13C-NMR studies of the effects of the carcinogen acetylaminofluorene on the conformation of dinucleoside monophosphate

13C-NMR spectra are obtained in aqueous solution of dinucleoside monophosphates (ApG and GpA) and of their adducts formed by the addition of the carcinogen acetylaminofluorene (AAF) to the C8 position of the guanine. The base and sugar carbons of all dimers and adducts are assigned. The task of assigning base and carbohydrate resonances was accomplished using a series of reference compounds. Significant changes in many of the carbon resonances of the adducts are observed suggesting three general conformational changes, namely: (1) chemical shift changes are noted in base carbon atom resonances as a function of temperature and adduct formation which are indicative of stacking effects; (2) large upfield shifts of the furanose C2' resonance of the guanosine-adduct indicate a shift to higher populations of the syn conformation. Other shifts of carbohydrate resonances are indicative of a change in conformation of the carbohydrate itself. (3) Large temperature effects on linewidth of several fluorine and furanose resonances indicate interconversion of various conformers in the dimer adduct.     

Increased -1 ribosomal frameshifting efficiency by yeast prion-like phenotype [PSI+]

Programmed -1 ribosomal frameshifting (-1RF) is usually regulated by a slippery sequence and an RNA secondary structure but can be affected by the slippery sequence combined with translational perturbation. Dramatic increases of -1RF efficiencies arise from the slippery sequences in [PSI+], an epigenetic modifier of translation termination fidelity. Curing of [PSI+] abolished such increases of -1RF efficiency. Enhanced -1RF frequency at the slippery sequences could be another physiological effect induced directly or indirectly by the perturbation of the translation process in [PSI+] cells.     

Hydroxylation of carbazoles by Aspergillus flavus VKM F-1024

Carbazole was metabolized by Aspergillus flavus VKM F-1024 forming few monohydroxylated products. The structure of metabolites was determined by TLC, GC, MS and (1)H NMR analyses. 3-Hydroxycarbazole was revealed as a major bioconversion product, 1-hydroxy- and 2-hydroxycarbazoles were observed as minor products. In the presence of 1-benzoylindole, the hydroxylation position shifted toward preferable accumulation of 2-hydroxycarbazole and the formation of 2,6- and 2,7-dihydroxycarbazoles. This effect and microbial formation of these metabolites have never been reported before. At the conversion of N-acetyl- and N-benzoylcarbazoles, carbazole was the major product, while 1-, 2- and 3-monohydroxycarbazoles were formed in small amounts.     

14-3-3γ蛋白协同mTOR信号通路影响奶牛乳腺上皮细胞生理功能

14-3-3γ作为蛋白质与蛋白质之间的"桥梁蛋白",广泛参与细胞凋亡、细胞分裂、信号转导和蛋白质合成等生物体所有的重要生命活动的调节。以体外培养的荷斯坦奶牛(Vaccas)乳腺上皮细胞(dairy cow mammary gland epithelial cells,DCMECs)为模型,研究14-3-3γ对奶牛乳腺上皮细胞乳蛋白、乳脂合成的影响。瞬时和稳定转染p GCMV/IRES/EGFP/14-3-3γ即14-3-3γ过表达的奶牛乳腺上皮细胞,应用CASY细胞活力分析仪分析细胞活力的变化,测定细胞中甘油三脂(triglyceride,TG)和β-酪蛋白(β-Casein,CSN2)的合成,并且通过Western blot检测DCMECs中m TOR(mammalian target of rapamycin,m TOR)和p-m TOR的表达量,探究14-3-3γ影响奶牛乳腺上皮细胞生理功能的机制。结果显示,瞬时和稳定转染p GCMV/IRES/EGFP/14-3-3γ的奶牛乳腺上皮细胞相对正常培养的奶牛乳腺上皮细胞,细胞活力极显著增强(P0.01),CSN2和TG表达量极显著增加(P0.01),同时m TOR和p-m TOR的表达量也显著增加(P0.01)。研究结果表明14-3-3γ参与泌乳关键信号通路即m TOR信号通路,能够增强乳腺上皮细胞活力,促进奶牛乳腺上皮细胞CSN2和TG的合成,丰富了奶牛泌乳信号通路,为乳品质研究提供了新的理论依据。     

