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PubMed2000

NMR spectroscopy based metabonomic studies on the comparative biochemistry of the kidney and urine of the bank vole (Clethrionomys glareolus), wood mouse (Apodemus sylvaticus), white toothed shrew (Crocidura suaveolens) and the laboratory rat.

The metabolic profiles of three wild mammals that vary in their trophic strategies, the herbivorous bank vole (Clethrionomys glareolus), the granivorous wood mouse (Apodemus sylvaticus), and the insectivorous white-toothed shrew (Crocidura suaveolens), were compared with that of a widely used strain of laboratory rat (Sprague Dawley). In conjunction with NMR spectroscopic investigations into the urine and blood plasma composition for these mammals, high resolution magic angle spinning (HRMAS) 1H-nuclear magnetic resonance (NMR) spectroscopy was applied to investigate the composition of intact kidney samples. Adaptation to natural diet affects both renal metabolism and urinary profiles, and while these techniques have been used to study the metabolism of the laboratory rat little is known about wild small mammals. The species were readily separated by their urinary profiles using either crude metabolite ratios or statistical pattern recognition. Bank vole urine contained higher concentrations of aromatic amino acids compared with the other small mammals, while the laboratory rats produced relatively more hippurate. HRMAS 1H-NMR demonstrated striking differences in both lipid concentration and composition between the wild mammals and Sprague Dawley rats. Bank voles contained high concentrations of the aromatic amino acids phenylalanine, tyrosine and tryptophan in all tissue and biofluids studied. This study demonstrates the analytical power of combined NMR techniques for the study of inter-species metabolism and further demonstrates that metabolic data acquired on laboratory animals cannot be extended to wild species.

Animals↗

PubMed1998

High-throughput solid-phase extraction for the determination of cimetidine in human plasma.

For the implementation and validation of an automated 'high-throughput' solid-phase extraction (SPE) system, using microtiter solid-phase technology and a pipetting robot, a SPE method previously validated manually for cimetidine in human plasma was adapted. Sample cleanup was performed by means of SPE using Microlute extraction plates in the 96-well format, each well filled with 50 mg of Varian C18 sorbent. Separation was performed by reversed-phase high-performance liquid chromatography (HPLC) with UV detection at 234 nm. The validated calibration range was from 0.100 to 5.00 mg/l, with an inaccuracy and imprecision below 20% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. Cross-check analysis of samples from a clinical trial showed that there is a good correlation between results obtained by the automated method and results obtained by the manual method. The average sample preparation time for a technician decreased from approximately 4 min per sample to 0.6 min. A sample throughput of at least 160 samples per day can be achieved, the HPLC analysis time being the rate-limiting step.

Chromatography, High Pressure Liquid↗

PubMed1997

Radioimmunoassay for the measurement of S9788 in serum and microdialysis samples.

S9788, 6-[4-(2,2-di-(fluorophenyl)-ethylaminol-1-piperdinyl]-N,N'-d i-2-propenyl-1, 3,5-triazine- 2, 4-diamine, is a novel compound designed to reverse tumour multidrug resistance associated with cancer chemotherapy. A specific and sensitive radioimmunoassay has been developed for the analysis of S9788 in serum samples and adapted for samples obtained by microdialysis. The limit of quantitation is 0.2 ng ml-1 in perfusion medium and there is no cross reactivity of the antibody with known metabolites of the parent compound or with certain cytotoxic compounds likely to be coadministered with S9788. Maximum probe recovery during microdialysis was 66% at a flow of 1 microliter min-1, using Ringer/BSA (70 mg ml-1) as the perfusion medium. The assay has sufficient sensitivity, precision, accuracy and specificity for the analysis of rat and human serum and microdialysis perfusate samples. The assay has been successfully applied to the determination of S9788 in rat plasma (total concentration) and the microdialysate of the same samples.

Animals↗

PubMed1995

Reconstitution of the endogenous plastoquinone pool in photosystem II (PS II) membrane fragments, inside-out-vesicles, and PS II core complexes from spinach.

