Time squared: repeated measures on phylogenies

Studies of gene expression profiles in response to external perturbation generate repeated measures data that generally follow nonlinear curves. To explore the evolution of such profiles across a gene family, we introduce phylogenetic repeated measures (PR) models. These models draw strength from 2 forms of correlation in the data. Through gene duplication, the family's evolutionary relatedness induces the first form. The second is the correlation across time points within taxonic units, individual genes in this example. We borrow a Brownian diffusion process along a given phylogenetic tree to account for the relatedness and co-opt a repeated measures framework to model the latter. Through simulation studies, we demonstrate that repeated measures models outperform the previously available approaches that consider the longitudinal observations or their differences as independent and identically distributed by using deviance information criteria as Bayesian model selection tools; PR models that borrow phylogenetic information also perform better than nonphylogenetic repeated measures models when appropriate. We then analyze the evolution of gene expression in the yeast kinase family using splines to estimate nonlinear behavior across 3 perturbation experiments. Again, the PR models outperform previous approaches and afford the prediction of ancestral expression profiles. To demonstrate PR model applicability more generally, we conclude with a short examination of variation in brain development across 4 primate species.     

Nonparametric,GINI-like Measures of Location and Dispersion

GINI's nonparametric measure of dispersion, defined as the mean of (n/2) pairs of a sample of n measurements, is modified for triplets. That way a GINI-like measure of location G* is defined as the mean of the (n/3) triplet medians, and a GINI-like measure of dispersion is defined as the mean of the (n/3) triplet ranges. G* and g* are less sensitive to outlier measurements than are the corresponding parametric measures, arithmetic mean and standard deviation, in small samples. Applications and application-related problems are discussed.     

Comparing Two Samples of Response Curves by Nonparamètric Tests of Predicted Order

Starting from MOSTELLER'S (1955) one-sample test of predicted order, some two-sample versions are proposed for comparing nonparametrically a treatment group (m response curves) with a control group (n response curves) as to a predicted shape represented by a specified rank order. The tests are applied to two groups of learning curves.     

Luminescence decay from copper(I) complexes of metallothionein

Luminescence in the 600 nm region from aqueous solutions of Zn₇metallothionein (ZnMT) has been studied as a function of the extent of substitution of the ZnMT with Cu⁺. The steady state emission intensity shows a strong correlation with cluster formation. Analysis of the emission decay curves obtained by single photon-counting techniques using a multiexponential decay function, indicates that three components are responsible for the observed emission intensity. A short lived component exhibits a lifetime of 20 ns, while the two longer lived components exhibit lifetimes in the 1 μ range at room temperature. The longer-lived emission is assigned to the excited triplet states of the CuS chromophore, most probably the metal-centered 3d⁹4s¹.     

Neurophysiological Indicators of Voluntary and Involuntary Visual Attention in a Human

The visual evoked potentials (EPs) in response to lateralized and central visual symbols under the conditions of involuntary (passive viewing) and selective attention (when one of the symbols was a target and required a rapid and precise motor reaction) are considered. The evoked potentials in the occipital, parietal, and frontal derivations were recorded in 20 healthy subjects. It was shown that the EP during selective attention are most pronounced and more alike in the parietal derivations. A strong positive correlation was revealed between the EP amplitude ([N1–P3] component) and the EP stability (correlation between the repeated EP). The involuntary and voluntary forms of attention supplement each other: the more expressed the involuntary attention (assessed by the [N1–P3] component) the higher the EP to target stimuli during voluntary attention and the shorter the reaction time. It is suggested that the role of visual attention consists in the increase and stabilization of cortical activity (primarily, the parietal regions) engaged in solving a visual task.     

Studies of DNA dumbbells. V. A DNA triplex formed between a 28 base-pair DNA dumbbell substrate and a 16 base linear single strand

CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T₄ single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5′-AGGAAGGAGGAAAGAG-3′. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5′-TCCTTCCTCCTTTCTC-3′ were also prepared and studied. At 22°C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 μM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 μM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts ～20°C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying. On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (～ 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand. From the model analysis a straightforward derivation of f_T(T), the fraction of triplex as a function of temperature, was obtained. Analysis of f_T vs temperature curves, in effect melting curves of the triplexes, provided evaluation of thermodynamic parameters of the melting transition. For the triplex formed with the dumbbell substrate, the total transition enthalpy is ΔH_T = 118.4 ± 12.8 kcal/mol (7.4 ± 0.8 kcal/mol per triplet unit) and the total transition entropy is ΔS_T = 344 ± 36.8 cal/K · mol (eu) (21.5 ± 2.3 eu per triple unit). The transition curves of the triplex formed with the linear duplex substrate displayed two distinct regions. A broad pretransition region from f_T = 0 to 0.55 and a higher, sharper transition above f_T = 0.55. The transition parameters derived from the lower temperature region of the curve are ΔH′_T = 44.8 ± 9.6 kcal/mol and ΔS′_T = 112 ± 33.6 eu (or ΔH′ = 2.8 ± 0.6 kcal/mol and ΔS′ = 7.0 ± 2.1 eu per triplet). These values are probably too small to correspond to actual melting of the triplex but instead likely reveal effects of end fraying of the duplex substrate on triplex stability. Transition parameters of the upper transition are ΔH′_T = 128.0 ± 2.3 kcal/mol and ΔS′_T = 379.2 ± 6.4 eu (ΔH′ = 8.0 ± 0.2 kcal/mol and ΔS′ = 23.7 ± 0.4 eu per triplet) in good agreement (within experimental error) with the transition parameters of the triplex formed with the dumbbell substrate. Supposing this upper transition reflects actual dissociation of the third strand from the linear duplex substrate this triplex is comparable in thermodynamic stability to the triplex formed with a dumbbell substrate. Even so, the biphasic melting character of the linear triplex obscures the whole analysis, casting doubt on its absolute reliability. Apparently triplexes formed with a dumbbell substrate offer technical advantages over triplexes formed from linear or hairpin duplex substrates for studies of DNA triplex stability. © 1993 John Wiley & Sons, Inc.     

Three-stranded helix-coil equilibrium in polyuridylic acid-adenosine complex

Interaction of poly U (polyuridylic acid) and adenosine is studied by following the changes in ultraviolet absorbance in the wavelength region near the isochromic wave-length for the complex formation. The interaction is studied as a function of temperature, concentration of adenosine, and ionic strength, while the concentration of poly U was held constant. It is confirmed that only the three-stranded complex with the stoichiometry 1A to 2U is formed and that it dissociates directly into free poly U and adenosine. No discontinuity of any kind was apparent in the melting curves, and poly U was found to possess no ordered structure above 10°C under the conditions used. The results were, therefore, analyzed in terms of an exact helix–coil equilibrium theory using the mismatching model, i.e., assuming that either completely formed base triplet or completely free unbonded bases only exist, and that the two sections of the polymer chains forming closed loops need not contain the same number of unbonded bases. Self-association of free adenosine was taken into consideration. (Base triplet is analog of base pair for a three-stranded helical complex. It refers to a unit of three coplanar bases, in this case two uracils and one adenine, hydrogen bonded to one another to form a triplet. Such triplets may stack over one another along the helical axis, and when they are so stacked the bases of two triplets next to each other may have stacking interactions between them.) The values for enthalpy and entropy changes, both per mole of base triplets, were obtained for the following processes at neutral pH and moderate to high salt concentrations. (1) Growfh: Binding of one adenosine molecule to two uracil residues (one from each poly U strand) to form a base triplet next to an already formed base triplet with which it has stacking interactions, a process that involves both hydrogen bonding and base stacking interactions, ΔH_s, = ?19 ± 2 kcal, ΔS_s = ?55 ± 6 clausius; (2) Initiation: Binding of one adenosine molecule to two uracil residues (one from each poly U strand) to form an isolated base triplet, a process that involves only hydrogen bonding interactions, ΔH_b* = 4.5 ± 2 kcal, ΔS_b* = 6.6 ± 3 clausius; and (3)Interruption: Unstacking of two stacked base triplets initially next to each other by formation of an interruption (viz. a closed loop) between them, a process that involves only base stacking interactions, ΔH_b = 23.5 ± 3 kcal, ΔS_b = 61.6 ± 7 clausius, where the entropy changes include contributions other than the configurational entropy of closed loops. The discrepancy between our results and the calorimetric ΔH_s of ?13 kcal is attributed to (i) the possible effects of salt arid polymer on the self-association of free adenosine, (ii) the uncertainty in the value of the parameter for the probability of ring closure, and (iii) the contributions due to the partial molal enthalpy of the solvent and the unstacking of any poly U structure to the calorimetric enthalpy.     

