Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI.

A physical map of the bacteriophage T5 genome was constructed by ordering the fragments produced by cleavage of T5 DNA with the restriction endonucleases SalI (4 fragments), SmaI (4 fragments), BamI (5 fragments), and HpaI (28 fragments). The following techniques were used to order the fragments. (i) Digestion of DNA from T5 heat-stable deletion mutants was used to identify fragments located in the deletable region. (ii) Fragments near the ends of the T5 DNA molecule were located by treating T5 DNA with lambda exonuclease before restriction endonuclease cleavage. (iii) Fragments spanning other restriction endonuclease cleavage sites were identified by combined digestion of T5 DNA with two restriction endonucleases. (iv) The general location of some fragments was determined by isolating individual restriction fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. (v) Treatment of restriction digests with lambda exonuclease before digestion with a second restriction enzyme was used to identify fragments near, but not spanning, restriction cleavage sites. (vi) Exonucleases III treatment of T5 DNA before restriction endonuclease cleavage was used to locate fragments spanning or near the natural T5 single-chain interruptions. (vii) Analysis of the products of incomplete restriction endonuclease cleavage was used to identify adjacent fragments.     

A cell pattern generation model based on an extended artificial regulatory network

Cell pattern generation has a fundamental role in both artificial and natural development. This paper presents results from a model in which a genetic algorithm (GA) was used to evolve an artificial regulatory network (ARN) to produce predefined 2D cell patterns through the selective activation and inhibition of genes. The ARN used in this work is an extension of a model previously used to create simple geometrical patterns. The GA worked by evolving the gene regulatory network that was used to control cell reproduction, which took place in a testbed based on cellular automata (CA). After the final chromosomes were produced, a single cell in the middle of the CA lattice was allowed to replicate controlled by the ARN found by the GA, until the desired cell pattern was formed. The model was applied to the problem of generating a French flag pattern.     

Following the biochemical and morphological changes of Bacillus atrophaeus cells during the sporulation process using Bioaerosol Mass Spectrometry

Bioaerosol Mass Spectrometry (BAMS), a real-time single cell analytical technique, was used to follow the biochemical and morphological changes within a group of Bacillus atrophaeus cells by measuring individual cells during the process of sporulation. A mutant of B. atrophaeus that lacks the ability to produce dipicolinic acid (DPA) was also analyzed. Single cell aerodynamic sizing was used to follow gross morphological changes, and chemical analysis of single cells by mass spectrometry was used to follow some biochemical changes of B. atrophaeus cells during endospore formation.     

An improved method for anti-glycolipid antibody and glycolipid determination by an enzyme-linked immunosorbent assay using polystyrene beads

Sensitive determination of anti-glycolipid antibody titer and glycolipid content by an enzyme-linked immunosorbent assay (ELISA) using polystyrene beads was achieved. Glycolipid-coated polystyrene beads were used as the immobilized antigen. As antigen glycolipids, gangliotetraosylceramide (GA1), gangliotriosylceramide (GA2) and neolactotetraosylceramide (paragloboside) were used. Concentrations of 1-500 ng glycolipid in liposomes/ml or 0.1-100 micrograms glycolipid/ml could be used for the glycolipid determination. Glycolipid determination by the competitive inhibition method was not influenced by the presence of other glycolipids. A great advantage of this method is that the glycolipid-coated beads can be used repeatedly by washing the used beads with 3M NaSCN solution. The method was applied to the detection of auto-antibody against GA1 in ascitic fluid from cancer patients.     

Bioassay-guided isolation of an alkaloid with antiangiogenic and antitumor activities from the extract of Fissistigma cavaleriei root