The state transition mechanism—simply depending on light-on and -off in Spirulina platensis

The state transition in cyanobacteria is a long-discussed topic of how the photosynthetic machine regulates the excitation energy distribution in balance between the two photosystems. In the current work, whether the state transition is realized by “mobile phycobilisome (PBS)” or “energy spillover” has been clearly answered by monitoring the spectral responses of the intact cells of the cyanobacterium Spirulina platensis. Firstly, light-induced state transition depends completely on a movement of PBSs toward PSI or PSII while the redox-induced one on not only the “mobile PBS” but also an “energy spillover”. Secondly, the “energy spillover” is triggered by dissociation of PSI trimers into the monomers which specially occurs under a case from light to dark, while the PSI monomers will re-aggregate into the trimers under a case from dark to light, i.e., the PSI oligomerization is reversibly regulated by light switch on and off. Thirdly, PSI oligomerization is regulated by the local H⁺ concentration on the cytosol side of the thylakoid membranes, which in turn is regulated by light switch on and off. Fourthly, PSI oligomerization change is the only mechanism for the “energy spillover”. Thus, it can be concluded that the “mobile PBS” is a common rule for light-induced state transition while the “energy spillover” is only a special case when dark condition is involved.     

Photoactivation of the manganese catalyst of O2 evolution. I. Biochemical and kinetic aspects

Photosynthetic O₂ evolution requires a Mn complex which is activated by light. An analysis of this activation process yielded the following results:

1.

1. In any given illumination, the time course is first order, the rate being proportional to the number of inactive O₂-evolving System II trapping centers (the quantum yield being invariant).     

Heterogeneity of hepatic microsomal UDP-glucuronosyltransferase(s) activities: comparison between human and mammalian species activities

The first comparative profiles of UDP-glucuronosyltransferase(s) (UDPGT) activities obtained under standard conditions in vitro in mammals (man, rat [Wistar and Gunn], mouse, monkey [Papio papio and Cynomolgus], pig, guinea pig, rabbit, dog) are presented for 16 aglycones. A decreasing scale of these activities was obtained from planar to bulky molecules. The scale was identical for each of the mammals studied, including man. Statistical analysis of the results revealed a division of the aglycones into three groups, one being correlated with the molecular form called GT1 the two others with the GT2 form. The profile of activities in the Gunn rat revealed very weak activity towards planar molecules (GT1). These results provide evidence that under standard conditions, human UDPGT activities are comparable to those from other animals.     

Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer-344 rats

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ring-U-14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2-6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by greater than 96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.     

Dual inhibition of cyclooxygenase and lipoxygenase by human haptoglobin: its polymorphism and relation to hemoglobin binding

Haptoglobin (Hp) binds hemoglobin (Hb) specifically and stoichiometrically. Since Hb stimulates prostaglandin (PG biosynthesis), we investigated if Hp effects arachidonic acid (AA) metabolism. The results showed that Hp (50-250 microg protein) inhibited the biosynthesis of PGs via cyclooxygenase (COX) and 12-HETE via lipoxygenase pathway in human platelets. Additional evidence was obtained by the loss of Hp inhibitory activity upon removal of Hp by affinity chromatography on hemoglobin sepharose and by inhibition of AA or bradykinin-induced bronchoconstriction in the guinea pig. Hb reduced the inhibitory effect of Hp in a concentration-related manner such that all its inhibitory activity was lost when completely bound by Hb. Of the three Hp phenotypes, Hp 1-1 showed maximum binding capacity to Hb indicating its greater protective role. These findings implicate Hp in the regulation of COX and lipoxygenase pathways and show Hp involvement in the body's endogenous defense system against inflammation. This indicates that mammals have dual defense system, i.e., a specific immune system and non-specific Hp defense system.     