Febrile tolerance develops in response to repeated administration of bacterial lipopolysaccharide but not of interleukin-1β in rats

Changes of abdominal temperature in response to intraperitoneal injections of 100 μg/kg bacterial lipopolysaccharide (LPS), repeated three times in intervals of 3 days were measured in rats. Only after the first injection of LPS a pronounced fever developed, a small fever was recorded after the second injection, while the response to the 3rd injection of LPS was almost the same as to an administration of solvent (0.9 % saline) to naive rats. Levels of bioactive cytokines (tumor necrosis factor α, TNFα, and interleukin-6, IL-6), measured 60 min after the 1st, 2nd or 3rd injection of LPS, were progressively attenuated. Changes of abdominal temperature in response to intraperitoneal injections of recombinant rat-interleukin-1β (rr IL-1β), also repeated three times in intervals of 3 days were measured in another group of rats. The febrile responses to the 1st, 2nd and 3rd injection of rr IL-1β were amost identical. The results of this study show that febrile tolerance develops in response to repeated injections of LPS, but not of its putative endogenous mediator, IL-1β, in rats.     

Spatial distributions of foliar nitrogen and phosphorus in crowns of Eucalyptus grandis

Summary Eucalyptus grandis trees were grown in plantations with and without added fertiliser to examine the effects of plant nutrition on photosynthesis and growth. Leaves were sampled from known locations within canopies of selected trees and leaf N and P concentrations were measured. Contour maps of N and P distributions were then produced for crowns of trees aged between 6 and 16 months. Gas exchange measurements on sample leaves were used to estimate parameters of a model of C₃ photosynthesis as a function of leaf N and P contentrations. Linear relationships were obtained between model parameters and leaf N concentration, but P appeared to be present in excess, since no correlation was found with P contentration. Photosynthetic light response curves were calculated for model leaves with differing N concentrations. The curves show that optimal concentrations of N in leaves depend on mean levels of irradiance during growth.     

Climatic signals in tree rings of <Emphasis Type="Italic">Burkea africana</Emphasis> and <Emphasis Type="Italic">Pterocarpus angolensis</Emphasis> from semiarid forests in Namibia

Tree-ring studies contribute worldwide to the understanding of climate and its relation to tree growth. Long tree-ring chronologies serve as climate proxies for the reconstruction of past, pre-instrument climate and its recent change. In tropical regions, the availability of exactly dated tree-ring chronologies is limited. The dendroclimatic potential of two dominant species from dry forests in northern Namibia was examined in the study presented in this paper. Both species (Burkea africana Hook and Pterocarpus angolensis DC) were sampled at two sites (ca. 900 km apart), and the response to several climatic variables, including ENSO indices, is studied. All specimens showed distinct growth rings and cross-dating between radii was successful for all trees. Species-specific mean curves were built for both sites. The mean curves of different species of the same site synchronised significantly, allowing the construction of a site-specific chronology. Synchronisation between sites was not possible, but spectral analysis of the chronologies implied that both show similar long-term (6.7 year) oscillation patterns. B. africana is more sensitive to rainfall variation than P. angolensis at both sites. Growth response to rainfall was positive, but a time-lag in the reaction occurred between the sites, corresponding to the time-lag of the beginning of the rainy season. Air temperature showed a negative correlation with stem increment at both sites. The response at the westernmost site to two ENSO indices indicates a tree growth decrease during El Niño years, which are generally dry in southern Africa.     

Intra‐population genetic variation in diapause incidence of adult‐diapausing Tetranychus pueraricola (Acari: Tetranychidae)

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Long lasting effect of a single audiogenic seizure on GABA turnover rates and steady-state levels

GABA turnover rates (TOR) and steady-state levels (SSL) were determined, 16–18 h after a single acoustic stimulation, in 15 brain areas of 3 mouse sublines. Each subline differs in its response to an acoustic stimulation (Rb1 mice are clonic-tonic seizure-prone, Rb2: clonic seizure-prone, Rb3: seizure-resistant). TOR and SSL were compared to those of unstimulated control mice and to those of repeatedly stimulated mice of the same subline. Following a single acoustic stimulation long-lasting alterations of GABA metabolism, mainly large alterations of GABA TOR, are observed. Most of the effects elicited after repeated stimulations, either on SSL or TOR, are not those of the last stimulation and repeated seizures (and/or stimulations) strengthen the effect of a single one. It appears that, for each of the Rb sublines, a specific and quite simple profile of the alterations of GABA metabolism in response to a single or repeated audiogenic seizures (and/or stimulations) can be given. The global analysis through the correlation of GABA TOR and SSL gives an indication that the alterations of the parameters of the correlation observed are to be allocated to the audiogenic seizures. Furthermore the tonic and clonic components of the audiogenic seizures can be distinguished.     

