网织红百分数法测定rHuEPO体内生物学活性的研究

本文通过对网织红百分数法测定rHuEPO体内生物学活性进行研究,找出网织红细胞百分数与rHuEPO浓度线性相关的范围。从而确定了用计数网织红细胞百分数的方法测定rHuEPO体内生物学活性。提出在rHuEPO体内生物学活性测定中选择合适的稀释液至关重要。对(3,3)法测定rHuEPO体内生物学活性进行了统计,批间CV、批内CV均接近10%,单次实验FL%平均数为3126%,并提出鼠间差异是造成FL%较大的主要原因;对(3,3)法与(2,2)法测定rHuEPO体内生物学活性进行比较,发现(2,2)法较(3,3)法更适合用于rHuEPO生产过程中rHuEPO体内生物学活性的测定。     

Apical surface of the epithelial cells in the gallbladder of the rainbow trout and the tench

Summary The aluminium-formaldehyde (ALFA) histofluorescence method was used to study the innervation of the gill of the marine bivalve mollusc Mytilus edulis and the results were contrasted with those obtained with the standard formaldehyde-induced-fluorescence (FIF) method. The ALFA method produced more fluorescing structures than the FIF method, thus revealing fine branches of the branchial nerve running beneath the gill epithelium which previously remained undetected. This study demonstrates the usefulness of the ALFA histofluorescence method in the study of marine invertebrates.This study was supported in part by Grants 1506RR08171 from NIMH and 5T32GM07641 from the MARC Program of NIGMS. I wish to thank E. Aiello for thoughtful discussions of the work     

Comparison of methods for the identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99% of the CNS species, respectively, while this rate was 84% for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2%), S. hominis (25%), S. haemolyticus (37.5%), and S. warneri (47.1%). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100% sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.     

Evaluating a new method to estimate the rate of leaf respiration in the light by analysis of combined gas exchange and chlorophyll fluorescence measurements

Day respiration (R(d)) is an important parameter in leaf ecophysiology. It is difficult to measure directly and is indirectly estimated from gas exchange (GE) measurements of the net photosynthetic rate (A), commonly using the Laisk method or the Kok method. Recently a new method was proposed to estimate R(d) indirectly from combined GE and chlorophyll fluorescence (CF) measurements across a range of low irradiances. Here this method is tested for estimating R(d) in five C(3) and one C(4) crop species. Values estimated by this new method agreed with those by the Laisk method for the C(3) species. The Laisk method, however, is only valid for C(3) species and requires measurements at very low CO(2) levels. In contrast, the new method can be applied to both C(3) and C(4) plants and at any CO(2) level. The R(d) estimates by the new method were consistently somewhat higher than those by the Kok method, because using CF data corrects for errors due to any non-linearity between A and irradiance of the used data range. Like the Kok and Laisk methods, the new method is based on the assumption that R(d) varies little with light intensity, which is still subject to debate. Theoretically, the new method, like the Kok method, works best for non-photorespiratory conditions. As CF information is required, data for the new method are usually collected using a small leaf chamber, whereas the Kok and Laisk methods use only GE data, allowing the use of a larger chamber to reduce the noise-to-signal ratio of GE measurements.     

Determination of the parameters of the single 2-allele autosomal locus model with incomplete penetrance

A method for estimating the parameters of the single autosomal two-allele locus model with incomplete penetrance (the gene frequency and penetrances of the three genotypes) is presented. The method requires the presence of (1) the population frequency of the trait; (2) any set of incidences in relatives of probands that would allow to obtain the estimations of additive and dominant genetic variances and (3) segregation frequencies determined by the generalized Weinberg's method. The method proposed yields the uniquel estimations of the model parameters for traits which have additive genetic variance differed from zero. Some aspects of practical use of the method are discussed.     

The demonstration of the influenza virus by the method of the immune electron microscopy

The direct and indirect method of the Immune electron microscopy (IEM) was used for the identification of two influenza viruses of the type A--[strain A/Chabarovsk/77 (H1N1) and A/Texas/77 (H3N2)]--by means of reference specific rat- and human- convalescent sera. In the cross-testing different dilutions of the virus in the allantoic fluid and of the sera were examined for the determination of the appropriate rates for the formation of the immunocomplexes. In the demonstration of the immunocomplexes the higher sensitivity of the indirect method was proved, by using the pig-anti-human-IgG serum,--in comparing with the direct method.     

The relative efficiency of the Hardy-Weinberg equilibrium-likelihood and the conditional on parental genotype-likelihood methods for candidate-gene association studies.