The possibility of reconstituting a functionally competent endogenous plastoquinone pool in photosystem II (PS II) membrane fragments, inside-out-vesicles (ISO-vesicles), and PS II core complexes was analyzed by measuring (i) the characteristic period four oscillation of the oxygen yield due to excitation of dark-adapted samples with a train of short flashes and (ii) laser flash-induced transients of the relative quantum yield of chlorophyll fluorescence. The data obtained revealed that (a) an endogenous pool capacity comparable to that of intact thylakoids can be restored in PS II membrane fragments and ISO-vesicles by a sonication treatment using native plastoquinone-9, (b) a more pronounced oxygen oscillation pattern arises in PS II core complexes after application of the same reconstitution procedure, (c) the extent of the endogenous pool restoration at a ratio of 15 quinone molecules per PS II in the reconstitution assay strongly depends on the nature of the quinone molecule [maximum effects can be only achieved with PQ-9, while at the same concentration ubiquinone-45 (UQ-9) is almost inefficient], and (d) a sonication step is required for stable insertion of PQ-9 into PS II preparations. Measurements of the reconstruction degree as a function of the structure of different quinones with selected properties lead to the conclusion that specific binding domains exist in PS II in addition to the QB site. These domains exhibit a surprisingly high specificity for the type of quinone that can be bound. On the basis of a comparison of the results obtained, the structure of the quinone head group seems to be more important than the large hydrophobic side chain and/or the general lipophilicity of the compound.

Fluorescence↗

PubMed1993

Structure-function relations in photosystem II. Effects of temperature and chaotropic agents on the period four oscillation of flash-induced oxygen evolution.

The characteristic period four oscillation patterns of oxygen evolution induced by a train of single-turnover flashes were measured in dark-adapted samples as a function of temperature and upon addition of chaotropic agents. The following results were obtained: (a) Within the range of 0 < theta < 35 degrees C, the ratio of the oxygen yield induced by the 4th and 3rd flashes of the train, Y4/Y3, and the oxygen yield induced by the 2nd flash, Y2, exhibit similar dependencies on the temperature in isolated thylakoids, PS II membrane fragments, and inside-out vesicles. (b) Below a characteristic temperature theta c of 20-25 degrees C, the values of Y4/Y3 and Y2, which reflect (at constant S0 dark population) the probabilities of misses and double hits, respectively, remain virtually independent of temperature, whereas above theta c these parameters increase. (c) The dark decays of S2 and S3 via fast and slow kinetics due to reduction of the water oxidase by YD and other endogenous electron donor(s), respectively, exhibit comparatively strong temperature dependencies in thylakoids with the following activation energies: EA(S2fast) = 55 kJ/mol, EA(S3fast) = 50 kJ/mol, EA(S2slow) = 85 kJ/mol, and EA(S3slow) = 75 kJ/mol. The activation energy of S0 oxidation to S1 by YDox was found to be markedly smaller with a value of EA(S0) = 30 kJ/mol. (d) Incubation with chaotropic agents at concentrations which do not significantly impair the oxygen evolution capacity leads to modifications of the oscillation pattern with remarkable differences for various types of agents: Tris and urea are practically without effect; guanidine hydrochloride affects Y4/Y3 in a similar way as elevated temperature but without significant changes of Y2 and the decay kinetics of S2 and S3; and anions of the Hofmeister series (SCN-, ClO4-, I-) cause a drastic destabilization of YDox. Possible structure-function relations of the PS II complex are discussed on the basis of these findings.

Guanidine↗

PubMed2001

Time-resolved monitoring of flash-induced changes of fluorescence quantum yield and decay of delayed light emission in oxygen-evolving photosynthetic organisms.

The present contribution describes a new experimental setup that permits time-resolved monitoring of the rise kinetics of the relative fluorescence yield, Phi(rel)(t), and simultaneously of the decay of delayed light emission, L(t), induced by strong actinic laser flashes. The results obtained by excitation of dark-adapted samples with a train of eight flashes reveal (a) in suspensions of spinach thylakoids, Phi(rel)(t) exhibits a typical period four oscillation that is characteristic for a dependence on the redox states S(i)() of the water oxidizing complex (WOC), (b) the relative extent of the unresolved "instantaneous" rise to the level (100 ns) at 100 ns and the maximum values of Phi(rel)(t) attained at about 45 s after each actinic flash, (45 s) synchronously oscillate and exhibit the largest values at flash nos. 1 and 5 and minima after flash nos. 2 and 3, (c) opposite effects are observed for the normalized extent of the rise kinetics in the 100 ns to 5 s time domain of relative fluorescence yield, Phi(rel)(5 s) - Phi(rel)(100 ns), i.e., both parameters attain minimum and maximum values after the first/fifth and second/third flash, respectively, and (d) analogous features for the "fast" and "slow" ns-kinetics of the fluorescence rise were observed in suspensions of Chlamydomas reinhardtii cells. A slight phase shift by one flash is ascribed to physiological differences. The applicability of this noninvasive technique to study reactions of photosystem II, especially the reduction kinetics of P680(*)(+) and their dependence on the redox state S(i)() of the WOC, is discussed.

