Inductance plethysmography measurement of CPAP-induced changes in end-expiratory lung volume

The respiratory inductance plethysmograph (RIP) has recently gained popularity in both the research and clinical arenas for measuring tidal volume (VT) and changes in functional residual capacity (delta FRC). It is important however, to define the likelihood that individual RIP measurements of VT and delta FRC would be acceptably accurate (+/- 10%) for clinical and investigational purposes in spontaneously breathing individuals on continuous positive airway pressure (CPAP). Additionally, RIP accuracy has not been compared in these regards after calibration by two commonly employed techniques, the least squares (LSQ) and the quantitative diagnostic calibration (QDC) methods. We compared RIP with pneumotachographic (PTH) measurements of delta FRC and VT during spontaneous mouth breathing on 0-10 cmH2O CPAP. Comparisons were made after RIP calibration with both the LSQ (6 subjects) and QDC (7 subjects) methods. Measurements of delta FRC by RIPLSQ and RIPQDC were highly correlated with PTH measurements (r = 0.94 +/- 0.04 and r = 0.98 +/- 0.01 (SE), respectively). However, only an average of 30% of RIPQDC determinations per subject and 31.4% of RIPLSQ determinations per subject were accurate to +/- 10% of PTH values. An average of 55.2% (QDC) and 68.8% (LSQ) of VT determinations per subject were accurate to +/- 10% of PTH values. We conclude that in normal subjects, over a large number of determinations, RIP values for delta FRC and VT at elevated end-expiratory lung volume correlate well with PTH values. However, regardless of whether QDC or LSQ calibration is used, only about one-third of individual RIP determinations of delta FRC and one-half of two-thirds of VT measurements will be sufficiently accurate for clinical and investigational use.     

Measurement of cytokines by bioassays: theory and application

A large number of cytokines have been characterized, of which several have proved successful in the clinic as biotherapeutic agents for malignant, infectious or autoimmune diseases. As biologically active proteins, they cannot be fully characterized by physicochemical methods alone. Thus, biological assays (bioassays) have become increasingly important for their biological characterization and potency determinations. Since cytokines exert various biological activities in vitro, cultured cell line-based bioassay methods have mainly been developed to quantify potency. Such bioassays, like all biological systems, are inherently variable. Thus, measurement of potency of a particular cytokine must be made relative to a common, stable, reference preparation of the same cytokine to permit valid inter-assay and inter-laboratory comparisons. The development and establishment of appropriate primary reference preparations as World Health Organization (WHO) International standards (IS) and reference reagents (RR) is essential for the standardization of bioassays. This review addresses the practical and statistical considerations for the development of valid bioassays, the preparation and use of WHO IS and RR and, in brief, the types of bioassay methods applicable to potency measurements of individual cytokines. More extensive details for the potency determinations of tumor necrosis factor-alpha (TNF-alpha), related cytokines, and biotherapeutic anti-TNF-alpha products are provided.     

Deformation of erythrocytes under shear: A small-angle light scattering study

In this study, sharp small-angle light scattering (SALS) images of erythrocytes under increasing shear stresses in a Couette flow were obtained, and accurate measurements of the angular positions of the two first minima and maxima have been carried out. The deformed cells were assumed to be three-axis ellipsoids of constant volume for all shear stresses. Application of the Physical Optics Approximation (POA) then permitted the determination of the cell dimensions as a function of the applied shear stress. Our results agree with determinations obtained by other methods.     

A simple assay to estimate the acetylcholinesterase molecular forms in crude extracts of rat skeletal muscle

All the current methods available for analyzing the acetylcholinesterase (AChE) molecular forms are time consuming and require the use of expensive equipment. We have found that by using the differential inactivation of globular (G4 + G1) and asymmetric AChE forms by high Mg2+ concentration, we can set up a very easy and quick assay that allows us to determine the relative proportions of AChE molecular forms present in rat skeletal muscles. This assay will be of great help in estimating changes in the muscle AChE forms under experimental conditions that require several simultaneous determinations.     

