A versatile platform for single-molecule enzymology of restriction endonuclease

Enzymes are the major players for many biological processes.Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level.Yet,these single-molecule experiments are technically di±cult and generally require complicated experimental design.Here,we develop a Holliday junction(HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET(sm-FRET).We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events.Thanks to the multiple-arms structure of HJ,the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm.Therefore,it is possible to independently change the substrate arm to study different enzymes with similar functions.Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations.Moreover,this method can be easily extended to study other types of DNA-binding enzymes without too much modi fication of the design.We anticipate it can find broad applications in single-molecule enzymology.

Enzymology properties of two different xylanases and their impacts on growth performance and intestinal microflora of weaned piglets

The enzyme xylanase is more and more widely used in feed production, but different xylanase have different properties, mechanism and application effects. To provide a theoretical basis for choosing more suitable xylanase in feed production, we selected bacterial xylanase(BX), labeled enzyme A, and trichoderma xylanase(TX), labeled enzyme B, and studied the enzymology properties and application effects on growth performance and gut flora in weaned piglets. The results showed that the activity levels of both appear parabolic along with increasing pH or temperature, but the amplitude of enzyme activity changing curves and the pH/temperature of optimal activity level are different, where enzyme A has the optimal activity level at 50 ℃ with a pH value of 5.0. The optimal activity level of enzyme B was achieved at 70 ℃ with a pH around 6.0. Enzyme B suffered very little activity loss with moisture level at16% and temperature from 80℃ to 90 ℃. Enzyme A suffered a big drop in activity level when processed with high temperature from around 80 ℃ to 90 ℃, and it was even completely inactivated at 90 ℃,Enzyme A has very low activity level after being processed in acid environment, but enzyme B has minor changes in activity level with respect to changes in acid level, indicating significantly different enzymatic properties between the two different sources of xylanases. In feeding experiment, the control group, was fed the basal diet, and the BX group and TX group were fed basal diets supplemented with 0,01% bacterial and fungal xylanases, respectively. The results showed that ADG of the BX group and TX group increased by 3.25%(P> 0.05) and 8.22%(P < 0.05), respectively, and the feed conversion ratio decreased by 6.74% and 7.86%(P > 0.05), respectively compared with the control group; TX group had significantly higher(P < 0.05) ADG compared with BX group; BX group and TX group had significantly lower ileum Escherichia coli level than the control group, which were reduced by up to 12.98%(P < 0.05) and 11.68%(P < 0.05), respectively, but the ileal lactic acid bacteria levels were significantly increased by 16.21%(P < 0.01) and 27.02%(P < 0.01),respectively. There were no significant differences(P > 0.05) between BX group and TX group in terms of lactic acid bacteria E. coli level. We concluded that fungal xylanase(enzyme B) has better performances in improving weaned piglet growth and in increasing ileal lactic acid bacteria level compared with bacterial xylanase(enzyme A).

木聚糖酶产生菌的筛选及其酶学性质初步研究

采用富集培养、平板划线分离、透明圈平板筛选的方法,从长期堆放玉米芯的土壤中分离到9株木聚糖酶产生菌,然后采用自然选育的方法从这些菌株中筛选出一株优良木聚糖酶菌株,用该菌株发酵产酶并对其酶学性质进行了初步研究.结果表明：在温度28-30℃、pH初始值5.0左右、摇床转速150 r/min、发酵48 h所产的木聚糖酶酶活较高,木聚糖酶活达到13.68 U/mL;酶促反应的较适温度为53℃,pH值为5.3左右;该酶较耐热,温度在60℃下,120 min后酶活仍残留60.82%.

Stress kinase inhibition modulates acute experimental pancreatitis

AIM To examine the role of p38 during acute experimental cerulein pancreatitis.METHODS Rats were treated with cerulein with or without a specific JNK inhibitor (CEP1347)andy or a specific p38 inhbitor (SB203380) and pancreatic stress kinase activity wasdetermined. Parameters to assess pancreatitis included trypsin, amylase, lipase, pancreatic weight and histology.RESULTS JNK inhibition with CEP1347ameliorated pancreatitis, reducing pancreatic edema. In contrast, p38 inhibition with SB203580aggravated pancreatitis with higher trypsinlevels and, with induction of acinar necrosis not normally found after cerulein hyperstimulation.Simultaneous treatment with both CEP1347 and SB203580 mutually abolished the effects of either compound on cerulein pancreatitis.CONCLUSION Stress kinases modulatepancreatitis differentially. JNK seems to promote pancreatitis development, possibly by supporting inflammatory reactions such as edema formation while its inhibition ameliorates pancreatitis. In contrast, p38 may help reduce organ destruction while inhibition of p38 during induction of cerulein pancreatitis leads to the occurrence of acinar necrosis.