Animals↗

PubMed1992

Photooxidation of cytochrome b559 in oxygen-evolving photosystem II.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome b Group↗

PubMed1994

Analyses of pH-induced modifications of the period four oscillation of flash-induced oxygen evolution reveal distinct structural changes of the photosystem II donor side at characteristic pH values.

This study presents a thorough analysis of the reaction pattern of flash-induced oxygen evolution in spinach thylakoids as a function of pH (4.5 < or = pH < or = 9) and the redox state of tyrosine YD in polypeptide D2. Evaluation of the experimental data within the conventional Kok model [Kok, B., Forbush, B., & McGloin, M. (1970) Photochem. Photobiol. 11, 457-475] led to the following results: (1) the probability of the miss factor is strongly pH dependent (with a pronounced minimum near neutral pH) while the double hit factor is less affected; (2) a marked increase of the apparent S0 population arises at alkaline pH in dark-adapted samples where most of the YD is reduced, but this effect is absent if the percentage of PS II containing the oxidized form YDox is high; and (3) the lifetimes of S2 and S3 exhibit a characteristic pH dependence that is indicative of conformational changes of functional relevance within the water-oxidizing complex and its environment; (4) the kinetic interaction of redox states S2 and S3 with YD is characterized by a change of its behavior at a threshold pH of 6.5-7.0; and (5) at acidic pH values the extent of S2 and S3 reduction by YD decreases concomitant with the occurrence of a very fast decay kinetics. On the basis of a detailed discussion of these results and data from the literature, the water oxidase is inferred to undergo structural changes at pH values of 5-5.5 and 6.5-7.0. These transitions are almost independent of the redox state Si and modify the reaction coordinates of the water oxidase toward endogenous reductants.

Hydrogen-Ion Concentration↗

PubMed1998

Nonphotochemical quenching of excitation energy in photosystem II. A picosecond time-resolved study of the low yield of chlorophyll a fluorescence induced by single-turnover flash in isolated spinach thylakoids.

Chlorophyll a fluorescence emission is widely used as a noninvasive measure of a number of parameters related to photosynthetic efficiency in oxygenic photosynthetic organisms. The most important component for the estimation of photochemistry is the relative increase in fluorescence yield between dark-adapted samples which have a maximal capacity for photochemistry and a minimal fluorescence yield (F0) and light-saturated samples where photochemistry is saturated and fluorescence yield is maximal (Fm). However, when photosynthesis is saturated with a short (less than 50 micro(s)) flash of light, which induces only one photochemical turnover of photosystem II, the maximal fluorescence yield is significantly lower (Fsat) than when saturation is achieved with a millisecond duration multiturnover flash (Fm). To investigate the origins of the difference in fluorescence yield between these two conditions, our time-resolved fluorescence apparatus was modified to allow collection of picosecond time-resolved decay kinetics over a short time window immediately following a saturating single-turnover flash (Fsat) as well as after a multiturnover saturating pulse (Fm). Our data were analyzed with a global kinetic model based on an exciton radical pair equilibrium model for photosystem II. The difference between Fm and Fsat was modeled well by changing only the rate constant for quenching of excitation energy in the antenna of photosystem II. An antenna-based origin for the quenching was verified experimentally by the observation that addition of the antenna quencher 5-hydroxy-1,4-naphthoquinone to thylakoids under Fm conditions resulted in decay kinetics and modeled kinetic parameters very similar to those observed under Fsat conditions in the absence of added quinone. Our data strongly support the origin of low fluorescence yield at Fsat to be an antenna-based nonphotochemical quenching of excitation energy in photosystem II which has not usually been considered explicitly in calculations of photochemical and nonphotochemical quenching parameters. The implications of our data with respect to kinetic models for the excited-state dynamics of photosystem II and the practical applications of the fluorescence yield parameters Fm and Fsat to calculations of photochemical yield are discussed.

Chlorophyll↗

PubMed2001

Dose escalation of cytotoxic drugs using haematopoietic growth factors: a randomized trial to determine the magnitude of increase provided by GM-CSF.

