A new method for assessing relative dynamic motion of vertebral bodies during cyclic loading in vitro

A new experimental technique for measuring generalized three-dimensional motion of vertebral bodies during cyclic loading in vitro is presented. The system consists of an orthogonal array of three lasers mounted rigidly to one vertebra, and a set of three mutually orthogonal charge-coupled devices mounted rigidly to an adjacent vertebra. Each laser strikes a corresponding charge-coupled device screen. The mathematical model of the system is reduced to a linear set of equations with consequent matrix algebra allowing fast real-time data reduction during cyclic movements of the spine. The range and accuracy of the system is well suited for studying thoracolumbar motion segments. Distinct advantages of the system include miniaturization of the components, the elimination of the need for mechanical linkages between the bodies, and a high degree of accuracy which is not dependent on viewing volume as found in photogrammetric systems. More generally, the spectrum of potential applications of systems of this type to the real-time measurement of the relative motion of two bodies is extremely broad.     

A new method for measurement of cyclic AMP phosphodiesterase activity.

The extreme sensitivity of chicken muscle fructose 1,6-bisphosphatase to inhibition by 5'-AMP has been utilized to develop a new method for the assay of cAMP phosphodiesterase activity. In this method, the substrate (cAMP) is first incubated with phosphodiesterase and the amount of 5'-AMP formed is then determined by measuring the degree of inhibition of fructose 1'6-bisphosphatase activity. The present method conveniently employs the spectrophotometric technique and is sensitive enough to detect the conversion of 50 pmol of cAMP to 5'-AMP in 1 ml of reaction mixture. This method is considered particularly valuable for those laboratories that are not equipped with facilities for measuring radioactivity.     

A new method for the determination of 14C radioactivity in ketone bodies.

A reaction vessel is described consisting of a simple reaction chamber fixed to an ordinary liquid scintillation-counting vial which allows the direct determination of radioactivity in acetone as well as separately in the carboxyl and acetone moieties of acetoacetate and 3-hydroxybutyrate. Radioactivity and its distribution between carbon 1 and carbons 2 through 4 can be determined in total ketone bodies as well as sequentially in acetone, acetoacetate, and 3-hydroxybutyrate. Recoveries with labeled ketone bodies under a variety of analytical conditions are presented.     

A new method of assessing anxiety-phobic states in rats]

A new multiparameter method for evaluating anxiety-phobic states in rats is elaborated. The method is based on the ranged scale consisting of parameters which characterize the species-specific rat response to the series of ethologically adequate test-stimuli inducing manifestation of the anxiety-phobic states. The method makes it possible to evaluate individual anxiety-phobic level in rats, to form experimental groups with rats of known individual anxiety-phobic level, to conduct repeated observations in the same animals. The method is easy to perform in laboratory and makes it possible to get the results quickly.     

A dynamic real-time method for monitoring epithelial barrier function in vitro

The intestinal epithelium functions as a physical barrier against the harmful environment of the lumen, which usually becomes impaired in the presence of intestinal diseases. In this work, we introduce an electronic impedance-based analysis using a real-time xCELLigence system to record the dynamic processes of ethanol-induced intestinal barrier dysfunction. In terms of analyzing morphological alterations in the paracellular junction complex and the organization of pericellular F-actin, this novel, real-time, cell-based technology shows considerable correlations with the standard transepithelial electrical resistance endpoint assay. In addition, monitoring barrier functions in real time allows unbiased screening and characterization of biochemical agents in the lumen that affect epithelial integrity. This functional assay further identifies the in vitro roles of the inducible nitric oxide synthase inhibitor, epithelial growth factor, tyrosine kinases, and phosphatases in regulating epithelial barrier function in response to ethanol administration. Taken together, our findings suggest that this novel, real-time, high-throughput method offers a promising tool for monitoring epithelial barrier functions in situations with more physiological relevance.     

A new method for estimating joint parameters from motion data

Joint centers and axes of rotation (joint parameters) are central to all branches of movement analysis. In gait analysis, the standard protocol used to determine hip and knee joint parameters is prone to errors arising from palpation, anthropometric regression equations, and misplaced alignment devices. Several alternative methods have been proposed, but to date none have been shown to be accurate and reliable enough for use in the clinical setting. This article describes a new method for joint parameter estimation. The new method can be summarized as follows: (i) the motions of two adjacent segments spanning a single joint are tracked, (ii) the axis of rotation between every pair of observed segment configurations is computed, (iii) the most likely intersection of all axes (effective joint center) and most likely orientation of the axes (effective joint axis) is found. Initial validation of the method was conducted on a hinged mechanical analog and a single healthy adult subject. For the analog, the center was found to be within 3.8 mm of the geometric center and 2.0 degrees of the geometric axis (standard deviation). For the adult subject, hip centers varied on the order of 1-3 mm, knee centers by 3-9 mm, and knee axes by 2.0 degrees. The results suggest that the new method is an objective, precise, and practical alternative to the standard clinical approach.     

A method for assessing phagocyte clearance rates in man.

By comparing the clearance rates of ICG and 99mTc-sulphur colloid from the blood stream, it has been possible to provide a method for assessing the phagocytic activity of the reticulo-endothelial system in man.     

Orientation of actin filaments during motion in in vitro motility assay.

