四氧嘧啶糖尿病大鼠主动脉零应力状态的变化

目的和方法：将沿径向切开的糖尿病大鼠及对照大鼠主动脉坏分别转正圩Ｋｒｅｂｓ液中,向其中分别加入缩血管物质及舒血管物质达各种浓度;观察其角度变化。用Ｓ－Ｐ法对大鼠主动脉壁肌动蛋白进行染色。结果：四氧嘧啶糖尿病大鼠病程４周时主动脉环展开角显著大于对照（Ｐ〈０．００１）。使用药物后大鼠主动脉坏展开角与使用前相比无明显差异（Ｐ〉０．０５）。糖尿病大鼠主动脉壁肌动蛋白色较对照组明显加深、染色的光密度显著大于     

Comparison of different methods for protein determination inAspergillus niger mycelium

Summary Protein contents were determined in submerged as well as in surface-grown citric acid producingAspergillus niger mycelia. Various methods (Kjeldahl, Biuret, Lowry and Coomassie Blue) for protein determination were compared. The Biuret method seemed to be more suitable than the others for true protein determination in mycelia. The Lowry method gave lower results in all cases. The Coomassie Blue method did not prove suitable for the material used.     

Quantification of biofilm accumulation by an optical approach

Methods for non-invasive, in situ, measurements of biofilm optical density and biofilm optical thickness were evaluated based on Pseudomonas aeruginosa experiments. Biofilm optical density, measured as intensity reduction of a light beam transmitted through the biofilm, correlates with biofilm mass, measured as total carbon and as cell mass. The method is more sensitive and less labor intensive than other commonly used methods for determining extent of biofilm mass accumulation. Biofilm optical thickness, measured by light microscopy, is translated into physical thickness based on biofilm refraction measurements. Biofilm refractive index was found to be close to the refractive index of water. The P. aeruginosa biofilms studied reached a pseudo steady state in less than a week, with stable liquid phase substrate, cell and TOC concentrations and average biofilm thickness. True steady state was, however, not reached as both biofilm density and roughness were still increasing after 3 weeks.     

A simple, rapid, and sensitive method of determining CSF IgG, using latex

A simple, rapid and sensitive method of determining cerebrospinal fluid IgG is presented. The procedure depends upon the fact that spinal fluid gamma globulin, and two of its components (IgG and IgM) will precipitate latex particles in proportion to their concentration, and that the optical density of the supernatant solution containing unprecipitated latex particles after centrifugation is inversely proportional to the concentration of these immunoglobulins. The method is sensitive to 50 nanograms, is inexpensive, requires 0.1 ml. or less of spinal fluid, and can be performed in minutes. Preliminary studies show that it compares favorably with results obtained by radial immunodiffusion.     

A comparison of five methods for obtaining the intrinsic viscosity of bovine serum albumin

Intrinsic viscosity [η] is a characteristic of proteins and other molecules related directly to their ability to disturb flow and indirectly to their size and shape. It is usually determined by extrapolating reduced viscosity to zero concentration. Four other methods for deriving [η] have been utilized by previous investigators. Studies of the intrinsic viscosity of bovine serum albumin had been carried out two years apart as a test of viscometry technique; the data obtained were used to compare the five methods. Four of the five produced [η] values ranging from 3.92 to 4.21 ml/g. Agreement was good between the two studies. The five methods were compared to each other using linearity of regression, statistical error of determination, effect of varying solvent time, and result obtained in different concentration ranges. By these four criteria, use of the regression of specific fluidity (1 ? 1/η_rel) with concentration was found superior to other methods. Its only deficiency was a requirement that solution density be corrected for at each concentration studied rather than applying a single correction for density after using kinematic viscosity data. All methods for deriving intrinsic viscosity are based on one of three equations; flow is expressed either in terms of reduced viscosity (Huggins), inherent viscosity (Kraemer), or specific fluidity. Of these three equations, specific fluidity is the most closely related both to theoretical analyses and to experimental studies of rigid spheres. There is abundant evidence in past reports that in contrast to rigid spheres, flexible polymers do not produce a linear rise in specific fluidity as their concentration increases, strongly suggesting that their molecular conformation is changing with concentration. A linear relation between fluidity and concentration has been observed for almost all proteins and protein mixtures studied. When this linear relation is present it indicates both that molecular conformation during flow is independent of concentration in the range studied and that the specific fluidity method for deriving intrinsic viscosity is the most appropriate.     

