Rotational and translational motions of human spermatozoa: angle dependence of dynamic laser light scattering

We have studied how the dynamic components of laser light scattered from human spermatozoa depend on the scattering angle. This was done by investigating the halfwidth of the intensity autocorrelation function. A model of the spermatozoa as freely rotating and translating linear objects was adequate to describe the scattered light. Rotational motions determined the halfwidth of the intensity autocorrelation function at very small scattering angles and contribution from translational motions was dominant at scattering angles larger than 20 degrees. The contribution from translational motions increased with increasing scattering angle. We found a nearly linear relationship between the translation speed and the rotation frequency. However, the ratio between the two properties varied more than expected from the methodological error. Therefore we introduced a propelling efficacy as a concept to describe the swimming efficiency. This property might contain important information about the swim characteristics.Abbreviations ACF Autocorrelation function - _1/2 halfwidth - RGD Rayleigh-Gans-Debye - SD Standard deviation Correspondence to: P. Thyberg     

A static laser light scattering assay for surfactant-induced tau fibrillization

Static light scattering is an important solution-based method for assaying spontaneous protein aggregation reactions. But the reliability of the measurements when conducted in the presence of fibrillization inducers has been questioned. Here the utility of static laser light scattering for quantitative assay of anionic micelle-induced protein fibrillization was characterized using tau protein, the major component of neurofibrillary lesions of Alzheimer's disease. Both inducer micellization and tau fibrillization made significant contributions to light scattering intensity. The intensity arising solely from micellization was quantified using proteins that promoted inducer micellization but could not fibrillize, such as mixed histones and assembly-incompetent mutant htau40(I277P/I308P). When corrected for micellization, reaction progress curves for wild-type tau fibrillization were sigmoidal and correlated well with measurements of total filament length made by transmission electron microscopy. The utility of the improved laser light scattering assay was demonstrated by quantifying the effect of inducer concentration on tau assembly kinetics using a three-parameter Gompertz growth function. Results showed that alkyl sulfate detergent accelerated tau nucleation as reflected by shorter lag times and modulated pre-nuclear equilibria to yield more filament mass at reaction equilibrium.     

The onset of amelogenin nanosphere aggregation studied by small-angle X-ray scattering and dynamic light scattering

Proteins with predominantly hydrophobic character called amelogenins play a key role in the formation of the highly organized enamel tissue by forming nanospheres that interact with hydroxyapatite crystals. In the present investigation, we have studied the temperature and pH-dependent self-assembly of two recombinant mouse amelogenins, rM179 and rM166, the latter being an engineered version of the protein that lacks a 13 amino acid hydrophilic C-terminus. It has been postulated that this hydrophilic domain plays an important role in controlling the self-assembly behavior of rM179. By small-angle X-ray and neutron scattering, as well as by dynamic light scattering, we observed the onset of an aggregation of the rM179 protein nanospheres at pH 8. This behavior of the full-length recombinant protein is best explained by a core-shell model for the nanospheres, where hydrophilic and negatively charged side chains prevent the agglomeration of hydrophobic cores of the protein nanospheres at lower temperatures, while clusters consisting of several nanospheres start to form at elevated temperatures. In contrast, while capable of forming nanospheres, rM166 shows a very different aggregation behavior resulting in the formation of larger precipitates just above room temperature. These results, together with recent observations that rM179, unlike rM166, can regulate mineral organization in vitro, suggest that the aggregation of nanospheres of the full-length amelogenin rM179 is an important step in the self-assembly of the enamel matrix.     

Microscope laser light scattering spectroscopy of single biological cells

A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 μm)³. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method.     

Size distribution of pressure-decomposed casein micelles studied by dynamic light scattering and AFM

Reversible and irreversible states of pressure-dissociated casein micelles were studied by in situ light scattering techniques and ex situ atomic force microscopy. AFM experiments performed at ambient pressure reveal heterogeneities across the micelle, suggesting a sub-structure on a 20 nm scale. At pressures between 50 and 250 MPa, the native micelles disintegrate into small fragments on the scale of the observed sub-structure. At pressures above 300 MPa the micelles fully decompose into their monomeric constituents. After pressure release two discrete populations of casein aggregates are observed, depending on the applied initial pressure: Between 160 and 240 MPa stable micelles with diameters near 100 nm without detectable sub-structures are formed. Casein micelles exposed to pressures above 280 MPa re-associate at ambient pressure yielding mini-micelles with diameters near 25 nm. The implications concerning structural models are discussed.     

