A tyrosinase inhibitor, Daedalin A, from mycelial culture of Daedalea dickinsii

A chromene-type compound, daedalin A (1), was isolated from mycelial culture broth of Daedalea dickinsii. Based on spectroscopic data, the structure of 1 was found to be (2R)-6-hydroxy-2-hydroxymethyl-2-methyl-2H-chromene. Daedalin A (1) strongly inhibited the activity of tyrosinase (IC(50): 194 muM). In addition, 1 also showed 1,1-diphenyl-2-picrylhydrazyl scavenging activity (IC(50): 16.9 microM) and superoxide anion scavenging activity (IC(50): 28.5 microM).     

Quercinol, an anti-inflammatory chromene from the wood-rotting fungus Daedalea quercina (Oak Mazegill)

The fungus Daedalea quercina (oak mazegill) was examined for its capability of producing antioxidative and anti-inflammatory compounds. Bioactivity guided fractionation of the extract from a mycelial culture led to the isolation of quercinol, which was identified as (-)-(2S)-2-hydroxymethyl-2-methyl-6-hydroxychromene 1 by NMR and X-ray analyses. The cryptic hydroquinone 1 shows a broad anti-inflammatory activity against cyclooxygenase 2 (COX-2), xanthine oxidase (XO), and horseradish peroxidase (HRP) at micromolar concentrations.     

Genetic and nongenetic influences on the ABH and Lea antigen levels of saliva and milk.

The saliva and milk of 250 parturient women were studied in relation to ABH antigen levels; part of the sample was also investigated for the Lewis (Lea) substance. The levels of A and B are higher in saliva, and those of H and Lea higher in milk. The H average salivary titers presented the relationship O greater than A2 greater than A1 greater than B greater than AB, but these differences were not present in milk. In addition, the salivary levels of A and B are similar in individuals of these groups but B greater than A in AB persons, and A1 greater than A2; while in milk A greater than B in A, B and AB subjects, and A1 approximately equal to A2. The amount of Lea substance depends of the ABH secretor status in both secretions; but independently of this difference, the average titers were always higher in milk. Correlation coefficients between the levels observed in the two secretions are statistically significant for the A substance in A persons (0.46), H in B (0.58) and Lea in all subjects tested (0.47). A stepwise multiple regression analysis performed to verify the influence of four genetic and six nongenetic variables in the ABH levels of both fluids indicated only one consistent modifying factor: ABO type.     

Blood group A and H determinants in polyglycosyl peptides of A1 and A2 erythrocytes

Polyglycosyl peptides were isolated from delipidated erythrocyte membranes of human blood-group A1 and A2 erythrocytes by extensive pronase digestion and gel filtration. As estimated by the amounts of N-acetylgalactosamine and 2-O-substituted galactose residues about 85% of the possible acceptor sites (H determinants) were saturated with A determinants in A1 polyglycosyl peptides whereas only 25% of H sites were filled in A2 glycopeptides. The distribution of A and H determinants in the glycopeptides was studied by affinity chromatography with Sepharose-bound Bandeiraea simplicifolia I-lectin (binds blood-group A and B determinants) and Ulex Europeaus I-lectin (binds blood-group H determinants). About 55% of the polyglycosyl peptides contained A, H, or A and H determinants in both A1 and A2 blood subgroups. 48% of the polyglycosyl peptides of blood group A1 and 10% of A2 bound to Bs I-lectin. 25% of the polyglycosyl peptides in A1 and 53% in A2 carried H determinants. The molecular size, monosaccharide composition and the substitution pattern of the monosaccharides in the Bs-I-bound polyglycosyl peptides were very similar in both A1 and A2 blood groups. The only difference was the amount of N-acetylgalactosamine which was on the average 3.7 mol/mol in A1 and 2.5 mol/mol in A2. The active fraction was found to be heterogeneous with respect to the amount of A determinants, which varied from 1 to 6 per glycopeptide in A1 and A2 polyglycosyl peptides. The findings do not indicate a structural difference between blood-group A1 and A2 polyglycosyl peptides and state chemically that A1 glycopeptides contain more A determinants than A2 glycopeptides.     

