Differential laws of left ventricular isovolumic pressure fall

An attempt has been made to test for a reliable method of characterizing the isovolumic left ventricular pressure fall in isolated ejecting hearts by one or two time constants, tau. Alternative nonlinear regression models (three- and four-parametric exponential, logistic, and power function), based upon the common differential law dp(t)/dt = - [p(t)-P ]/ tau(t) are compared in isolated ejecting rat, guinea pig, and ferret hearts. Intraventricular pressure fall data are taken from an isovolumic standard interval and from a subinterval of the latter, determined data-dependently by a statistical procedure. Extending the three-parametric exponential fitting function to four-parametric models reduces regression errors by about 20-30%. No remarkable advantage of a particular four-parametric model over the other was revealed. Enhanced relaxation, induced by isoprenaline, is more sensitively indicated by the asymptotic logistic time constant than by the usual exponential. If early and late parts of the isovolumic pressure fall are discarded by selecting a subinterval of the isovolumic phase, tau remains fairly constant in that central pressure fall region. Physiological considerations point to the logistic model as an advantageous method to cover lusitropic changes by an early and a late tau. Alternatively, identifying a central isovolumic relaxation interval facilitates the calculation of a single ("central") tau; there is no statistical justification in this case to extend the three-parametric exponential further to reduce regression errors.     

Spontaneous and GABA-induced single channel currents in cultured murine spinal cord neurons

Intracellular and patch clamp recordings were made from embryonic mouse spinal cord neurons growing in primary cell culture. Outside-out membrane patches obtained from these cells usually showed spontaneous single channel currents when studied at the resting potential (-56 +/- 1.5 mV). In 18 out of 30 patches tested, spontaneous single channel activity was abolished by making Tris+ the major cation on both sides of the membrane. The remaining patches continued to display spontaneous single channel currents under these conditions. These events reversed polarity at a patch potential of 0 mV and displayed a mean single channel conductance of 24 +/- 1.2 pS. Application of the putative inhibitory transmitter gamma-aminobutyric acid (0.5-10 microM) to outside-out patches of spinal cord cell membrane induced single channel currents in 10 out of 15 patches tested. These channels had a primary conductance of 29 +/- 2.8 pS in symmetrical 145 mM Cl- solutions. Frequency distributions for the open times of these channels were well fit by the sum of a fast exponential term ("of") with a time constant tau of = 4 +/- 1.3 ms and a slow exponential term ("os") with a time constant tau os = 24 +/- 8.1 ms. Frequency distributions for channel closed times were also well fit by a double exponential equation, with time constants tau cf = 2 +/- 0.2 ms and tau cs = 62 +/- 20.9 ms.     

Costs of parturition and rearing in female sika deer (Cervus nippon)

The costs of parturition and lactation of female sika deer on Kinkazan Island (9.6 km(2) in size), northern Japan, which live at a high density (about 50 deer/km(2)), were evaluated by comparison of body weights of 481 females measured during a 15 year study (1993-2008). Weight data were chosen from only females that did not give birth in the preceding year. The mean body weight of females that did not give birth ("yelds") was significantly lower (P < 0.001) than that of females who gave birth ("milks"); yelds' body weight was 93.1% and 83.5% that of milks in the preceding and parturition years, respectively. The yelds increased in body weight by the following March by 8.2% (P < 0.001), whereas milks did not. Among the milks, those whose fawns survived until the following May ("rearing milks") lost body weight by 14.9% (P < 0.001). Milks who lost fawns within a week after birth ("early fawn-less milks") did not lose body weight (P = 0.583), while those whose fawns died after the first autumn but died before May ("late fawn-less milks") lost body weight by 19.9% (P < 0.001). These results indicate that sika deer females do not enter estrus unless they are heavy enough, and that both parturition and rearing are costly for sika deer mothers living in high-density conditions.     