Quantitation of Protein Content by Biuret Method During Production of Yellow Fever Vaccine

Protein content of 60 batches of Yellow Fever Vaccine was measured by Biuret method and was compared to the values obtained by Kjeldahl method. Statistical analysis did not show any difference between the two methods. The Biuret method is specific, easy to carry out and takes little time for protein estimation during production of Yellow Fever Vaccine.     

Comparison of four methods for quantification of biofilms in biphasic cultures

Three methods for determining the total biofilm amount in biphasic cultures have been compared: dry weight by filtration after solvent treatment, optical density with a biomass probe and protein content. The activity of the biofilm was estimated through mineral nitrogen consumption. Calculation of the coefficients of variation shows that these parameters could be used to characterise such a biofilm. The optical density by biomass probe was the most reliable one (repeatability <0.5%) to quantify total biofilm and a linear relation was verified against dry weight. © Rapid Science Ltd. 1998     

Influence of semiflexible structural features of actin cytoskeleton on cell stiffness based on actin microstructural modeling

A new actin cytoskeleton microstructural model based on the semiflexible polymer nature of the actin filament is proposed. The relationship between the stretching force and the mechanical properties of cells was examined. Experiments on deforming hematopoietic cells with distinct primitiveness from normal and leukemic sources were conducted via optical tweezer manipulation at single-cell level. The modeling results were demonstrated to be in good agreement with the experimental data. We characterized how the structural properties of the actin cytoskeleton, such as prestress, density of cross-links, and actin concentration, affect the mechanical behavior of cells based on the proposed model. Increasing prestress, actin concentration, and density of cross-links reduced cell deformation, and the cell also exhibited strain stiffening behavior with an increase in the stretching force. Compared with existing models, the proposed model exhibits a distinct feature in probing the influence of semiflexible polymer nature of the actin filament on cell mechanical behavior.     

A comparison of different methods to determine the end of exponential growth in CHO cell cultures for optimization of scale-up

Maximizing cell growth rate and cell yield are among the most important features of a successful mammalian cell culture production process. To minimize time and resources needed to scale up cell mass it is important to maintain the cultures in exponential growth at every scale. Here we report results comparing viable cell counts, packed cell volume, intracellular nucleotide ratios, cell cycle analysis, and on-line oxygen uptake rates (OUR) and optical density for the determination of the end of exponential growth to optimize transfer times during scale-up of CHO cell cultures. Viable cell concentration, packed cell volume, and relative abundance of cells in S-phase were not very reliable at determining the end of exponential growth during the process. In contrast, on-line determination of OUR and off-line determination of intracellular nucleotide ratios (U-ratio) were very sensitive to changes in growth rate, enabling clear determination of the end of exponential growth within a short time. Although on-line OUR was found to be the most convenient and fastest method, it is restricted to instrumented and continuously monitored cultures. In contrast the nucleotide method can be applied with any culture scale and condition but needs the availability of an operator running an HPLC system and takes about an hour from sampling to result. Optical density showed an inflection along with OUR and U-ratio but was less sensitive in determining the end of exponential growth.     

Evaluation of a new system for the fixation, concentration, and staining of intestinal parasites in fecal specimens, with critical observations on the trichrome stain

Proto-fix (Alpha-Tec Systems, Inc., Vancouver, WA) is a new single vial, environmentally safe, parasitology (pathogenic and nonpathogenic protozoans and helminths) fixative and transport solution. It is used in conjunction with a new concentration/sedimentation reagent, CONSED, (Alpha-Tec Systems, Inc. Vancouver, WA) as a replacement to the formalin-ethyl acetate (FEA) concentration procedure using Lugol's iodine. The newly adopted procedure was tested against the FEA concentration samples using split proficiency testing samples supplied by the American Association of Bioanalysts (AAB). Routinely, patient samples collected, fixed, and transported in Proto-Fix were processed and tested at Diagnostic Labs, Inc. (DLI), Phoenix, AZ. Detected parasites were documented using a video camera-printer system attached to the optical equipment. The quality of the fixative and stain were found to be superior to that of the FEA-Lugol's method and the yield of detected parasites was considerably higher. Eighty-five percent of 39 unknown parasite species tested were correctly detected using the Proto-fix-CONSED system compared to 46% using the FEA-Lugol's method. Of all the other methods and stains used at DLI, the trichrome stain (a popular modification of Gomori's trichrome stain for tissue sections) was found to be least reliable.     