Fissistigma cavaleriei (Levl) Rehd (Annonaceae) is used as a folklore medicine for treatment of inflammation, arthritis, and tuberculosis by Miao people in China. In the present study, the antiangiogenic activity of F. cavaleriei was investigated. The chorioallantoic membrane of the fertilized hen's egg (CAM assay) was used to determine antiangiogenic activity of the plant extract. Compound (1), a compound with antiangiogenic activity, was isolated by bioassay-guided fractionation from F. cavaleriei for the first time. The structure of compound (1) was elucidated on the basis of spectroscopic methods. Colorimetric COX (ovine) inhibitor screening assay was used to determine its inhibitory effect on COX-1 and COX-2. MTT and Sulforhodamine B assays were used to investigate its cytotoxic effects on tumor cell lines. As a result, compound (1) showed a selectively inhibiting effect on COX-2 and could inhibit the growth of tumor cells in vitro. The antitumor activity of compound (1) was further confirmed by the observation that compound (1) administration significantly inhibited the growth of S-180 cells in mice. Moreover, compound (1) was able to enhance the antitumor activity of doxorubicin in the mice bearing with S-180 cells while combined with doxorubicin. In conclusion, compound (1) is a multi-target molecule and further experimental investigations are needed to determine whether it can be used as a lead molecule for tumor treatment.     

Synergistic effect of salt mixture on the gelation temperature and morphology of methylcellulose hydrogel

Gelation temperature of methylcellulose (MC) can be altered by adding different additives. Pure MC showed sol-gel transition at 60°C. Sodium citrate and sodium tartrate were used alone and in combination to see the effect of individual salt and combination of salts on the gelation temperature of MC. The gelation temperature of all the binary and ternary combinations of MC and salts were measured with different methods such as test tube tilting method (TTM), UV-vis spectroscopy, viscometry, and by rheometer and also the morphology of gels were characterized with the help of environmental scanning electron microscopy (ESEM). It was observed that when 0.1M sodium citrate (NaC) and 0.1M sodium tartrate (NaT) were used separately, the gelation temperature of MC was reduced up to 44°C and 47°C respectively but when mixture of NaC and NaT (0.1(M) NaC and 0.1(M) (NaT)) were used the gelation temperature was further reduced to 36°C. It was clear from ESEM images that when NaC and NaT were used separately the formation of network was not distinguishable. But, well-connected network structure was observed when a mixture 0.1M NaC and 0.1M NaT was used.     

排卵前期卵泡颗粒细胞端粒酶的表达及其影响因素

用端粒酶重复扩增酶联免疫吸附分析法（telomeric repeat amplification protocol-enzyme linked immunoadsordent assay,TRAP-ELISA）观察体外培养的大鼠排卵前期卵巢颗粒细胞中端粒酶活性的表达及其影响因素,并用放射免疫分析法（radioimmunoassay,RIA）同步测定培养液中雌二醇（estradiol,E2）、孕西阿（progesterone,P0）含量的变化及MTT（四甲基偶氮唑盐）法测定颗粒细胞增殖指数,分析颗粒细胞中端粒酶活性的表达以及端粒酶活性表达的影响因素。本实验中大鼠排卵前期卵巢颗粒细胞中有端粒酶活性表达,且在人绒毛膜促性腺激素（human chorionic gonadotropin,HCG）、卵泡刺激素（follicle-stimu1ating hormone,FSH）、二丁酰环磷腺苷（dbcAMP）及维拉帕米（verapamil）作用下活性明显升高,而在反义c-myb作用下活性明显降低。RIA测定培养液中雌激素及孕激素含量发现,在verapamil及FSH作用下E2与P0分泌量明显升高,在dbcAMP及HCG作用下分泌量无明显改变,而在反义c-myb作用下分泌量明显降低,在不同作用因素下的端粒酶活性与它相对应的E2及P0分泌量无相关性。MTT法测定显示,反义hTERT能明显抑制颗粒细胞的增殖。由此可以证实,排卵前期卵巢的颗粒细胞中表达有端粒酶活性,其活性受FSH、HCG、verapamil、dbcAMP及癌基因的影响,并且端粒酶活性与颗粒细胞增殖功能相关。     

A comparison of the rate of DNA synthesis in myeloblasts from peripheral blood and bone marrows of patients with acute nonlymphocytic leukemia

Durations of S-phase (Ts) and total cell cycle times (Tc) were measured from the peripheral blood (PB) and bone marrow aspirates (BM) of five patients with acute nonlymphocytic leukemia (ANLL). Intravenous bromodeoxyuridine (BrdU) was used as the first label for S-phase cells and a monoclonal anti-BrdU antibody was used to detect the positive cells. Tritiated thymidine [( 3H]Tdr) was used as a second label in vitro, and the Ts was calculated by counting the number of cells labeled either by BrdU or by [3H]Tdr or by both. Our data demonstrate that the duration of S-phase in myeloblasts obtained from BM is quite similar to that of circulating leukemic cells. Finally, the most accurate assessment of percentage of myeloblasts actively engaged in DNA synthesis can be obtained only from bone marrow biopsies following in vivo labeling.     

