A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues

The conversion of xanthine dehydrogenase to a free radical producing oxidase is an important component of oxygen-mediated tissue injury. Current assays for these enzymes are of limited sensitivity, making it difficult to analyze activities in organ biopsies or cultured cells. The xanthine oxidase-catalyzed conversion of pterin (2-amino-4-hydroxypteridine) to isoxanthopterin provides the basis for a fluorometric assay which is 100-500 times more sensitive than the traditional spectrophotometric assay of urate formation from xanthine. Enzyme activity as low as 0.1 pmol min-1 ml-1 can be measured with the fluorometric pterin assay. Xanthine oxidase is assayed in the presence of pterin only, while combined xanthine dehydrogenase plus oxidase activity is determined with methylene blue which replaces NAD+ as an electron acceptor. The relative proportions and specific activities of xanthine oxidase and dehydrogenase determined by the fluorometric pterin assay are comparable with the spectrophotometric measurement of activities present in rat liver, intestine, kidney, and plasma. The assay has been successfully applied to brain, human kidney, and cultured mammalian cells, where xanthine dehydrogenase and oxidase activities are too low to detect spectrophotometrically.     

Advancements and future directions in enzyme technology for biomass conversion

Enzymatic hydrolysis of pre-treated lignocellulosic biomass is an ideal alternative to acid hydrolysis for bio-ethanol production, limited primarily by pre-treatment requirements and economic considerations arising from enzyme production costs and specific activities. The quest for cheaper and better enzymes has prompted years of bio-prospecting, strain optimization through genetic engineering, enzyme characterization for simple and complex lignocellulosic feedstock, and the development of pre-treatment strategies to mitigate inhibitory effects. The recent shift to systematic characterizations of de novo mixtures of purified proteins is a promising indicator of maturation within this field of study, facilitating progression towards feedstock assay-based rapid enzyme mixture optimization. It is imperative that international standards be developed to enable meaningful comparisons between these studies and the construction of a database of enzymatic activities and kinetics, aspects of which are explored here-in. Complementary efforts to improve the economic viability of enzymatic hydrolysis through process integration and reactor design are also considered, where membrane-confinement shows significant promise despite the associated technological challenges. Significant advancements in enzyme technology towards the economic conversion of lignocellulosic biomass should be expected within the next few years as systematic research in enzyme activities conforms to that of traditional reaction engineering.     

The preference for an additional child among married women in Seoul, Korea

South Korea reported a total fertility rate (TFR) of 1.08 in 2005. This is the lowest level of all nations in the Organisation for Economic Co-operation and Development (OECD). Recently, the decline in the fertility rate has been a dominant phenomenon in Korea's major cities. This study investigated the relationship between social environmental factors and fertility intentions for married women in Seoul, the capital of Korea, using a sample of 2211 married women who responded to the Seoul Citizens Health and Social Indicators Survey, 2005. Here, the effects of selected social environmental characteristics on fertility intentions are explored using multivariate logistic regression models. The relationships among a woman's age, number of living children, job type, housing type, and social group participation were strong indicators of the intention to have additional children. Younger women living with fewer children generally have a higher intention to have additional children. Among women's job types, blue-collar workers have a lower preference for additional children than white-collar workers and housewives. Married women participating in social groups have a lower preference for additional children than non-participants. Women's participation in social activities appears to have various benefits, both individually and socially. However, whereas women's participation in economic activities has been linked to questions of fertility in previous studies, the relationship between fertility and social activities has been downplayed. Women's participation in social activities has increased over the past several decades, and the trend continues to grow. Therefore, women's participation in social activities must be accepted as the status quo, and compatibility between women's participation in social activities and childrearing needs to be increased. Consequently, a strong foundation for a fertility-friendly environment is needed, focusing on blue-collar workers and participation in social activities by married women.     