Expression and alterations of different molecular form γ-glutamyl transferase and total RNA concentration during the carcinogenesis of rat hepatoma

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Changes in erythrocyte membrane ATPases and plasma lipid peroxides in upper abdominal surgery under intravenous procaine-balanced anesthesia

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Theoretical and experimental biology in one<br>—A symposium in honour of Professor Kuo-Chen Chou’s 50th anniversary and Professor Richard Giegé’s 40th anniversary of their scientific careers

It has been a dream that theoretical biology can be extensively applied in experimental biology to accelerate the understanding of the sophiscated movements in living organisms. A brave assay and an excellent example were represented by enzymology, in which the well-established physico-chemistry is used to describe, to fit, to predict and to improve enzyme reactions. Before the modern bioinformatics, the developments of the combination of theoretical biology and experimental biology have been mainly limited to various classic formulations. The systematic use of graphic rules by Prof. Kuo-Chen Chou and his co-workers has significantly facilitated to deal with complicated enzyme systems. With the recent fast progress of bioinformatics, prediction of protein structures and various protein attributes have been well established by Chou and co-workers, stimulating the experimental biology. For example, their recent method for predicting protein subcellular localization (one of the important attributes of proteins) has been extensively applied by scientific colleagues, yielding many new results with thousands of citations. The research by Prof. Chou is characterized by introducing novel physical concepts as well as powerful and elegant mathematical methods into important biomedical problems, a focus throughout his career, even when facing enormous difficulties. His efforts in 50 years have greatly helped us to realize the dream to make “theoretical and experimental biology in one”. Prof. Richard Giege is well known for his multi-disciplinary research combining physics, chemistry, enzymology and molecular biology. His major focus of study is on the identity of tRNAs and their interactions with aminoacyl-tRNA synthetases (aaRS), which are of critical importance to the fidelity of protein biosynthesis. He and his colleagues have carried out the first crystallization of a tRNA/aaRS complex, that between tRNAAsp and AspRS from yeast. The determination of the complex structure contributed significantly to under- stand the interaction of protein and RNA. From his fine research, they have also found other biological function of these small RNAs. He has developed in parallel appropriate methods for his research, of which the protein crystallogenesis, a name he has coined, is an excellent example. Now macromolecular crystallogenesis has become a developed science. In fact, such contribution has accelerated the development of protein crystallography, stimulating the study of macromolecular structure and function.

Purification and Characterization of a Novel Lipase from Antarctic Krill

Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl S-100),resulting in 5.2%recovery with a 22.4-fold purification ratio.The optimal pH and temperature for enzyme activity were 8.0 and 45℃,respectively.Purified lipase had Km and Vmax values of 3.27mmolL−1 and 2.4Umg−1,respectively,using p-nitrophenyl laurate as the substrate.Lipase activity was enhanced by adding Ca2+and Mg2+ions in the concentration ranges of 0–0.5mmolL−1 and 0–0.3mmolL−1,respectively,while the activity was inhibited by a further increase in these ion concentrations.Fe3+and Cu2+ions showed obvious inhibitory effects on enzyme activity,and the inhibition rates were 71.8%and 53.3%when the ion concentrations were 0.5mmolL−1.