Selecting a control group that is perfectly matched for ethnic ancestry with a group of affected individuals is a major problem in studying the association of a candidate gene with a disease. This problem can be avoided by a design that uses parental data in place of nonrelated controls. Schaid and Sommer presented two new methods for the statistical analysis using this approach: (1) a likelihood method (Hardy-Weinberg equilibrium [HWE] method), which rests on the assumption that HWE holds, and (2) a conditional likelihood method (conditional on parental genotype [CPG] method) appropriate when HWE is absent. Schaid and Sommer claimed that the CPG method can be more efficient than the HWE method, even when equilibrium holds. It can be shown, however that in the equilibrium situation the HWE method is always more efficient than the CPG method. For a dominant disease, the differences are slim. But for a recessive disease, the CPG method requires a much larger sample size to achieve a prescribed power than the HWE method. Additionally, we show how the relative risks for the various candidate-gene genotypes can be estimated without relying on iterative methods. For the CPG method, we represent an asymptotic power approximation that is sufficiently precise for planning the sample size of an association study.     

Quantitation of the cells of the seminiferous epithelium of the human testis employing the Sertoli cell as a constant

Summary Two variants of a method for determining the quantitative structure of the human seminiferous epithelium are described. Both versions (counting cells in whole tubules and in CA's) utilize specific criteria for the identification of cells, tubules and /or CA's. Both methods utilize the Sertoli cell as a constant with the cell types expressed as numbers of cells per Sertoli cell. One hundred biopsies were quantitated by the tubular method and 100 by the CA method. Counts were found to be reproducible and consistent. Comparisons were made between the two methods, different individuals doing the quantitation, quantitations of the right and left testes of the same subject and between biopsies from the same subject taken at different times. No difference was found between the right and left testes or biopsies from the same subject taken at different times. Quantitation of biopsies from one individual before and after testicular X-ray irradiation show how the method can be applied.This investigation was supported in part by U.S. Atomic Energy Commission Contract AT(45-1)1780 and Grant No. 680-0806 from The Ford Foundation.The authors wish to express their thanks and appreciation to Dr. Yves Clermont (Montreal) and Dr. Eugene Oakberg (Oak Ridge) for their aid in the conception of the project and the general outline of the protocol, and again to the latter for personally performing some of the tubular counts once the method had been established, to Mr. Malcom Hobbs for assistance with the subjects, to Dr. Daniel DiIaconi for performing the biopsy technique and to Dr. Kathleen O'Keefe for the statistical analysis.     

A method for determining the apoplastic water volume of conifer needles

A method for direct estimation of percentage apoplastic water volume (% APO) in conifer needles is described. The method presented here, and designated the pressure-needle (P-N) method, measures the relative water content of the needles to develop a curve similar to the pressure-volume (P-V) curve. P-V and P-N curves were developed for Picea pungens Engelm. cv. Hoopsi, Pinus sylvestris L., Abies gradis (Dougl.) L., and Pseudotsuga menziesii (Mirb) Franco. The % APO estimated by the two procedures varied as much as 2-fold, while other parameters were similar. The P-V method generated consistently higher and more variable % APO than the P-N method, due to the inclusion of the apoplastic water of the stem in the P-V method. For conifers, the P-N method offers a more accurate and precise method for determining % APO.     

An aid to the determination of the ventilatory threshold

Detection of the ventilatory threshold during an incremental load exercise test by eye can be difficult. Although various alternative methods employing information other than the ventilation can be used to assist in determining the ventilatory threshold, they rely on underlying assumptions about the physiological basis for the ventilatory threshold. The method presented here (CUSUM) uses only the ventilation data, and therefore avoids such assumptions. Twelve subjects performed a total of 47 incremental exercise tests to exhaustion. Determinations of the ventilatory thresholds made by eye from the ventilation data (mean of three independent observers) were used as a standard for comparison with determinations using the modified V-slope method and the CUSUM method. A mean (SD) difference of 0.6 (2.84) ml·min^–1·kg^–1 was found between the standard ventilatory thresholds and those determined using the modified V-slope method. A similar comparison between the standard ventilatory thresholds and those determined using the CUSUM method yielded a difference of –0.11 (2.35) ml min^–1·kg^–1. It was concluded that the CUSUM method was a useful aid for the detection of the ventilatory threshold using the ventilation data alone.     