Mitogen activated protein kinase-dependent activation of c-Jun and c-Fos is required for neuronal differentiation but not for growth and stress response in PC12 cells

MAPK-dependent activation of AP-1 protein c-Jun is involved in PC12 cell differentiation and apoptosis. However, the role of other AP-1 proteins and their connection to MAPKs during growth, differentiation and apoptosis has remained elusive. Here we studied the activation of AP-1 proteins in response to ERK, JNK, and p38 signaling upon NGF, EGF and anisomycin exposures. All treatments caused different kinetics and strength of MAPK and AP-1 activities. NGF induced persistent ERK and AP-1 activities, whereas upon EGF and anisomycin exposures, their activities were only weakly and transiently induced. The sustained AP-1 activity was associated with concomitant c-Fos and c-Jun expression and phoshorylation, which were JNK and ERK dependent. While inhibition of the ERK, JNK, and p38 activities partially prevented AP-1 activity and suppressed differentiation, none of them was required for anisomycin-induced apoptosis. The importance of c-Fos and c-Jun as mediators of differentiation was demonstrated by the findings that the corresponding siRNAs suppressed NGF-induced neurite outgrowth. However, the capacity of c-Fos to promote differentiation required cooperation with Jun proteins. In contrast, Fra-2 expression was not required for the differentiation response. Together, the results show that sustained c-Jun and c-Fos activities mediate MAPK signaling and are essential for differentiation of PC12 cells.     

An improved method for heterozygote identification in feline and human mucopolysaccharidosis VI, arylsulfatase-B deficiency

An improved method has been developed for the detection of heterozygotes for feline and human mucopolysaccharidosis VI. Arylsulfatase-A and -B activities were assayed in leukocyte extracts following separation of the enzymes by batch chromatography on DEAE cellulose. Determination of arylsulfatase-B specific activities did not permit accurate heterozygote identification, whereas the arylsulfatase-A to arylsulfatase-B activity ratio discriminated all 16 obligate heterozygotes for the feline and human disorders.     

重大工程建设中生态安全格局构建基本原则和方法

随着人类活动的日益加强,生态安全成为全球关注的话题.生态安全的状态受到人类干扰活动的影响,而区域生态安全演变也会影响人类活动的功效.20世纪90年代以来,我国先后开展了长江三峡大坝、青藏铁路、西气东输、西电东送、南水北调等重大工程项目的建设.这些重大工程建设对区域生态安全的影响,以及区域生态系统演变对工程安全运行的影响成为我国政府关注的重要课题.如何在强度干扰下开展生态恢复与区域生态安全格局的构建,不仅关系到区域生态环境的保护,也关系到各项重大工程的顺利运行.本文系统分析了工程建设的类型、特征及其对区域生态环境的影响,在此基础上,提出了重大工程建设中生态恢复与安全格局构建的基本原则和方法.认为在工程建设中构建生态安全格局需要从6个方面着手：区域生态环境现状分析与评价、工程建设生态干扰与风险评价、生态安全格局预案构建、区域生态环境效应情景分析、区域生态恢复与安全格局再优化和生态系统管理方案的建立.讨论了我国重大工程建设中生态安全格局构建面临的突出问题.     

New in vitro reporter gene bioassays for screening of hormonal active compounds in the environment

Identification of chemicals with endocrine-disrupting activities in the past two decades has led to the need for sensitive assays for detection and monitoring of these activities in the environment. In vitro reporter gene assays represent a relatively fast and easy-to-perform method for detection of compounds that are able to bind to hormonal receptors and stimulate or silence their transactivation activity, thus interfering with the hormone signaling pathways. This paper reviews upgrades on reporter gene assays performed during the last decade. The utilization of new reporter genes (luciferase and green fluorescent protein coding genes) significantly improved the sensitivity of the tests and made them faster. Reporter gene assays now represent a high-throughput system for screening chemicals for hormonal activity. Finally, modification of test set-ups for testing anti-hormonal activities also enabled measurements of endocrine-disrupting activities in complex environmental samples such as sediments and wastewater treatment plant effluents.     