Timing the start of division in E. coli: a single-cell study

We monitor the shape dynamics of individual E. coli cells using time-lapse microscopy together with accurate image analysis. This allows measuring the dynamics of single-cell parameters throughout the cell cycle. In previous work, we have used this approach to characterize the main features of single-cell morphogenesis between successive divisions. Here, we focus on the behavior of the parameters that are related to cell division and study their variation over a population of 30 cells. In particular, we show that the single-cell data for the constriction width dynamics collapse onto a unique curve following appropriate rescaling of the corresponding variables. This suggests the presence of an underlying time scale that determines the rate at which the cell cycle advances in each individual cell. For the case of cell length dynamics a similar rescaling of variables emphasizes the presence of a breakpoint in the growth rate at the time when division starts, tau(c). We also find that the tau(c) of individual cells is correlated with their generation time, tau(g), and inversely correlated with the corresponding length at birth, L(0). Moreover, the extent of the T-period, tau(g) - tau(c), is apparently independent of tau(g). The relations between tau(c), tau(g) and L(0) indicate possible compensation mechanisms that maintain cell length variability at about 10%. Similar behavior was observed for both fast-growing cells in a rich medium (LB) and for slower growth in a minimal medium (M9-glucose). To reveal the molecular mechanisms that lead to the observed organization of the cell cycle, we should further extend our approach to monitor the formation of the divisome.     

Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filament. The rapidly growing ("barbed") end has higher association and dissociation rate constants than the slowly growing ("pointed") end. The values of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 microM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.     

On one-dimensional nucleation and growth of "living" polymers. II. Growth at constant monomer concentration

An analytical solution is given for the kinetics of reversible homogeneous one-dimensional growth, assuming that all association rate constants have the same value k, that all dissociation rate constants are likewise equal to k, and that the monomer concentration has a constant value, C. Such growth tends to generate a maximally polydisperse ("white") distribution of cluster concentrations ci, all approaching a limiting value equal to that of the critical nucleus, cn. Continued growth merely increases the range of cluster sizes over which this white distribution applies. A simple expression is obtained for the flux sigma infinity i = n dci/dt, which becomes constant and equal to (kC - k)cn. The monomer uptake increases with time, and is given approximately by (kC - k)2cnt.     

Growth rate of potato tubers and endogenous contents of indolylacetic acid and abscisic acid

Potato plants ( Solanum tuberosum L. ev. Ostara) were grown in water culture and the growth rate of individual tubers was measured daily or at two day intervals. Tubers of different growth rate and/or different age (days after tuberization) were harvested and analysed for indolylacetic acid (IAA) and abscisic acid (ABA). Within individual tubers the IAA content decreases from the apical to the basal part of the tuber. Tuber age and corresponding fresh weight are negatively correlated with the endogenous IAA content. If, however, individual tubers of comparable age but different growth rates are compared, a significant positive correlation between growth rate and IAA content is revealed, while ABA showed a significant negative correlation with growth rate. Removal of all fast-growing tubers from individual plants causes an increase in the growth rate of the remaining tubers within 3–4 days. This coincides with a particularly steep increase in IAA content. The data support the idea that endogenous IAA content may be one factor responsible for controlling the growth rate ("sink-activity ") of individual tubers.     

Transient studies of nutrient uptake, growth, and indole alkaloid accumulation in heterotrophic cultures of hairy roots of Catharanthus roseus

The kinetics of growth, the uptake of macronutrients, and the accumulation of indole alkaloids were investigated in long-term, heterotrophically cultured transgenic ("hairy") roots of Catharanthus roseus.Tabersonine, ajmalicine, and serpentine were monitored over a 70-day period. The doubling time [dry-weight (DW) basis] of C. roseus hairy roots in B5/2 nutrients supplemented with 3% sucrose was 3.6 days. NH(4) (+), NO(3),(-) and P(i) were depleted sequentially from culture medium by hairy roots, while sugars remained undepleted. The growth-limiting nutrient was inorganic nitrogen, NH(4) (+) and NO(3) (-), with exponential-phase overall biomass yields of 34.1 and 5.0 g DW/g nutrient, respectively. Extracellular pH decreased to 4.8 in early exponential phase of culture growth from the initially adjusted value of 5.7, increased subsequently to a maximum of 7.7 in late exponential phase of growth coincident with the maximum of fresh weight (FW)/DW ratio, before decreasing to 5.5-5.0. The organic acids, pyruvate, formate, lactate, and succinate were excreted by hairy roots starting in late phase of exponential growth, possibly resulting in the late-culture pH decrease. Tabersonine accumulation was distinctly growth associated with maximum specific and total yields of 1.15 mg/g DW and 5.6 mg/L, respectively, in late-exponential phase of growth. Serpentine accumulation was non growth associated with increasing specific and total levels in stationary growth phase: 1.3 mg/g DW and 10.5 mg/L, respectively. The accumulation of ajmalicine also appeared growth associated. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 527-534, 1997.     