Efficacy of the cytobrush versus the cotton swab in the collection of endocervical cells

Cervical smears should contain endocervical cells to be accepted as adequate for a cytologic diagnosis. Before this study was undertaken, one-third of the smears received in the Cytology Laboratory of Odense University Hospital were inadequate. In an attempt to increase the rate of adequate smears, a randomized study was undertaken to compare the efficacy of the combined spatula-Cytobrush method to that of the spatula-cotton swab method traditionally used by doctors in Denmark. The incidence of smears containing cylindrical and/or metaplastic cells was 89% by the spatula-Cytobrush method as compared to 62% by the spatula-cotton swab method (P less than .001). There were large differences between the rates of adequate smears from the various doctors when using the spatula-cotton swab method (range, 14% to 82%); these differences were far less when using the spatula-Cytobrush method (range, 75% to 100%). A large-scale application of the spatula-Cytobrush method should result in fewer repeat smears required and fewer false-negative smears.     

Modification of the Recalde method for the isolation of human monocytes

A modification of the method for monocyte isolation reported by Recalde (1984. J. Immunol. Methods. 69: 71-77) is described. Application of the modified method to 36 consecutive healthy adult donors gave a monocyte purity of 73 +/- 8% monocytes with less than 1% polymorphonuclear leukocytes and a yield of 3.44 +/- 0.93 x 10(5) monocytes/ml blood. While the monocyte purity of the modified Recalde method was lower than that obtained by elutriation (method BB in Fogelman et al. 1981. J. Lipid Res. 22: 1131-1141) in 15 donors (71 +/- 10% vs. 83 +/- 6%) the monocyte yield and the viability of the cells before and after culture were similar in both methods. The modified Recalde method does not require the expensive or complicated equipment required for elutriation and permits the isolation of human monocytes for culture in autologous serum from multiple donors in a single day.     

相机陷阱法在潮间带底栖蟹类群落生态学中的应用研究

潮间带大型底栖动物调查是滨海湿地生物多样性监测的重要环节。目前普遍应用的传统侵入性调查方法（挖掘法、手捕法和陷阱诱捕法）存在干扰强、耗时久和劳动密集等问题,无法客观反映活动性较强的蟹类等重要功能类群的群落结构和物种多样性。对陆地野生动物研究中常用的相机陷阱法进行了符合潮间带应用条件的便携式设计,并以红树林蟹类为例,通过与3种传统侵入性调查方法进行对比,探讨相机陷阱法在潮间带底栖蟹类群落生态学研究中的应用。结果表明：（1）相机陷阱法在种群密度测定方面比挖掘法提高2.1倍,比手捕法提高10.3倍,比陷阱诱捕法提高16.3倍;（2）相机陷阱法在调查物种数方面均高于3种传统侵入性调查方法,适用于沙蟹科（Ocypodidae）和方蟹科（Grapsidae）蟹类调查;（3）相机陷阱法对个体大小选择性弱,采样更加全面;（4）相机陷阱法在群落alpha多样性（Shannon-Wiener指数）、beta多样性（样方-多度矩阵总方差Var （Y））和功能多样性（Rao二次熵和功能离散度）调查方面整体不弱于侵入性调查方法。此外,相较于侵入性调查方法,相机陷阱法对沉积物结构破坏小、不干扰蟹类群落结构,在兼顾数据丰富性的同时又具有高效性。然而,由于相机陷阱法基于蟹类底表活动性,无法适用于冬季调查、夜行性蟹类和游泳型蟹类调查。未来,相机陷阱法的发展将与基于深度学习的图像处理技术结合以满足数据处理需求。研究揭示了相机陷阱法在潮间带底栖蟹类群落中的应用优越性与局限性,为潮间带大型底栖动物群落生态学的研究提供技术支撑。     

Comparison of the SCoRE and HA methods for locating in vivo the glenohumeral joint centre

A recent paper has described a new functional method, the symmetrical centre of rotation (SCoRE), for locating joint centre position [Ehrig, R.M., Taylor, W.R., Duda, G.N., Heller, M.O., 2006. A survey of formal methods for determining the centre of rotation of ball joints. Journal of Biomechanics 39 (15), 2798-2809]. For in vitro analyses, the SCoRE method showed better precision than helical axis (HA) or sphere fitting methods. Despites HA determination is very sensitive to small angular velocity, the International Society of Biomechanics has recommended to use HA for locating the glenohumeral joint centre. This paper aims at comparing the SCoRE method with the HA method for locating in vivo the glenohumeral joint centre according to the movement characteristics. Nine subjects performed 10 cycles of three different movements at two different velocities. For each test (combination of movements) the location of the centre of rotation was estimated with both methods (SCoRE and HA). Analyses focused on the 3D location of the glenohumeral joint centre and on the repeatability of location (standard deviation). This study showed that SCoRE and HA methods yielded the same GH location. Nevertheless, with SCoRE method, the location of the glenohumeral joint centre was different according to the test. This study evidenced that the SCoRE method was more precise than HA method (error of 3 mm versus 4.6 mm) and that the GH location with the SCoRE method was not affected by movements with slow velocities.     