Fibrin structures during tissue-type plasminogen activator-mediated fibrinolysis studied by laser light scattering: relation to fibrin enhancement of plasminogen activation

The aim was to relate fibrin structure and the stimulatory effect of fibrin on plasminogen activation during t-PA-mediated fibrinolysis using Lys⁷⁸-plasminogen as activator substrate. Structural studies were undertaken by static and dynamic laser light scattering, cryo transmission electron microscopy and by the measurement of conversion of fibrin to X-, Y- and D-fragments. The kinetics of plasmin formation were monitored by measurement of the rate of pNA-release from Val-LeuLys-pNA. The process of fibrin formation and degradation comprised three phases. In the first phase, protofibrils with an average length of about 10 times that of fibrinogen were formed. The duration of this phase decreased with increasing t-PA concentration. The second phase was characterized by a sudden elongation and lateral aggregation of fibrin fibers, most pronounced at low levels of t-PA, and by formation of fragment X-polymer. The third phase was dominated by fragmentation of fibers and by formation of Y- and D-fragments: Plasmin degraded the fibers from within, resulting in the formation of long loose bundles, which subsequently disintegrated into thin filaments with a length of less than 10 and a mass per length close to one relative to fibrinogen. Plasmin generation at high t-PA concentrations sets in just prior to (and at low t-PA concentrations shortly after) the onset of the rapid second phase of elongation and lateral aggregation of fibrin fibers. The maximal rate of plasmin formation per mol t-PA was the same at all concentrations of activator and was achieved close to the time of the peak level of fragment X-polymer. Plasmin formation ceased after formation of substantial amounts of Y- and D-fragments. At this stage the length was between 300 and 3 and the mass per length close to 1, both relative to fibrinogen. In conclusion our results indicate that (1) formation of short fibrin protofibrils is the minimal requirement for the onset of the stimulatory effect of fibrin on plasminogen activation by t-PA, (2) formation of fragment X protofibrils is sufficient to induce optimal stimulation of plasminogen activation, and (3) plasmin degrades laterally aggregated fibrin fibers from within, resulting in the conversion of the fibers into long loose bundles, which later disintegrate into thin filaments.Abbreviations t-PA tissue-type plasminogen activator - Lys⁷⁸-plasminogen plasmin-modified plasminogen, mainly with NH₂-terminal lysine (residues 78-791, residue numbering according to Forsgren et al. 1987) - Val-Leu-Lys-pNA H-D-valyl-L-leucyl-L-lysine-4-nitroanilide - Phe-Pip-Arg-pNA H-D-phenylalanyl-L-arginine-4-nitroanilide - pNA p-nitroanilide - SDS sodium dodecyl sulphate The present work has been supported by the Danish Natural Science Research Council and the Danish Agricultural and Veterinary Research CouncilDeceased on August 2, 1991 Correspondence to: R. Bauer     

Self-assembly of melanin studied by laser light scattering

The unknown molecular weight and chemical structure of melanin place the study of these pigments outside the range of the classical biochemical techniques; thus in this paper the problem of characterizing these heterogeneous biopolymers was approached by means of light scattering techniques, static and dynamic. The static technique allowed us to identify the macromolecular properties (MW and R(g)(2)(1/2)) of melanin extracted from sepia inksac and of two synthetic analogues: L-Dopa melanin obtained by autooxidation and by enzymatic oxidation by Tyrosinase. By dynamic light scattering (DLS), the hydrodynamic radius R(h) was measured to monitor the temporal behaviour of the polymerization and aggregation processes and R(h) variation by changing the chemical constraints of the polymerization medium, such as pH and ionic strength. The fractal dimension d of the aggregates of melanin, both natural and synthetic, in the past only recognized during the aggregation of the synthetic one by lowering the pH of the medium, was a useful parameter to further investigate and compare the structure of melanin granules of differing origins, revealing for the natural sample, a structure with clusters that are spherical, not largely hydrated and self-assembled, following a reaction limited aggregation kinetics (d=2.38).     