Responses of bioenergy sorghum cell wall metabolism to agronomic practices

Maximum lignocellulose yield of biomass sorghum [Sorghum bicolor L. (Moench.)] is hampered by complex biological phenomena related to rotation, nitrogen (N) fertilization, soil tillage, and excessive biomass removal. The biochemical basis of the effects of agronomic practices on sorghum production was studied by the enzymology of the active peroxidase (EC 1.11.1.7) isoenzymes that synthesize lignin. All studied practices altered the peroxidase pI values. Control sorghum without rotation and without N fertilization had the most inhibited peroxidase with very low maximum velocity (Vmax) value (3.10 mmol·min﹣1), and very low lignin (857 kg·ha﹣1) yield, which could decrease soil organic carbon possibly leading to adverse changes in soil chemistry. Corn-sorghum rotations with and without N fertilization increased the Vmax values of peroxidase and lignin and cellulose yields. Rotated sorghum subjected to 50% residue return (the percentage of crop residue was returned to the plot immediately after grinding at harvest) and 280 kg·ha﹣1 N fertilization possessed very active peroxidase (Vmax value 66.4 mmole·min﹣1) and the highest lignin (1387 kg·ha﹣1) yield. The 25% residue return rate without N fertilization induced high lignin (1125 kg·ha﹣1) and cellulose (11,961 kg·ha﹣1) but the 25% residue return rate with 280 kg·ha﹣1 N fertilization induced lower lignin (1046 kg·ha﹣1) yield. Continuously cropped sorghum treated with 336 kg·N·ha﹣1 produced active peroxidase that shared competitive inhibition relationship with the peroxidase of the 84 kg·N·ha﹣1 treatment. Ridge tillage combined with 280 kg·ha﹣1 N fertilization under continuous sorghum resulted in inhibited peroxidase possessing low Vmax value (13.0 μmole·min﹣1). Changing to conventional tillage combined with 280 kg·ha﹣1 N fertilization relieved the inhibition and increased the Vmax value to 23.7 mmol·min﹣1. These biological anomalies of sorghum cell wall related to agronomic practices originated from doubly inhibited sorghum peroxidases. This understanding may guide the choice of sustainable agronomic practices for maximizing lignocellulose yields for the bioenergy industry while protecting the environment.

Electrophoretic Purification and Characterization of Human NADH-Glutamate Dehydrogenase Redox Cycle Isoenzymes Synthesizing Nongenetic Code-Based RNA Enzyme

NADH-glutamate dehydrogenase (GDH) is active in human tissues, and is chromatographically purified, and studied because it participates in synthesizing glutamate, a neurotransmitter. But chromatography dissociates the GDH isoenzymes that synthesize nongenetic code-based RNA enzymes degrading superfluous mRNAs thereby aligning the cellular reactions with the environment of the organism. The aim was to electrophoretically purify human hexameric GDH isoenzymes and to characterize their RNA enzyme synthetic activity as in plants. The outcome could be innovative in chemical dependency diagnosis and management. Multi metrix electrophoresis including free solution isoelectric focusing, and through polyacrylamide and agarose gels were deployed to purify the redox cycle isoenzymes of laryngeal GDH, and to assay their RNA enzyme synthetic activities. The laryngeal GDH displayed the 28 binomial isoenzymes typical of higher organisms. Isoelectric focusing purification produced pure GDH. Redox cycle assays of the GDH isoenzymes produced RNA enzymes that degraded human stomach total RNA. In the reaction mechanism, the Schiff-base intermediate complex between α-ketoglutarate and GDH is the target of nucleophiles, resulting to the disruption of synthesis of glutamate, and RNA enzyme. The strongest nucleophiles are the psychoactive alkaloids of tobacco, cocaine, opium poppy, cannabis smoke because they are capable of reacting with GDH Schiff base intermediate to stimulate synthesis of aberrant RNA enzymes that degrade cohorts of mRNAs thereby changing the biochemical pathways and exacerbating drug overdose and chemical dependency. Electrophoretic purification, and characterization of the RNA enzyme synthetic activity set the forecourt for innovative application of GDH redox cycles in the diagnostic management of chemical dependency.

小鼠Mmp-9 shRNA重组慢病毒包装、筛选及干扰效果检测

目的:设计、筛选、构建并鉴定Mmp-9小干扰RNA(small interfering RNA,siRNA),并构建其慢病毒表达载体,为血管化疾病的防治提供理论依据及实验室数据。方法:针对基质金属蛋白酶9靶基因设计siRNA序列,并合成小发卡RNA(short hairpin RNA,shRNA)结构的DNA,与经AgeΙ和EcoRΙ酶切的pSIL-RFP连接、转化大肠杆菌感受态细胞,构建成携带Mmp9-shRNA慢病毒质粒载体(pSIL-RFP/MMP9-shRNA),PCR及测序鉴定后,Western blot筛选出携带最佳siRNA的慢病毒质粒载体,并利用慢病毒包装质粒(pHelper1.0和pHelper2.0)将其制备成MMP9-shRNA慢病毒,并测定病毒滴度为1×1010pfu/l。结果:PCR和DNA测序证实合成的含基质金属蛋白酶9短发卡RNA慢病毒载体寡核苷酸链正确插入pSIL-RFP载体,表明实验成功构建基质金属蛋白酶9RNA干扰慢病毒表达载体。结论:成功的构建并筛选出针对小鼠Mmp9基因沉默的特异性shRNA重组慢病毒,为血管化性疾病的防治研究提供基础。

