Flow cytometry reveals different lag times in rapid cytoplasmic calcium elevations in human neutrophils in response to N-formyl peptide

Flow cytometric analyses were performed to study intracellular single-cell calcium transients ([Ca²⁺]_i) in suspended human neutrophils during the initial phase of N-formyl peptide stimulation. Thereby, two neutrophil populations became apparent. Early maximally Ca²⁺-responding (high fluorescence) neutrophils and not-yet Ca²⁺-responding (low fluorescence) neutrophils, but no neutrophils with intermediate levels of [Ca²⁺]_i, were detected. Within 7 s the number of low fluorescence neutrophils decreased and the number of high fluorescence neutrophils increased maximally. This suggests that [Ca²⁺]_i transients occurred abruptly in individual neutrophils within a time interval below 1 s. At lower N-formyl peptide concentrations the lag times of individual neutrophils and the interval time of maximal activation of the [Ca²⁺]_i-responding neutrophil population increased, however the percentage of [Ca²⁺]_i-responding cells decreased. Surprisingly, no influence of the N-formyl peptide concentration on the [Ca²⁺]_i-induced fluorescence signal of the individual cell was observed: it was always in an almost maximal range or not responding. In parallel, binding studies performed with fluorescein-labeled N-formyl peptide revealed that the heterogeneity of [Ca²⁺]_i-responding cells cannot be explained by different receptor occupancy. In summary, this study demonstrates that [Ca²⁺]_i transients induced by N-formyl peptides in suspended individual human neutrophils occur very rapidly in an almost “all-or-none manner” and that the mean increasing fluorescence signal of a calcium indicator within a whole neutrophil population results from varying lag times of the individual cells, rather than from the mean simultaneous progress of many cells. © 1993 Wiley-Liss, Inc.     

TRANSIT TIMES THROUGH THE CYCLE PHASES OF JEJUNAL CRYPT CELLS OF THE MOUSE ANALYSIS IN TERMS OF THE MEAN VALUES AND THE VARIANCES

Mean transit times as well as variances of the transit times through the individual phases of the cell cycle have been determined for the crypt epithelial cells of the jejunum of the mouse. To achieve this the fraction of labelled mitoses (FLM) technique has been modified by double labelling with [³H] and [¹⁴C]thymidine. Mice were given a first injection of [³H]thymidine, and 2 hr later a second injection of [¹⁴C]thymidine. This produces a narrow subpopulation of purely ³H-labelled cells at the beginning of G₂-phase and a corresponding subpopulation of purely ¹⁴C-labelled cells at the beginning of the S-phase. When these two subpopulations progress through the cell cycle, one obtains FLM waves of purely ³H- and purely ¹⁴C-labelled mitoses. These waves have considerably better resolution than the conventional FLM-curves. From the temporal positions of the observed maxima the mean transit times of the cells through the individual phases of the cycle can be determined. Moreover one obtains from the width of the individual waves the variances of the transit times through the individual phases. It has been found, that the variances of the transit times through successive phases are additive. This indicates that the transit times of cells through successive phases are independently distributed. This statistical independence is an implicit assumption in most of the models applied to the analysis of FLM curves, however there had previously been no experimental support of this assumption. A further result is, that the variance of the transit time through any phase of the cycle is proportional to the mean transit time. This implies that the progress of the crypt epithelial cells is subject to an equal degree of randomness in the various phases of the cycle.     

TfM mutation and masculinization versus feminization of the mouse central nervous system

The sexual behavior of mice of various genotypes has been studied in our stock in which X-linked genes, Tfm and (O^hv), as well as an autosomal dominant gene, Sxr, are maintained. Since the absence of neonatal imprinting in Tfm (O^hv)/Y^ leads to no sexual behavior, we conclude that neonatal imprinting is the prerequisite for feminization as well as masculinization of the central nervous system. Since individual components of sexual behavior may become feminine or absent instead of being masculine in sex-reversed Tfm (O^hv)/+(O⁺, Sxr/+^♂ with mosaic brains, we conclude that neonatal imprinting directly involves individual neurons, and that different degrees of imprinting by the same agent lead to masculinization or feminization. In accordance with recent views, we believe estradiol to be this imprinting agent.We envisage the role of the Tfm locus in the central nervous system as follows: within individual neurons for sexual behavior, the synthesis of aromatizing enzymes is normally inducible by androgens; these enzymes are therefore noninducible in Tfm (O^hv)/Y^. In normal neonates, exposure to testosterone leads to the induced intracellular conversion of testosterone to estradiol, self imprinting by estradiol causing masculinization. Feminization may normally be due to the direct exposure of these neurons to a low circulating concentration of estradiol. An alternative explanation might be that the estradiol-receptor protein in these neurons also is normally inducible by testosterone. In this case, neonatally testosterone-exposed neurons would become inherently more responsive to estradiol than neonatally estradiol-exposed neurons.     

