Identification of a System N-Like Na+-Dependent Glutamine Transport Activity in Rat Brain Neurons

Abstract: Glutamine is a primary precursor for the biosynthesis of the neurotransmitters glutamate and γ-aminobutyric acid. It is proposed that glutamine, synthesized and released by astrocytes, is transported into the neuron for subsequent conversion to neurotransmitters. To provide a more complete characterization of this process, we have delineated the transport systems for glutamine uptake in primary cultures of brain neuronal cells from 1-day-old rats. The Na⁺-dependent glutamine entry is mediated by system A, system ASC, and a third, previously unidentified, activity that has been tentatively designated as system N^b. System N^b activity can be monitored by assaying Na⁺-dependent [³H]glutamine uptake in the presence of 2 m M concentrations of both 2-(methylamino)isobutyric acid and threonine to block uptake by systems A and ASC, respectively. The newly identified transport activity exhibits an apparent substrate specificity that is unique compared with the hepatic system N, because it is inhibited by glutamine and asparagine, but not by histidine. Also, the affinity of system N^b for glutamine, as estimated from K _m values, is significantly greater than that observed for the hepatic and muscle Na⁺-dependent glutamine transporters, systems N and N^m. In sharp contrast to the hepatic system N transporter, system N^b exhibits a relative insensitivity to pH and does not permit Li⁺ substitution for Na⁺ as the cosubstrate. The substrate specificity, kinetic analysis, pH sensitivity, and cation dependence of this transport activity indicate that it represents a glutamine transport system not previously identified.     

Rheological characterization of photochemical changes of ethyl(hydroxyethyl)cellulose dissolved in water in the presence of an ionic surfactant and a photosensitizer

The effects of addition of the photosensitizer riboflavin (RF) to semidilute solutions of the systems ethyl(hydroxyethyl)cellulose (EHEC)/water, EHEC/sodium dodecyl sulfate (SDS), and EHEC/cetyltrimethylammonium bromide (CTAB) on the turbidity and the linear viscoelasticity are studied. The turbidity behavior and the cloud point (CP) are influenced by the addition of RF to the EHEC/SDS system, whereas no discernible change is observed for the other systems. The rheological features of all systems are affected by the presence of RF at lower temperatures, whereas at temperatures close to the CP, only a slight effect is detected. Both the EHEC/SDS and EHEC/CTAB systems evolve thermoreversible gels at the same temperature (37.5 degrees C), but in the presence of RF, the EHEC/CTAB system does not form a gel, whereas the gel temperature for the EHEC/SDS system is depressed (32.5 degrees C). Light irradiation of RF in the EHEC/SDS/RF system causes fragmentation of the network and a higher temperature is required to re-form the incipient gel network. The photochemical degradation of EHEC gives rise to a decrease in the dynamic moduli and the complex viscosity for all of the three systems. The effect is strengthened at higher temperatures and it is most pronounced for the EHEC/SDS system.     

Active Uptake of Substance P Carboxy-Terminal Heptapeptide (5–11) into Rat Brain and Rabbit Spinal Cord Slices

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.     

植物蛋白质SUMO化修饰体外高效检测系统

蛋白质SUMO化修饰是一种调控蛋白命运的关键修饰方式, 广泛参与植物生长发育及逆境胁迫响应。SUMO化修饰过程主要由激活酶(E1)-结合酶(E2)-连接酶(E3)组成的级联酶促反应催化, 其关键酶组分将SUMO分子缀合至底物蛋白的赖氨酸残基, 形成共价异肽键以完成SUMO化修饰过程。该文报道了1种植物蛋白质SUMO化修饰体外高效检测系统, 通过在大肠杆菌(Escherichia coli)中构建拟南芥(Arabidopsis thaliana) SUMO化修饰的关键通路实现对底物蛋白的SUMO化修饰, 结果可通过免疫印迹进行检测。该系统可以简化植物蛋白质SUMO化修饰的检测流程, 为植物细胞SUMO化修饰的功能研究提供了有力工具。     

Construction and co-expression of plasmid encoding xylitol dehydrogenase and a cofactor regeneration enzyme for the production of xylitol from D-arabitol