A microbial biosensor for hydrogen sulfide monitoring based on potentiometry

In this study, a microbial biosensor for hydrogen sulfide (H₂S) detection based on Thiobacillus thioparus immobilized in a gelatin matrix was developed. The T. thioparus was immobilized via either surface adsorption on the gelatin matrix or entrapment in the matrix. The bacterial and gelatin concentration in the support were then varied in order to optimize the sensor response time and detection limit for both methods. The optimization was conducted by a statistical analysis of the measured biosensor response with various bacterial and polymer concentrations. According to our experiments with both immobilization methods, the optimized conditions for the entrapment method were found to be a gelatin concentration of 1% and an optical density of 82. For the surface adsorption method, 0.6% gelatin and an optical density of 88 were selected as the optimal conditions. A statistical model was developed based on the extent of the biosensor response in both methods of immobilization. The maximum change in the potential of the solution was 23.16 mV for the entrapment method and 34.34 mV for the surface absorption method. The response times for the entrapment and adsorption methods were 160 s and 105 s, respectively. The adsorption method is more advantageous for the development of a gas biosensor due to its shorter response time.     

Electron microscopic visualization of the filament binding mode of actin-binding proteins

A large number of actin-binding proteins (ABPs) regulate various kinds of cellular events in which the superstructure of the actin cytoskeleton is dynamically changed. Thus, to understand the actin dynamics in the cell, the mechanisms of actin regulation by ABPs must be elucidated. Moreover, it is particularly important to identify the side, barbed-end or pointed-end ABP binding sites on the actin filament. However, a simple, reliable method to determine the ABP binding sites on the actin filament is missing. Here, a novel electron microscopic method for determining the ABP binding sites is presented. This approach uses a gold nanoparticle that recognizes a histidine tag on an ABP and an image analysis procedure that can determine the polarity of the actin filament. This method will facilitate future study of ABPs.     

Structural analysis of muscle thin filament

Thin sheets of Ac-Tm-Tn paracrystals were prepared in the presence of high concentration of Ca²⁺ ion and three-dimensional image analysis was performed. The optical diffraction pattern of an electron micrograph showed spots up to 1/1.6 nm⁻¹ in the radial direction and up to 1/2.5 nm⁻¹ in the axial direction, the best resolution ever obtained so far. The translationally filtered image showed clear polarity of filament which looked like a “spearhead” per each crossover repeat of actin helix.The three-dimensionally reconstructed model looked very similar to the inner regions (A+B domains) of the Ac-Tm-S1 complex obtained by Toyoshima and Wakabayashi (14, 15) when they were placed so that the “spearhead” pattern of the Tc-Tm-Tn complex and the “arrowhead” pattern of the Ac-Tm-S1 complex pointed in the same direction. The myosin-binding site of actin was identified by comparison of two structures.The model of actin molecule cut out from the thin filament model had a low density region within itself, which was located about 2.5 nm from the helix axis. That low density region divided actin molecule into two domains, a large and a small domain. A dense “pillar” was detected which connected two neighboring actin molecules along a left-handed generic helix 1 nm from the helix axis. Two actin-actin binding sites which were responsible for the connection through the “pillar” were located on the inner surface of actin molecule.To obtain better crystalline arrays of actin, we tried a method utilizing adsorption to lipid. A positively-charged monolayer of lipids was formed on the surface of a small volume of buffer solution which was put in a microwell. Solution of negatively-charged F-actin was then injected into the buffer solution and was allowed to be joined to the lipid monolayer by electrostatic attraction. Fluidity of the lipid monolayer enabled the two-dimensional crystallization of actin. Electron microscopy revealed that larger paracrystalline arrays were formed more rapidly (< 1 hr) than those formed within solution, which demonstrated the advantage of this adsorption method.     

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.     