Detection of methyl parathion using immuno-chemiluminescence based image analysis using charge coupled device

A novel method based on immuno-chemiluminescence and image analysis using charge coupled device (CCD) for the qualitative detection of methyl parathion (MP) with high sensitivity (up to 10 ppt) is described. MP antibodies raised in poultry were used as a biological sensing element for the recognition of MP present in the sample. The immuno-reactor column was prepared by packing in a glass capillary column (150 microl capacity) MP antibodies immobilized on Sepharose CL-4B through periodate oxidation method. Chemiluminescence principle was used for the detection of the pesticide. Light images generated during the chemiluminescence reaction were captured by a CCD camera and further processed for image intensity, which was correlated with pesticide concentrations. K(3)Fe(CN)(6) was used as a light enhancer to obtain detectable light images. Different parameters including concentrations of K(3)Fe(CN)(6), luminol, urea H(2)O(2), antibody, addition sequence of reactants and incubation time to obtain best images were optimized. The results obtained by image analysis method showed very good correlation with that of competitive ELISA for methyl parathion detection. Competitive ELISA method was used as a reference to compare the results obtained by CCD imaging.     

Effects of electron transport inhibitors on iron reduction in Aeromonas hydrophila strain KB1

The aim of this study was to determine the influence of respiratory chain inhibitors upon iron (III) reduction in Aeromonas hydrophila strain KB1. Optimal conditions of the reduction process were established by determining the amount of biomass, optimal pH, temperature and substrate concentration. The obtained results allowed us to determine Hill equation coefficients (K(m)=1.45+/-0.18 mM; V(max)=83.40+/-2.70 microM/min, and h=0.7+/-0.03). The value of h points to Michaelis-like kinetics of the process. The substrate concentration used in our study was such as to allow the maximum iron reduction rate. The reaction was mesophilic. The participation of electron carriers in the iron reduction process was investigated using respiratory chain inhibitors. Rotenone and capsaicin were used to study Q sites of the respiratory chain complex I. Dicumarol was used as an inhibitor of the quinone loop, while quinacrine was used to inhibit alloxazine centers. Additionally, complex III inhibitors, such as antimycin A, myxothiazole and 2-heptyl-4-hydroxy-quinoline N-oxide (HQNO) were used. Azide was used to inhibit complex IV. The observed inhibition of iron reduction by rotenone and capsaicin may suggest the existence of Q sites in formate reductase, analogous to those in complex I. Inhibition of quinones, isoalloxazine centers and complex III suggests participation of these carriers in the electron transport during iron reduction. Lack of inhibition of iron reduction by azide suggests that complex IV does not participate in this process.     

A quantum dot-based optical immunosensor for human serum albumin detection

In this study, a CdSe/ZnS quantum dot (QD)-based immunosensor using a simple optical system for human serum albumin (HSA) detection is developed. Monoclonal anti-HSA (AHSA) immobilized on 3-aminopropyltriethoxysilane (APTES)-modified glass was used to capture HSA specifically. Bovine serum albumin (BSA) was used to block non-specific sites. The solution, containing AHSA-QD complex prepared by mixing biotinylated polyclonal anti-HSA and streptavidin coated QD, was used to conjugate with the HSA molecules captured on AHSA/BSA/APTES-modified glass for the modification of HSA with QD. A simple optical system, comprising a diode laser (405 nm), an optical lens, a 515-nm-long pass filter, and an Si-photodiode, was used to detect fluorescence and convert it to photocurrent. The current intensity was determined by the amount of QD specifically conjugated with HSA, and was therefore HSA-concentration-dependent and could be used to quantify HSA concentration. The detection limit of the pure QD solution was ~3.5×10(-12) M, and the detection limit for the CdSe/ZnS QD-based immunosensor developed in this study was approximately 3.2×10(-5) mg/ml. This small optical biosensing system shows considerable potential for future applications of on-chip liver-function detection.     