Ergothioneine and its congeners:anti-ageing mechanisms and pharmacophore

Ergothioneine,Ovothiol,and Selenoneine are sulfur/selenium-containing histidine-derived natural products widely distributed across different organisms.They exhibit significant antioxidant properties,making them as potential lead compounds for promoting health.Increasing evidence suggests that Ergothioneine is positively correlated with healthy ageing and longevity.The mechanisms underlying Ergothioneine's regulation of the ageing process at cellular and molecular levels are beginning to be understood.In this review,we provide an in-depth and extensive coverage of the anti-ageing studies on Ergothioneine and discuss its possible intracellular targeting pathways.In addition,we highlight the recent efforts in elucidating the biosynthetic details for Ergothioneine,Ovothiol,and Selenoneine,with a particular focus on the study of their pharmacophore-forming enzymology.

Telomerase activity in cervical intraepithelial neoplasia

Background It was reported that telomerase expression is closely associated with cellular immortality and cancer. This study was designed to investigate the relationship between telomerase expression and the carcinogenesis of cervical cancer, the possible use of telomerase as a marker of cervical intraepithelial neoplasia (CIN) progression or regression, and the natural history of CIN.Methods Telomeric repeat amplification protocol (TRAP) assay was used to measure telomerase activity in cervical scrapings and biopsy samples obtained from 105 cases affected with various cervical conditions, including chronic cervicitis (n =20), CIN (n =64, 16 cases of CIN Ⅰ , 20 cases of CIN Ⅱ , and 28 cases of CIN Ⅲ), and invasive squamous cell carcinoma (n =21).Results In exfoliated cell samples, telomerase activity was detected in 5 of 20 (25. 0%) cases of cervicitis, 10 of 16 (62. 5%) cases of CIN Ⅰ , 11 of 20 (55. 0%) cases of CIN Ⅱ , 23 of 28 (82.1%) cases of CIN Ⅲ, and 13 of 21 (61. 9%) cases of carcinoma. In cervical biopsy samples, telomerase activity was detected in 6 of 20 (30. 0%) cases of cervicitis, 8 of 16 (50. 0%) cases of CIN Ⅰ , 9 of 20 (45. 0%) cases of CIN Ⅱ , 27 of 28 (96. 4%) cases of CIN Ⅲ, and 20 of 21 (95. 2%) cases of carcinoma. Telomerase activation was significantly higher in CIN samples than in cervicitis samples. Telomerase activity was detected at similar frequency in samples from cervical scrapings and cervical biopsies.Conclusion These results seem to suggest that telomerase expression may be associated with carcinogenesis of the cervix. TRAP assay of cervical scraping samples could be used to monitor and predict the development of CIN in clinical practice.

Tyrosine metabolic enzymes from insects and mammals：A comparative perspective

Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess finetuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modem genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precur sor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mam malian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects.

Sensing Sulfur Conditions： Simple to Complex Protein Regulatory Mechanisms in Plant Thiol Metabolism

Sulfur is essential for plant growth and development, and the molecular systems for maintaining sulfur and thiol metabolism are tightly controlled. From a biochemical perspective, the regulation of plant thiol metabolism high- lights nature＇s ability to engineer pathways that respond to multiple inputs and cellular demands under a range of con- ditions. In this review, we focus on the regulatory mechanisms that form the molecular basis of biochemical sulfur sensing in plants by translating the intracellular concentration of sulfur-containing compounds into control of key metabolic steps. These mechanisms range from the simple （substrate availability, thermodynamic properties of reactions, feedback inhi- bition, and organelle localization） to the elaborate （formation of multienzyme complexes and thiol-based redox switches）. Ultimately, the dynamic interplay of these regulatory systems is critical for sensing and maintaining sulfur assimilation and thiol metabolism in plants.

Prompt and Easy Activation by Specific Thioredoxins of Calvin Cycle Enzymes of Arabidopsis thaliana Associated in the GAPDH/CP12/PRK Supramolecular Complex

The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and phosphoribulokinase （PRK） can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins （TRXs）. We have investigated the interactions of eight different chloroplast thioredoxin isoforms （TRX f1, m1, m2, m3, m4, y1, y2, x） with GAPDH （A4, B4, and B8 isoforms）, PRK and CP12 （isoform 2）, all from Arabidopsis thaliana. In the complex, both A4-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs ml and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B4- and Bs-GAPDH were absolutely specific for TRX fl. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A4-GAPDH in the complex was much faster （and less demanding in terms of reducing potential） than activation of free oxidized B4- or BB-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH （B- containing isoforms） would be more suited to conditions of very active photosynthesis.