Flavonol glycosides from Asplenium foreziense and its five related taxa and A. incisum

The flavonoids of Asplenium foreziense, A. fontanum subsp. fontanum and subsp. pseudofontanum, A. obovatum subsp. obovatum var. obovatum and var. protobillotii, A. obovatum subsp. lanceolatum, and A. incisum were isolated and identified for chemotaxonomic survey. A major constituent of all taxa was kaempferol 3-O-gentiobioside. As minor compounds, kaempferol 3,7-O-glycoside and/or kaempferol 3-O-glycoside were found in A. fontanum, A. obovatum and A. foreziense, and kaempferol 3-O-gentiobioside-4'-O-glucoside, kaempferol 3-O-glucoside and quercetin 3-O-diglucoside in A. incisum. It was suggested that A. foreziense, A. fontanum including subsp. pseudofontanum and A. obovatum including subsp. lanceolatum are not only morphologically but also chemotaxonomically related. The East Asian A. incisum was chemically and geographically different from these taxa.     

Lipid peroxidation and physical properties of smooth and rough hepatoma microsomes

Smooth and rough endoplasmic reticulum of two Morris hepatomas, the slow growing 9618A and the fast growing 3924A, have been isolated, and their biochemical composition, supramolecular organization, and response to the action of peroxidative agents have been studied. Cytochrome P450 content and lipid availability are the limiting factors of their peroxidizability. The hemoprotein content is reduced about 80% in hepatoma 9618A and is virtually absent in hepatoma 3924A. The peroxidizability decreases with increasing growth rate of the tumor. The protein, phospholipid, and cholesterol content, the fatty acid composition as well as the double bond index, and the saturated and unsaturated fatty acid content are reported. Differences have been found between normal liver and tumors and between the fractions within a given tumoral tissue. The molecular order, as determined by fluorescence anisotrophy decay of DPH, increases in total microsomes and in the smooth fraction going from liver 9618A to 3924A, whereas for the rough fraction it is the same in liver and hepatoma 9618A; in 3924A it increases of about 30%. Fluidity decreases in total microsomes going from liver to 3924A, to 9618A. In both the purified fractions it decreases with increasing deviation of the tumor.     

Lipid peroxidation and physical properties of smooth and rough hepatoma microsomes

Smooth and rough endoplasmic reticulum of two Morris hepatomas, the slow growing 9618A and the fast growing 3924A, have been isolated, and their biochemical composition, supramolecular organization, and response to the action of peroxidative agents have been studied. Cytochrome P₄₅₀ content and lipid availability are the limiting factors of their peroxidizability. The hemoprotein content is reduced about 80% in hepatoma 9618A and is virtually absent in hepatoma 3924A. The peroxidizability decreases with increasing growth rate of the tumor. The protein, phospholipid, and cholesterol content, the fatty acid composition as well as the double bond index, and the saturated and unsaturated fatty acid content are reported. Differences have been found between normal liver and tumors and between the fractions within a given tumoral tissue. The molecular order, as determined by fluorescence anisotrophy decay of DPH, increases in total microsomes and in the smooth fraction going from liver 9618A to 3924A, whereas for the rough fraction it is the same in liver and hepatoma 9618A; in 3924A it increases of about 30%. Fluidity decreases in total microsomes going from liver to 3924A, to 9618A. In both the purified fractions it decreases with increasing deviation of the tumor.     