Characterization and engineering of glycosyltransferases responsible for steroid saponin biosynthesis in Solanaceous plants

Solanaceous plants contain steroid saponins that have diverse biological and pharmacological activities. The structures of their sugar chains play an important role in their activities. A functional glucosyltransferase SaGT4A from Solanum aculeatissimum glucosylates both steroidal sapogenins and steroidal alkaloids. A potato (S. tuberosum) glycosyltransferase StSGT, which has a high degree of sequence homology with SaGT4A, exhibits the same substrate specificity toward steroidal compounds as SaGT4A. To identify the residues or domain structures responsible for these enzymatic activities, we determined the residues that are essential for SaGT4A activity, compared the specific activities of SaGT4A and StSGT, and constructed several SaGT4A/StSGT chimeric proteins, focusing on the donor-sugar recognition domain. These proteins were heterogeneously expressed in E. coli and purified, and their glycosyltransferase activities were evaluated using a coupled assay. His369 and Glu377, located in the consensus motif for plant glycosyltransferases, and Cys121, Cys247, and Cys370 were shown to be important for SaGT4A activity. StSGT exhibited more activity with UDP-galactose as a sugar donor than with UDP-glucose, whereas SaGT4A exhibited glucosyltransferase activity exclusively. The sugar selectivities of SaGT4A and StSGT were not altered by exchanging their domains, and some of the chimeric proteins showed no activity. These results suggest that the differences in the SaGT4A and StSGT amino acid sequences do not simply reflect their distinct sugar-donor specificities. We also successfully converted the non-functional SaGT4A homolog, SaGT4R, into an active glucosyltransferase.     

Control enzyme levels in the plasma, brain and liver from wild birds and mammals in Britain

Brain from 47 avian and 17 mammalian species and the liver from 19 avian and 7 mammalian species has been examined for acetyl cholinesterase and nitrophenyl acetate esterase activities. Plasma from 27 avian and 7 mammalian species has been examined for acetyl cholinesterase, cholinesterase, nitrophenyl acetate esterase, glutamate, oxaloacetate transaminase, glutamate pyruvate transaminase, glutamate dehydrogenase, sorbitol dehydrogenase and lactate dehydrogenase activities. The studies have revealed that variations in enzyme activities occur between species but that there are discernible trends within families. The results indicate that comprehensive control enzyme data is necessary in order to assess the effects of exposure to agricultural chemicals in wildlife.     

Evaluating changes in land cover and their importance for global change

During recent years, much progress has been made in integrating traditional natural science disciplines and in the developmnet of multidisciplinary models. This is crucial for an increased understanding of the dynamics of the Earth system. The domination of human activities in altering these dynamics is still increasing. However, few research projects have focused directly on understanding the motives for such intensification. It has only recently been acknowledged that improved understanding of human driving forces of global change is required to enable meaningful projections of plausible future states of the Earth system.     

Distribution of creatine, guanidinoacetate and the enzymes for their biosynthesis in the animal kingdom. Implications for phylogeny

1. The distribution of creatine and the creatine-synthesizing enzymes in the animal kingdom has been investigated. Creatine was found in tissues of all vertebrates examined, and in various invertebrates from phyla Annelida, Echinodermata, Hemichordata and Chordata, subphylum Cephalochordata. The activities of the creatine-synthesizing enzymes, arginine-glycine transamidinase and guanidinoacetate methylpherase, were not detected in the hagfish or in any of the invertebrates, including those in which creatine was found, with the exception that transamidinase activities were detected in the amphioxus and salt water clam; however, these activities are considered to be artifacts for reasons mentioned in the text. Additional evidence that the hagfish and various creatine-containing invertebrates could not synthesize creatine was the observation that these animals did not convert one or the other of the likely precursors of creatine (arginine and glycine) into creatine, in vivo. Further, the inability of these animals to synthesize creatine is correlated with the observations that all animals tested were able to abstract creatine from their aqueous environment. 2. The activities of the creatine-synthesizing enzymes were detected in the sea lamprey and in all but a few of the other vertebrates examined. Neither activity could be detected in the sharks and rays (cartilaginous fish), buffalo fish (bony fish) or the snapping turtle. Transamidinase or guanidinoacetate methylpherase activity could not be found in the salamander or garter snake, respectively. 3. The results obtained with the lamprey are in direct contrast with those obtained with the hagfish (both subphylum Agnatha, class Cyclostomata). The lamprey had the ability to synthesize creatine and did not abstract creatine from lake water. The hagfish did not have any apparent ability to synthesize creatine and did abstract creatine from sea water. The present report thus supports the theory that the myxinoid (hagfish) and petromyzoid (lamprey) agnathans are only distantly related. 4. The lack of creatine-synthesizing enzyme activities in the cartilaginous fishes may have phylogenetic significance, but may also be explained by the availability of creatine in the diet of these animals. The lack of one or both enzyme activities in vertebrates other than the hagfish and the cartilaginous fish is suggested to be the result of creatine in the diet.     