BACKGROUND: The magnitude of chemotherapy dose escalation made possible by the use of recombinant haematopoietic growth factors has not been quantified in a randomized trial. PATIENTS AND METHODS: Patients with refractory or relapsing Hodgkin's disease were randomized to receive the Dexa-BEAM regimen with escalating etoposide doses supported by placebo or granulocyte-macrophage colony-stimulating factor (GM-CSF). Using an adaptive sampling method independently in both arms, the etoposide dose was escalated until the maximal tolerated dose for the first cycle was reached. RESULTS: Thirty patients were randomized to GM-CSF and thirty to placebo. The etoposide dose could be escalated considerably in both treatment arms. Maximal etoposide dose for the first cycle was 1920 mg/m2 for patients receiving GM-CSF and 1160 mg/m2 for patients receiving placebo (P = 0.045 one-sided), corresponding to a 65% higher etoposide dose and a 13% higher dose intensity with GM-CSF. Dose-limiting events were similar in both arms, consisting mainly of prolonged neutropenia and consecutive infections. Treatment efficacy was not different in the two treatment groups. CONCLUSIONS: While GM-CSF permits a somewhat higher dose escalation than placebo, the increase in dose intensity provided by GM-CSF is small. The use of CSF for interval reduction rather than dose escalation is the more effective strategy for dose intensification.

Adolescent↗

PubMed2000

Strain and sex differences on olfactory discrimination learning in C57BL/6J and DBA/2J inbred mice (Mus musculus).

In this study, the authors explored potential strain and sex differences in nonspatial cognitive ability. Beginning around 90 days of age, male and female C57BL/6J (C57) and DBA/2J (DBA) inbred mice (Mus musculus) were tested on a task of simple odor discrimination learning with 3 repeated reversals. Males learned the task more readily than females, and DBA mice learned the task more readily than C57 mice. All differences became evident after repeated testing. Similarity of perseveration measures indicated the differences were not due to inhibitory deficits. Instead, a phase analysis localized differences to a transitional period of reversal learning. Females increased transitional errors that more likely indicated adaptive sampling strategies than memory failures. C57 females used this strategy indiscriminately, but DBA females sampled as a function of environmental uncertainty.

Animals↗

PubMed1985

A neutron diffraction study on the location of the polyene chain of retinal in bacteriorhodopsin.

We report on the location of the chain part of the retinylidene chromophore in the projected density of bacteriorhodopsin as determined by neutron diffraction from the two-dimensional purple membrane lattice. For this purpose, partially deuterated retinal was synthesized containing 10 deuterons at positions C-8, C-10, C-12, C-14, C-19(3), and C-20(3) of the polyene chain. Two sets of dark-adapted samples were prepared in entirely different ways: (i) Deuterated retinal was incorporated biosynthetically during growth of the bacteria by using the mutant JW5, which is deficient in the synthesis of retinal. (ii) The chromophore was converted to retinal oxime, the resulting colorless apomembrane was regenerated with deuterated retinal, and the residual retinal oxime was removed by washing with bovine serum albumin. Characterization of these samples by x-ray diffraction, absorption, and flash spectroscopy showed that they were identical to native purple membrane samples as judged by these criteria. Fourier difference maps were calculated from the differences in inplane diffraction from the deuterated membranes and from protonated samples that were prepared in exactly the same way. At 8.7 A resolution, both maps show a single major peak at the same position with the center of mass of the labeled part of the chain (C-11) between helices 6 and 3 but closer to helix 6. It appears likely that the COOH-terminal helix G, to which retinal is attached at lysine-216, is either helix 2 or 6.

Apoproteins↗

PubMed1994

Angular orientation of the stable tyrosyl radical within photosystem II by high-field 245-GHz electron paramagnetic resonance.

The 4 K 245-GHz/8.7-T electron paramagnetic resonance spectrum of the stable tyrosyl radical in photosystem II, known as TyrD., has been measured. Illumination at 200 K enhances the signal intensity of TyrD. by a factor of > 40 compared to the signal obtained from dark-adapted samples. This signal enhancement and the unusual line shape of the TyrD. resonance result from the magnetic dipolar coupling of the radical to the manganese cluster involved in oxygen evolution. The relative angular orientation of the manganese cluster with respect to TyrD. has been determined from line-shape analysis. The resonance arising from TyrD. in Tris-washed manganese-free photosystem II sample is also distorted. This effect probably originates from the influence of the nonheme iron on the spin relaxation of the tyrosyl radical. The relative angular orientation of the nonheme iron has also been determined. Oriented samples were used to determine the angular orientation of TyrD. with respect to the membrane plane. Combining angular data with published distances, we have constructed a three-dimensional picture of the relative positions of TyrD., the manganese cluster, and the nonheme iron. The data suggest a more symmetrical placement of the manganese relative to TyrD. and TyrZ, the tyrosine involved in electron transfer, than is usually assumed in current models of photosystem II.