Transient and steady state phase response curves of limit cycle oscillators

The experiment of phase shifts resulting from discrete perturbations of stable biological rhythms has been carried out to study entrainment behavior of oscillators. There are two kinds of phase response curves, which are measured in experiments, according to as one measures the phase shifts immediately or long after the perturbation. The former is the first transient phase response curve and the latter is the steady state phase response curve. We redefine both curves within the framework of dynamical system theory and homotopy theory. Several topological properties of both curves are clarified. Consequently, it is shown that we must compare the shapes of both two phase response curves to investigate the inner structures of biological oscillators. Moreover, we prove that a single limit cycle oscillator involving only two variables cannot simulate transient resetting behavior reported by Pittendrigh and Minis (1964). In other words, the circadian oscillator of Drosophila pseudoobscura does not consist of a single oscillator of two variables. Finally we show that a model which consists of two limit cycle oscillators is able to simulate qualitatively the phase response curves of Drosophila.     

High-performance liquid chromatography using on-line solid-phase extraction: determination of furosemide in human serum

An on-line solid-phase extraction technique based on column switching (heart-cutting) was developed for direct injection analysis of Furosemide in human serum. In order to minimize the influence of deterioration in pre-treatment column efficiency, which was caused by protein precipitation with repeated injections of serum, furosemide was completely enriched at the top of the analytical column by ion-pair formation with tetra-n-butylammonium ion during heart-cutting. The robustness of the established on-line solid-phase extraction system was confirmed under routine conditions, As a result, almost comparable chromatograms could be obtained even though 50 repeated injections of a 100-μl volume of serum were carried out using one pre-treatment column. The linearity of the calibration curves was demonstrated by the correlation coefficient which was greater than 0.99999 (5–1000 ng/ml). The relative errors and C.V of quality control samples were within 4.00 and 5.88%, respectively (furosemide concentration: 5, 100 and 1000 ng/ml).     

CIRCADIAN PHASE AND PERIOD RESPONSES TO LIGHT STIMULI IN TWO NOCTURNAL RODENTS

We report period response curves (τRC) for two nocturnal Murid species from India, Mus booduga and Mus platythrix. We further discuss the method of phase shift estimation in the presence of τ-changes, because such changes pose a serious methodological problem in the estimation of phase shifts. Although the τRC indicates that most of the phase shifts are associated with small changes in τ, the period changes across all the phases showed a significant positive correlation with the phase shifts. We conclude that τRCs are a reality even in nocturnal mammals, although their amplitude is less than what is usually found in diurnal mammals, and requires a larger data set to be distinguished from noise.     

Molecular analysis of the genomic organization of the Hymenoptera Diadromus pulchellus and Eupelmus vuilleti

The genomic organization of two parasitic wasps was analyzed by DNA reassociation. Cot curves revealed a pattern with three types of components. A highly repetitive DNA, accounting for 15 to 25% of the genome, was identified as satellite DNA. The moderately repetitive DNA corresponds to 26 to 42% of the genome in both species, and shows large variations in complexity, repetitive frequency and a number of sub-components between males and females. These variations are seen as resulting from DNA amplification during somatic and sexual differentiation. Dot blot analyses show that such DNA amplifications concern several types of structural and regulatory genes. The presence of repeated mobile elements was studied by the Roninson method to compare the repeated sequence patterns of Diadromus pulchellus and Eupelmus vuilleti with those of Drosophila melanogaster. The occurrence and organization of mobile elements in these Hymenoptera differ from those of the neighboring order of Diptera. The repetitive and unique components define very large genomes (1 to 3 × 10⁹ base pairs). The genomic organization in Parasitica appears to be an extreme drosophilan type. We propose that the germinal genome of these parasitic wasps is primarily composed of satellite DNA blocks and very long stretches of unique sequences, separated by a few repeated and/or variously deleted, interspersed elements of each mobile element family.     

Ultraviolet photoproducts at the ochre suppressor mutation site in the glnU gene of Escherichia coli: relevance to "mutation frequency decline"

Summary Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA. Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly (mutation frequency decline or MFD), whereby post-irradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold. We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 by region of glnU which includes the anticodon-encoding triplet. We found that four different photolesions are produced within the 3 by sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6–4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6–4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6–4) photoproduct is formed, apparently at the G in the mutation site. We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants.     

