Cytochrome P-450 mediated interaction between hydroperoxide and molecular oxygen. A proposed method for P-450 kinetic studies

Rat liver cytochrome P-450 mediates a novel reaction between equimolar quantities of dissolved oxygen and organic hydroperoxides. The reaction shares some of the properties of both NADPH-O2 dependent hydroxylation and NADPH-O2 independent peroxidase reactions, but does not require either NADPH, phosphatidylcholine, or any substrates other than hydroperoxide and oxygen. It proceeds at a rate approximately 100 times faster than other well known P-450 hydroxylation reactions. Monitoring the rate of O2 consumption in this novel reaction may be a simple and rapid means for studying the kinetics of cytochrome P-450.     

Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell

Fluorescence lifetime imaging microscopy (FLIM) is a technique in which the mean fluorescence lifetime of a chromophore is measured at each spatially resolvable element of a microscope image. The nanosecond excited-state lifetime is independent of probe concentration or light path length but dependent upon excited-state reactions such as fluorescence resonance energy transfer (FRET). These properties of fluorescence lifetimes allow exploration of the molecular environment of labelled macromolecules in the interior of cells. Imaging of fluorescence lifetimes enables biochemical reactions to be followed at each microscopically resolvable location within the cell.     

Oxygen participation in the in vivo and in vitro aging of collagen fibres

We have examined, as a function of oxygen concentration, the in vivo and in vitro aging rates of tendon from animal species with widely differing life-spans. The results suggest that concomitant with the genetically determined aldimine type cross-linking reactions which probably are complete by the time the animal has reached physiological maturity, there is an on-going oxygen-mediated system of reactions which also effectively cross-link the structure. These reactions appear to proceed with their own intrinsic rate dependent only upon oxygen concentration, and independent of the particular species involved. They may not be required by connective tissues, but are an unavoidable consequence of the presence of free oxygen in tissues with a low rate of turnover.     

Rate-independent constructs for chemical computation

This paper presents a collection of computational modules implemented with chemical reactions: an inverter, an incrementer, a decrementer, a copier, a comparator, a multiplier, an exponentiator, a raise-to-a-power operation, and a logarithm in base two. Unlike previous schemes for chemical computation, this method produces designs that are dependent only on coarse rate categories for the reactions ("fast" vs. "slow"). Given such categories, the computation is exact and independent of the specific reaction rates. The designs are validated through stochastic simulations of the chemical kinetics.     

Dickeya dadantii pectic enzymes necessary for virulence are also responsible for activation of the Arabidopsis thaliana innate immune system

Soft‐rot diseases of plants attributed to Dickeya dadantii result from lysis of the plant cell wall caused by pectic enzymes released by the bacterial cell by a type II secretion system (T2SS). Arabidopsis thaliana can express several lines of defence against this bacterium. We employed bacterial mutants with defective envelope structures or secreted proteins to examine early plant defence reactions. We focused on the production of AtrbohD‐dependent reactive oxygen species (ROS), callose deposition and cell death as indicators of these reactions. We observed a significant reduction in ROS and callose formation with a bacterial mutant in which genes encoding five pectate lyases (Pels) were disrupted. Treatment of plant leaves with bacterial culture filtrates containing Pels resulted in ROS and callose production, and both reactions were dependent on a functional AtrbohD gene. ROS and callose were produced in response to treatment with a cellular fraction of a T2SS‐negative mutant grown in a Pels‐inducing medium. Finally, ROS and callose were produced in leaves treated with purified Pels that had also been shown to induce the expression of jasmonic acid‐dependent defence genes. Pel catalytic activity is required for the induction of ROS accumulation. In contrast, cell death observed in leaves infected with the wild‐type strain appeared to be independent of a functional AtrbohD gene. It was also independent of the bacterial production of pectic enzymes and the type III secretion system (T3SS). In conclusion, the work presented here shows that D. dadantii is recognized by the A. thaliana innate immune system through the action of pectic enzymes secreted by bacteria at the site of infection. This recognition leads to AtrbohD‐dependent ROS and callose accumulation, but not cell death.     

Biogenesis of β-Carotene in Mycobacterium kansasii

The biogenesis of beta-carotene in the photochromogen Mycobacterium kansasii consists of two reactions. The first reaction is photochemical, and is dependent on the wavelength of the incident light and on oxygen but is independent of temperature. The second reaction does not require illumination, and is dependent on the temperature and on oxygen. The latter, or dark reaction, requires the synthesis of new protein, and was shown to have the characteristics of an inducible system. Carotenogenesis was stimulated by incident light of wavelengths of 420, 540, and 650 nm. Immediately after illumination there was an increase in the synthesis of ribonucleic acid and beta-carotene accumulation started after a lag of 8 to 10 min. The synthesis of beta-carotene exhibited temperature dependence with an optimum of about 36 C.     