脑红蛋白ELISA检测试剂盒的研发与应用

目的:利用ELISA技术研发一种灵敏、快速检测人血清中脑红蛋白(Ngb)含量的检测试剂盒,并探讨其在正常人血清样本检测中的应用。方法:采用热诱导的原核表达体系获得重组人源脑红蛋白(rhNgb),通过凝胶过滤和阴离子交换层析等制备rhNgb纯品;将适量rhNgb纯品免疫动物获得抗rhNgb单克隆抗体和多克隆抗体;用双抗体夹心ELISA技术制备rhNgb-ELISA检测试剂盒;用该试剂盒对410例正常人血清中Ngb的含量进行检测,其中包括1.5岁以下婴儿血清55例、19～70岁成人血清355例。结果:用制备的rhNgb-ELISA试剂盒检测发现Ngb在正常人血清中的分布与年龄存在明显的相关性,表现为"两头高、中间低"的趋势,婴儿和老年人血清中Ngb的含量分别为1.6708±0.4945和2.1962±0.6703μmol/L,而中年人血清中Ngb的含量为0.3622±0.0716μmol/L。结论:采用双抗夹心ELISA技术并结合严格的质量控制,研发了高效灵敏的rhNgb-ELISA检测试剂盒,并初步获得了正常人血清中Ngb的含量分布情况,为临床缺血及缺氧性脑损伤等相关疾病的辅助诊断治疗提供了参考信息。     

Diffuse reflectance infrared Fourier transform spectroscopic characterization of a silica-immobilized N-hydroxysuccinimide active ester crosslinking agent and its precursors

Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy was used to characterize the product of each step in the preparation of a silica-immobilized N-hydroxysuccinimide (NHS) active ester. The preparation of this NHS active ester linkage was based on a literature procedure for the immobilization of proteins. The DRIFT method was used to guide modification of this literature procedure. The DRIFT method also was used to indicate an impurity entrapped in the 60-A diameter pores of the silica support during the formation of the immobilized active ester. Degradation of the immobilized NHS active ester, stored under either argon or dioxane, can be followed by the DRIFT method. Myoglobin and glycine were allowed to react with the active ester, and the result for this silica support was evaluated by the DRIFT method. Elemental analysis was used to provide information on the loading of the silica-immobilized moieties that were presented for DRIFT analysis.     

Cometabolic degradation of trichloroethylene in a bubble column bioscrubber

A bubble column bioreactor was used as bioscrubber to carry out a feasibility study for the cometabolic degradation of trichloroethylene (TCE). Phenol was used as cosubstrate and inducer. The bioreactor was operated like a conventional chemostat with regard to the cosubstrate and low dilution rates were used to minimize the liquid outflow. TCE degradation measurements were carried out using superficial gas velocities between 0.47and 4.07 cm s(-1) and TCE gas phase loads between 0.07 and 0.40 mg L(-1) Depending on the superficial gas velocity used, degrees of conversion between 30% and 80% were obtained. A simplified reactor model using plug flow for the gas phase, mixed flow for the liquid phase, and pseudo first order reaction kinetics for the conversionof TCE was established. The model is able to give a reasonable approximation of the experimental data. TCE degradation at the used experimental conditions is mainly limited by reaction rate rather than by mass transfer rate. The model can be used to calculate the reactor volume and the biomass concentration for a required conversion. (c) 1995 John Wiley & Sons Inc.     