The biotransformation of D-arabitol into xylitol was investigated with focus on the conversion of D-xylulose into xylitol. This critical conversion was accomplished using Escherichia coli to co-express a xylitol dehydrogenase gene from Gluconobacter oxydans and a cofactor regeneration enzyme gene which was a glucose dehydrogenase gene from Bacillus subtilis for system 1 and an alcohol dehydrogenase gene from G. oxydans for system 2. Both systems efficiently converted D-xylulose into xylitol without the addition of expensive NADH. Approximately 26.91 g/L xylitol was obtained from around 30 g/L D-xylulose within system 1 (E. coli Rosetta/Duet-xdh-gdh), with a 92% conversion yield, somewhat higher than that of system 2 (E. coli Rosetta/Duet-xdh-adh, 24.9 g/L, 85.2%). The xylitol yields for both systems were more than 3-fold higher compared to that of the G. oxydans NH-10 cells (7.32 g/L). The total turnover number (TTN), defined as the number of moles of xylitol formed per mole of NAD(+), was 32,100 for system 1 and 17,600 for system 2. Compared with that of G. oxydans NH-10, the TTN increased by 21-fold for system 1 and 11-fold for system 2, hence, the co-expression systems greatly enhanced the NADH supply for the conversion, benefiting the practical synthesis of xylitol.     

Effective switch-on fluorescence sensing of zinc(II) ion by 8-aminoquinolino-beta-cyclodextrin/adamantaneacetic acid system in water

A water-soluble 8-aminoquinolino-beta-cyclodextrin/1-adamantaneacetic acid (1/ADA) system is prepared in situ and exhibits a unique switch-on fluorescence response to Zn(2+) over other common metal ions. Spectrophotometric studies demonstrate that this system can strongly coordinate Zn(2+) through a cyclodextrin/substrate/metal triple recognition mode, and the resulting 1/ADA/Zn(2+) ternary complex emits the blue-green fluorescence (lambda=490nm) that can be easily distinguished by eyes in aqueous solution. Significantly, the switch-on fluorescence response of 1/ADA to Zn(2+) is barely affected by various metal ions except Cu(2+). As a result, this system can behave as an efficient supramolecular fluorescence sensor for Zn(2+) in water.     

Wavelength-dependent quantum yield of ATP synthesis and NADP reduction in normal and dichlorodimethylphenylurea-poisoned chloroplast

At short wavelengths (525–690 mμ) the direct measurement of the quantum yield of the photoreduction of NADP⁺ in normal O₂-evolving spinach chloroplasts is constant ( approx. 0.3 equiv/hv). At short wavelengths (<690 mμ) the quantum yield for NADP⁺ reduction in 3(3,4-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts supplied with the ascorbate-2,6-dichlorophenolindophenol couple (donor system) is approx. half as efficient as the normal system. At long wavelengths the quantum yield of NADP⁺ reduction in the donor system increases by a factor of 2 ( approx. 0.3 equiv/hv) when compared with the corresponding yield for the donor system at short wavelengths ( approx. 0.15 equiv/hv).

Between 525 and 690 mμ, the phosphorylation yield for the normal system is constant ( = 0.15 ATP/hv), maintaining a constant P/2e ratio of unity. The P/2e ratios indicate a tight coupling between phosphorylation and electron transport encompassing a single phosphorylation site for the transfer of two electrons.

Between 525 and 680 mμ, the phosphorylation yield for the donor system is constant ( approx. 0.04 ATP/hv), maintaining a P/2e ratio of approx. 0.5. At longer wavelengths (>690 mμ) the phosphorylation yield of the donor system rises ( approx. 0.07–0.08 ATP/hv) concomitant with the rise in the yield of electron flow.

These experiments suggest the possibility that two types of phosphorylation processes operate in chloroplasts, (1) a short-wavelength process coupled to the normal O₂-evolving activity, and (2) a long-wavelength process coupled to the electron-donor activity of reagents such as DCIP.     