The contribution of induction of temperate phages to the numbers of free somatic coliphages in waters is not significant

Somatic coliphages have been proposed as indicators of water quality. But several factors have been considered a drawback for their use as indicators. We evaluated the contribution of temperate phages to the numbers of somatic coliphages detected in water by ISO (International Standards Organization) standardised methods. Prophage induction from naturally occurring bacteria was assayed with mitomycin C, ciprofloxacin and UV irradiation. Results indicate that the presence of prophages will not influence the determinations of somatic coliphages in water.     

DNA ploidy in breast lesions. A comparative study using two commercial image analysis systems and flow cytometry

DNA ploidy determinations on a series of 24 breast specimens were performed independently utilizing flow cytometry (FCM) and two separate commercially available computerized image analysis systems for image cytometry (ICM). The tissues analyzed were obtained from 20 carcinomas, 2 benign neoplasms and 2 benign reductive procedures. The results showed a close correlation between the DNA indices (DIs) obtained by all methods in 14 of the 24 cases. In four cases, all methods showed aneuploid peaks, but with differing DIs. In six cases (two benign and four malignant) FCM showed diploidy while ICM showed peridiploid cell populations. The results obtained with the two image analysis systems were in agreement for 20 of the 24 cases. ICM is an acceptable alternative to FCM for reproducible ploidy analysis. ICM-based measurements have the advantage of the visual discrimination of abnormal cells and therefore may have a greater sensitivity in identifying small aneuploid populations. Populations with DIs in the range of 1.0 to 1.3 need to be assessed carefully in ICM-based determinations due to the potential that these "aneuploid" peaks may represent shifted diploid populations.     

Single-image diffusion coefficient measurements of proteins in free solution

Diffusion coefficient measurements are important for many biological and material investigations, such as studies of particle dynamics and kinetics, and size determinations. Among current measurement methods, single particle tracking (SPT) offers the unique ability to simultaneously obtain location and diffusion information about a molecule while using only femtomoles of sample. However, the temporal resolution of SPT is limited to seconds for single-color-labeled samples. By directly imaging three-dimensional diffusing fluorescent proteins and studying the widths of their intensity profiles, we were able to determine the proteins' diffusion coefficients using single protein images of submillisecond exposure times. This simple method improves the temporal resolution of diffusion coefficient measurements to submilliseconds, and can be readily applied to a range of particle sizes in SPT investigations and applications in which diffusion coefficient measurements are needed, such as reaction kinetics and particle size determinations.     

A Proposal for the Use of Standardized Methods for Chlorophyll Determinations in Ecological and Eco-physiological Investigations

The various methods for chlorophyll determination, found in the literature, may give chlorophyll values differing by as much as 20%. Not only the choice of solvent and equations but also the routines and conditions for processing of samples can be large sources of errors. In the present paper some recommendations are given concerning sampling and storage of plant material, extraction and determination of chlorophylls. A simple and fast method for chlorophyll determinations is described and recommended as a suitable method for most ecological and eco-physiological investigations in which chlorophylls are determined. The principle of the method is that the total amount of chlorophylls is determined in a colorimeter calibrated for chlorophyll measurements. Results from a prototype of the “Chlorophyllometer” are presented and compared with spectrophotometric determinations.     

Comparison of liposome entrapment parameters by optical and atomic absorption spectrophotometry

Methods for the complete characterization of liposomes prepared by ether-injection are described in detail. The validity of atomic absorption spectrophotometry Ior measuring markers of trapped volume was checked by comparative determinations of markers with established optical spectrophotometrical methods. The favorable results usingl atomic absorption spectrophotometry to quantitate the marker Mn²⁺ are of particular relevance as manganese ion is also the paramagnetic probe in n.m.r, measurements of water permeability of lipo-somes; our results indicate that in such measurements no other marker need be incorporated.     

Class-directed structure determination: foundation for a protein structure initiative.

The recent sequencing of many complete genomes, combined with the development of methods that allow rapid structure determination for many proteins, has changed the way in which protein structure determinations can be approached. One-by-one determinations of individual protein structures will soon be augmented by class-directed structure analyses in which a group of proteins is targeted and structures of representative members are determined and used to represent the entire group. Such a shift in approach would be the foundation for a broad protein structure initiative targeting classes of proteins important for biotechnology and for a fundamental understanding of protein function.     