The Analysis of Intracellular and Intercellular Calcium Signaling in Human Anterior Lens Capsule Epithelial Cells with Regard to Different Types and Stages of the Cataract

In this work we investigated how modifications of the Ca²⁺ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca²⁺ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca²⁺ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca²⁺ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca²⁺ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca²⁺ activity. The presence of spontaneous activity was also found to affect the stimulated Ca²⁺ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca²⁺ signaling of LECs in cataractogenesis.     

Letter: Nuclear magnetic resonance of protein-nucleic acid interactions. II. The E. coli tRNA-Glu complex with glutamyl-tRNA synthetase

We have observed the 300 MHz high-resolution proton nuclear magnetic resonance spectrum of the Escherichia coli tRNA^Glu complex with the glutamyl-tRNA synthetase. The observations are fitted very well by a computer-simulated spectrum of the E. coli tRNA^Glu itself, with the individual resonances broadened from 45 Hz to 150 Hz because of the increased molecular weight. This indicates that no helical arms open upon complex formation, nor is there evidence for any additional Watson-Crick base-pairs in the complex.     

不同放牧制度下呼伦湖流域草原植被冠层截留

冠层截留是降雨过程中的水量分配和流域水平衡的一个重要组成部分,通过水浸泡法和降雨模拟实验研究呼伦湖流域草原3种放牧制度下(休牧、轮牧、自由放牧(超载放牧))植被冠层截留量的变化规律,并利用遥感解译植被归一化指数(NDVI),确定3种放牧制度下草原面积,估算呼伦湖流域草原降雨截留量。研究表明:在休牧、轮牧、自由放牧3种制度下,水浸泡法测定的截留量分别是0.468、0.320、0.271 mm。降雨模拟实验法测得的结果分别是0.957、0.613、0.431 mm。休牧、轮牧、自由放牧草场叶面积指数、盖度、容重、生物量等指标差异显著(P0.05),且单株植被高度、鲜重对截留量影响显著呈线性正相关关系。呼伦湖流域草原一次降雨量为大于等于30 mm全流域降雨,其植被截留量为6.462×106m3。     

1H, 13C and 15N resonance assignments of domain 1 of receptor associated protein

The 39 kDa receptor associated protein (RAP) is a modular protein consisting of multiple domains. There has been no x-ray crystal structure of RAP available and the full-length protein does not behave well in a NMR tube. To elucidate the 3D structure of the RAP, we undertook structure determination of individual domains of the RAP. As the first step, here we report the nearly complete assignments of the ¹H, ¹³C and ¹⁵N chemical shift signals of domain 1 of the RAP.     

Time Course of Individual Ca2+ Sparks in Frog Skeletal Muscle Recorded at High Time Resolution

Discrete Ca²⁺ release events (Ca²⁺ “sparks”) were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 μs per line). Fibers loaded with the Ca²⁺ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at −90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca²⁺ sparks at ∼30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5–2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 ± 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca²⁺ ions are being released by the sarcoplasmic reticulum Ca²⁺ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca²⁺ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.     