Studies on nucleic acid reassociation kinetics: V. Effects of disparity in tracer and driver fragment lengths.

Measurements are described of the kinetics of nucleic acid strand pair reassociation where the complementary strands are of different lengths and are present in different concentrations. Rate constants for the reaction of labelled fragments ("tracer") with excess complementary strands ("driver") were determined, both for driver fragment length greater than tracer fragment length and for the reverse case. Second order reactions and pseudo-first order reactions utilizing strand separated drivers and tracers were studied. The nucleic acids which served for this investigation were phiX174 DNA and RNA, plasmid RSF2124 DNA and E. coli DNA. Approximate empirical expressions relating driver and tracer fragment lengths with the observed rate constants were obtained for practical use. In long tracer-short driver reactions the observed rate constant for the tracer reaction increases proportionately with tracer length. In long driver-short tracer reactions the rate of tracer reaction is retarded. The latter result is unexpected and appears to represent a departure from standard interpretations of the renaturation reaction.     

Lateral mobility in membranes as detected by fluorescence recovery after photobleaching.

The evaluation of lateral diffusion coefficients of membrane components by the technique of fluorescence recovery after photobleaching (FRAP) is often complicated by uncertainties in the values of the intensities F(O), immediately after bleaching, and F(infinity), after full recovery. These uncertainties arise from instrumental settling time immediately after bleaching and from cell, tissue, microscope, or laser beam movements at the long times required to measure F(infinity). We have developed a method for precise analysis of FRAP data that minimizes these problems. The method is based on the observation that a plot of the reciprocal function R(tau) = F(infinity)/[F(infinity)-F(tau)] is linear over a large time range when (a) the laser beam has a Gaussian profile, (b) recovery involves a single diffusion coefficient, and (c) there is no membrane flow. Moreover, the ratio of intercept to slope of the linear plot is equal to tau 1/2, the time required for the bleached fluorescence to rise to 50% of the full recovery value, F(infinity). The lateral diffusion coefficient D is related to tau 1/2 by tau 1/2 = beta w2/4D where beta is a defined parameter and w is the effective radius of the focused laser beam. These results are shown to indicate that the recovery of fluorescence F(tau) can be represented over a large range of percent bleach, and recovery time tau by the relatively simple expression F(tau) = [ F(o) + F(infinity) (tau/tau 1/2)]/[1 + tau/tau 1/2)]. FRAP data can therefore be easily evaluated by a nonlinear regression analysis with this equation or by a linear fit to the reciprocal function R(tau). It is shown that any error in F(infinity) can be easily detected in a plot of R(tau) vs. tau which deviates significantly from a straight line when F(infinity) is in error by as little as 5%. A scheme for evaluating D by linear analysis is presented. It is also shown that the linear reciprocal plot provides a simple method for detecting flow or multiple diffusion coefficients and for establishing conditions (data precision, differences in multiple diffusion coefficients, magnitude of flow rate compared to lateral diffusion) under which flow or multiple diffusion coefficients can be detected. These aspects are discussed in some detail.     

