Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope

We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to approximately 10(5) independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10(-7) cm(2)/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.     

Contributions to the mathematical biophysics of organic form III. Deformation of shell shaped cellular aggregates

The average concentrations of a substance diffusing into or from an open spherical shell, in which it is consumed or produced at a constant rate, are calculated by the approximation method. An application of the result to the problem of deformation of such a shell under the influence of diffusion forces is indicated.     

Diffusion and simultaneous chemical reactions: I. A method for solving the equations of some systems in which a fixed concentration exists at a boundary

Many solutions are available to the differential equations for systems consisting of a space region with a boundary at which the concentration is fixed, diffusion occurring across this boundary. A method is described for readily transforming these solutions into results for similar systems in which the diffusing substance is removed by a first-order reaction and also removed or produced at a rate which is expressible as a polynomial in the time variable. Subsidiary transformations and steady-state conditions are also discussed. An indication is given of biological applications of the results made available by this method.     

Substance P-like immunoreactivity in the nervous system of hydra

Summary Using immunocytochemistry we find substance P-like material in nerve cells of hydra. These nerve cells are situated in the ectoderm of the basal disk and tentacles. Radioimmunoassay of hydra extracts gives dilution curves parallel to that of synthetic substance P, from which it can be calculated that one animal contains at least 0.6 fmol substance P-like immunoreactivity. After chromatography on Biogel P-100, the substance P-like immunoreactivity elutes as a peak in the void volume and a peak at the position of synthetic substance P.     

Substance P-like immunoreactivity in the nervous system of hydra

Using immunocytochemistry we find substance P-like material in nerve cells of hydra. These nerve cells are situated in the ectoderm of the basal disk and tentacles. Radioimmunoassay of hydra extracts gives dilution curves parallel to that of synthetic substance P, from which it can be calculated that one animal contains at least 0.6 fmol substance P-like immunoreactivity. After chromatography on Biogel P-100, the substance P-like immunoreactivity elutes as a peak in the void volume and a peak at the position of synthetic substance P.     

Effect of convective flow on the oxygen tension profile in the cornea

The effect of convective flow on the distribution of a diffusing substance that is being consumed is analyzed. For the mammalian cornea, one of the few tissues where convection, diffusion and consumption simultaneously exist, the effect of convection on the distribution of oxygen is shown to be small.     

Lateral diffusion of rhodopsin in photoreceptor cells measured by fluorescence photobleaching and recovery.

Frog rod outer segments were labeled with the sulfhydryl-reactive label iodoacetamido tetramethylrhodamine. The bulk of the label reacted with the major disk membrane protein, rhodopsin. Fluorescence photobleaching and recovery (FPR) experiments on labeled rods showed that the labeled proteins diffused rapidly in the disk membranes. In these FPR experiments we observed both the recovery of fluorescence in the bleached spot and the loss of fluorescence from nearby, unbleached regions of the photoreceptor. These and previous experiments show that the redistribution of the fluorescent labeled proteins after bleaching was due to diffusion. The diffusion constant, D, was (3.0 +/- 10(-9) cm2 s-1 if estimated from the rate of recovery of fluorescence in the bleached spot, and (5.3 +/- 2.4) x 10(-9) cm2 s-1 if estimated from the rate of depletion of fluorescence from nearby regions. The temperature coefficient, Q10, for diffusion was 1.7 +/- 0.5 over the range 10 degrees--29 degrees C. These values obtained by FPR are in good agreement with those previously obtained by photobleaching rhodopsin in fresh, unlabeled rods. This agreement indicates that the labeling and bleaching procedures required by the FPR method did not significantly alter the diffusion rate of rhodopsin. Moreover, the magnitude of the diffusion constant for rhodopsin is that to be expected for an object of its diameter diffusing in a bilayer with the viscosity of the disk membrane. In contrast to the case of rhodopsin, FPR methods applied to other membrane proteins have yielded much smaller diffusion constants. The present results help indicate that these smaller diffusion constants are not artifacts of the method but may instead be due to interactions the diffusing proteins have with other components of the membrane in addition to the viscous drag imposed by the lipid bilayer.     

STUDIES ON THE GROWTH HORMONE OF PLANTS : VI. THE DISTRIBUTION OF THE GROWTH SUBSTANCE IN PLANT TISSUES

1. It is shown that when plant tissues are ground with water the growth substance contained therein is inactivated by the oxidizing enzymes. 2. A simple method of extraction is described which enables the quantitative determination of growth substance in such tissues. 3. The amount and distribution of growth substance in the Avena coleoptile is determined by this method, and it is shown that while the substance does not diffuse out from the lower parts of the coleoptile, it is nevertheless present in considerable amounts, the concentration decreasing steadily with the distance from the tip. 4. Growth substance is also present in considerable amounts in Avena roots, and here also its concentration decreases steadily with distance from the tip. 5. The amount of growth substance diffusing out of root tips into dextrose agar, even during long periods of time, is not greater than the amount obtainable by direct extraction. Actual production in the root tip therefore either does not take place at all, or else takes place under quite different conditions from the production in the tip of the coleoptile.     