Inactivity of YGL082W in vitro due to impairment of conformational change in the catalytic center loop

MINDY-1 is a recently discovered new family of deubiquitinating enzymes(DUB),but one of its yeast homologs,YGL082 W,does not show any DUB activity in vitro.Sequence alignment shows that YGL082 W possesses the correct catalytic triad,and yet did not catalyze either the hydrolysis of di-ubiquitin,crosslinking with C-terminally propargylated ubiquitin,or hydrolysis of ubiquitin-7-amino-4-methylcoumarin.After obtaining a crystal structure of the catalytic domain of YGL082 W,we identified an interesting difference between the catalytic center loop of YGL082 W and that of its human homolog MINDY-1.Because the conformation of the catalytic center loop was previously reported to be important for the deubiquitination activity of MINDY-1,we hypothesized that Glu27(instead of the corresponding Pro136 in MINDY-1) of the catalytic center loop of YGL082 W may impair the conformational change and account for the lack of activity.This hypothesis was supported by homology modeling and molecular dynamics simulations,which showed that the Pro-to-Glu mutation(P136 E mutation for MINDY-1) creates a hydrogen bond that inhibits the conformation change of the catalytic center loop of MINDY-1.Further experiments through site-directed mutation validated this hypothesis,showing that the P27 E mutation caused MIY1(a homologous active DUB from yeast) to lose activity.

Complex Regulation of Prolyl-4-Hydroxylases Impacts Root Hair Expansion

Root hairs are single cells that develop by tip growth, a process shared with pollen tubes, axons, and fungal hyphae. However, structural plant cell walls impose constraints to accomplish tip growth. In addition to polysaccharides, plant cell walls are composed of hydroxyproline-rich glycoproteins （HRGPs）, which include several groups of O-glycoproteins, including extensins （EXTs）. Proline hydroxylation, an early post-translational modification （PTM） of HRGPs catalyzed by prolyl 4-hydroxylases （P4Hs）, defines their subsequent O-glycosylation sites. In this work, our genetic analyses prove that P4H5, and to a lesser extent P4H2 and P4H13, are pivotal for root hair tip growth. Second, we demonstrate that P4H5 has in vitro preferred specificity for EXT substrates rather than for other HRGPs. Third, by P4H promoter and protein swapping approaches, we show that P4H2 and P4H13 have interchangeable functions but cannot replace P4H5. These three P4Hs are shown to be targeted to the secretory pathway, where P4H5 forms dimers with P4H2 and P4H13. Finally, we explore the impact of deficient proline hydroxylation on the cell wall architec- ture. Taken together, our results support a model in which correct peptidyl-proline hydroxylation on EXTs, and possibly in other HRGPs, is required for proper cell wall self-assembly and hence root hair elongation in Arabidopsis thaliana.

The Interaction of Spinach Nitrite Reductase with Ferredoxin： A Site-Directed Mutation Study

A series of site-directed mutants of the ferredoxin-dependent spinach nitrite reductase has been characterized and several amino acids have been identified that appear to be involved in the interaction of the enzyme with ferredoxin. In a complementary study, binding constants to nitrite reductase and steady-state kinetic parameters of site-directed mutants of ferredoxin were determined in an attempt to identify ferredoxin amino acids involved in the interaction with nitrite reductase. The results have been interpreted in terms of an in-silico docking model for the 1：1 complex of ferredoxin with nitrite reductase.

The Presence and Localization of Thioredoxins in Diatoms, Unicellular Algae of Secondary Endosymbiotic Origin

Diatoms are unicellular algae of great ecological importance. So far, very little is known about the regulation of carbon fixation in these algae; however, there are strong indications that in diatom plastids, the ferredoxin/thioredoxin system might play a minor role in redox regulation of the photosynthetic reactions compared to land plants. Until now, it is unknown whether there are fewer or other target enzymes of thioredoxins in diatoms. Only a single potential target enzyme for thioredoxin, the plastidic fructose-l,6-bisphosphatase, has yet been identified. Nevertheless, during the annotation of the genome of the diatom Phaeodactylum tricornutum, we identified several genes encoding different thioredoxins. Utilizing in vivo expression of GFP：presequence fusion proteins in P. tricornutum, we were able to show that these thioredoxins are targeted either into plastids, mitochondria, or remain in the cytosol. Surprisingly, two of the three usually cytosolic thioredoxin h proteins are apparently plastid associated and, together with a thioredoxin reductase, putatively located in the periplastidic compartment. This is one of the few indications for so far unknown enzymatic reactions in the space between the two pairs of diatom plastid envelope membranes.