Fitting kinetic data for two independent saturable terms by MULTIFIT II, a general purpose curve fitting program in FORTRAN IV

The present report describes multifit ii, a fortran program for fitting data to non-linear equations and its application to biphasic kinetic data. Biphasic plots are encountered, e.g. in cases of enzymatic reactions and in the area of amino acid transport across biological membranes. This program gives a least squares fit of an equation to data, and obviates the need for non-statistical approximations, with their inherent bias. Equations to be fit are written in the form (numerator)/(denominator), where each may contain up to four terms, and each term may be a maximum of fourth order, including up to four independent variables. Data may be fit by the present program in either direct or reciprocal form. The approach taken is an iterative procedure (Newton's method). Scaling and weighting of data are incorporated. Parameters used to generate ideal test data in biphasic kinetic plots were recovered to four significant figures. The utility of the program in fitting real biphasic data obtained from amino acid transport experiments is demonstrated.     

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP‐dependent enzymes

Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real‐time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler‐assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'‐phosphate‐dependent enzymes is presented using SEC for direct monitoring of enzyme‐bound and free reaction intermediates. Time‐resolved changes of the different cofactor states, e.g. pyridoxal 5'‐phosphate, pyridoxamine 5'‐phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate‐independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP‐dependent enzymes.     

A novel software tool for high throughput measurements of interconversion barriers: DCXplorer

The software program DCXplorer is introduced to directly access interconversion rate constants in dynamic chromatography and electrophoresis. The program utilizes the unified equation of chromatography which can evaluate reaction rate constants of all kinds of first order reactions of processes taking place during a separation process. Evaluations with DCXplorer are facilitated by a graphical user interface which allows zooming into the area of interest of an interconversion profile and calculating reaction rate constants without a time consuming simulation process. DCXplorer was applied to determine the enantiomerization barrier of the diuretic drug chlorthalidone by pressure supported dynamic capillary electrokinetic chromatography (DEKC) under acidic conditions at pH 5.00 and pH 3.75. Activation parameters DeltaH(++) and DeltaS(++) were obtained from temperature dependent measurements between 15.0 and 35.0 degrees C in 5 K steps at pH 3.75 and between 30.0 and 50.0 degrees C in 10K steps at pH 5.00.     

PhosTShunter: a fast and reliable tool to detect phosphorylated peptides in liquid chromatography Fourier transform tandem mass spectrometry data sets

A database independent search algorithm for the detection of phosphopeptides is described. The program interrogates the tandem mass spectra of LC-MS/MS data sets regarding the presence of phosphorylation specific signatures. To achieve maximum informational content, the complementary fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD) are used independently for peptide fragmentation. Several criteria characteristic for peptides phosphorylated on either serine or threonine residues were evaluated. The final algorithm searches for product ions generated by either the neutral loss of phosphoric acid or the combined neutral loss of phosphoric acid and water. Various peptide mixtures were used to evaluate the program. False positive results were not observed because the program utilizes the parts-per-million mass accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Additionally, false negative results were not generated owing to the high sensitivity of the chosen criteria. The limitations of database dependent data interpretation tools are discussed and the potential of the novel algorithm to overcome these limitations is illustrated.     

Kinetics of apoptotic markers in exogeneously induced apoptosis of EL4 cells

We investigated the time-dependence of apoptotic events in EL4 cells by monitoring plasma membrane changes in correlation to DNA fragmentation and cell shrinkage. We applied three apoptosis inducers (staurosporine, tubericidine and X-rays) and we looked at various markers to follow the early-to-late apoptotic events: phospholipid translocation (identified through annexin V-fluorescein assay and propidium iodide), lipid package (via merocyanine assay), membrane fluidity and anisotropy (via fluorescent measurements), DNA fragmentation by the fluorescence-labeling test and cell size measurements. The different apoptotic inducers caused different reactions of the cells: staurosporine induced apoptosis most rapidly in a high number of cells, tubercidine triggered apoptosis only in the S phase cells, while X-rays caused a G2/M arrest and subsequently apoptosis. Loss of lipid asymmetry is promptly detectable after one hour of incubation time. The phosphatidylserine translocation, decrease of lipid package and anisotropy, and the increase of membrane fluidity appeared to be based on the same process of lipid asymmetry loss. Therefore, the DNA fragmentation and the cell shrinkage appear to be parallel and independent processes running on different time scales but which are kinetically inter-related. The results indicate different signal steps to apoptosis dependent on inducer characteristics but the kinetics of "early-to-late" apoptosis appears to be a fixed program.     