Comparison of the quick Gram stain method to the B&M modified and favor methods

The Gram stain is an established method for bacterial identification, but the time needed to carry out this stain is 2-3 min. We attempted to shorten this time and stained a total of 70 clinical specimens isolated from using the Bartholomew & Mittwer (B&M) modified or Favor methods with a 3 s duration for washing and staining steps. Results were plotted and analyzed using a Hue Saturation Intensity (HSI) model. The range based on a plot of the two methods with the HSI model was presented as a reference interval. Our results indicated that 100% (35/35) of strains were Gram positive and 97.1% (34/35) were Gram negative for the quick B&M modified method. In the quick Favor method, 80.0% (28/35) were Gram positive and 68.6% (24/35) of strains were Gram negative. We propose that the quick B&M modified method is equivalent to the standard Gram staining method and is superior to the quick Favor method.     

Determination of the fraction composition of blood lipoproteins by the small-angle X-ray scattering technique.

A new method for analyzing the fraction composition of blood lipoproteins (LP) was developed based on the small-angle X-ray scattering (SAXS) technique. The method allows quantitative determination of the contents of basic LP fractions (high-density LP, low-density LP, very low-density LP and their subfractions) in the blood plasma or serum. The results of LP analysis by the new method were compared with electron microscopy, ultracentrifugation and gel electrophoresis data. The results obtained by SAXS correlated with those obtained by traditional methods. The new method for the determination of the LP fraction composition in the blood is rapid (1-1.5 h), uses only one reagent (e.g., sucrose) and features a high accuracy and resolution up to LP subfractions. A total of 0.05 ml of the blood plasma or serum is required for an assay. The assays can be carried out in purified preparations or in the blood plasma or serum. The method developed can be used in clinical practice for diagnostics and in scientific research.     

Reevaluation of the phenol-sulfuric acid reaction for the estimation of hexoses and pentoses

Evidence is provided to show that in the conventional phenol-sulfuric acid reaction procedure, phenol underwent sulfonation in situ and the phenolsulfonic acid formed decreased the color intensity for hydroxymethyl furfural (HMF), furfural, and many hexoses and pentoses tested. A modified method is described to overcome this problem in which phenol was added after the dehydration of carbohydrates by sulfuric acid and after cooling the system. The color intensity around 475-485 nm for different compounds was fairly proportional to the amount of furfural derivatives (absorption at 310-320 nm) formed from the sugars in the modified method unlike in the conventional procedure. The studies also show that for condensation of HMF derivatives with phenol, heat is not necessary. The color intensity in the modified method also increased compared to that in the conventional method. The increase in the modified method compared to that in the conventional method was 6.0-fold for furfural, 9.1-fold for hydroxymethyl furfural, 3.7-fold for fructose, 2.3-fold for xylose, and 2.0-fold for glucose and arabinose. The possible reasons for this differential increase are discussed.     

A method for measuring the oxygen consumption of intact cell monolayers

This report describes an open-air method for measuring the O(2) consumption (QO(2)) of intact monolayers of cultured cells. This method is based on Fick's second law of diffusion. It requires only a micromanipulator and a miniature O(2) electrode to measure the PO(2) gradient in the culture medium in the well. It was compared with the conventional oxygraph chamber method. Both methods gave the same value for QO(2) in freshly isolated rat type II cells: 166 +/- 15.3 nmol. h(-1). 10(6) cells(-1) for the open-air method and 151 +/- 11.6 nmol. h(-1). 10(6) cells(-1) for the oxygraph chamber method (n = 11 experiments). But the open-air method gave significantly larger values for QO(2) in cells cultured for 2 days (236 +/- 8.8 nmol. h(-1). 10(6) cells(-1)) than the oxygraph method (71 +/- 15.2 nmol. h(-1). 10(6) cells(-1); P < 0.001; n = 12 experiments). This suggests that the way cells are detached from their substratum to be placed in the oxygraph chamber affects their QO(2). The open-air method may be useful for studies on the metabolic properties of monolayers because the cells do not risk being damaged.     

A Comparative Study on the Efficiency of Two Enucleation Methods in Pig Somatic Cell Nuclear Transfer: Effects of the Squeezing and the Aspiration Methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72–78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.     

Use of the loss-tangent function in dielectric spectroscopy

A new method for finding the dielectric parameters of biological substances is presented. The method makes use of the loss-tangent as a function of frequency to identify the dominating relaxation process. The method was tested for a few cell suspensions (blood and lymphocytes) and two tissues (liver and spleen). The obtained parameters agree well with those calculated from Maxwell-Wagner theory (beta dispersion).