Rhodamine-phalloidin was added to F-actin, and the orientation of transition dipoles of the dye was measured in single actin filaments by polarization of fluorescence. Rhodamine-phalloidin was well immobilized on the surface of actin, indicating that changes in orientation of the dye reported changes in orientation of actin monomers. In stationary filaments the dipoles were inclined at 49.3 degrees with respect to the filament axis. The disorganization of dipoles in stationary filaments was insignificant. When the filaments were made to translate, the average orientation of the dye did not change, but disorganization slightly increased. Disorganization increased significantly when filaments were free in solution. We concluded that, within the accuracy of our measurements (approximately 18%), actin monomers did not undergo major reorientations during motion, but that binding of myosin heads deformed the structure of filaments.     

The feasibility of modal testing for measurement of the dynamic characteristics of goat vertebral motion segments

Structural vibration testing might be a promising method to study the mechanical properties of spinal motion segments as an alternative to imaging and spinal manipulation techniques. Structural vibration testing is a non-destructive measurement technique that measures the response of a system to an applied vibration as a function of frequency, and allows determination of modal parameters such as resonance frequencies (ratio between stiffness and mass), vibration modes (pattern of motion) and damping. The objective of this study was to determine if structural vibration testing can reveal the resonance frequencies that correspond to the mode shapes flexion-extension, lateroflexion and axial rotation of lumbar motion segments, and to establish whether resonance frequencies can discriminate specific structural alterations of the motion segment. Therefore, a shaker was used to vibrate the upper vertebra of 16 goat lumbar motion segments, while the response was obtained from accelerometers on the transverse and spinous processes and the anterior side of the upper vertebra. Measurements were performed in three conditions: intact, after dissection of the ligaments and after puncturing the annulus fibrosus. The results showed clear resonance peaks for flexion-extension, lateral bending and axial rotation for all segments. Dissection of the ligaments did not affect the resonance frequencies, but puncturing the annulus reduced the resonance frequency of axial rotation. These results indicate that vibration testing can be utilised to assess the modal parameters of lumbar motion segments, and might eventually be used to study the mechanical properties of spinal motion segments in vivo.     

A new quantitative in vitro microculture method for Giardia duodenalis trophozoites

A reliable, rapid and low-cost method for drug sensitivity determination of Giardia duodenalis trophozoites (WB-strain) was developed in a 96-well plate. Using a standard inoculum of 5 x 10(4) trophozoites per well (300 microl), good growth was obtained after sealing the plate with an air-tight adhesive tape and incubation at 37 degrees C for 72 h in modified TYI-S-33 medium. Viable burdens were quantified using the formazan dyes MTT (100 microg/well) and XTT (20 microg/well) and the fluorescent substrate resazurin (2.5 microg/well). Prior removal of the culture medium is required since it causes spontaneous reduction of the substrate. Resazurin proved to be far superior to MTT and XTT with a level of sensitivity of about 3 x 10(4) trophozoites. Inhibitory concentrations (IC(50)) of several anti-giardial reference drugs were within the range of published values: metronidazole 2.25 microM, tinidazole 1.75 microM, albendazole 0.10 microM, furazolidone 2.00 microM and quinacrine 0.32 microM. The broad-spectrum antibiotics chloramphenicol, rifampicin, penicillin G+streptomycin and gentamycin were devoid of any inhibitory activity and are considered suitable for decontamination during excystation experiments.     

A new alternative in vitro method for quantification of Toxoplasma gondii infectivity

An in vitro method to determine the infectious potency of an unknown suspension of the protozoan parasite Toxoplasma gondii based on kinetics of host cells lysis was developed. Mic1-3KO a mutant strain of T. gondii RH tachyzoites was inoculated in 25-cm2 flasks containing a 90% confluent monolayer of human foreskin fibroblasts. Lysis kinetics was monitored for infection ratios ranging from 1∶10? to 1∶10; we defined 10? tachyzoites/ml?1 as the threshold value for parasite egress. Results allowed us to build a calibration curve relating the initial infection ratios to the time needed to reach 10? tachyzoites/ml?1. Finally, we validated the method using a known mixture of dead and live parasites. This method was found to estimate with accuracy the initial ratio of infection of the unknown parasite suspension. This easy-to-use method is reproducible and can be applied to any T. gondii tachyzoite RH strain, genetically modified or not. This method is also suitable for testing promising candidates for an effective live vaccine.     

Increase in levels of cyclic AMP during avian limb chondrogenesis in vitro.

In the present study the level of cAMP was measured during in vitro chondrogenesis of wing mesenchyme of stage 24 chick embryos and was found to increase significantly from 6.3 pmol/mg protein at the end of the first day of culture to 9.7 pmol/mg protein on the second day, when chondrogenic expression is first detected by the appearance of an Alcian blue staining extracellular matrix. Nonchondrogenic cultures derived from wings of stage 19 embryos had a lower level of cAMP (4.4 +/- 0.07 pmol/mg protein). The level of cAMP in intact wings was 4.5 +/- 0.4 pmol/mg protein and did not change between stages 19 through 25. The correlatin between increased levels of cAMP and the onset of chondrogenesis is consistent with a role of cAMP in the expression of differentiated functions in chondrocytes, as well as in some other cell types.