LGA: A method for finding 3D similarities in protein structures

We present the LGA (Local-Global Alignment) method, designed to facilitate the comparison of protein structures or fragments of protein structures in sequence dependent and sequence independent modes. The LGA structure alignment program is available as an online service at http://PredictionCenter.llnl.gov/local/lga. Data generated by LGA can be successfully used in a scoring function to rank the level of similarity between two structures and to allow structure classification when many proteins are being analyzed. LGA also allows the clustering of similar fragments of protein structures.     

Computer aided evaluation of differences in host reactions between isolates of turnip mosaic virus fromSisymbrium loeselii

Twenty three isolates of turnip mosaic virus (TuMV) obtained from spontaneously infected plants ofSiaymbrivm loeaelii JUSL. were serologically related, but differed in reactions of eleven host plants. Susceptibility and sensitivity of each host for each TuMV isolate were classified by one of six degrees (0 to 5) respectively. Similarities of isolates (as compared in 253 pairs) were evaluated by means of a computer; for this purpose the source program MINDIF has been elaborated using the universal program language Algol 60. A table of differences between isolates was obtained and distribution of isolate pairs dependent upon the difference values was stated. The fitness of hosts for differentiating TuMV isolates was ascertained quantitatively. No relation of isolate similarity to geographical indices could be established.     

Kinetic analysis of chemical reactions coupled to an enzymic step. Application to acid phosphatase assay with Fast Red.

Acid phosphatase assay with alpha-naphthyl phosphate as substrate and the use of diazonium salt (Fast Red TR) for chromophore formation was kinetically analysed as a system of two chemical reactions coupled to an enzymic reaction. This system follows a mechanism defined as enzymic-chemical-chemical (EzCC). The accumulation of chromophore with reaction time presented a marked lag period, which was only dependent on the rate constants of the chemical reactions and was independent of the enzymic step. The specific rate constants of each chemical step were determined in 3.8-5.0 pH and 10-35 degrees C temperature ranges. Thermodynamic parameters of the chemical steps were also obtained. Measurement of acid phosphatase activity can be carried out in the pH range 3.8-5.0 (4.8 was optimal pH) without the need to eliminate the lag period.     

Analysis of multivariate recurrent event data with time‐dependent covariates and informative censoring

Multivariate recurrent event data are usually encountered in many clinical and longitudinal studies in which each study subject may experience multiple recurrent events. For the analysis of such data, most existing approaches have been proposed under the assumption that the censoring times are noninformative, which may not be true especially when the observation of recurrent events is terminated by a failure event. In this article, we consider regression analysis of multivariate recurrent event data with both time‐dependent and time‐independent covariates where the censoring times and the recurrent event process are allowed to be correlated via a frailty. The proposed joint model is flexible where both the distributions of censoring and frailty variables are left unspecified. We propose a pairwise pseudolikelihood approach and an estimating equation‐based approach for estimating coefficients of time‐dependent and time‐independent covariates, respectively. The large sample properties of the proposed estimates are established, while the finite‐sample properties are demonstrated by simulation studies. The proposed methods are applied to the analysis of a set of bivariate recurrent event data from a study of platelet transfusion reactions.     

The translational requirement for complete La Crosse virus mRNA synthesis is cell-type dependent.

The translational requirement to prevent premature termination during La Crosse virus S mRNA synthesis was found to be cell-type dependent. This requirement was present in the BHK, HEL, and Vero cell lines we examined, but not in C6/36 mosquito cells. The cell-dependent translational requirement could be reproduced in vitro by using either cell extracts or purified virions of BHK and C6/36 cells. In the BHK reactions, the polymerase terminated predominantly at nucleotide 175 in the absence of concurrent translation and required translation to read through this position. In the C6/36 reactions, however, the polymerase reads through nucleotide 175 efficiently independent of translation. Reconstitution studies suggested that the translational requirement was due to a factor(s) present in BHK, but not in C6/36, cells.     

Changes in hepatic dolichol and dolichyl monophosphate caused by treatment of rats with inducers of the endoplasmic reticulum and peroxisomes and during ontogeny

Rats were treated with various inducers of the endoplasmic reticulum and peroxisomes and the properties and distributions of dolichol and dolichyl phosphate analyzed. The treatment of rats with carcinogenic agents 2-acetylaminofluorene, N-nitrosodiethylamine and 3-methylcholanthrene and with the compounds such as phenobarbital, terpentine, cholestyramine and di(2-ethylhexyl)phthalate have all caused changes in the microsomal or lysosomal contents of dolichol to various extents, but only the latter group influenced dolichyl-P concentration. Shortly after birth, the hepatic content of dolichyl-P reaches the adult level, whereas the level of the free alcohol is low at birth but increases continuously thereafter. Incorporation of [3H]mevalonate into dolichol was also dependent on factors other than de novo synthesis, e.g., the pool size. Rates of glycosylation reactions dependent on dolichyl-P exhibit considerable changes but are independent of the existing levels of lipid intermediate. GDP-mannosyl transferase activity increases greatly with birth, but the enzyme activity returns to the adult level within a day after birth. These results demonstrate that structural and functional modifications induced with drugs can greatly influence the content and distribution of dolichol which are independent of the existing levels of dolichyl-P.     