Diffusion of H-meromyosin in F-actin plus ATP solution at a very low electrolyte concentration

The translational diffusion coefficient (D) of H-meromyosin in actin (F-actin) and ATP solution was measured under conditions wherein the actin-activated ATPase activity is close to its maximal value at a very low electrolyte concentration. The results were compared with similar data obtained with 0.1 M KCl, where H-meromyosin and actin were almost completely dissociated. With 0.1 M KCl, it was found that there was no dependence of the D of H-meromyosin on actin concentration. On the other hand, at a very low electrolyte concentration, it was found that the D of H-meromyosin did depend on actin concentration; at a rather high actin concentration (and activation of ATPase), it was slightly larger than at low or zero actin concentrations. This behavior of D at a low electrolyte concentration is interpreted on the assumption that even in solution, H-meromyosin molecules can actively slide on actin filaments due to the ATPase activity.     

山蕗菜根酶活性的初步研究

通过蛋白质、多糖和纤维素含量的变化,研究了新鲜山蕗菜根所含内源蛋白酶、多糖水解酶和纤维素酶的活力。结果表明,新鲜山蕗菜根匀浆后1 h蛋白质水解程度为41.58%,2 h多糖水解程度约为26%,3.5 h纤维素水解程度约为3.8%。说明山蕗菜根蛋白酶和多糖水解酶具有较高活力,而纤维素酶活力较小。通过福林法和DNS法分别测得其粗提液蛋白酶和多糖水解酶的活力分别为24 221.57U/g粗酶蛋白和45 018.65U/g粗酶蛋白。     

Cell traction force and measurement methods

Cell traction forces (CTFs) are crucial to many biological processes such as inflammation, wound healing, angiogenesis, and metastasis. CTFs are generated by actomyosin interactions and actin polymerization and regulated by intracellular proteins such as alpha-smooth muscle actin (α-SMA) and soluble factors such as transforming growth factor-β (TGF-β). Once transmitted to the extracellular matrix (ECM) through stress fibers via focal adhesions, which are assemblies of ECM proteins, transmembrane receptors, and cytoplasmic structural and signaling proteins (e.g., integrins), CTFs direct many cellular functions, including cell migration, ECM organization, and mechanical signal generation. Various methods have been developed over the years to measure CTFs of both populations of cells and of single cells. At present, cell traction force microscopy (CTFM) is among the most efficient and reliable method for determining CTF field of an entire cell spreading on a two-dimensional (2D) substrate surface. There are currently three CTFM methods, each of which is unique in both how displacement field is extracted from images and how CTFs are subsequently estimated. A detailed review and comparison of these methods are presented. Future research should improve CTFM methods such that they can automatically track dynamic CTFs, thereby providing new insights into cell motility in response to altered biological conditions. In addition, research effort should be devoted to developing novel experimental and theoretical methods for determining CTFs in three-dimensional (3D) matrix, which better reflects physiological conditions than 2D substrate used in current CTFM methods.     

A critical reappraisal of Waddell's technique for ultraviolet spectrophotometric protein estimation

Waddell's method of estimating protein concentration by the difference between spectrophotometric absorptions (215-225 nm) has been reexamined. Over limited ranges of total protein, a linear relation was found for ten purified proteins; the narrowest range was between 5 and 25 micrograms/ml. Using published extinction coefficients at 280 nm for these ten proteins, protein concentration at 280 nm correlated closely with the 215 nm/225 nm difference measurements (mean difference of 2.6%). Waddell's method also accurately determined the total protein in a mixture of proteins with widely varying individual 280-nm extinction coefficients. Biuret estimates of total protein in plasma or serum gave poor correlation with measurements by Waddell's method. Within protein concentration limits, Waddell's method was linear, narrow, and more variable, both for individual proteins and for protein mixtures, than previously reported. Within these limits, the method is probably as accurate a measure of total protein as measurement by nitrogen analysis, with the advantage of being nondestructive.     

Conversion of Woodward's Reagent K in an aqueous medium: mathematical analysis applied to enzyme modification

A change in the optical density of Woodward's Reagent K solution at 340 nm has been shown. It is observed after the reagent has been dissolved in a weakly acidic medium. The optical density correlates with the reagent's ability to inhibit transketolase. A method for assay of the inhibitor concentration changes in the medium during enzyme modification is suggested.