Biogenesis of chloroplast membranes X. Changes in the photosynthetic specific activity and the relationship between the light harvesting system and photosynthetic electron transfer chain during greening of Chlamydomonas reinhardi y-1 cells

Specific activities of photophosphorylation and light-dependent pH rise at different stages of the greening process, have been measured as a function of the illumination intensity.     

Physiological functions of the TRPM4 channels via protein interactions

Transient Receptor Potential, Melastatin-related, member 4 (TRPM4) channels are Ca²⁺-activated Ca²⁺-impermeable cation channels. These channels are expressed in various types of mammalian tissues including the brain and are implicated in many diverse physiological and pathophysiological conditions. In the past several years, the trafficking processes and regulatory mechanism of these channels and their interacting proteins have been uncovered. Here in this minireview, we summarize the current understanding of the trafficking mechanism of TRPM4 channels on the plasma membrane as well as heteromeric complex formation via protein interactions. We also describe physiological implications of protein-TRPM4 interactions and suggest TRPM4 channels as therapeutic targets in many related diseases. [BMB Reports 2015; 48(1): 1-5]     

The polarity-inducing kinase Par-1 controls Xenopus gastrulation in cooperation with 14-3-3 and aPKC

Par (partitioning-defective) genes were originally identified in Caenorhabditis elegans as determinants of anterior/posterior polarity. However, neither their function in vertebrate development nor their action mechanism has been fully addressed. Here we show that two members of Par proteins, 14-3-3 (Par-5) and atypical PKC (aPKC), regulate the serine/threonine kinase Par-1 to control Xenopus gastrulation. We find first that Xenopus Par-1 (xPar-1) is essential for gastrulation but not for cell fate specification during early embryonic development. We then find that xPar-1 binds to 14-3-3 in an aPKC-dependent manner. Our analyses identify two aPKC phosphorylation sites in xPar-1, which are essential for 14-3-3 binding and for proper gastrulation movements. The aPKC phosphorylation-dependent binding of xPar-1 to 14-3-3 does not markedly affect the kinase activity of xPar-1, but induces relocation of xPar-1 from the plasma membranes to the cytoplasm. Finally, we show that Xenopus aPKC and its binding partner Xenopus Par-6 are also essential for gastrulation. Thus, our results identify a requirement of Par proteins for Xenopus gastrulation and reveal a novel interrelationship within Par proteins that may provide a general mechanism for spatial control of Par-1.     

Characterization of the purified microsomal FAD-containing monooxygenase from mouse and pig liver

The FAD-containing monooxygenase (FMO) has been purified from both mouse and pig liver microsomes by similar purification procedures. Characterization of the enzyme from these two sources has revealed significant differences in catalytic and immunological properties. The pH optimum of mouse FMO is slightly higher than that of pig FMO (9.2 vs. 8.7) and, while pig FMO is activated 2-fold by n-octylamine, mouse FMO is activated less than 20%. Compounds, including primary, secondary and tertiary amines, sulfides, sulfoxides, thiols, thioureas and mercaptoimidazoles were tested as substrates for both the mouse and pig liver FMO. Km- and Vmax-values were determined for substrates representative of each of these groups. In general, the mouse FMO had higher Km-values for all of the amines and disulfides tested. Mouse FMO had Km-values similar to those of pig FMO for sulfides, mercaptoimidazoles, thioureas, thiobenzamide and cysteamine. Vmax-values for mouse FMO with most substrates was approximately equal, indicating that as with pig FMO, breakdown of the hydroxyflavin is the rate limiting step in the reaction mechanism. Either NADPH or NADH will serve as an electron donor for FMO, however, NADPH is the preferred donor. Pig and mouse FMOs have similar affinity for NADPH (Km = 0.97 and 1.1 microM, respectively) and for NADH (Km = 48 and 73 microM, respectively). An antibody, prepared by immunizing rabbits with purified pig liver FMO, reacts with purified pig liver FMO but not with mouse liver FMO, indicating structural differences between these two enzymes. This antibody inhibited pig FMO activity up to 60%.     