The CRISPR/Cas Adaptive Immune System of Pseudomonas aeruginosa Mediates Resistance to Naturally Occurring and Engineered Phages

Here we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer region of phage DMS3 to make it a target of resistance by the CRISPR/Cas system and screening for mutants that escape CRISPR/Cas-mediated resistance, we show that nucleotides within the PAM and seed sequence and across the non-seed-sequence regions are critical for the functioning of this CRISPR/Cas system. We also demonstrate that P. aeruginosa can acquire spacer content in response to lytic phage challenge, illustrating the adaptive nature of this CRISPR/Cas system. Finally, we demonstrate that the P. aeruginosa CRISPR/Cas system mediates a gradient of resistance to a phage based on the level of complementarity between CRISPR spacer RNA and phage protospacer target. This work introduces a new in vivo system to study CRISPR/Cas-mediated resistance and an additional set of tools for the elucidation of CRISPR/Cas function.     

Alpha-Fetoprotein Promoter-Driven Cre/LoxP-Switched RNA Interference for Hepatocellular Carcinoma Tissue-Specific Target Therapy

Backgroud

RNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.

Methods

Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3) and non-HCC cell lines (L-02, Hela and SW1116) were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.

Results

The AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.

Conclusions

An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.     

Anaerobic fumarate transport in Escherichia coli by an fnr-dependent dicarboxylate uptake system which is different from the aerobic dicarboxylate uptake system.

Escherichia coli grown anaerobically with fumarate as electron acceptor is able to take up C4-dicarboxylates by a specific transport system. The system differs in all tested parameters from the known aerobic C4-dicarboxylate transporter. The anaerobic transport system shows higher transport rates (95 mumol/g [dry weight] per min versus 30 mumol/g/min) and higher Kms (400 versus 30 microM) for fumarate than for the aerobic system. Mutants lacking the aerobic dicarboxylate uptake system are able to grow anaerobically at the expense of fumarate respiration and transport dicarboxylates with wild-type rates after anaerobic but not after aerobic growth. Transport by the anaerobic system is stimulated by preloading the bacteria with dicarboxylates. The anaerobic transport system catalyzes homologous and heterologous antiport of dicarboxylates, whereas the aerobic system operates only in the unidirectional mode. The anaerobic antiport is measurable only in anaerobically grown bacteria with fnr+ backgrounds. Additionally, the system is inhibited by incubation of resting bacteria with physiological electron acceptors such as O2, nitrate, dimethyl sulfoxide, and fumarate. The inhibition is reversed by the presence of reducing agents. It is suggested that the physiological role of the system is a fumarate/succinate antiport under conditions of fumarate respiration.     

Aqueous two-phase protein partitioning using textile dyes as affinity ligands.

A simple and inexpensive aqueous two-phase system for the affinity partitioning of proteins is introduced. An aqueous solution consisting of maltodextrin (M100; molecular mass, 1800) and polyvinylpyrrolidone (PVP360; molecular mass, 360,000) formed two phases at 4 degrees C when the concentration of the polymers was 22.5% (w/w) and 4.0% (w/w), respectively. When the amino derivatives of chlorotriazine textile dyes or other azo textile dyes were added to the two-phase system they partitioned asymmetrically, favoring the upper, less dense, PVP360-rich phase. The association of the textile dyes with PVP360 did not prevent them from acting as affinity ligands for proteins. Three of the dyes screened increased the partition coefficient of purified lysozyme nearly 50-fold over a control containing no dye. Parameters such as pH, ionic strength, and dye concentration modulated the affinity-partitioning effect of the system. The partition coefficient of lysozyme in an egg white protein mixture increased severalfold as the total protein content of the system approached 4% (w/w), indicating that protein concentration is also important in determining the partitioning characteristics of this two-phase system. Proteins were efficiently freed of PVP360 and textile dye by recovery in a high-salt solution when another two-phase system was formed upon the addition of a solution of concentrated potassium phosphate to the isolated upper phase of a PVP360/M100/textile dye two-phase system. The affinity-partitioning system presented here allows one to screen large numbers of potentially useful protein ligands to optimize protein separation, followed by direct scaleup to a system size determined by the user.     

Flash detection/identification of pathogens, bacterial spores and bioterrorism agent biomarkers from clinical and environmental matrices.