Glucuronidation of oxidized fatty acids and prostaglandins B1 and E2 by human hepatic and recombinant UDP-glucuronosyltransferases

Arachidonic acid (AA) can be metabolized to various metabolites, which can act as mediators of cellular processes. The objective of this work was to identify whether AA, prostaglandin (PG) B1 and E2, and 15- and 20-hydroxyeicosatetraenoic acids (15- and 20-HETE) are metabolized via glucuronidation. Assays with human recombinant UDP-glucuronosyltransferase 1A (UGT1A) isoforms revealed that AA and 15-HETE were glucuronidated by UGT1A1, 1A3, 1A4, 1A9, and 1A10, whereas 20-HETE was glucuronidated by UGT1A1 and 1A4 and PGB1 was glucuronidated by UGT1A1, 1A9, and 1A10. All substrates were glucuronidated by recombinant UGT2B7, with AA and 20-HETE being the best substrates. Kinetic analysis of UGT1A1 and 1A9 with AA resulted in Km values of 37.9 and 45.8 microM, respectively. PGB1 was glucuronidated by UGT1A1 with a Km of 26.3 microM. The Km values for all substrates with UGT2B7 were significantly higher than with the UGT1A isoforms. Liquid chromatography-mass spectrometry of glucuronides biosynthesized from PGB1 and 15-HETE showed that hydroxyl groups were the major target of glucuronidation. This work demonstrates a novel metabolic pathway for HETEs and PGs and the role of UGT1A isoforms in this process. These results indicate that glucuronidation may play a significant role in modulation of the availability of these fatty acid derivatives for cellular processes.     

Purification of rat kidney arginases A1 and A4 and their subcellular distribution

Arginase A1 and arginase A4 were isolated from rat kidney. Arginase A4, which is the main form of arginase in rat kidney, was obtained at a highly purified preparation; its specific activity was 1057 mumoles ornithine . min-1 . mg-1 protein. The two forms differed in subcellular localization. Form A1 was restricted to the cytosol while form A4 occurred mainly in the mitochondrial matrix. Kidney arginases A1 and A4 were found to differ in immunological properties. Kidney arginase A1, in contrast to arginase A4, precipitated with antibodies against arginase A1 from rat liver. Arginase A1 from kidney was shown to differ from arginase A1 from the liver. The two enzymes could be distinguished by double diffusion test and immunoelectrophoresis.     

阳生植物和阴生植物的叶黄素循环与非辐射能量耗散X

自然条件下阳生植物和阴生植物的光合速率存在着明显的差距,它们拥有不同的适应强光胁迫的能力,前者明显强于后者。从叶黄素组分来看,阳生植物拥有更大的叶黄素库[紫黄质（V）＋单环环氧玉米黄质（A）＋玉米黄质（Z）],其中Z和[Z＋A]的含量更明显高于阴生植物;从阳生植物或阴生植物内部来看,不同物种间,Z1[Z＋A]和[V＋A＋Z]含量的差异相对较小,A则基本相同;不论是阳生植物还是阴生植物,非光化学猝灭值与Z、[Z＋A]及[V＋A＋Z]含量均呈现较好的正相关关系,后三者含量越高,非光化学猝灭值越大,而且[V＋A＋Z]库的大小与Z含量基本上是成比例增另的。说明在不同植物种间,[Z＋]（主要是Z）仍然是影响非辐射能量耗能的主要因素。     

Multilocus analysis of variation and speciation in the closely related species Arabidopsis halleri and A. lyrata

Nucleotide variation in eight effectively unlinked genes was surveyed in species-wide samples of the closely related outbreeding species Arabidopsis halleri and A. lyrata ssp. petraea and in three of these genes in A. lyrata ssp. lyrata and A. thaliana. Significant genetic differentiation was observed more frequently in A. l. petraea than in A. halleri. Average estimates of nucleotide variation were highest in A. l. petraea and lowest in A. l. lyrata, reflecting differences among species in effective population size. The low level of variation in A. l. lyrata is concordant with a bottleneck effect associated with its origin. The A. halleri/A. l. petraea speciation process was studied, considering the orthologous sequences of an outgroup species (A. thaliana). The high number of ancestral mutations relative to exclusive polymorphisms detected in A. halleri and A. l. petraea, the significant results of the multilocus Fay and Wu H tests, and haplotype sharing between the species indicate introgression subsequent to speciation. Average among-population variation in A. halleri and A. l. petraea was approximately 1.5- and 3-fold higher than that in the inbreeder A. thaliana. The detected reduction of variation in A. thaliana is less than that expected from differences in mating system alone, and therefore from selective processes related to differences in the effective recombination rate, but could be explained by differences in population structure.     