NMR and CD studies on the interaction of Alzheimer beta-amyloid peptide (12-28) with beta-cyclodextrin

Polymerization of the amyloid beta-peptide (Abeta) has been identified as a major feature of the pathogenesis of Alzheimer's disease (AD). Inhibition of the formation of these toxic polymers of Abeta has emerged as an approach for developing therapeutics for AD. NMR and CD spectra were used to investigate the interaction between cyclodextrin and Abeta(12-28) peptide, which was reported to be an important region for forming amyloid fibrils. CD spectral analyses show that of the alpha-, beta- and gamma-cyclodextrins only beta-cyclodextrin inhibits the aggregation of Abeta(12-28) at pH 5.0. Analysis of the one-dimensional proton NMR spectra of Abeta(12-28) and the mixture of Abeta(12-28) with beta-cyclodextrin clearly indicates that there are chemical shift changes in the aromatic ring of Phe19 and the methyl groups of Val18 in the peptide. The NOESY spectra show cross-peaks between H-3 and H-5 of beta-cyclodextrin and the aromatic protons of Phe19 and Phe20. These chemical shift differences and NOEs demonstrate that there is an interaction between Abeta(12-28) and beta-cyclodextrin. Analysis of the cross-peak intensity in the NOESY spectra reveals that the aromatic rings of Phe19 and 20 are generally inserted into beta-cyclodextrin at the broad side and are oriented toward the narrow side of the cavity.     

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO2 fixation in the presence of dihydroxyacetone phosphate

1. Dihydroxyacetone phosphate in concentrations ? 2.5 mM completely inhibits CO₂-dependent O₂ evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of P_i, but not by addition of 3-phosphoglycerate. In the absence of P_i, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the ¹⁴C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70–95%. Based on these results the mechanism of the inhibition of O₂ evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts.2. Although O₂ evolution is completely suppressed by dihydroxyacetone phosphate, CO₂ fixation takes place in air with rates of up to 65μ mol · mg^?1 chlorophyll · h^?1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation.3. Under anaerobic conditions, the rates of CO₂ fixation in presence of dihydroxyacetone phosphate are low (2.5–7 μmol · mg^?1 chlorophyll · h^?1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2 · 10^?7 M) reaching values of up to 60 μmol · mg^?1 chlorophyll · h^?1. As under these conditions the ATP necessary for CO₂ fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO₂ fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded that only under anaerobic conditions an “overreduction” of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5 · 10^?7 M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO₂ fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO₂ fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation.4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO₂ fixation in presence of dihydroxyacetone phosphate by dibromothymoquinone under anaerobic conditions indicates that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.     

Transport of mercury compounds across bimolecular lipid membranes: effect of lipid composition, pH and chloride concentration

The use of bimolecular lipid membranes (BLM) as model membrane allows the analysis of the transport of mercury compounds across the lipidic barriers of biological membranes. The results of flux measurements show that two mercury compounds--HgCl2 and CH3HgCl--cross the BLM but the overall permeabilities are dependent on the pH of the aqueous media, and are not apparently influenced by the different phospholipid constituents of the bilayers. On the other hand, electrical measurements show that, function of the chemical speciation, the transport of this metal is done essentially in the neutral form.     

The structures of garcinones a,b and c: Three new xanthones from Garcinia mangostana

Three new tetraoxygenated xanthones (garcinones A, B and C), each disubstituted with C₅-units, have been isolated from the chloroform extract of the fruit-hulls of Garcinia mangostana. Their structures were established by a combination of spectral interpretation and chemical correlation.