Particle motion in single acinar cells observed by microscope laser light scattering spectroscopy

We report here the direct observation of the motion of cytoplasmic subcellular organelles and macromolecules within single, metabolically active, pancreatic acinar cells using microscope laser-light scattering spectroscopy (MLLSS). The relative amount and the effective diffusion coefficient of the mobile particles shows a marked difference in magnitude between the apical and basal regions of the cell. Secretory stimulation evoked by the cholinergic agonist bethanechol induces changes in the relative motion in the cytoplasm of approximately one fifth of the acinar cells. This study demonstrates the feasibility and utility of a novel technique, MLLSS, for the analysis of intracellular events in regions as small as (2 m)³ in single, granule secreting cells. It also shows MLLSS to be a powerful tool for the detection and measurement of altered motion in disrete subcellular regions of small mammalian cells after biochemical and pharmacological manipulations.     

Characterization of reacting solutions of macromolecules by laser light scattering and Airfuge centrifugation

Simple, fast and accurate measurements combining centrifugation in a table top Airfuge and laser light scattering in the Airfuge tube are described. The procedure achieves quantitative separation of particles according to their sedimentation coefficient in microliter volumes. By scanning through the sedimenting boundary association equilibrium constants are evaluated.     

Solution properties of starch nanoparticles in water and DMSO as studied by dynamic light scattering

Solution properties of starch nanoparticles dispersed in DMSO and in water were studied using dynamic light scattering. The particle size distribution had two main peaks in both solvents at all scattering angles studied. They were at around 40 and 300 nm, ascribed to isolated starch nanoparticles and their aggregates, respectively. From the excess scattering intensity by the 40-nm particles, the molecular weight of the nanoparticle was estimated as 2.2–2.6×10⁶ g/mol. When the concentration was increased, another peak appeared at around 1 μm. Raising the temperature from 25 to 65 °C did not change the distribution, indicating a purely entropic process in dynamic equilibrium of the aggregation. In DMSO, an oscillatory behavior was observed in the autocorrelation function at high temperatures.     

鱼类精子活力研究进展

鱼类精子在精巢和精浆中一般不活动,只有当精子被排到体外并被外界环境的溶液稀释后才能活动．鱼类精子活力受渗透压、离子、ｐＨ 值、温度及ＣＯ２ 等因子的调节和影响, 不同的鱼类其精子活力有不同的调节方式;外界因子对鱼类精子活力的影响, 是通过影响ｃＡＭＰ－ＡＴＰ－Ｍｇ２＋ 系统来影响鞭毛的活动而实现的． 精子活力的评价指标主要有：精子激活后的运动时间、精子激活比例、精子运动速度及精子鞭毛摆动频率等． 大多数鱼类的精子,其活动能力是在生殖管道中获得的．     

Properties of pancreatic zymogen granules studied by quasi-elastic light scattering

Physico-chemical properties of isolated zymogen granules of the mouse pancreas were studied by means of quasi-elastic light scattering. The average diameter of the granules in 0.3 M sucrose was found to be 1.1 ± 0.1 μm from the correlation time of intensity fluctuation of the scattered light. The average diameter altered depending on the osmolality of the medium in a manner that the alteration was smaller than that expected from the van't Hoff relation. Aggregation of the granules induced by the increase of Ca²⁺ concentration or the decrease of pH in the medium was also detected. The aggregation started at a critical level of 1 mM CaCl₂ or at pH 5.4.     

Motility analysis of circularly swimming bull spermatozoa by quasi-elastic light scattering and cinematography.

The Rayleigh-Gans-Debye approximation is used to predict the electric field autocorrelation functions of light scattered from circularly swimming bull spermatozoa. Using parameters determined from cinematography and modeling the cells as coated ellipsoids of semiaxes a = 0.5 micrometers, b = 2.3 micrometers, and c = 9.0 micrometers, we were able to obtain model spectra that mimic the data exactly. A coat is found to be a necessary attribute of the particle. It is also clear that these model functions at 15 degrees may be represented by the relatively simple function used before by Hallett et al. (1978) to fit data from circularly swimming cells, thus giving some physical meaning to these functional shapes. Because of this agreement the half-widths of experimental functions can now be interpreted in terms of an oscillatory frequency for the movement of the circularly swimming cell. The cinematographic results show a trend to chaotic behavior as the temperature of the sample is increased, with concomitant decrease in overall efficiency. This is manifested by a decrease in oscillatory frequency and translational speed.     

Observation of flagellation of spermatozoa by depolarized laser light scattering.

