4 cooperates with MDM2-mediated p53 degradation, directly inhibiting p53 transcription by binding to its transactivation domain. Our previous study reported that the simultaneous inhibition of MDM2 and MDM4 using nutlin-3 (an inhibitor of the MDM2-p53 interaction) and chimeric small interfering RNA with DNA-substituted seed arms (named chiMDM2 and chiMDM4) more potently activated p53 than the MDM2 or MDM4 inhibitor alone and synergistically augmented antitumor effects in various types of cancer cells with the wild-type (wt) TP53. Recently, the synergism of MDM2 and mitogen-activated protein kinase kinase (MEK) inhibitors has been demonstrated in wt TP53 colorectal and non-small cell lung cancer cells harboring mutant-type (mt) KRAS. The current study examined whether chiMDM4 augmented the synergistic antitumor effects of MDM2 and MEK inhibition using chiMDM2 or nutlin-3 and trametinib, respectively. ChiMDM2 and trametinib used in combination demonstrated a synergistic antitumor activity in HCT116 and LoVo colon cancer cells, and SNU-1 gastric cancer cells harboring wt TP53 and mt KRAS. Furthermore, chiMDM4 synergistically enhanced this combinational effect. Similar results were observed when nutlin-3 was used instead of chiMDM2. MDM4/MDM2 double knockdown combined with trametinib treatment enhanced G1 arrest and apoptosis induction. This was associated with the accumulation of p53, suppression of phosphorylated-extracellular signal-regulated kinase 2, inhibition of retinoblastoma phosphorylation, suppression of E2F1-activated proteins, and potent activation of pro-apoptotic proteins, such as Fas and p53 upregulated modulator of apoptosis. The results inidcated that the triple inhibition of MDM4, MDM2 and MEK exerted a potent antitumor effect in wt TP53 colon and gastric cancer cells with mt KRAS. Simultaneous activation of p53 and inhibition of aberrant KRAS signaling may be a rational treatment strategy for gastrointestinal tumors.

Stereoselective C3-substituent modification and substrate channeling by oxidoreductase BchC in bacteriochlorophyll a biosynthesis.

We report the in vitro activity of recombinant BchC oxidoreductase involved in bacteriochlorophyll a biosynthesis. BchC of Rhodobacter capsulatus preferentially oxidizes 31 R-3-(1-hydroxyethyl)-chlorophyllide a and 31 R-3-(1-hydroxyethyl)-bacteriochlorophyllide a in the presence of NAD+ to 3-acetyl-chlorophyllide a and bacteriochlorophyllide a, respectively, leaving the unreacted 31 S-epimers. In the reverse reaction, BchC with NADH predominately produces 31 R-epimeric alcohols from the 3-acetyl-(bacterio)chlorins. BchC of Chlorobaculum tepidum demonstrates the same 31 R-selectivity, suggesting that utilization of 31 R-epimers in BchC-catalyzed reductions may be conserved across different phyla of photosynthetic bacteria. Additionally, the presence of BchC accelerates the 3-vinyl hydration by BchF hydratase of Chlorobaculum tepidum during conversion of chlorophyllide a to 3-acetyl-chlorophyllide a through 3-(1-hydroxyethyl)-chlorophyllide a, indicating that these enzymes work cooperatively to promote efficient bacteriochlorophyll a biosynthesis.

Synthesis of submicrometer-sized titania spherical particles with a sol-gel method and their application to colloidal photonic crystals.

A synthetic method for preparing submicrometer-sized titania particles is proposed, which is based on hydrolysis of titanium alkoxide with the use of a cosolvent and an amine catalyst for alkoxide hydrolysis. The preparation was performed with different amines of ammonia, methylamine (MA), and dimethylamine (DMA) in different solvents of ethanol/acetonitrile, ethanol/methanol, ethanol/acetone, ethanol/acetonitrile, and ethanol/formamide for 0.1-0.3 M water and 0.03 M titanium tetraisopropoxide (TTIP) at temperatures of 10-50 degrees C. The use of the ethanol/acetonitrile solvent with MA was required for preparing monodispersed, spherical particles. The number average of the titania particle sizes and their coefficient of variation were varied from 143 to 551 nm and from 5.7 to 20.6%, respectively, with reaction temperature and concentrations of water and MA. Colloidal crystals of titania particles fabricated with a sedimentation method revealed reflection peaks attributed to Bragg's diffraction. Annealing at 100-1000 degrees C led to shrinkage and crystallization of titania particles followed by an increase in the refractive index of titania particles.