A micro-enzymatic method to measure cholesterol and triglyceride in lipoprotein subfractions separated by density gradient ultracentrifugation from 200 microliters of plasma or serum

A micro-enzymatic method was developed to measure total cholesterol (CHOL) and triglyceride (TG) in lipoproteins and their subfractions separated by density gradient ultracentrifugation. This method had a detection limit and sensitivity below 2 mg/dl and accuracy (bias to reference sera) and imprecision (coefficient of variation) of less than 3% between 2 and 30 mg/dl for both CHOL and TG. In addition, the method was in good agreement with standardized Abell-Kendall CHOL (r = 0.98) and enzymatic TG (r = 0.99) methods. Lipoproteins from 200 microliters of plasma or serum were separated by either equilibrium (EQ)- or rate zonal (RZ)-density gradient ultracentrifugation and the resulting fractions were analyzed for CHOL and TG by the micro-enzymatic method. Lipoprotein measurements by these micro-enzymatic/density gradient methods were highly correlated with standardized Lipid Research Clinic (LRC) procedures and preparative ultracentrifugation. The EQ-density gradient procedure also allowed determination of CHOL and TG in LDL and HDL subfractions within any desired density interval. These methods will facilitate the measurements and study of lipoproteins and their subfractions especially in infants, children, the elderly, and small animals. In addition, the micro-enzymatic method may be adapted to other modes of lipoprotein separation such as liquid chromatography, electrophoresis, and precipitation. CHOL or TG determinations could be made on approximately 500 density gradient fractions per hour.     

The utilization of bicarbonate ions by the macroalga Ascophyllum nodosum (L.) Le Jolis

Abstract A comparison of some of the methods used to determine whether aquatic plants have the ability to utilize bicarbonate ions as a source of inorganic carbon for photosynthesis has been applied to the intertidal macroalga Ascophyllum nodosum. These include: observing photosynthesis at a high pH (below the alga's CO₂ compensation point), pH compensation point determinations, comparing the photosynthetic characteristics at low pH (5.20) and at high pH (7.95), estimating the maximal rates at which CO₂ can diffuse through the unstirred layer and the rate at which CO₂ can be produced from bicarbonate dehydration in the unstirred layer. All indicated that Ascophyllum nodosum can use bicarbonate ions for photosynthesis, though some were not always consistent. Calculating the total inorganic carbon concentration from pH measurements and acidification CO₂ determinations revealed that the assumption that the alkalinity remains constant during pH drift experiments is not always valid.     

Relative molecular weight and concentration determination of sodium hyaluronate solutions by gel-exclusion high-performance liquid chromatography

Gel-exclusion chromatography coupled with HPLC instrumentation can be used to determine the molecular weight of highly purified sodium hyaluronate in solution. The method is very reproducible, precise, and rapid, and allows molecular weight determinations up to 2 million to be done in the presence of considerable impurities. This technique offers considerable advantages over traditional light-scattering, sedimentation equilibrium, and viscometry methods for molecular weight determinations, in that HPLC-gel exclusion is rapid and not subject to errors arising from impurities. Simultaneous with molecular weight measurements, sodium hyaluronate concentrations can be determined with a lower range of 0.1 to 0.3 mg/ml dependent upon the sensitivity of the refractive index-detecting system. Unlike the carbazole reaction, this technique is unaffected by low-molecular-weight impurities such as monosaccharides or substances with relative molecular weights less than 18,000.     

A comparison of methods for assessing toxicant effects on algal growth

Summary Ankistrodesmus braunii was used as the test organism to assess the effects of mercuric chloride on the growth of this species. Optical density measurements correlated highly with dry weight determinations, chlorophyll content, total cell counts, and respiring cell counts. Any one of the above methods may be used alone or in combination with other methods to assess the effects of toxicants on algal growth and survival.     