Depolarized laser light-scattering theory was applied to derive the autocorrelation function of laser light scattered by motile spermatozoa, assuming that each spermatozoon is a chain of rotatable rigid ellipsoids of revolution and also that the rotational velocity about an axis perpendicular to the symmetry axis of the ellipsoid is constant for times of the order of the characteristic decay time of the autocorrelation function. The rotations are produced by flagellar movements of the spermatozoa. The correlation function thus obtained was related to the second-order coefficient of a Legendre polynomial expansion of the rotation of the direction angle of the ellipsoidal axis. The experimental fact that the correlation function for dead spermatozoa of sea urchin resembled that for flagella mechanically separated from spermatozoa indicated to us that the depolarized light was scattered mainly by flagella. The rotational velocity distribution of the flagella was determined by comparing the theoretical analysis with the experimentally obtained correlation functions for the motile and dead spermatozoa. The value of the average velocity caused by the flagellation, 230 rad/s, was in good agreement with that measured under an optical microscope.     

Effects of cyclic AMP on the motility of mature and immature hamster epididymal spermatozoa studied by reactivation of the demembranated cells

Hamster spermatozoa from the caput and cauda epididymides were demembranated with 0.04% Triton X-100 and reactivated with 1 mM ATP. Motility parameters were analysed by video recording and stroboscopic photography. In the absence of added cAMP, reactivated cauda sperm showed percentage motility and forward swimming patterns similar to those of intact cells, but velocities were lower. When 2 or 20 μM cAMP was present, the velocities were increased but there was no effect on beat frequencies or percentage of forward progressing sperm. Cyclic AMP also markedly increased the percentage of cauda sperm which at first displayed nonprogressive “looping” movement. Addition of cAMP to the reactivation medium greatly improved the otherwise feeble and irregular motility of the demembranated caput sperm by increasing the percentage motility and beat frequencies of nonprogressive cells. It also induced forward motility with beat frequencies and velocities similar to cauda sperm reactivated in the absence of cAMP, but looping was never seen, indicating a change in the flagellar apparatus with maturation. The time required for the exhibition of the cAMP effects was reduced when caput sperm were reactivated in extracts of another previously maximally reactivated caput sperm preparation. The results suggest the production of some potent compound(s) by the axonemes for the manifestation of the cAMP effects.     

Determination of proteins with fullerol by a resonance light scattering technique

Fullerol has been synthesized through the reaction of fullerene C60 with NaOH in aqueous solution by means of ultrasonic agitation and characterized by infrared and 1H-nuclear magnetic resonance spectroscopy. The fullerol obtained shows good solubility and excellent stability in water. A weak resonance light scattering (RLS) spectrum of fullerol was observed in aqueous solution. However, the intensity of the RLS signal could be enhanced in the presence of proteins, including bovine serum albumin (BSA), human serum albumin (HSA), pepsin (Pep), and lysozyme (Lys). Based on the enhancement of the RLS, a sensitive method for the determination of proteins has been established. The quantitative conditions were considered with regard to the effects of the pH, the ion strength, and the concentration of the fullerol. Under the optimum conditions, the intensity of the RLS was proportional to the concentration of proteins with the limits of detection of 9.7, 10.9, 57.4, and 8.5 ng mL(-1) for BSA, HSA, Pep, and Lys, respectively. Almost no interference can be observed from some amino acids, nucleic acids, and most of the metal ions. The model samples and human serum samples were determined satisfactorily with the proposed method.     

Continuous assay for DNA polymerization by light scattering

The time course of DNA polymerization, catalyzed by DNA polymerase I, can be monitored by following changes in light scattering that result from increases in molecular weight of the polymer. Increases in mass of 10% result in an easily measurable change in light scattering. It has been shown that the light scattering change is correlated with the incorporation of radioactive nucleotide, as measured by the filter binding method. Continuous monitoring of the time dependence of polymerization should prove useful in kinetic analysis of the polymerase reaction and related enzyme-DNA interactions.     

Cytoplasmic streaming in Chara corallina studied by laser light scattering.

An apparatus is described by means of which the power versus frequency spectrum of the photomultiplier current can be obtained for laser light scattered by streaming cytoplasm in the algal cell Chara corallina. A Doppler peak is noted in the spectrum which is abolished when cytoplasmic streaming is arrested by electrical stimulation. For 5 cells of Chara, this simple laser-Doppler velocimeter gave streaming velocities (46-7 mum s-1, S.D. +/- 4-8 at 20 degrees C) similar to those obtained for the same cells using the light microscope (44-3 mum s-1, S.D. +/- 5-3 at 20 degrees C). A narrow distribution of streaming velocities is indicated. The technique described provides a rapid, quantitative assay of the in vivo rheological properties of cytoplasm.