森林生态效益的线性联立方程组模型的研究

在森林生态效益似乎不相关模型的基础上,分析众多生态效益中的相互依存和从属关系,引入内生变量Y1、Y2作为另一个生态方程的自变量,构造联立方程组模型．该模型比较深入地刻画了在森林生态效益因变量集中,以森林涵养水源效益为主导。与其它生态效益间的支配关系．从森林生态效益自身规律出发。构造联立方程组结构参数矩阵B和T,根据其中隐含的参数间约束条件,生成线性限制方程HA=L。是确保本森林生态效益联立方程组模型成功的关键．特别是对森林生态效益因变量与自变量非线性关系的分析,为森林生态效益联立方程组模型的构造奠定科学基础．本模型是过度可识别且误差结构矩阵不是对角矩阵的联立方程组模型．用三步最小二乘估计法的Matlab程序,得森林涵养水源、森林固土、森林保肥和森林防风固沙效益联立方程组模型,平均精度可达80％以上．由此估计出全国2000年的森林涵养水源生态效益量为4227×10^8t;森林固土效益量为3．9×10^8t;森林保肥效益量为4．7×10^8t;森林防风固沙量为22．8×10^8t．     

The effect of transfer from low to high light intensity on electron transport in Rhodospirillum rubrum membranes

The effects of transfer from low to high ligh intensity on membrane bound electrontransport reactions of Rhodospirillum rubrum were investigated. The experiments were performed with cultures which did not form bacteriochlorophyll (Bchl) for about two cell mass doublings during the initial phase of adaptation to high light intensity. Lack of Bchl synthesis causes a decrease of Bchl contents of cells and membranes. Also, the cellular amounts of photosynthetically active intracytoplasmic membranes decrease.In crude membrane fractions containing both cytoplasmic and intracytoplasmic membranes the initial activities of NADH oxidizing reactions increase only slightly (about 1.2 times) per protein, but the initial activities of succinate oxidizing reactions decrease (multiplied by a factor of 0.7). On a Bchl basis activities of NADH oxidizing reactions increase 3.4 times while activities of succinate dependent reactions increase 1.9 times. With isolated intracytoplasmic membranes activities of NADH as well as succinate dependent reactions increase to a comparable extent on a Bchl basis (about 1.8 times) and stay nearly constant on a protein basis. Cytochrome c oxidase responds like succinate dependent reactions. The data indicate that in cells growing under the conditions applied NADH oxidizing electron transport systems are incorporated into both, cytoplasmic and intracytoplasmic membranes, while incorporation of succinate oxidizing systems is confined to intracytoplasmic membranes only.Activities of photophosphorylation and succinate dependent NAD⁺ reduction in the light increase per Bchl about 1.8 times. On a Bchl basis increases of the fast light induced on reactions at 422 nm and increases of soluble cytochrome c ₂ levels are comparable to increases of photophosphorylations and succinate dependent activities. But increases of slow light off reactions at 428 nm and of b-type cytochrome levels become three times greater then increases of cytochrome c ₂ reactions and levels. These results infer that although electrontransport reactions of intracytoplasmic membranes change correlated to each other, Bchl, cytochrome c ₂ and b-type cytochromes cellular levels are independent of each other. Furthermore, the data indicate that cytochrome c ₂ rather than b-type cytochrome is involved with steps rate limiting for photophosphorylation.Abbreviations Bchl bacteriochlorophyll - DCIP 2,6-dichlorophenolindophenol     

The nature of the effector cells mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC).

The nature of the cell types capable of mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) was investigated utilizing effector cells from athymic nude and euthymic heterozygous control littermate mice as well as Sephadex anti-Fab immunoabsorbent column purified spleen cell populations from normal (CS7BL/6) mice. Chicken erythrocytes (CRBC) and the mouse lymphoma, EL-4, were used as target cells in both cytotoxicity assays. MICC utilizing CRBC targets was mediated by several effector cell types whereas MICC utilizing EL-4 lymphoma targets was T-cell dependent. ADCC against both CRBC and EL-4 lymphoma targets occurred independently of the presence of T-cells. In addition, effector cell populations incapable of mediating MICC against EL-4 lymphoma targets were capable of mediating ADCC against the same EL-4 targets. Thus, utilizing the appropriate target cells, EL-4 but not CRBC, a sharp distinction can be made between the effectors for ADCC and MICC: ADCC is T-cell independent while MICC is dependent on the presence of mature thymus-derived cells. Furthermore these studies demonstrate that the nature of the target cell employed in MICC and ADCC reactions plays a critical role in defining the types of effector cells capable of mediating these cytotoxicity reactions.