Oxygen isotope fractionations across individual leaf carbohydrates in grass and tree species

Almost no δ¹⁸O data are available for leaf carbohydrates, leaving a gap in the understanding of the δ¹⁸O relationship between leaf water and cellulose. We measured δ¹⁸O values of bulk leaf water (δ¹⁸O_LW) and individual leaf carbohydrates (e.g. fructose, glucose and sucrose) in grass and tree species and δ¹⁸O of leaf cellulose in grasses. The grasses were grown under two relative humidity (rH) conditions. Sucrose was generally ¹⁸O‐enriched compared with hexoses across all species with an apparent biosynthetic fractionation factor (ε_bio) of more than 27‰ relative to δ¹⁸O_LW, which might be explained by isotopic leaf water and sucrose synthesis gradients. δ¹⁸O_LW and δ¹⁸O values of carbohydrates and cellulose in grasses were strongly related, indicating that the leaf water signal in carbohydrates was transferred to cellulose (ε_bio = 25.1‰). Interestingly, damping factor p_exp_x, which reflects oxygen isotope exchange with less enriched water during cellulose synthesis, responded to rH conditions if modelled from δ¹⁸O_LW but not if modelled directly from δ¹⁸O of individual carbohydrates. We conclude that δ¹⁸O_LW is not always a good substitute for δ¹⁸O of synthesis water due to isotopic leaf water gradients. Thus, compound‐specific δ¹⁸O analyses of individual carbohydrates are helpful to better constrain (post‐)photosynthetic isotope fractionation processes in plants.     

METABOLIC RELATIONSHIPS BETWEEN MYELIN SUBFRACTIONS: ENTRY OF GALACTOLIPIDS AND PHOSPHOLIPIDS 1

Abstract— Partially purified myelin from the brains of 17-day-old rats was separated into 4 subfractions on a three-step sucrose gradient by virtue of heterogeneity in density and particle size. Precursor-product relationships between different membrane fractions were investigated by determining the specific radioactivity of individual lipids in each subcellular fraction 15 min after intracranial injection of an appropriate precursor. Rats were injected with [2-³H]glycerol. myelin subfractions prepared, and individual lipids separated by TLC. For choline and ethanolamine phospholipids, specific radioactivity was highest in the densest fraction (D), intermediate in the next densest fraction (C), and lowest in the lighter fractions (B and A). Similar results were observed for cerebroside and sulphatide when [³H]galactose was the precursor. These data are consistent with (but do not prove) a precursor-product relationship for individual lipids from the densest to the lightest subfraction. Another experimental design involving time staggered injections of [³H] and [¹⁴C] precursors was developed which enables a more definitive result with regard to precursor-product relationships to be obtained. A precursor-product relationship between a given lipid in a dense myelin membrane fraction, and the same lipid in a lighter subfraction, would be indicated by a change in isotope ratio. If there is no precursor-product relationship. Ihe isotope ratio should be constant. Such experiments were done with [³H] and [¹⁴C]glycerol. The data indicated that phosphatidyl ethanolamine and its plasmalogen analog were added first to the densest subfraction and then in turn to the lighter subfractions. In contrast, phosphatidyl choline and its plasmalogen analog were added “simultaneously” (i.e. with delays of much less than 15min) to each of the subfractions. Similar experiments with [³H] and [¹⁴C]galactose showed that cerebroside, sulphatide and galactosyl diglyceride also entered the subfractions simultaneously rather than in sequential order. Thus the assembly of the myelin sheath involves an obligate order of addition of certain lipids. while other lipids are probably added in a random order.     

Effect of C4 Depletion on the Utilization of the Terminal Components of Guinea-pig Complement by Endotoxin

ENDOTOXIN and antigen-antibody complexes (Ag-Ab) are potent inflammatory agents which induce marked pathophysiological effects in a number of animal species. Both these agents lead to increased vascular permeability, local accumulations of polymorphonuclear leucocytes and alterations in the coagulability of blood¹. There is mounting evidence that these similarities derive from endotoxins and Ag-Ab being activators of the complement (C) system, an important mediator of inflammation¹'². When endotoxins or preformed Ag-Ab are reacted with normal guinea-pig (GP) serum so as to induce equivalent consumption of whole haemolytic C, there are, however, striking differences in the consumption profiles of the individual C components³. Although interaction of Ag-Ab with GP serum results in the consumption of CI, C4 and C2 as well as C3 to C9, the interaction of endotoxin with GP serum leads to a marked depletion of C3 to C9 with little or no detectable loss of C1,C4 and C2 (ref. 3).     

Alteration of Racial Differences in Melanosome Distribution in Human Epidermis after Exposure to Ultraviolet Light

CAREFUL studies of human epidermis have revealed that there is no significant difference between the number of melanocytes in the various racial groups, although there are regional differences in the population density of DOPA-positive melanocytes in various areas of the body (for example, on the forehead, 2,310 mm^?2; abdomen, 800 mm^?2; and back, 1,100 mm^?2)¹. These differences in the number of melanocytes may explain the colour differences in various areas of the body, but the colour of skin in the various races is apparently due to variations in the number of melanin granules, or melanosomes, in the melanocytes and in the epidermal keratinocytes, as well as to the degree of melanization of the individual melanosomes.     