Carboxylated lysine is required for higher activities in Hydantoinases

Hydantoinases are industrial enzymes with varying degree of activities on variable substrates to form different products. Although, few of the hydantoinase structures were known recently, the functional details and active site mechanism were not clearly understood yet. In a structure determination effort we reported that Bacillus sp. AR9 hydantoinase contains uncarboxylated lysine in the active site, whereas all the other hydantoinases have a carboxylated active site lysine. Here we describe the importance of carboxylated lysine for differential activities by making lysine mutations as well as carboxylating the lysine in a D-hydantoinase from Bacillus sp. AR9. The lysine to alanine and lysine to arginine mutations showed reduced activities whereas carboxylation of the lysine has enhanced the activity. Theoretical studies involving the calculation of electrostatic potentials for the hydroxide ion between the two metal ions present in the active site suggest that the presence of carboxylated lysine increases the nucleophilicity of the hydroxide.     

A radiochemical assay for N-acetyl-L-aspartate amidohydrolase (EC 3.5.1.15) and its occurrence in the tissues of the chicken

A simple and rapid radiochemical method for the determination of N-acetyl-L-aspartic acid amidohydrolase (EC 3.5.1.15) activity using ion exchange chromatography has been developed. The activity of this enzyme in the developing brain and some non-nervous tissues of the chicken has been determined. No activity of the enzyme could be detected in the brains of chick embroys prior to 14 days of gestation; activities gradually increased thereafter to adult levels which are about 60% of that found in the adult rat. In non-nervous system tissues of the adult chicken, activities varied from high levels in the kidney to low levels in heart and breast muscle. Treatment of the homogenates of the adult tissues with a detergent significantly increased the enzyme activity, suggesting that a portion of the enzyme is membrane bound.     

Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis

The Rho subfamily of GTPases has been shown to regulate cellular morphology. We report the discovery of a new member of the Rho family, named RhoL, which is equally similar to Rac, Rho, and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary caused nurse cells to collapse and fuse together. Mutant forms of Cdc42 mimicked this effect. Expression of constitutively active RhoL led to nurse cell subcortical actin breakdown and disruption of nurse cell- follicle cell contacts, followed by germ cell apoptosis. In contrast, Rac activity was specifically required for migration of a subset of follicle cells called border cells. All three activities were necessary for normal transfer of nurse cell cytoplasm to the oocyte. These results suggest that Rho protein activities have cell type-specific effects on morphogenesis.     

Functional analyses for tRNase Z variants: an aspartate and a histidine in the active site are essential for the catalytic activity

We performed functional analyses for various single amino-acid substitution variants of Escherichia coli, Bacillus subtilis, and human tRNase Zs. The well-conserved six histidine, His(I)-His(VI), and two aspartate, Asp(I) and Asp(II), residues together with metal ions are thought to form the active site of tRNase Z. The Mn(2+)-rescue analysis for Thermotoga maritima tRNase Z(S) has suggested that Asp(I) and His(V) directly contribute the proton transfer for the catalysis, and a catalytic mechanism has been proposed. However, experimental evidence supporting the proposed mechanism was limited. Here we intensively examined E. coli and B. subtilis tRNase Z(S) variants and human tRNase Z(L) variants for cleavage activities on pre-tRNAs in the presence of Mg(2+) or Mn(2+) ions. We observed that the Mn(2+) ions cannot rescue the activities of Asp(I)Ala and His(V)Ala variants from each species, which are lost in the presence of Mg(2+). This observation may support the proposed catalytic mechanism.     

