Titration of human coronaviruses using an immunoperoxidase assay.

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV).Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth,viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides are liable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids [corrected].

Titration of human coronaviruses, HcoV-229E and HCoV-OC43, by an indirect immunoperoxidase assay.

Calculation of infectious viral titers represents a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for strains 229E and OC43 of human coronavirus (HCoV). An alternative indirect immunoperoxidase assay (IPA) is herein described for the detection and titration of these viruses. Susceptible cells are inoculated with serial logarithmic dilutions of samples in a 96-well plate. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as "Tissue Culture Infectious Dose" (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain replicating virus. This technique is a reliable method for the titration of HCoV in biological samples (cells, tissues, or fluids).

Mechanical properties of tracheal smooth muscle are impaired in the rabbit with experimental cardiac pressure overload.

UNLABELLED: STUDY OBJECTIVES/DESIGN: Impaired function of striated and arterial smooth muscle is known to occur in humans and animals with various forms of cardiac diseases, but limited information is available on the mechanical behavior of airway smooth muscle. We tested the hypothesis that the baseline mechanical properties of tracheal smooth muscle (TSM) were impaired at an early stage of cardiac overload. ANIMALS: We used a model of cardiac hypertrophy induced by surgical abdominal aortic stenosis (AS) in adult rabbits. Twelve animals with AS and 8 sham-operated control rabbits were studied 12 weeks after surgery. In rabbits with AS, the heart weight/body weight ratio was higher than in control rabbits (2.36 +/- 0.43 g/kg vs 1.98 +/- 0.20 g/kg, p < 0.05) [mean +/- SD], attesting to moderate cardiac hypertrophy. No clinical signs of congestive heart failure were observed. MEASUREMENTS: Isolated TSM strips were electrically stimulated at 37 degrees C, 2.5 mM [Ca(2+)](0), against 8 to 10 load levels, from zero load to full isometry. Force-velocity relationship was elicited using the conventional afterloaded isotonic method. RESULTS: Peak isometric tension was lower in rabbits with AS than in control rabbits (25 +/- 11 mN/mm(2) vs 34 +/- 14 mN/mm(2), p < 0.05), whereas maximum unloaded shortening velocity, maximum extent of muscle shortening, and relaxation parameters did not differ between groups. The curvature of the force-velocity relationship (which reflects the myothermal economy of force generation) and peak mechanical efficiency were lower in rabbits with AS than in control rabbits. CONCLUSIONS: These results indicate that the contraction of isolated rabbit TSM was less powerful and less economical in cardiac hypertrophy, attesting to early impairment of the mechanical properties of TSM during cardiac overload.

[Optical tweezers in biology and medicine]. / Les pinces optiques en biologie et en médecine.

Optical trapping techniques provide unique means to manipulate biological particles such as virus, living cells and subcellular organelles. Another area of interest is the measurement of mechanical (elastic) properties of cell membranes, long strands of single DNA molecule, and filamentous proteins. One of the most attractive applications is the study of single motor molecules. With optical tweezers traps, one can measure the forces generated by single motor molecules such as kinesin and myosin, in the piconewton range and, for the first time, resolve their detailed stepping motion.

Velocity of actomyosin sliding in vitro is reduced in dystrophic mouse diaphragm.

It has recently been suggested that dystrophin deficiency in mdx diaphragm muscle is associated with quantitative changes in the myosin molecular motor. In vitro motility assays were used to study the kinetics of actomyosin interactions between purified actin filaments and myosin molecules. Monomeric myosin was obtained from the diaphragm and limb (semitendinosus) muscles of 9-mo-old male mdx (mdx) and age-matched control mice. The sliding velocity (vo, microm/s) of fluorescent-labeled actin filaments moving over a myosin-coated surface (40 microg/ml) was measured. In diaphragm, vo was significantly slower in mdx than in control mice (1.2 +/- 0.1 microm s(-1) versus 1.9 +/- 0.1 microm s(-1), p < 0.001). Conversely, there was no significant difference in vo between control and mdx semitendinous muscles (2.4 +/- 0.1 microm s(-1) versus 2.5 +/- 0.1 micro(-1)). As compared with control mice, mdx diaphragm exhibited a shift from IIX-MHC to IIA-MHC (p < 0.001) and a reduction in IIB-MHC (p < 0.01). Semitendinous muscle from control and mdx mice contained almost exclusively type IIB MHC. Our results are in good agreement with the proposal that myosin is altered in dystrophic mouse diaphragm.