黑线姬鼠与大林姬鼠组织中超氧化物酶和过氧化物酶的分布与活性比较

以黑线姬鼠(Apodemus agrarius)和大林姬鼠(A. peninsulae)为研究对象,采用聚丙烯酰胺凝胶电泳(PAGE)不连续体系的方法,比较分析了心、肝、肾、肌肉、脑、肺6种器官和组织中超氧化物酶(SOD)和过氧化物酶(POD)活性,并建立了2种酶的电泳图谱。结果显示,上述2种酶在黑线姬鼠和大林姬鼠的6种器官和组织中均有表达并表现出明显的特异性,其中,2种鼠中超氧化物酶共分离出迁移率由0.15～0.66的9条电泳谱带,过氧化物酶共分离出迁移率由0.09～0.83的20条电泳谱带。在肝和肺中酶的活性最强,黑线姬鼠6种器官和组织中超氧化物酶活性均强于大林姬鼠,2种鼠组织中过氧化物酶的活性和分布相似,但在同一物种不同器官和组织间过氧化物酶的活性及分布存在明显差异。     

Natural and induced polyploidy in Acacia dealbata Link. and Acacia mangium Willd

Seeds were obtained from seven natural populations of Acacia dealbata, three natural populations of A. mangium and a seed orchard of A. mangium, representing the natural range of the two species. Polyploids were discovered in two of the seven populations of A. dealbata. The 2C DNA amount for diploid A. dealbata (2n = 2x = 26) was 1.74 pg, and for diploid A. mangium (2n = 2x = 26) was 1.30 pg. A naturally occurring tetraploid of A. dealbata (2n = 4x = 52) had a 2C DNA amount of 3.41 pg and a naturally occurring triploid genotype had a 2C DNA amount of 2.53 pg. The use of colchicine and oryzalin was investigated as a means of producing higher frequencies of tetraploids of both A. mangium and A. dealbata for incorporation into breeding programmes. Colchicine treatment gave tetraploid frequencies up to 29% for A. dealbata seedlings, and up to 18% for A. mangium seedlings. In contrast, no tetraploid A. mangium was detected following oryzalin treatment, and the low frequencies of tetraploids observed in A. dealbata could be attributed to their natural occurrence.     

Preparation and properties of alpha- and epsilon-amino-protected porcine relaxin derivatives

The chemical modification of the amino groups of B29 porcine relaxin resulted in pure derivatives of N alpha A1-citraconyl-B29 relaxin, N epsilon A7, N epsilon A16, N epsilon B8-tris [[[(methylsulfonyl)ethyl]oxy]carbonyl]-B29 relaxin (Msc3-relaxin), and N alpha A1, N epsilon A7, N epsilon A16, N epsilon B8-tetrakis [[[(methylsulfonyl)ethyl]oxy]carbonyl]-B29 relaxin (Msc4-relaxin). N alpha A1-Citraconyl-B29 relaxin was obtained after selective deprotection of fully acylated B29 relaxin derivatives. The quantitative reaction of N alpha A1-citraconylrelaxin with [[(methylsulfonyl)ethyl]-oxy]carbonyl succinimide ester followed by deprotection of the citraconyl group resulted in N epsilon A7, N epsilon A16, N epsilon B8-Msc3-B29 relaxin, the starting material for selective chemical modifications at the N terminus of the relaxin A chain. In mouse interpubic ligament assay both Msc3 and Msc4 derivatives of relaxin showed a bioactivity of 30%, while in the case of N alpha A1-citraconyl-B29 relaxin the bioactivity was reduced to 15%. When compared with unmodified relaxin, only the circular dichroic spectrum of N alpha A1-citraconyl-B29 relaxin revealed significant differences. Therefore, the loss in bioactivity of the N alpha A1-citraconyl-B29 relaxin seems to be related to the structural changes caused by the introduction of a negative charge at the N terminus of the A chain.     