Diffucison of OXYGEN, Nitrogen and water in hyluronate solutions.

A method is reported for the measurement of the diffusion coefficients in water of some sparingly soluble gases. The results obtained for the diffusion coefficients of oxygen and nitrogen gas through water at 25 degrees C are 2.12. 10-5 and 2.61 . 10-5 cm-2 . s-1, respectively. A check on the accuracy of the teachnique using tritiated water as the diffusing substance gave a value of 2.15 . 10-5 cm-2 . s-1 which agrees within 3% with recent values from the literature. The method was applied to the measurement of oxygen, nitrogen, and tritiated water diffusion coefficients through agarose gels and through agarose gels containing hyluronate. The results indicate that the hyaluronate had only a small effect as a barrier to the diffusion of such low molecular weight substance.     

竹红菌光敏作用的初步探讨*

用纸片法作为筛选光敏物质及光敏作用的研究方法。结果证实了(1)竹红菌Hypo crella bambusae中含有对革兰氏阳性细菌具光敏活性的成分;(2)从中筛选到一种红色的光敏色素,属苝醌的一个新的衍生物,取名竹红菌甲素(Hypocrellin A简称“甲素”);(3)揭示了“甲素”是对可见光敏感、作用光谱较宽的光动力学(photodynamic)物质。从化合物的类型、光敏特性、治疗方法、治疗对象及其效果看,可以认为甲素是一新型的光化学疗法药物。     

竹红菌光敏作用的初步探讨

用纸片法作为筛选光敏物质及光敏作用的研究方法。结果证实了(1)竹红菌Hypo crella bambusae中含有对革兰氏阳性细菌具光敏活性的成分;(2)从中筛选到一种红色的光敏色素,属苝醌的一个新的衍生物,取名竹红菌甲素(Hypocrellin A简称“甲素”);(3)揭示了“甲素”是对可见光敏感、作用光谱较宽的光动力学(photodynamic)物质。从化合物的类型、光敏特性、治疗方法、治疗对象及其效果看,可以认为甲素是一新型的光化学疗法药物。     

苏云金杆菌发酵上清液中生物活性物质的研究

对苏云金芽胞杆菌MP-342发酵上清液中生物活性物质的抑菌作用及化学结构进行初步研究。经717树脂脱色和732树脂吸附、氨水洗脱、真空浓缩及冷冻干燥,得到了纯度较高的精制品。然后进行了抑菌活性试验,结果表明该物质对苹果轮纹菌(Dothiorella gregaria)和苹果褐斑病菌(Marssonin mali)等植物病害微生物有明显的抑菌作用。最后通过质谱分析,表明该活性物质的分子量为396,推测该物质与国外文献报道的zwittermicin A可能为同一物质。     

Germ cell cluster formation and oogenesis in the hymenopteran Coleocentrotus soldanskii

During first stages of oogenesis in an ichneumonid wasp Coleocentrotus soldanskii. in each 32 cell cluster, as many as 8 cystocytes enter meiosis. Only one differentiates as an oocyte, the rest are gradually transdetermined to become nurse cells. Significantly, the nurse cells that have passed the first stages of meiosis retain some features characteristic of the oocyte. Namely the nuclei of these cells, as the oocyte nucleus, produce accessory nuclei (AN). Gradual transdetermination of pro-oocytes into nurse cells and the occurrence of ANs in transdetermined cells suggest that a diffusing substance, forming a concentration gradient, is responsible for the determination of the oocyte within the cystocyte groups in hymenopterans.     

Interaction of substance P with tubulin

Binding of the peptide neurotransmitter substance P to brain tubulin in vitro inhibits self-assembly of the protein into microtubules and disrupts preassembled microtubules. This cooperative inhibition of the maximum extent of self-assembly by substance P is explicable in terms of preferential binding to the protomer state as compared to the polymer state of tubulin. The inhibition is relieved by the microtubule-associated protein MAP2, which evidently acts in a mixed competitive-noncompetitive fashion. Substance P interacts directly with the isolated C-terminal 4-kDa peptide fragment of tubulin, which appears to contain the specific binding area for MAP2, but is without effect on the self-assembly of the larger (48-kDa) part of the tubulin molecule called S-tubulin. The results are consistent with the C-terminal fragment having a binding site for the cationic substance P as well as for MAP2. However, factors other than electrostatic interaction must be operative, since the sulfoxide of substance P, a derivative with oxidized methionine but similar electrostatic characteristics, is inactive in inhibiting the extent of microtubule assembly.     

