Characterization of anti-complementary acidic heteroglycans from the seed of Coix lacryma-jobi var. ma-yuen

The anti-complementary polysaccharides, CA-1 and CA-2, were purified from the seed of Coix lacryma-jobi L. var. ma-yuen. CA-1 consists of rhamnose, arabinose, xylose, galactose, galacturonic acid and glucuronic acid in molar ratios of 1.8:43.8:10.8:33.2:3.2:7.2, and CA-2 consists of rhamnose, arabinose, xylose, mannose, galactose, glucose, galacturonic acid and glucuronic acid in molar ratios of 2.4:37.0:11.8:1.7:35.6:2.9:2.6:6.0. CA-1 and CA-2 contained 8 ∼ 11% protein. Their M_rs were estimated to be 160 000 in CA-1 and 70 000 in CA-2 by gel filtration. CA-2 showed more potent anti-complementary activity than CA-1 in low dose.Methylation analysis of CA-1, its carboxyl-reduced products (reduced CA-1a and CA-1b) and CA-2 were carried out by the use of GC/MS and the results suggested that CA-1 has a very complicated and highly branched structure, and CA-2 is also composed of the same glycosidic linkages as CA-1 in different molar proportions. The results of exo α-L-arabinofuranosidase treatment and partial acid hydrolysis suggested that CA-1 and CA-2 contained arabino 3,6-galactan moiety and most of the arabinose was present as an α-L-furanosyl residues in the non-reducing terminals and (1 → 5)-linked side chains which mostly attached to the O-3 of (1 → 6)-linked galactopyranosyl residues. The results also suggested that CA-1 and CA-2 contained rhamnogalacturonan moiety which has a main chain consisting of (1 → 4)-linked galacturonic acid and (1 → 2)-linked rhamnose, and arabino 3,6- and 4-galactan might be attached to the rhamnosyl residue at the O-4. All glucuronic acid residues were present at the non-reducing terminals.     

Mechanism of the phosphorylase reaction. Utilization of D-gluco-hept-1-enitol in the absence of primer

alpha-Glucan phosphorylases from rabbit skeletal muscle, potato tubers and Escherichia coli catalyze the utilization of 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) in the presence of arsenate or phosphate. 1H-NMR analysis in the presence of 2H2O and arsenate indicated formation of 1-[1-2H]deoxy-alpha-D-glucoheptulose with rates comparable to the arsenolysis of poly- or oligosaccharides. The reaction depends on the presence of a dianionic 5'-phosphate group of pyridoxal in the active conformation of the phosphorylases. Heptenitol is the first known substrate of alpha-glucan phosphorylases which does not require a primer. This is explained by the finding that heptenitol is exclusively used as substrate for the degradative pathway of the phosphorylase reaction where it competes with polysaccharide substrates. In the presence of phosphate the reaction product is 1-deoxy-alpha-D-gluco-heptulose 2-phosphate (heptulose-2-P), which subsequently inhibits the reaction. This characterizes heptulose-2-P as an enzyme-derived inhibitor. The Ki = 1.9 X 10(-6) M with potato phosphorylase suggests the formation of a transition-state-like enzyme-ligand complex. These findings, together with the fact that the phosphates of heptulose-2-P and pyridoxal 5'-phosphate are linked by hydrogen bridges [Klein, H. W., Im, M. J., Palm, D. & Helmreich, E. J. M. (1984) Biochemistry 23, 5853-5861], make it likely that both phosphates are involved in phosphorylase catalysis. A catalytic mechanism of phosphorylase action is proposed in which a 'mobile' phosphate anion plays a versatile role. It serves as proton carrier for the substrate activation, it stabilizes the intermediate and acts as a nucleophile which can accept a glycosyl residue reversibly.     

Structural basis of the catalytic reaction mechanism of novel 1,2-alpha-L-fucosidase from Bifidobacterium bifidum

1,2-alpha-L-fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fucalpha1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2'-fucosyllactose (substrate), and L-fucose and lactose (products) at 1.12-2.10 A resolution. The AfcA Fuc domain is composed of four regions, an N-terminal beta region, a helical linker, an (alpha/alpha)6 helical barrel domain, and a C-terminal beta region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of L-fucose moiety in 2'-fucosyllactose, and its location is nearly identical near the O1 atom of beta-L-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA.     