Use of creatine in the elderly and evidence for effects on cognitive function in young and old

The ingestion of the dietary supplement creatine (about 20 g/day for 5 days or about 2 g/day for 30 days) results in increased skeletal muscle creatine and phosphocreatine. Subsequently, the performance of high-intensity exercise tasks, which rely heavily on the creatine-phosphocreatine energy system, is enhanced. The well documented benefits of creatine supplementation in young adults, including increased lean body mass, increased strength, and enhanced fatigue resistance are particularly important to older adults. With aging and reduced physical activity, there are decreases in muscle creatine, muscle mass, bone density, and strength. However, there is evidence that creatine ingestion may reverse these changes, and subsequently improve activities of daily living. Several groups have demonstrated that in older adults, short-term high-dose creatine supplementation, independent of exercise training, increases body mass, enhances fatigue resistance, increases muscle strength, and improves the performance of activities of daily living. Similarly, in older adults, concurrent creatine supplementation and resistance training increase lean body mass, enhance fatigue resistance, increase muscle strength, and improve performance of activities of daily living to a greater extent than resistance training alone. Additionally, creatine supplementation plus resistance training results in a greater increase in bone mineral density than resistance training alone. Higher brain creatine is associated with improved neuropsychological performance, and recently, creatine supplementation has been shown to increase brain creatine and phosphocreatine. Subsequent studies have demonstrated that cognitive processing, that is either experimentally (following sleep deprivation) or naturally (due to aging) impaired, can be improved with creatine supplementation. Creatine is an inexpensive and safe dietary supplement that has both peripheral and central effects. The benefits afforded to older adults through creatine ingestion are substantial, can improve quality of life, and ultimately may reduce the disease burden associated with sarcopenia and cognitive dysfunction.     

Comparison of two reference preparations for horse chorionic gonadotrophin in four in-vivo and in-vitro assays

A number of horse chorionic gonadotrophin (CG) preparations of different purities and from diverse sources have been compared in radioimmuno-, radioreceptor, in-vitro cell culture, and in-vivo assays. The relative activities of the great majority of the preparations tested were consistent in the 4 assay systems. Moreover, their relative activities in the 4 assays were consistent with those found for unfractionated plasmas. These preparations were therefore considered to represent the native form of hormone. The second International Reference Preparation (IRP2) was among the few preparations exhibiting discordant relative activities in the different assay systems. Its relative in-vivo activity was almost 50% lower than that found in the 3 other assays. This could be due to denaturation of the hormone during its preparation or to selection of isoform(s) not representative of the whole population of molecules. For standardization of horse CG preparations by in-vivo assay, IRP2 has proved to be a reliable standard. However, for the standardization of preparations by in-vitro methods, a standard giving consistent results in in-vivo and in-vitro assays must be used. The present report indicates that the NIH standard, but not IRP2, fulfils these requirements.     

The use of potassium iodide equilibrium density gradient centrifugation in the purification of RNA for hybridization with nonreiterated DNA sequences.

A new instrument has been developed that is able to monitor enzyme activities in chromatography column eluents automatically. The instrument consists of a simple stopped-flow instrument, equipped with a multishutoff valve to direct reagents and eluent flow. Assays of the eluate may be done at present intervals. The instrument may also be used for monitoring enzyme activities in perfasates as well as for monitoring the elution of metabolites.     

Last decade update for three-finger toxins: Newly emerging structures and biological activities

Three-finger toxins(TFTs) comprise one of largest families of snake venom toxins. While they are principal to and the most toxic components of the venoms of the Elapidae snake family, their presence has also been detected in the venoms of snakes from other families. The first TFT, α-bungarotoxin, was discovered almost 50 years ago and has since been used widely as a specific marker of the α7 and muscle-type nicotinic acetylcholine receptors. To date, the number of TFT amino acid sequences deposited in the UniProt Knowledgebase free-access database is more than 700, and new members are being added constantly.Although structural variations among the TFTs are not numerous, several new structures have been discovered recently; these include the disulfide-bound dimers of TFTs and toxins with nonstandard pairing of disulfide bonds. New types of biological activities have also been demonstrated for the well-known TFTs, and research on this topic has become a hot topic of TFT studies. The classic TFTs α-bungarotoxin and α-cobratoxin, for example, have now been shown to inhibit ionotropic receptors of γ-aminobutyric acid, and some muscarinic toxins have been shown to interact with adrenoceptors. New, unexpected activities have been demonstrated for some TFTs as well, such as toxin interaction with interleukin or insulin receptors and even TFT-activated motility of sperm. This minireview provides a summarization of the data that has emerged in the last decade on the TFTs and their activities.