Direct evidence of "damage accumulation" in cement mantles surrounding femoral hip stems retrieved at autopsy: cement damage correlates with duration of use and BMI

The "damage accumulation" phenomenon has not been quantitatively demonstrated in clinical cement mantles surrounding femoral hip stems. We stained transverse sections of 11 postmortem retrieved femoral hip components fixed with cement using fluorescent dye-penetrant and quantified cement damage, voids, and cement-bone interface gaps in epifluorescence and white light micrographs. Crack density (Cr.Dn), crack length-density (Cr.Ln.Dn), porosity, and cement-bone interface gap fraction (c/b-gap%) were calculated, normalized by mantle area. Multiple regression tests showed that cement damage (Cr.Ln.Dn. & Cr.Dn.) was significantly positively correlated (r(2)=0.98, p<0.001) with "duration of use" and body mass index ("BMI") but not cement mantle "porosity". There were significant interactions: "duration of use"*"BMI" was strongly predictive (p<0.005) of Cr.Dn.; and "duration of use"*"porosity" was predictive (p=0.04) of Cr.Ln.Dn. Stem related cracks accounted for approximately one fifth of Cr.Dn and one third of Cr.Ln.Dn. The mean c/b-gap% was 13.8% but it did not correlate (r(2)=0.01, p=0.8) with duration of use. We concluded that duration-dependent fatigue damage accumulation occurred during in vivo use. BMI strongly influenced cement crack length and the rate of new crack formation over time. Voids did not increase the rate of crack initiation but appeared to have promoted crack growth over time. Although not progressive, substantial bone resorption at the cement-bone interface appeared to be common.     

Environmental and sibling resemblance components of variance and covariance in traits of early child development

The multiple regression analyses were undertaken to elucidate the significance and relative importance of different potential determinants of several child development traits (i.e. age at which child 1. turns himself over; 2. sits up; 3. stands up; 4. walks, and 5. cuts his first tooth). Despite the fact that we used a relatively wide gamut of potential determinants (such as parental age, geographic origin, occupation, current residence, or family size) of trait variability, the results of multiple regression analysis (N = 300 families) indicate an almost complete absence of significant factors and studied variables of development. Sibling resemblance component of variance and covariance for five mentioned traits of development and some morphological characters (weight, length and head circumference at day of birth and at 16 months) were studied in 66 families. The transmissibility ("heritability") values for the development traits were small, ranging from 0 ("Turn") to 0.42 ("Walked"). The Findings suggest that there are significant inverse relationship between some of the developmental traits and inborn morphological characters.     

Length growth of two Escherichia coli B/r substrains.

Length growth of synchronized Escherichia coli B/r substrain A (ATCC 12407) and B/r substrain F26 (Thy his) was followed with an electron microscope. Cells were grown with doubling times (tau) of 60 min (B/rA) and of 82 and 165 min (B/rF26). Different length growth patterns were found for the two substrains. In B/rF, the length growth rate increased about midway in the cell cycle. For tau = 165 min, the rate increase was preceded by a short period of slow growth. For B/r A (r = 60 min), this period seemed to occur at the beginning of the cell cycle. The possibility is raised that the different length growth patterns are related to different deoxyribonucleic acid replication patterns of the respective strains.     

Domain flexibility in ligand-free and inhibitor-bound Escherichia coli adenylate kinase based on a mode-coupling analysis of 15N spin relaxation

Adenylate kinase from Escherichia coli (AKeco), consisting of a 23.6-kDa polypeptide chain folded into domains CORE, AMPbd, and LID catalyzes the reaction AMP + ATP <--> 2ADP. The domains AMPbd and LID execute large-amplitude movements during catalysis. Backbone dynamics of ligand-free and AP(5)A-inhibitor-bound AKeco is studied with slowly relaxing local structure (SRLS) (15)N relaxation, an approach particularly suited when the global (tau(m)) and the local (tau) motions are likely to be coupled. For AKeco tau(m) = 15.1 ns, whereas for AKeco*AP(5)A tau(m) = 11.6 ns. The CORE domain of AKeco features an average squared order parameter, , of 0.84 and correlation times tau(f) = 5-130 ps. Most of the AKeco*AP(5)A backbone features = 0.90 and tau(f) = 33-193 ps. These data are indicative of relative rigidity. Domains AMPbd and LID of AKeco, and loops beta(1)/alpha(1), alpha(2)/alpha(3), alpha(4)/beta(3), alpha(5)/beta(4), and beta(8)/alpha(7) of AKeco*AP(5)A, feature a novel type of protein flexibility consisting of nanosecond peptide plane reorientation about the C(i-1)(alpha)-C(i)(alpha) axis, with correlation time tau(perpendicular) = 5.6-11.3 ns. The other microdynamic parameters underlying this dynamic model include S(2) = 0.13-0.5, tau(parallel) on the ps time scale, and a diffusion tilt beta(MD) ranging from 12 to 21 degrees. For the ligand-free enzyme the tau(perpendicular) mode was shown to represent segmental domain motion, accompanied by conformational exchange contributions R(ex) < or = 4.4 s(-1). Loop alpha(4)/beta(3) and alpha(5)/beta(4) dynamics in AKeco*AP(5)A is related to the "energetic counter-balancing of substrate binding" effect apparently driving kinase catalysis. The other flexible AKeco*AP(5)A loops may relate to domain motion toward product release.     