MiR-423-5p inhibition alleviates cardiomyocyte apoptosis and mitochondrial dysfunction caused by hypoxia/reoxygenation through activation of the wnt/β-catenin signaling pathway via targeting MYBL2

MicroRNA (miR) plays an integral role in cardiovascular diseases. M-iR-423-5p is aberrantly expressed in patients with myocardial infarction and heart failure. The aim of the present study was to study the roles and mechanisms of miR-423-5p in hypoxia/reoxygenation (H/R) mediated cardiomyocytes injury. H9C2 cells were transfected with negative control, miR-423-5p mimic, and inhibitor for 48 hr, followed by exposed to H/R condition. Cell apoptosis rate, caspase 3/7 activities, Bax and cleaved-caspase 3 (c-caspase 3) protein levels were assayed by flow cytometry, Caspase-Glo 3/7 Assay kit, western blot analysis, respectively. Furthermore, the mitochondrial membrane potential, adenosine triphosphate (ATP) content, reactive oxygen species (ROS) production, and Drp1 expression were also investigated. Furthermore, the dual-luciferase reporter assay was used to evaluate the relationship between miR-423-5p and Myb-related protein B (MYBL2). The roles of miR-423-5p in wnt/β-catenin were assessed by western blot analysis. The results revealed that H/R triggered miR-423-5p expression. Overexpression of miR-423-5p promoted cardiomyocyte apoptosis, enhanced the activities of caspase 3/7, upregulated the expression of Bax and c-caspase 3. miR-423-5p upregulation caused the loss of mitochondrial membrane potential and the reduction of ATP content, the augment of ROS production and Drp1 expression. However, the opposite trends were observed upon suppression of miR-423-5p. In addition, miR-423-5p could target the 3′ untranslated region of MYBL2. miR-423-5p depletion led to the activation of the wnt/β-catenin signaling pathway via targeting MYBL2. Knockdown of MYBL2 was obviously reversed the roles of miR-423-5p in apoptosis and mitochondrial dysfunction. Taken together, miR-423-5p suppression reduced H/R-induced cardiomyocytes injury through activation of the wnt/β-catenin signaling pathway via targeting MYBL2 in cardiomyocytes.     

Synthesis and biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin

Two new aryl azides, (Z)-1-(3'-azido-4'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 9 and (Z)-1-(4'-azido-3'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 5, modeled after the potent antitumor, antimitotic agent combretastatin A-4 (CA-4), have been prepared by chemical synthesis as potentially useful photoaffinity labeling reagents for the colchicine site on beta-tubulin. Aryl azide 9, in which the 3'-hydroxyl group of CA-4 is replaced by an azido moiety, demonstrates excellent in vitro cytotoxicity against human cancer cell lines (NCI 60 cell line panel, average GI50 = 4.07 x 10(-8) M) and potent inhibition of tubulin polymerization (IC50 = 1.4+/-0.1 microM). The 4'-azido analogue 5 has lower activity (NCI 60 cell line panel, average GI50 = 2.28 x 10(-6) M, and IC50 = 5.2+/-0.2 microM for inhibition of tubulin polymerization), suggesting the importance of the 4'-methoxy moiety for interaction with the colchicine binding site on tubulin. These CA-4 aryl azide analogues also inhibit binding of colchicine to tubulin, as does the parent CA-4, and therefore these compounds are excellent candidates for photoaffinity labeling studies.     

Trypanosoma brucei: chemical evidence that cathepsin L is essential for survival and a relevant drug target