Strontium metabolism in teeth and enamel dose assessment: analysis of the Techa river data

People living on the banks of the Techa river were exposed to ⁹⁰Sr in the early 1950s. Data obtained by radiochemical measurements of extracted permanent teeth, ⁹⁰Sr autopsy measurements in bone and tooth samples, in vivo measurements of surface beta activity of the anterior teeth and whole-body counter (WBC) measurements of ⁹⁰Sr in the skeleton have been analyzed. Surface beta activity measurements indicate a biological half-life of ⁹⁰Sr of about 35 years in enamel. The WBC measurements have been performed since 1974 and a model for the age-dependent strontium retention in human bone has been used to extrapolate to previous time periods when the other measurement results were obtained. For the first decade after the intake, the ratio of the ⁹⁰Sr concentrations in teeth and bones were found to decrease with age at the time of major intake, from about 10 for 1-year-old children to about 0.3 for adults. There was a considerable variability of individual data within each age group. For adults, the correlation between ⁹⁰Sr in skeleton and teeth was not high at 0.47 according to radiochemical data for posterior teeth (molars and premolars) and 0.43 according to measurements of surface beta activity for anterior teeth. For children and adolescents there was no correlation between individual measurements in the skeleton and teeth. The absorbed dose in enamel due to ⁹⁰Sr in dentine has been calculated by Monte Carlo simulations of the electron transport. The results are in agreement with EPR measurements of the absorbed dose in the enamel of persons exposed, mainly due to ⁹⁰Sr ingestion. Received: 19 July 1999 / Accepted: 5 May 2000     

Characterization and comparisons of five N2-fixing Azolla-Anabaena associations,I. Optimization of growth conditions for biomass increase and N content in a controlled environment*

Abstract Biomass increase, C and N content, C₂H₂ reduction, percentage dry weight and chlorophyll a/b ratios were determined for clones of Azolla caroliniana Willd., A. filiculoides Lam., A. mexicana Presl., and A. pinnata R. Br. as a function of nutrient solution, pH, temperature, photoperiod, and light intensity in controlled environment studies. These studies were supplemented by a glasshouse study. Under a 16 h, 26°C day at a light intensity of 200 μmol m^?2 s^?1 and an 8 h, 19° C dark period, there was no significant difference in the growth rates of the individual species on the five nutrient solutions employed. Growth was comparable from pH 5 to pH 8, but decreased at pH 9. Using the same photoperiod and light intensity but constant growth temperatures of 15–40°C, at 5°C intervals, the individual species exhibited maximum growth, nitro-genase (N²ase) activity and N content at either 25° or 30°C. There was no difference in the temperature optima at pH 6 and pH 8. The tolerance of the individual species to elevated temperature was indicated to be A. mexicana> A. pinnata> A. caroliniana> A.filiculoides. At the optimum temperature, growth rates increased with increasing photoperiod at both pH 6 and pH 8 but N₂ase activity was usually highest at a 16 h light period. At photon flux densities of 100, 200, 400 and 600 μmol m^?2 s^?1, during a 16 h light period and optimum growth temperature of the individual species, N₂ase activity was saturated at less than 200 μmol m^?2 s^?1 and growth at 400 μmol m^?2 s^?1.No interacting effects of light and pH were noted for any species, nor were light intensities up to 1700 μmol m^?2 s^?1 detrimental to the growth rate or N content of any species in a 5 week glasshouse study with a natural 14.5 h light period and a constant temperature of 27.5°C. Using the optimum growth temperature, a 16 h light period, and a photon flux density of at least 400 μmol m^?2 s^?1, the Azolla species all doubled their biomass in 2 days or less and contained 5–6% N on a dry weight basis.     