Fast statistical tests for detecting heterotachy in protein evolution

The w statistic introduced by Lockhart et al. (1998. A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages. Mol Biol Evol. 15:1183-1188) is a simple and easily calculated statistic intended to detect heterotachy by comparing amino acid substitution patterns between two monophyletic groups of protein sequences. It is defined as the difference between the fraction of varied sites in both groups and the fraction of varied sites in each group. The w test has been used to distinguish a covarion process from equal rates and rates variation across sites processes. Using simulation we show that the w test is effective for small data sets and for data sets that have low substitution rates in the groups but can have difficulties when these conditions are not met. Using site entropy as a measure of variability of a sequence site, we modify the w statistic to a w' statistic by assigning as varied in one group those sites that are actually varied in both groups but have a large entropy difference. We show that the w' test has more power to detect two kinds of heterotachy processes (covarion and bivariate rate shifts) in large and variable data. We also show that a test of Pearson's correlation of the site entropies between two monophyletic groups can be used to detect heterotachy and has more power than the w' test. Furthermore, we demonstrate that there are settings where the correlation test as well as w and w' tests do not detect heterotachy signals in data simulated under a branch length mixture model. In such cases, it is sometimes possible to detect heterotachy through subselection of appropriate taxa. Finally, we discuss the abilities of the three statistical tests to detect a fourth mode of heterotachy: lineage-specific changes in proportion of variable sites.     

Classes of hydration sites at protein-water interfaces: the source of contrast in magnetic resonance imaging.

Immobilized protein solute, approximately 20 wt %, alters the longitudinal and transverse nuclear magnetic relaxation rates 1/T1 and 1/T2 of solvent water protons in a manner that makes their values indistinguishable from those of a typical human tissue. There is now a quantitative theory at the molecular level (S.H. Koenig and R. D. Brown III (1993) Magn. Reson. Med. 30:685-695) that accounts for this, as a function of magnetic field strength, in terms of several distinguishable classes of water-binding sites at the protein-water interface at which significant relaxation and solute-solvent transfer of proton Zeeman energy occur. We review the arguments that these several classes of sites, characterized by widely disparate values of the resident lifetimes tau M of the bound waters, are associated with different numbers of hydrogen bonds that stabilize the particular protein-water complex. The sites that dominate relaxation-and produce contrast in magnetic resonance imaging (MRI), which derives from 1/T1 and 1/T2 of tissue water protons-have tau M approximately 10(-6)s. These, which involve four hydrogen bonds, occupy < or = 1% of the protein-water interface. Sites that involve three bonds, although more numerous, have < or = 20% smaller intrinsic effect on relaxation. The greater part of the "traditional" hydration monolayer, with even shorter-lived hydrogen-bonded waters, has little influence on solvent relaxation and is relatively unimportant in MRI. Finally, we argue, from the data, that most of the protein of tissue (a typical tissue is mostly protein) must be rotationally immobile (with Brownian rotational relaxation times slower than that of a 5 x 10(7) Da (very heavy) globular protein). We propose a functional basis for this immobilization ("cytoplasmic order"), and then indicate a way in which this order can break down ("cytoplasmic chaos") as a result of neoplastic transformation (cancer) and alter water-proton rates of pathological tissue and, hence, image contrast in MRI.     