Electron Spin Resonance Spectroscopy↗

PubMed2000

Hierarchical Bayes estimation of hunting success rates with spatial correlations.

A Bayesian hierarchical generalized linear model is used to estimate hunting success rates at the subarea level for postseason harvest surveys. The model includes fixed week effects, random geographic effects, and spatial correlations between neighboring subareas. The computation is done by Gibbs sampling and adaptive rejection sampling techniques. The method is illustrated using data from the Missouri Turkey Hunting Survey in the spring of 1996. Bayesian model selection methods are used to demonstrate that there are significant week differences and spatial correlations of hunting success rates among counties. The Bayesian estimates are also shown to be quite robust in terms of changes of hyperparameters.

Animals↗

PubMed1990

Effect of frequent milkings on milk NAGase, plasmin, trypsin inhibitory capacity and the quality of whey as the growth medium for mastitis pathogens.

Unequal milking intervals affected milk somatic cell count and N-acetyl-beta-D-glucosaminidase (NAGase) activity. The total daily output of milk NAGase and plasmin decreased, if quarters were emptied frequently during the day. Mastitis pathogens showed stimulated growth in whey prepared from filled quarters as compared with growth in whey from quarters emptied frequently during the day. The quality of whey as growth medium for mastitis pathogens paralleled plasmin activity in respective milk samples. Adaptation of mastitis pathogens to grow in whey had an enhancing effect on bacterial growth during subsequent inoculations in whey. Bacteria probably "learn" to overcome the effect from endogenous antibacterial factors and to use nutrients present in whey.

Acetylglucosaminidase↗

PubMed1993

Where plants make oxygen: a structural model for the photosynthetic oxygen-evolving manganese cluster.

In the photosynthetic evolution of oxygen, water oxidation occurs at a catalytic site that includes four manganese atoms together with the essential cofactors, the calcium and chlorine ions. A structural model and a determination of the manganese oxidation states based on x-ray absorption spectroscopy are presented. The salient features, in both higher plants and cyanobacteria, are a pair of di-mu-oxo bridged manganese binuclear clusters linked by a mono-mu-oxo bridge, one proximal calcium atom, and one halide. In dark-adapted samples, manganese occurs in oxidation states (III) and (IV). Data from oriented membranes display distinct dichroism, precluding highly symmetrical structures for the manganese complex.

Bridged-Ring Compounds↗

PubMed2000

Inhibition of photosynthetic electron transport by UV-A radiation targets the photosystem II complex.

We have studied the inhibition of photosynthetic electron transport by UV-A (320-400 nm) radiation in isolated spinach thylakoids. Measurements of Photosystem II (PSII) and Photosystem I activity by Clark-type oxygen electrode demonstrated that electron flow is impaired primarily in PSII. The site and mechanism of UV-A induced damage within PSII was assessed by flash-induced oxygen and thermoluminescence (TL) measurements. The flash pattern of oxygen evolution showed an increased amount of the S0 state in the dark, which indicate a direct effect of UV-A in the water-oxidizing complex. TL measurements revealed the UV-A induced loss of PSII centers in which charge recombination between the S2 state of the water oxidizing complex and the semireduced Q(A)- and Q(B)- quinone electron acceptors occur. Flash-induced oscillation of the B TL band, originating from the S2Q(B)- recombination, showed a decreased amplitude after the second flash relative to that after the first one, which is consistent with a decrease in the amount of Q(B)- relative to Q(B) in dark adapted samples. The efficiency of UV-A light in inhibiting PSII electron transport exceeds that of visible light 45-fold on the basis of equal energy and 60-fold on the basis of equal photon number, respectively. In conclusion, our data show that UV-A radiation is highly damaging for PSII, whose electron transport is affected both at the water oxidizing complex, and the binding site of the Q(B) quinone electron acceptor in a similar way to that caused by UV-B radiation.

Electron Transport↗

PubMed2000

[Population projections: goals, methods, hypotheses].

Population projections generally use the component method which consists in adapting sample size by age and sex from mortality, fertility and migration hypotheses. This exercise is not always aimed a providing projection data, but may also serve to analyze factors changing in the population. Comparatively long-term demographic forecasts, particularly for mortality, can be made due to the major inertia of demographic phenomena. However, the quality of such predictions depends on the quality of knowledge of past evolution in the demographic factors and on the capacity to forecast their future evolution.

Adolescent↗