Effects of Substance P on Carbachol-Stimulated 45Ca2+ Uptake into Cultured Adrenal Chromaffin Cells

Substance P is known to modulate acetylcholine-induced catecholamine release from adrenal chromaffin cells. To investigate the mechanisms involved in this modulation, the present study examined the effects of substance P on net 45Ca2+ fluxes in cultures of bovine adrenal chromaffin cells. Two effects of substance P were observed: (1) Substance P inhibited carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux and (2) substance P protected against desensitization of carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux. Thus substance P modulates two other cholinergic responses, 45Ca2+ uptake and 45Ca2+ efflux, in a manner similar to its modulation of catecholamine release. The results also indicate that substance P's inhibition of net carbachol-induced 45Ca2+ uptake is due to inhibition of 45Ca2+ uptake rather than enhancement of 45Ca2+ efflux. Substance P almost completely inhibited carbachol-induced 45Ca2+ uptake in both Na+-containing and Na+-free media, suggesting that substance P can inhibit the uptake of 45Ca2+ induced by carbachol regardless of whether 45Ca2+ is taken up through voltage-sensitive or acetylcholine receptor-linked channels. However, substance P produced only a small inhibition of K+-induced 45Ca2+ uptake, indicating that substance P does not interact directly with voltage-sensitive Ca2+ channels. In addition, substance P's inhibition of carbachol-induced 45Ca2+ uptake was noncompetitive with respect to Ca2+, were unable to overcome substance P's inhibition of [3H]-norepinephrine ( [3H]NE) release. It is concluded that substance P does not interact directly with Ca2+ channels in bovine adrenal chromaffin cells.     

Antibody-quantum dot conjugates exhibit enhanced antibacterial effect<Emphasis Type="Italic">vs.</Emphasis> unconjugated quantum dots

The effect of a 20-min exposure to antibody-quantum dot (Ab-QD) conjugates on colony counts of Escherichia coli was assessed by the spread-plate method and compared with exposure to unconjugated QDs having only amine or carboxyl groups on their surfaces. Under these conditions, Ab-QD conjugates generally exhibited >90% reduction in colony-forming units as compared to untreated E. coli and E. coli treated with unconjugated QDs after incubation for as long as 41 h. The antibacterial effect of Ab-QD conjugates vs. unconjugated QDs on Salmonella enterica subsp. enterica serovar Typhimurium was also assessed by means of a disk-diffusion technique which demonstrated greater growth inhibition (approximately 3 mm greater) by Ab-QD conjugate-impregnated disks than by unconjugated-QD-only-impregnated disks at a 10-microg disk load. At a 25-microg disk load, both treatment groups exhibited nearly equal growth inhibition.     

Some quantitative characteristics of the frog retinal rod outer segments]

Retinal rod outer segments in frogs were studied by means of light microscopy, refractometry, microspectrophotometry, and electron microscopy. Analysis of the data obtained shows that an unidentified substance, which makes up about 50% of outer segment dry weight, is lost during routine biochemical investigations. The protein parts of the rhodopsin molecules make up 85% of the outer segments proteins and 25% of outer segment dry weight. Rhodopsin molecules can be arranged in a square array with a unit cell side of about 7 nm on one side of each disk membrane. Lipids in a single membrane occupy only 2 nm, and disk membranes are strongly hydrated.     

Competitive exclusion in a two-species chemotaxis model

We consider a mathematical model for the spatio-temporal evolution of two biological species in a competitive situation. Besides diffusing, both species move toward higher concentrations of a chemical substance which is produced by themselves. The resulting system consists of two parabolic equations with Lotka–Volterra-type kinetic terms and chemotactic cross-diffusion, along with an elliptic equation describing the behavior of the chemical. We study the question in how far the phenomenon of competitive exclusion occurs in such a context. We identify parameter regimes for which indeed one of the species dies out asymptotically, whereas the other reaches its carrying capacity in the large time limit.     

A finite element model of the L5-S1 functional spinal unit: development and comparison with biomechanical tests in vitro

The main objective of this work is to develop a three-dimensional finite element model of the L5-S1 segment that is able to simulate its passive mobility measured in vitro. Due to their limited role in segment mobility, an isotropic linear elastic constitutive law was used for cartilage, cancellous and cortical bone. The intervertebral disk ground substance was modeled with a non-linear hyperelastic polynomial law. Fibers of the disk, as well as ligaments, were modeled with piecewise linear springs. Flexion-extension, axial rotation, and lateral bending torques were applied to the model. A comparison with the experimental results obtained on the same segment for these three major motions was conducted. The compliance of the segment subjected to pure torques was found to be similar between numerical and experimental results for all major motions. Coupled motions and translations were also similar, even in their amplitude. For lateral bending, the normal coupled motions originate from the geometry of the disk and not from the facet geometry.     

A Finite Element Model of the L5-S1 Functional Spinal Unit: Development and Comparison with Biomechanical Tests In Vitro

The main objective of this work is to develop a three-dimensional finite element model of the L5-S1 segment that is able to simulate its passive mobility measured in vitro . Due to their limited role in segment mobility, an isotropic linear elastic constitutive law was used for cartilage, cancellous and cortical bone. The intervertebral disk ground substance was modeled with a non-linear hyperelastic polynomial law. Fibers of the disk, as well as ligaments, were modeled with piecewise linear springs. Flexion-extension, axial rotation, and lateral bending torques were applied to the model. A comparison with the experimental results obtained on the same segment for these three major motions was conducted. The compliance of the segment subjected to pure torques was found to be similar between numerical and experimental results for all major motions. Coupled motions and translations were also similar, even in their amplitude. For lateral bending, the normal coupled motions originate from the geometry of the disk and not from the facet geometry.