Three-dimensional analysis of the maxillary first molar crowns of Canadian Inuit

The maxillary first molar crowns of 48 male and 38 female Canadian Inuit (Eskimos) were analyzed three-dimensionally by using moiré contourography methods. Cusp heights were significantly higher in males while the mesial and distal marginal ridges were more proximally placed in males. The metacone and the oblique ridge appeared to be better developed in the males. Comparison of the present results with published results for Dutch and Japanese indicated that the Japanese and Inuit had lower cusps that were more widely spaced than the Dutch. Correlation coefficients suggest that height measurements were positively correlated as were linear measurements. However, there were few significant correlations between height and linear determinations, suggesting that the development of the "width" of a crown is nearly independent of the development of the height of the cusps and crown.     

Leaf water potentials measured with a pressure chamber

Leaf water potentials were estimated from the sum of the balancing pressure measured with a pressure chamber and the osmotic potential of the xylem sap in leafy shoots or leaves. When leaf water potentials in yew, rhododendron, and sunflower were compared with those measured with a thermocouple psychrometer known to indicate accurate values of leaf water potential, determinations were within ± 2 bars of the psychrometer measurements with sunflower and yew. In rhododendron. water potentials measured with the pressure chamber plus xylem sap were 2.5 bars less negative to 4 bars more negative than psychrometer measurements.

The discrepancies in the rhododendron measurements could be attributed, at least in part, to the filling of tissues other than xylem with xylem sap during measurements with the pressure chamber. It was concluded that, although stem characteristics may affect the measurements, pressure chamber determinations were sufficiently close to psychrometer measurements that the pressure chamber may be used for relative measurements of leaf water potentials, especially in sunflower and yew. For accurate determinations of leaf water potential, however, pressure chamber measurements must be calibrated with a thermocouple psychrometer.

     

Direct measurements of cell number using computer-aided video microscopy

Quantitative studies in cell culture require accurate measurements of cell density and kinetics. We have developed a direct, rapid, and noninvasive method for measuring cell number in monolayer culture. Using computer-aided video microscopy, cell number was measured without detaching or chemically destroying the cells, thereby allowing sequential measurements in the same cell population. Cell number measured by computer-aided microscopy closely correlated with hemocytometer counts and determinations of total cell protein. For high-density monolayers of mesenchymal cells, however, staining was required for accurate counts. Unlike other techniques for measuring cell density, computer-aided microscopy was especially accurate in medium- to low-density cultures (less than 6000 cells/cm2). In addition, we applied this technique to the construction of separate proliferation curves for glomerular mesangial and vascular endothelial cells in coculture. These measurements by cell type in coculture are impossible using conventional methods for determining cell number.     

Cortical thickness measurements and photon absorptiometry for determination of bone quantity.

Reproducibility of estimates of cortical bone quantity in the appendicular skeleton by two methods was studied in healthy individuals and patients undergoing long-term hemodialysis. Repeated measurements of cortical thickness (CT) at the midpoint of the second metacarpal were taken from single radiographs of both hands by two independent observers. Repeated measurements by the same observer were more reproducible and the degree of reproducibility was far greater in healthy subjects than in dialysis patients. Repeated measurements were made of bone mineral content (BMC) and bone width (W) of the distal radius by photon absorptiometry. Repeated BMC/W determinations were highly reproducible in both healthy subjects and dialysis patients. High correlation was found between BMC and cross-sectional cortical area and between both simple cortical thickness and cortical area/width. Thus the photon absorptiometric technique is superior for the serial monitoring of bone quantity, particularly in patients with uremic osteodystrophy, but results obtained by the two methods in group studies should be comparable.     

A systems-theory approach to the analysis of multiexponential fluorescence decay.

A mathematical model of the fluorescence decay experiment based on linear systems theory is presented. The model suggests an experimental technique that increases the probability of correctly determining the decay constants of a multicomponent system. The use of moment methods for data analysis improves accuracy by combining information obtained from several discrete experiments. Examples are presented to show that the analysis of a three component system composed of known standards is improved as the number of experimental determinations is increased from one to four. The discrete measurements are made by changing the excitation and emission wavelengths.     

Changes in the Resistance of the Mesophyll Tissue 1

Porometer measurements of the resistance offered to viscousflow of air by mesophyll tissue rings cut from leaves were carriedout simultaneously with determinations of changes in tissuewater content, Decreases in water content were accompanied byincreases in mesophyll resistance and these changes are discussedin relation to porometer measurements of total resistance offeredby leaves to viscous flow of air.