We propose to develop an integrated rapid, semiportable, prototype point microbial detection/identification system for clinical specimens that is also capable of differentiating microbial bioterrorism attacks from threats or hoaxes by defining the pathogen. The system utilizes "flash" extraction/analytical system capable of detection/identification of microbes from environmental and clinical matrices. The system couples demonstrated technologies to provide quantitative analysis of lipid biomarkers of microbes including spores in a system with near-single cell (amol/microl) sensitivity. Tandem mass spectrometry increases specificity by providing the molecular structure of neutral lipids, phospholipids, and derivatized spore-specific bacterial biomarker, 2,6-dipicolinic acid (DPA) as well as the lipopolysaccharide-amide-linked hydroxy-fatty acids (LPS-ALHFA) of Gram-negative bacteria. The extraction should take about an hour for each sample but multiple samples can be processed simultaneously.     

Enhanced sequence coverage in tryptic fragment analysis by two-dimensional HPLC/MS using a monolithic silica capillary column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

Functional characterization of PCFT/HCP1 as the molecular entity of the carrier-mediated intestinal folate transport system in the rat model

Proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characteristics of rat PCFT/HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH 5.5, with smaller Michaelis constant (K(m)) for the former (2.4 microM) than for the latter (5.7 microM), indicating its characteristic as a proton-coupled folate transporter that favors folate than methotrexate as substrate. rPCFT/HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characteristics of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the K(m) values were 1.2 and 5.8 microM for folate and methotrexate, respectively, in the rat small intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1, and we successfully provided several lines of evidence that indicate its role as the molecular entity of the intestinal folate transport system.     

Enhanced Sequence Coverage in Tryptic Fragment Analysis by Two-Dimensional HPLC/MS Using a Monolithic Silica Capillary Column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

Osmoregulation by potassium transport in Escherichia coli

Abstract Cell turgor pressure determines the extent of K⁺ accumulation by Escherichia coli cells. K⁺ influx is mediated both by a constitutive system with a low affinity for K⁺ (Trk) and by an inducible high affinity system (Kdp). K⁺ efflux is controlled by as yet unidentified but independent systems. Cell K⁺ concentration may be the link between growth in media of high osmolarity and the concomitant accumulation of compatible solutes such as betaine.     

Effects of 15N application frequency on nitrogen uptake efficiency in citrus trees

Two irrigation systems were used to compare nitrogen uptake efficiency in citrus trees and to evaluate the NO₃⁻ runoff in «Navelina» orange trees [Citrus sinensis (L.) Osbeck] on Carrizo citrange rootstock (Citrus sinensis × Poncirus trifoliata Raf.). These were fertilized with 125 g N as labelled K¹⁵NO₃ and grown outdoors in containers filled with a sand-loamy soil. Two groups of 3 trees received this N dose either in five equally split applications by a flooding irrigation system or in 66 applications by drip. Trees were harvested at the end of the vegetative cycle (December) and the isotopic ratios of ¹⁵N/¹⁴N were measured in the soil-plant system. The N uptake efficiency of the whole tree was higher with drip irrigation (75 percnt;) than with flooding system (64 percnt;). In the 0-90 cm soil profile, the N immobilized in the organic fraction was similar for both irrigation methods (around 13 percnt;), whereas the N retained as NO₃⁻ was 1 percnt; of the N applied under drip and 10 percnt; under flooding. In the last case, most of NO₃⁻ remained under root system and it could be lost to leaching either by heavy rainfalls or excessive water applications. These results showed that a drip irrigation system was more efficient for improving water use and N uptake from fertilizer, in addition to potentially reduced leaching losses.     

Peroxidase-Mediated Oxidation, a Possible Pathway for Activation of the Fungal Nephrotoxin Orellanine and Related Compounds. ESR and Spin-Trapping Studies

Orellanine is the tetrahydroxylated and di-N-oxidized bipyridine toxin extracted from several Cortinarius mushrooms among them C. orellanus. The pathogenic mechanism involved in the C. orellanus-poisoning by orellanine leading to kidney impairment is not yet fully understood until now. Electron spin resonance (ESR) spectroscopy has been used to study the activation of orellanine by horseradish peroxidase/H₂O₂ system at physiological pH. Evidence for a one-electron oxidation of the toxin by this enzymatic system to an ortho-semiquinone radical intermediate is presented.