The protozoan parasite causing human African trypanosomiasis, Trypanosoma brucei, displays cysteine peptidase activity, the chemical inhibition of which is lethal to the parasite. This activity comprises a cathepsin B (TbCATB) and a cathepsin L (TbCATL). Previous RNA interference (RNAi) data suggest that TbCATB rather than TbCATL is essential to survival even though silencing of the latter was incomplete. Also, chemical evidence supporting the essentiality of either enzyme which would facilitate a target-based drug development programme is lacking. Using specific peptidyl inhibitors and substrates, we quantified the contributions of TbCATB and TbCATL to the survival of T. brucei. At 100 μM, the minimal inhibitory concentration that kills all parasites in culture, the non-specific cathepsin inhibitors, benzyloxycarbonyl-phenylalanyl-arginyl-diazomethyl ketone (Z-FA-diazomethyl ketone) and (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline methyl ester (CA-074Me) inhibited TbCATL and TbCATB by >99%. The cathepsin L (CATL)-specific inhibitor, ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) (CAA0225), killed parasites with >99% inhibition of TbCATL but only 70% inhibition of TbCATB. Conversely, the cathepsin B (CATB)-specific inhibitor, (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074), did not affect survival even though TbCATB inhibition at >95% was statistically indistinguishable from the complete inhibition by Z-FA-diazomethyl ketone and CA-074Me. The observed inhibition of TbCATL by CA-074 and CA-074Me was shown to be facilitated by the reducing intracellular environment. All inhibitors, except the CATB-specific inhibitor, CA-074, blockaded lysosomal hydrolysis prior to death. The results suggest that TbCATL, rather than TbCATB, is essential to the survival of T. brucei and an appropriate drug target.     

Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer

The possibility of using aged porcine oocytes treated with caffeine, which inhibits the decrease in M-phase promoting factor activity, for pig cloning was evaluated. Cumulus-oocyte complexes (COCs) were cultured initially for 36 h and subsequently with or without 5 mM caffeine for 24 h (in total for 60 h: 60CA+ or 60CA- group, respectively). As a control group, COCs were cultured for 48 h without caffeine (48CA-). The pronuclear formation rates at 10 h after electrical stimulation in the 60CA+ and 60CA- groups decreased significantly (p < 0.05) compared with the 48CA- group. However, the fragmentation rate was significantly higher (p < 0.05) in the 60CA- group than in the 60CA+ and 48CA- groups. When the stimulated oocytes were cultured for 6 days, the 60CA+ group showed significantly lower blastocyst formation and higher fragmentation or degeneration rates (p < 0.05) than the 48CA- group. However, the number of total cells in blastocysts was not affected by maturation period or caffeine treatment. When somatic cell nuclei were injected into the non-enucleated oocytes and exposed to cytoplasm for a certain duration (1-11 h) before the completion of maturation (48 or 60 h), the rate of nuclear membrane breakdown after exposure to cytoplasm for 1-2 h in the 60CA- oocytes was significantly lower (p < 0.05) than in the other experimental groups. The rate of scattered chromosome formation in the same 60CA- group tended to be lower (p = 0.08) than in the other groups. After the enucleation and transfer of nuclei, blastocyst formation rates in the 60CA+ and 60CA- groups were significantly lower (p < 0.05) than in the 48CA- group. Blastocyst quality did not differ among all the groups. These results suggest that chromosome decondensation of the transplanted somatic nucleus is affected by both the duration of exposure to cytoplasm and the age of the recipient porcine oocytes, and that caffeine treatment promotes nuclear remodelling but does not prevent the decrease in the developmental ability of cloned embryos caused by oocyte aging.     

LncRNA CRNDE promotes cell proliferation,migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis

Ovarian cancer seriously threatens the health of women. LncRNA CRNDE is known to be upregulated in ovarian cancer. However, the mechanism by which CRNDE regulates the progress of ovarian cancer is largely unknown. MTT assay was applied to measure the cell viability. Colony formation assay was used to measure the cell proliferation. Cell migration was tested by wound healing, and Transwell assay was performed to detect cell invasion. In addition, the expression of miR-423-5p, CRNDE and FSCN1 were detected by RT-qPCR and western blotting, respectively. Meanwhile, dual-luciferase reporter assay and RIP assay were performed to explore the correlation between miR-423-5p and CRNDE (or FSCN1). CRNDE and FSCN1 were upregulated in ovarian cancer cells (SKOV3, CAOV-3, IGROV1, A2780 and C13K), while miR-423-5p was downregulated. Moreover, silencing of FSCN1/CRNDE significantly decreased proliferation, migration and invasion of ovarian cancer cells (SKOV3 and CI3K) via suppressing MMP-2 and MMP-9. In addition, CRNDE could sponge miR-423-5p, and FSCN1 was confirmed to be the direct target of miR-423-5p. Furthermore, CRNDE knockdown-induced inhibition of FSCN1 was notably reversed by miR-423-5p downregulation. Knockdown of CRNDE inhibited cell proliferation, migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis. Thus, CRNDE may serve a new target for ovarian cancer.