Incorporation of 14C-mevalonate into biliary cholesterol and bile acids by perfused rat liver: Effect of plasma and individual lipoproteins

Effect of plasma and individual lipoproteins on the incorporation of ¹⁴C-mevalonate into biliary bile acids and cholesterol by perfused rat liver was investigated. Use of plasma free perfysate (instead of whole blood) gave similar rates of incorporation of ¹⁴ C-mevalonate into biliary bile acids, but showed a decrease in percent distribution in cholic acid and a increase in β-muricholic acid. Addition of VLDL (isolated from Zucker rats) into the plasma free perfusate caused a significant increase in the incorporation of the label into bile acids, but LDL and HDL had no effect. HDL caused an increase in biliary excretion of ¹⁴C-cholesterol.     

How Does Intracellular Ca2+ Oscillate: By Chance or by the Clock?

Ca²⁺ oscillations have been considered to obey deterministic dynamics for almost two decades. We show for four cell types that Ca²⁺ oscillations are instead a sequence of random spikes. The standard deviation of the interspike intervals (ISIs) of individual spike trains is similar to the average ISI; it increases approximately linearly with the average ISI; and consecutive ISIs are uncorrelated. Decreasing the effective diffusion coefficient of free Ca²⁺ using Ca²⁺ buffers increases the average ISI and the standard deviation in agreement with the idea that individual spikes are caused by random wave nucleation. Array-enhanced coherence resonance leads to regular Ca²⁺ oscillations with small standard deviation of ISIs.     

An introduction to biological nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical techniques available to biology. This review is an introduction to the potential of this method and is aimed at readers who have little or no experience in acquiring or analyzing NMR spectra. We focus on spectroscopic applications of the magnetic resonance effect, rather than imaging ones, and explain how various aspects of the NMR phenomenon make it a versatile tool with which to address a number of biological problems. Using detailed examples, we discuss the use of ¹H NMR spectroscopy in mixture analysis and metabolomics, the use of ¹³C NMR spectroscopy in tracking isotopomers and determining the flux through metabolic pathways (‘fluxomics’) and the use of ³¹P NMR spectroscopy in monitoring ATP generation and intracellular pH homeotasis in vivo. Further examples demonstrate how NMR spectroscopy can be used to probe the physical environment of a cell by measuring diffusion and the tumbling rates of individual metabolites and how it can determine macromolecular structures by measuring the bonds and distances which separate individual atoms. We finish by outlining some of the key challenges which remain in NMR spectroscopy and we highlight how recent advances—such as increased magnet field strengths, cryogenic cooling, microprobes and hyperpolarisation—are opening new avenues for today's biological NMR spectroscopists.     

Differential effects of cobalt on the initiation of fast axonal transport

Effects of Co²⁺ on the fast axonal transport of individual proteins were examined in vitro in bullfrog spinal/sciatic nerves.³⁵S-methionine-labeled proteins, fast-transported in control and Co²⁺-treated preparations were separated via two-dimensional gel electrophoresis. While the overall amount of protein transported was reduced, no qualitative differences could be seen when gel fluorographic patterns were compared. Quantitative analyses of the 48 most abundantly transported species revealed two significantly different populations (p < 0.01) differentially sensitive to Co²⁺ and distinguishable to a large extent by molecular weight. Those proteins less sensitive to Co²⁺ ranged from ~20,000 to 35,000 daltons while those more sensitive to Co²⁺ were >~35,000 daltons. The finding that all proteins are affected by Co²⁺ supports the proposal that fast-transported proteins are subject to a common Co²⁺-sensitive, Ca²⁺-requiring step. The observed differential effects are consistent with more than one Ca²⁺-dependent step occurring during the initiation phase of fast transport.This research was supported by a Muscular Dystrophy Association postdoctoral fellowship to G.C.S., and by research grants from NSF (BNS 79-24125) and the National Multiple Sclerosis Society (RG 1296-A-1) to R.H.     

Influence of cycloheximide on the synthesis and utilization of amino acids in suspension cultures

Cells from 4-days old suspension cultures of Paul's Scarlet rose were incubated with acetate-U-¹⁴C for 10 minutes. After washing, cells were incubated for 2 hours in growth medium in the presence and absence of cycloheximide. The ¹⁴C content of individual amino acids in the soluble form and in protein were determined at the end of the 10 minute pulse and at intervals thereafter in control cells and those treated with cycloheximide. During the period following the pulse there was a 3-fold increase in the ¹⁴C content of protein in control cells; no such increase occurred in the presence of cycloheximide.