The orellanine ortho-semiquinone generated by the peroxidase/H₂O₂ system abstracts hydrogen from glutathione, generating the glutathionyl radical which is spin-trapped by 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and subsequently detected by ESR spectroscopy. Similarly, the ortho-semiquinone abstracts hydrogen from ascorbic acid to generate the ascorbyl radical which is detected by direct ESR. The peroxidatic oxidation of orellanine to semiquinone followed by its reduction by glutathione or ascorbic acid does not induce dioxygen uptake. The relationship between chemical structure and HRP oxidation of orellanine-related molecules, namely orelline and DHBPO₂ (the parent molecule lacking of hydroxyl groups in 3 and 3' position) has been investigated in absence or in presence of reducing agents. None of the orellanine-related compounds can be oxidized by the HRP/H₂O₂ system, showing that both catecholic moieties and aminoxide groups are necessary for observing the formation of the ortho-semiquinone form of orellanine. As shown for the (photo)chemical oxidation of orellanine, the mechanism of toxicity could be correlated with a depletion of glutathione and ascorbate levels which are implicated in the defence against oxidative damage.     

Tet-on和Cre/loxP系统双重调控表达丙型肝炎病毒NS3/4A蛋白酶三转基因小鼠的建立

目的:建立Tet-On调控系统和Cre/loxP基因剔除系统双重调控表达丙型肝炎病毒(HCV)NS3/4A丝氨酸蛋白酶三转基因小鼠。方法:选择适龄并经鉴定的在Tet-on系统调控下肝脏特异性表达Cre重组酶的双转基因小鼠Lap/LC-1与在Tet-on系统调控下肝脏特异性表达萤光素酶(Luc)的双转基因小鼠Lap/NS3/4A交配,子代小鼠经PCR检测、筛选基因组中NS3/4A、Lap、LC-1等3个转基因片段均阳性的小鼠。三阳性的NS3/4A/Lap/LC-1小鼠经多西环素(Dox)诱导1周后,以在体生物发光成像系统(BLI)检测报告基因Luc的表达,免疫组化检测小鼠体内Cre重组酶、HCV NS3/4A丝氨酸蛋白酶的表达状况。结果:NS3/4A/Lap/LC-1小鼠经Dox诱导后,BLI结果显示仅在小鼠肝脏部位有强烈的发光信号,表明这些小鼠肝细胞内报告基因Luc特异高效表达;免疫组化结果证实Cre重组酶、NS3/4A蛋白酶仅在经诱导后的小鼠肝细胞中特异性表达。结论:建立了Tet-On调控系统和Cre/loxP基因剔除系统双重调控下表达HCV NS3/4A丝氨酸蛋白酶的三转基因小鼠模型,为进一步研究HCV NS3/4A丝氨酸蛋白酶在HCV感染后与宿主相互作用的机制,以及抗NS3/4A丝氨酸蛋白酶特异性抑制剂的筛选奠定了基础。     

Proficiency of the Tradescantia-micronucleus image analysis system for scoring micronucleus frequencies and data analysis.

The Tradescantia-micronucleus (Trad-MCN) bioassay is an efficient short-term test for genotoxicity of pollutants. In order to increase the efficiency and to standardize the micronucleus (MCN) scoring process, an automated scoring system was developed using the principle of image analysis in computer science. This assemblage is called the Tradescantia-micronucleus image analysis (Trad-MCNIA) system. The MCN frequencies scored by this system were compared with those scored by human observation for its proficiency. A set of low MCN frequency (around 5 MCN/100 tetrads) slides prepared from a control group, a set of medium MCN frequency (around 20 MCN/100 tetrads) slides prepared from sodium azide treated plant cuttings and a set of high MCN frequency (around 50 MCN/100 tetrads) slides prepared from X-ray treated materials were used for this study. In the low MCN frequency slides, the Trad-MCNIA system scored about the same value as human observation. In the medium and high frequency slides, MCN frequencies scored by the system were lower than those scored by human observers. This discrepancy was corrected by increasing the power of the objective of the microscope in the system. The MCN frequencies scored by the system attained 90% congruity with those scored by human observers after the correction. The scoring speed of the system was about 3.5 times as fast as that by human observers, and the data could be statistically analyzed immediately after the data scores were recorded. Further improvements can be made by upgrading the video camera and the computer speed.