Synthesis and biological activity of tricyclic aryloimidazo-, pyrimido-, and diazepinopurinediones

Syntheses and physicochemical properties of N-aryl-substituted imidazo-, pyrimido-, and 1,3-diazepino[2,1-f]purinediones are described. These derivatives were synthesized by the cyclization of 7-haloalkyl-8-bromo-1,3-dimethyl- or 1,3-dipropyl-xanthine derivatives with corresponding arylamines. The obtained compounds (1-40), which can be envisaged as sterically fixed and configurationally stable analogs of 8-styrylxanthines, were evaluated for their affinity to adenosine A(1) and A(2A) receptors, the receptor subtypes that are predominant in the brain. Selected compounds were additionally investigated for affinity to the A(2B) and A(3) receptor subtypes. Many of the compounds showed adenosine A(2A) receptor affinity at micromolar or submicromolar concentrations and were A(2A)-selective, for example, compound 23 with p-fluoro substituent displayed K(i) value of 0.147 microM at the rat A(2A) receptor and more than 170-fold-A(2A) selectivity, compound 17 with naphthyl substituent had K(i) value of 0.219 microM and a more than 114-fold-A(2A) selectivity. The compounds were somewhat weaker and less selective at the human receptor subtypes. Elongation of the dimethyl substituent to dipropyl in xanthine moiety improved affinity but reduced selectivity. 1,3-Dimethylimidazo-, pyrimido-, and diazepinopurinediones were evaluated in vivo as anticonvulsants in MES, ScMet, TTE tests and examined for neurotoxicity in mice (ip). Substances with pyrimido ring displayed protective activity in ScMet or in MES and ScMet tests, showing also neurotoxicity. The pyrimidine annelated ring is beneficial for both receptor affinity and anticonvulsant activity.     

Kinetic studies on the association and dissociation of myosin subfragment 1 and actin.

The reactions of pyrene-labeled actin with myosin subfragment 1 (S1) and S1-ligand complexes at low ionic strength are described by the schemes [formula: see text] where M refers to a myosin head; A is actin; L is ligand; the asterisk refers to a high fluorescence state of actin; and K1 and K3 are association constants. K1 is reduced approximately 10-fold for M.ADP or M.pyrophosphate versus M alone. The rate constant of the isomerization step (k2) is 150-200 s-1 for A*M, A*M.ADP, and A*M-pyrophosphate (20 degrees C). The interaction between the ligand the actin binding sites reduces K2 from 2,000 for A*M to 50-100 for A*M.ADP and to approximately unity for A*M-pyrophosphate. The A*M.ADP state is equated with the AM'.ADP state of Sleep and Hutton (Sleep, J., A., and Hutton, R. L. (1980) Biochemistry 19, 1276-1283).     

Fermentation performance and intracellular metabolite patterns in laboratory and industrial xylose-fermenting Saccharomyces cerevisiae

Heterologous genes for xylose utilization were introduced into an industrial Saccharomyces cerevisiae, strain A, with the aim of producing fuel ethanol from lignocellulosic feedstocks. Two transformants, A4 and A6, were evaluated by comparing the performance in 4-l anaerobic batch cultivations to both the parent strain and a laboratory xylose-utilizing strain: S. cerevisiae TMB 3001. During growth in a minimal medium containing a mixture of glucose and xylose (50 g/l each), glucose was preferentially consumed. During the first growth phase on glucose, the specific growth rates were 0.26, 0.32, 0.27 and 0.30 h^–1 for strains TMB 3001, A (parental strain), A4, and A6, respectively. The specific ethanol productivities were 0.04, 0.13, 0.04 and 0.03 g/g^.per hour, for TMB 3001, A, A4 and A6, respectively. The specific xylose consumption rates were 0.06, 0.21 and 0.14 g/g^.per hour, respectively for strains TMB 3001, A4 and A6. Xylose consumption resulted mainly in the formation of xylitol, with biomass and ethanol being minor products. The metabolite profile of intermediates in the pentose phosphate pathway and key glycolytic intermediates were determined during growth on glucose and xylose, respectively. The metabolite pattern differed depending on whether glucose or xylose was utilized. The levels of intracellular metabolites were higher in the industrial strains than in the laboratory strain during growth on xylose. Electronic Publication     