Ultrastructural aspects of the somatic and buccal infraciliature ofColeps amphacanthus Ehrenberg 1833

Summary Somatic and buccal infraciliature ofColeps amphacanthus Ehrenberg 1833 were studied by light and electron microscopy. The somatic kineties are composed of monokinetids and 2 dikinetids at the anterior end of each kinety. The monokinetids are associated with postciliary microtubules at triplet 9, a kinetodesmal fiber at triplet 5 and 7 and nearly radially arranged transverse microtubules at triplet 4. The associated fibrillar systems of the posterior kinetosome of the dikinetids are like those of the monokinetids. The anterior kinetosome is associated with transverse microtubules at triplet 4 and one or few postciliary microtubules at triplet 9. The anterior kinetosome bears only a short cilium.The oral ciliature is composed of a kinety of nearly circumorally arranged paroral dikinetids and 3 adoral organelles at the ventral left side of the oral opening. Nematodesmata arising from the oral ciliature form the major component of the cytopharyngeal apparatus which is lined by microtubular ribbons of postciliary origin. The buccal cavity is surrounded by oral papillae which often contain toxicysts.     

ANALYSIS OF STARCH ACCUMULATION AND GERMINATION IN ONOCLEA SPORES

Previous studies have shown that low fluences of light accelerate starch accumulation and enhance germination of Onoclea spores. Fluence response curves for induction of starch accumulation were compared with fluence response curves for enhancement of germination in order to determine if the two photoresponses in Onoclea spores have a common photoreceptor. Fluence response curves indicate that both responses were proportional to the log of the fluence and that the relative fluence efficiencies of the four wavelength regions tested were similar for induction of both germination and starch accumulation. Red (600–720 nm) irradiation was the most efficient, while green (500–600 nm), blue (400–520 nm), and far-red (720–900 nm) irradiations showed a decreasing order of efficiency for induction of the responses. A correlation coefficient between the amount of starch accumulated as a result of red irradiation and the final percent germination was calculated to be 0.964. These results support the hypothesis that a common photoreceptor mediates both photoinduced germination and starch accumulation. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) inhibits photosynthesis by blocking the flow of electrons from Photosystem II to Photosystem I. At 0.1 mM DCMU failed to inhibit both photoinduced starch acumulation and germination. This result and the greater efficiency of red than blue light, the low fluence functional for induction, and the fluence dependency argue against the participation of photosynthesis in photoinduced starch accumulation. A similar conclusion has been previously drawn for photoenhancement of Onoclea spore germination. Additionally, the effects 0.01–1.0 mm cycloheximide and 100 μl/l ethylene on photoinduced starch accumulation were investigated. Neither agent inhibited starch accumulation, whereas both substances inhibited germination 70–90% when applied at a time coincidental with the period of rapid starch accumulation. These results indicate that the photoinduction of starch accumulation does not have an ethylene sensitive stage nor does it require protein synthesis as does photoenhancement of germination of Onoclea spores.     

Action spectra for light-induced germination in dormant lettuce seeds

Fluence response curves for red light-induced germination of thermodormant (TD) seeds of Lactuca sativa L. show two regions that differ in their light sensitivity. In the region of high sensitivity, the germination responses differ between seed batches and can be altered by dark storage or far red irradiation. Induction of germination in far red dormant (FRD) seeds requires far higher fluences. Action spectra for induction to 60% germination were determined for these various response types. Spectra for the regions of low sensitivity response are similar for TD and FRD seeds. In comparison, the action spectrum for the highly sensitive response in TD seeds is significantly shifted to longer wavelengths. Analogous differences exist in the action spectra for far red reversal of the red induced germination responses. Germination induction in the low sensitivity region shows repeated red-far red reversibility. Far red reversal of red induction in the high sensitivity region does not saturate even at the highest far red fluences available and requires increased red fluences for subsequent reinduction. A model quantitatively accounting for these observations is presented. It is pointed out that action spectra of processes involving photoreversible pigments with partly overlapping absorption spectra in general are not identical with the absorption spectra of the partners. They should depend upon the degree of phototransformation required to elicit a given physiological response. In the case of induction of lettuce seed germination the observed action spectra can be interpreted as reflecting different requirements for P _fr of the various response types. Our results do not necessitate the assumption of spectroscopically different forms of phytochrome in these seeds.Abbreviations TD thermodormant - FRD far red dormant - P phytochrome - P _r red absorbing form of P - P _fr far red absorbing form of P