Properties of microfiltration membranes: Mechanisms of flux loss in the recovery of an enzyme

The transmission and rate of filtration of the enzyme yeast alcohol dehydrogenase (YADH) has been studied at capillary pore microfiltration membranes. Photon correlation spectroscopy (PCS) with nanometer resolution showed that the enzyme existed as discreate molecules only for a narrow range of pH and ionic strength. Under such conditions, the transmission of the enzyme was high. However, the rate of filtration still decreased continuously with time. Analyssis of the time dependence of the rate of filtration indicated that this decrease was due to in-pore enzyme deposition at low concentration ("standard blocking model") and suface depositon at high concentration ("cake filtration model"). Use of atomic force microscopy (AFM) gave unequivocal and quantitative confirmation of these inferences. The work shows the great advantage of using advanced physical characterization techniques, both for the identification of the optimum conditions for filtration (PCS) and for the elucidation of mechanisms giving rise to inefficiencies in the filtration process (AFM). (c) 1995 John Wiley & Sons, Inc.     

Some properties of potassium-stimulated calcium influx in presynaptic nerve endings

Potassium-stimulated 45Ca entry into rat brain synaptosomes was measured at times ranging from 1 to 60 s. The K-rich solutions were used to depolarize the synaptosomes. Backflux of 45Ca from the synaptosomes was negligible during the first 10-20 s of incubation. An initial ("fast") phase of K-stimulated Ca entry, lasting from 1 to 2 s was observed. This phase was inhibited by low concentrations of La (KI approximately equal to 0.3 microM). It was also abolished ("inactivated") by incubating the synaptosomes in depolarizing solutions (containing veratridine, gramicidin, or elevated [K]o) before the addition of 45Ca. An additional long lasting ("slow") phase of K-stimulated Ca entry was also detected. This "slow" Ca entry was much less sensitive to La (KI > 100 microM) and was not affected by depolarizing the synaptosomes before the addition of 45Ca. The rate of influx during the fast phase was about four times the rate of Ca influx during the slow phase. Neither the fast nor slow phase of Ca entry was sensitive to tetrodotoxin (10 microM), a potent blocker of Na channels, but both phases were inhibited by Ni, Mn, Mg, and other agents that block Ca channels. The data are consistent with the presence of two distinct populations of voltage-regulated, divalent cation-selective pathways for Ca entry in presynaptic brain nerve endings.     

Transpiration efficiency of a tropical pioneer tree (Ficus insipida) in relation to soil fertility

The response of whole-plant water-use efficiency, termed transpiration efficiency (TE), to variation in soil fertility was assessed in a tropical pioneer tree, Ficus insipida Willd. Measurements of stable isotope ratios (delta(13)C, delta(18)O, delta(15)N), elemental concentrations (C, N, P), plant growth, instantaneous leaf gas exchange, and whole-plant water use were used to analyse the mechanisms controlling TE. Plants were grown individually in 19 l pots with non-limiting soil moisture. Soil fertility was altered by mixing soil with varying proportions of rice husks, and applying a slow release fertilizer. A large variation was observed in leaf photosynthetic rate, mean relative growth rate (RGR), and TE in response to experimental treatments; these traits were well correlated with variation in leaf N concentration. Variation in TE showed a strong dependence on the ratio of intercellular to ambient CO(2) mole fractions (c(i)/c(a)); both for instantaneous measurements of c(i)/c(a) (R(2)=0.69, P <0.0001, n=30), and integrated estimates based on C isotope discrimination (R(2)=0.88, P <0.0001, n=30). On the other hand, variations in the leaf-to-air humidity gradient, unproductive water loss, and respiratory C use probably played only minor roles in modulating TE in the face of variable soil fertility. The pronounced variation in TE resulted from a combination of the strong response of c(i)/c(a) to leaf N, and inherently high values of c(i)/c(a) for this tropical tree species; these two factors conspired to cause a 4-fold variation among treatments in (1-c(i)/c(a)), the term that actually modifies TE. Results suggest that variation in plant N status could have important implications for the coupling between C and water exchange in tropical forest trees.