     

Nucleoside Phosphorylases from Clostridium Perfringens in the Synthesis of 2′,3′-Dideoxyinosine

Four Clostridium perfringens phosphorylases were subcloned, overexpressed and analyzed for their substrate specificity. DeoD(1) and PunA could use a variety of purine substrates, including an antiviral drug 2′,3′-dideoxyinosine (ddI). In one-pot synthesis using Clostridium phosphorylases, 2′,3′-dideoxyuridine and hypoxanthine were converted to ddI at yield of about 30%.     

Modification of essential carboxyl group in rabbit muscle phosphorylase by water-soluble carbodiimide

Water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC) and glycine ethyl ester (GEE) as a nucleophile were used to modify the essential carboxyl group of phosphorylases. The inactive b form of the muscle phosphorylase was modified faster than the active a form and potato phosphorylases. Use of N,N,N',N'-tetramethyl-ethylenediamine (TEMED)-HCl buffer system (pH 6.2) resulted in a remarkable difference from the previous results obtained with phosphate and beta-glycerophosphate buffer systems. That is, the substrate glucose 1-phosphate gave the best protection of the three phosphorylase activities. Glucose and glycogen were also effective to retard the inactivation of muscle phosphorylases, though glycogen was not effective for the potato enzyme. The EDC-GEE-modified phosphorylase b retained the affinity for AMP-Sepharose, though partially modified enzyme completely lost the homotropic cooperativity. Phosphorylase b was subjected to differential labeling with [14C]GEE. A labeled peptide was obtained after CNBr cleavage and peptic digestions, and corresponded to the catalytic site sequence surrounding the GEE-substituted Asp 661 and Glu 664. Either or both of these EDC-modified carboxyl residues may have an important role in the catalytic reaction.     

Ligand effects on the dephosphorylation of heart and skeletal muscle specific phosphorylases

Pseudo first order rate constants were determined for the dephosphorylation of heart and skeletal muscle specific phosphorylase a isoenzymes isolated from rabbit and pig using rabbit muscle phosphorylase phosphatase (mol. wt 34,000). The rate constants determined in the absence of ligands, were 4-5 fold lower for heart specific phosphorylases than for skeletal muscle specific ones. Glucose 6-phosphate (0.5-1 mM) enhances the rate of dephosphorylation of heart specific isophosphorylases 3-fold and suspends inhibition by 10(-5) M AMP, however, it has no significant effect on the dephosphorylation of skeletal muscle specific enzymes under the same conditions. Our data support characteristic functional differences between heart and skeletal muscle specific phosphorylases both in rabbit and pig.     

A cathepsin L-specific inhibitor preferentially inhibits degradation of autophagosomal LC3 and GABARAP in HeLa and Huh-7 cells

CAA0225 ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) is a cathepsin L-specific inhibitor recently selected out by extensive screening of a series of new epoxysuccinyl peptides. CAA0225 inhibited rat liver cathepsin L with IC50 values of 1.9 nM, but not rat liver cathepsin B (IC50, >1000-5000 nM). We compared effects of CAA0225 on autophagy with those of CA-074 that was previously developed as a cathepsin B-specific inhibitor. In HeLa and Huh-7 cells cultured under nutrient-deprived conditions both CAA0225 and CA-074 significantly and comparably inhibited degradation of long-lived proteins. Meanwhile, CAA0225 effectively inhibited degradation of LC3-II and GABARAP, whereas CA-074-OMe had only a marginal effect on their levels. Therefore, cathepsin L does not seem to play a general role in the degradation of proteins in the lumen of autophagosomes, but is involved more specifically in the degradation of autophagosomal membrane markers.     