Structural determination of the substrate specificities and regioselectivities of the rat and human fatty acid omega-hydroxylases

The substrate and regiospecificities of the known CYP4A enzymes from rat (CYP4A1, -4A2, -4A3, and -4A8) and human (CYP4A11) have been determined using lauric (C12), myristic (C14), palmitic (C16), oleic (C18:1), and arachidonic (C20:4) acids. The CYP4A2 and CYP4A8 cDNAs required to complete the enzyme set were cloned from a rat kidney library. All five proteins were expressed in Escherichia coli and were purified with the help of a six-histidine tag at the carboxyl terminus. Two complementary CYP4A2-CYP4A3 chimeras fused at residue 119 (CYP4A2) and 122 (CYP4A3) were constructed to explore the roles of the 18 amino acid differences between the parent proteins in determining their catalytic profiles. The chimera in which the first 119 amino acids are from CYP4A2 indicates that the first 120 amino acids control the substrate specificity. The chimera in which the first 122 amino acids are from CYP4A3 is inactive due to a defect in electron transfer to the heme group. The highest activity for lauric acid was obtained with CYP4A1 and CYP4A8, but for all the proteins the activity decreased with increasing fatty acid chain length. The fact that none of the rat and human CYP4A enzymes exhibits a high activity with arachidonic acid appears to limit their role as catalysts for the physiologically important conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE).     

Aluminium effects on growth and Al,Ca, Mg,K and P levels in triticale,wheat and rye

Summary The effects of A1 on the growth and mineral composition of different cultivars of triticale (X Triticosecale, Wittmack), wheat (Triticum aestivum L.) and rye (Secale cereale L.) growing in 1/5 strength Steinberg solutions containing 0 or 6 ppm A1 were evaluated after 32 days. Aluminum increased the concentrations of P and K in the roots and K in the tops of most of the cultivars tested. A1 tolerant triticale retained a lower concentration of Mg in the roots and tops than the A1 sensitive triticale, when subjected to A1 stress. In addition, A1 treatments resulted in smaller increases in root P for the A1 tolerant triticale than for the A1 sensitive cultivars.The concentration of root Ca and P of the A1 tolerant wheat cultivars were significantly below that of the more sensitive plants. Aluminum tolerance in rye appeared to be associated with lower Ca and higher Mg concentrations in the tops. The accumulation of P and A1 in the roots was characteristic of sensitivity in triticale, wheat and rye.     

Activation mechanism of monkey and porcine pepsinogens A. One-step and stepwise activation pathways and their relation to intramolecular and intermolecular reactions

The activation of Sepharose-bound monkey pepsinogen A under acidic conditions proceeded by cleavage of the Leu47-Ile48 bond, indicating the occurrence of the intramolecular one-step activation, although the rate of cleavage was very slow. On the other hand the activation of monkey pepsinogen A in solution was highly dependent on pepsinogen concentration and the addition of exogenous pepsin A accelerated the rate of activation, indicating the predominance of intermolecular reaction. The cleavage site, however, was also restricted to the Leu47-Ile48 bond. Thus, apparently exclusive one-step activation occurred in monkey pepsinogen. The activation of porcine pepsinogen A in solution was also dependent on pepsinogen concentration and the addition of exogenous pepsin A accelerated the rate of activation. The major cleavage site by the exogenously added pepsin was the Leu44-Ile45 bond. Therefore the site most susceptible to the intermolecular attacks was the bond connecting the activation segment and the pepsin moiety in both monkey and porcine pepsinogens. In porcine pepsinogen, however, a part of the zymogen was activated through the intermediate form, and an intramolecular reaction was suggested to be involved in the generation of this form. These results showed that in both pepsinogens A the intramolecular reaction occurred, first yielding pepsin A or the intermediate form, which then acted intermolecularly on the remaining pepsinogen or the intermediate form to complete the activation in a short time. A molecular mechanism for the activation reaction was proposed to explain consistently the experimental results.