A monoclonal antibody to an oocyte-specific poly(A) RNA-binding protein

Xenopus oocyte-specific poly(A) RNA-binding proteins were isolated and used to prepare monoclonal antibodies. One antibody was used to characterize one particular antigen by immunoblot analysis. The antigen had a molecular weight of 56,000 was oocyte-specific, and decreased in amount during oogenesis. The antigen was localized in the cytoplasm throughout oogenesis and sedimented mainly at 40-60 S. The antigen also was shown to bind poly(A) RNA following chromatography of ribonucleoprotein particles on oligo(dT)-cellulose. The antibody was used to immunoadsorb nontranslating ribonucleoprotein particles. Fifty-five per cent of the poly(A) RNA sedimenting between 40-60 S was shown to be bound by the antigen. The further use of this antibody in attempting to examine other components of the ribonucleoprotein particle is discussed.     

Factors that determine the plasma-membrane potential in bloodstream forms of Trypanosoma brucei.

The plasma-membrane potential (Delta(psi)p) in bloodstream forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN- were used to report Delta(psi)p directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Delta(psi)p only when Delta(psi)m had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Delta(psi)p had a value of -82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (-76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Delta(psi)p together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl- by the gluconate anion on Delta(psi)p. The contribution to Delta(psi)p by electrogenic pumping of Na+/K+-ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Delta(psi)p.     

Scano-magneto immunoassay based on carbon nanotubes/gold nanoparticles nanocomposite for Salmonella enterica serovar Typhimurium detection

To improve sensitivity of S. enterica serovar Typhimurium detection, multiwalled carbon nanotubes (MWCNTs) and gold nanoparticles (AuNPs) were combined and used as a label to amplify signal in a scanometric based assay. In this study, the MWCNTs/AuNPs nanocomposite was fabricated by directly assemble of Au(3+) to MWCNTs and allowed growing of AuNPs along the MWCNTs surface. This MWCNTs/AuNPs nanocomposite was then attached to anti-S. typhimurium antibody (MWCNTs/AuNPs/Ab(1)) and used as a detecting molecule. Upon binding to Salmonella, they were pre-concentrated by magenetic beads/antibody (MBs/Ab(2)) forming a sandwich immuno-complex which is later spotted on a nitrocellulose membrane coated slide. Silver reduction was applied to amplify signal. The detection limit of 42CFU/ml was achieved when 2% BSA was used as a blocking agent. Given different types of real samples testing, chicken broth was found to give lowest detection limit, followed by orange juice low fat and whole milk. Selectivity testing was performed by using Escherichia coli as interference and found slightly cross-reactivity which could be due to specificity of the Ab used. By virtue of using a slide for multi-samples spotting and a flatbed scanner for signal-read out acquisition, this scano-magneto immunoassay could enable low-cost detection as well as high throughput screening.     

The influence of nitrate concentration upon the end-products of nitrate dissimilation by bacteria in anaerobic salt marsh sediment

Abstract Experiments were carried out with slurries of saltmarsh sediment to which varying concentrations of nitrate were added. The acetylene blocking technique was used to measure denitrification by accumulation of nitrous oxide, while reduction of nitrate to nitrite and ammonium was also measured. There was good recovery of reduced nitrate and at the smallest concentration of nitrate used (250 μM) there was approximately equal reduction to either ammonium or nitrous oxide (denitrification). Nitrite was only a minor end-product of nitrate reduction. As the nitrate concentration was increased the proportion of the nitrate which was denitrified to nitrous oxide increased, to 83% at the greatest nitrate concentration used (2 mM), while reduction to ammonium correspondingly decreased. This change was attributed either to a greater competitiveness by the denitrifiers for nitrate as the ratio of electron donor to electron acceptor decreased; or to the increased production of nitrite rather than ammonium by fermentative bacteria under high nitrate, the nitrite then being reduced to nitrous oxide by denitrifying bacteria.     

Noninvasive tumor volume measurement using magnetic resonance (MR) imaging and an open sided saddle coil

A magnetic resonance (MR) imaging scanner operated at 0.5 T with a specially constructed receiving coil was used to measure volumes of primary spontaneous tumors in rats and guinea pigs. The coil was used to improve the signal to noise ratio (S/N) of the MR images of tumors in these small animals. The tumor volume was determined by the summation of the volume of contiguous slices or ellipsoid approximation. The accuracy of the volume measurement was better when the numerical integration was used in calculating the slice volume. The open sided saddle (OSS) coil used as the receiving coil gave better S/N than that of the standard head coil.