Naphthalene,phenanthrene and surfactant biodegradation

The impact of surfactants on naphthalene and phenanthrene biodegradation and vice versa after surfactant flushing were evaluated using two anionic surfactants: sodium dodecyl sulfate (SDS) and sodium dodecyl benzene sulfonate (SDBS); and two nonionic surfactants: POE (20) sorbitan monooleate (T-maz-80) and octylphenol poly(ethyleneoxy) ethanol (CA-620). Naphthalene and phenanthrene biodegradation varied differently in the presence of different surfactants. Naphthalene biodegradation was not impacted by the presence of SDS. In the presence of T-maz-80 and CA-620, naphthalene biodegradation occurred at a lower rate (0.14 d^-1 for T-maz-80 and 0.19 d^-1 for CA-620) as compared to un-amended control (0.29 d^-1). Naphthalene biodegradation was inhibited by the presence of SDBS. In the presence of SDS, phenanthrene biodegradation occurred at a lower rate (0.10 d^-1 as compared to un-amended control of 0.17 d^-1) and the presence of SDBS, CA-620 and T-maz-80 inhibited phenanthrene biodegradation. The surfactants also responded differently to the presence of naphthalene and phenanthrene. In the presence of naphthalene, SDS biodegradation was inhibited; SDBS and T-maz-80 depleted at a lower rate (0.41d^-1 and 0.12 d^-1 as compared to 0.48 d^-1 and 0.22 d^-1). In the absence of naphthalene, CA-620 was not degradable, while in the presence of naphthalene, CA-620 began to degrade at a comparatively low rate (0.12 d^-1). In the presence of phenanthrene, SDS biodegradation occurred at a lower rate (1.2 d^-1 as compared to 1.68 d^-1) and a similar trend was observed for T-maz-80. The depletion of SDBS and CA-620 did not change significantly. The choice of SDS for naphthalene-contaminated sites would not adversely affect the natural attenuation of naphthalene, in addition, naphthalene was preferentially utilized to SDS by naphthalene-acclimated microorganisms. Therefore, SDS was the best choice. T-maz-80 was also found to be usable in naphthalene-contaminated sites. For phenanthrene contaminated sites, SDS was the only choice.     

Relationships between free-living protozoa, cultivable Legionella spp., and water quality characteristics in three drinking water supplies in the Caribbean

The study whose results are presented here aimed at identifying free-living protozoa (FLP) and conditions favoring the growth of these organisms and cultivable Legionella spp. in drinking water supplies in a tropical region. Treated and distributed water (±30°C) of the water supplies of three Caribbean islands were sampled and investigated with molecular techniques, based on the 18S rRNA gene. The protozoan host Hartmannella vermiformis and cultivable Legionella pneumophila were observed in all three supplies. Operational taxonomic units (OTUs) with the highest similarity to the potential or candidate hosts Acanthamoeba spp., Echinamoeba exundans, E. thermarum, and an Neoparamoeba sp. were detected as well. In total, 59 OTUs of FLP were identified. The estimated protozoan richness did not differ significantly between the three supplies. In supply CA-1, the concentration of H. vermiformis correlated with the concentration of Legionella spp. and clones related to Amoebozoa predominated (82%) in the protozoan community. These observations, the low turbidity (<0.2 nephelometric turbidity units [NTU]), and the varying ATP concentrations (1 to 12 ng liter(-1)) suggest that biofilms promoted protozoan growth in this supply. Ciliophora represented 25% of the protozoan OTUs in supply CA-2 with elevated ATP concentrations (maximum, 55 ng liter(-1)) correlating with turbidity (maximum, 62 NTU) caused by corroding iron pipes. Cercozoan types represented 70% of the protozoan clones in supply CA-3 with ATP concentrations of <1 ng liter(-1) and turbidity of <0.5 NTU in most samples of distributed water. The absence of H. vermiformis in most samples from supply CA-3 suggests that growth of this protozoan is limited at ATP concentrations of <1 ng liter(-1).     

CA-125 monitoring during chemotherapy for ovarian cancer

Serum CA-125 was determined in 60 patients with variously extended ovarian cancer and monitored during and after chemotherapy. The study indicates that: 1) prechemotherapy CA-125 shows the presence of an active disease with an accuracy greater than 85%. The antigen is elevated in 97% of patients with greater than 2 cm disease, but sensitivity is low (67%) in patients with minimal residual disease (less than 2 cm); 2) changes in CA-125 correspond well with the response to chemotherapy. CA-125 becomes negative in every patient having clinical CR and increases in every patient with progressive disease. These changes can indicate the type of response some months ahead of time; 3) CA-125 indicates in advance the recurrence of the disease after an objective remission: 4) In the conditions studied CA-125 basal levels do not seem to have prognostic value as regarding either response or survival.     

MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation

In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2.