Evidence for a role of proteinpolysaccharides in regulation of mineral phase separation in calcifying cartilage.

Our previous studies have indicated the presence of a macromolecular inhibitor of in vitro mineral growth, as well as a mineral nucleational agent in extracellular matrix fluid aspirated by micropuncture methods from epiphyseal hypertrophic cell cartilage. In this report, new miniaturized methods were used to extract proteinpolysaccharide complexes (PPC) from cartilage, to isolate a light fraction (PPL-C), and further, to separate it into R1, R2, and SR2 subfractions. These methods were applied to PPL-C complexes separated from microdissected epiphyseal cartilages and to cetylpyridinium chloride (CPC) precipitates of extracellular matrix fluid aspirated from similar cartilages. Most of all of the inhibitory action on an in vitro system of mineral growth shown by whole cartilage PPL-C and by cartilage fluid PPC obtained from noncalcifying sites was contained in the R2 fraction which represented (1/4)-[unk] of the total hexuronate. The R2 fraction was diminished or absent from calcified cartilage fluids and from whole calcified epiphyseal septa. The ratio R1 + R2: SR2 ranged from 0.37 to 0.71 in the fluids and whole tissue samples of noncalcified cartilages. The R2 fraction was distinguished from SR2 by a 2- to 3-fold higher protein: hexuronate ratio. These data are interpreted to indicate that the inhibitory R2 fraction was degraded or otherwise inactivated at the zone of provisional calcification and that this inhibitor participates in the physiological mechanism that regulates endochondral calcification.

Equilibrium and kinetic properties of the interaction between tetrodotoxin and the excitable membrane of the squid giant axon.

Squid giant axons were treated with tetrodotoxin (TTX) in concentrations ranging from 1 nM to 25 nM and the resulting decrease in sodium current was followed in time using the voltage clamp technique. The removal of TTX from the bathing solution produced only partial recovery of the sodium current. This suggests that the over-all interaction is more complex than just a reversible reaction. By correcting for the partial irreversibility of the decrease in sodium current, a dissociation constant of 3.31 x 10(-9)M was calculated for the reaction between TTX and the reactive site of the membrane. The data obtained fit a dose-response curve modified to incorporate the correction for partial irreversibility when calculated for a one-to-one stoichiometry. The fit disagreed with that calculated for a reaction between two molecules of TTX with a single membrane-reactive site, but neither supported nor disproved the possibility of a complex formed by two reactive sites with one molecule of TTX. Values of the rate constants for the formation and dissociation of the TTX-membrane complex, k(1) and k(2), respectively, were obtained from the kinetic data. The values are: k(1) = 0.202 x 10(8)M(-1), and k(2) = 0.116 min(-1). The magnitude of the dissociation constant derived from these values is 5.74 x 10(-9)M, which has the same order of magnitude as that obtained from equilibrium measurements. Arrhenius plots of the rate constants gave values for the thermodynamic quantities of activation.

A study of cardiorespiratory dynamics with step and ramp exercise tests in normoxia and hypoxia.

The ability of the cardiorespiratory system to adapt to the demands of increased exercise intensity was studied under the effects of hypoxia with two different submaximal cycling exercise protocols. A step transition in work rate from 25 W to 105 W and a ramp increase in work rate from a baseline of 25 W with a slope of 40 W.min-1 were used. Eight healthy male subjects each completed maximal exercise tests to exhaustion, as well as six repetitions of the step and the ramp, during normoxia and during hypoxia (FIO2 = 14%, balance N2). VO2 was measured breath by breath and cardiac output (Q) was estimated beat by beat by impedance cardiography. Kinetic analyses of the VO2 and Q data were performed to obtain the gain and the total lag time. With hypoxia, the gain of the VO2 response was no different from normoxia, at 10.6(SEM 0.1) ml.min-1.W-1, but total lag time increased from 23.0(2.2) s to 32.5(2.6) s (p less than 0.05). In contrast, with ramp exercise there was a significant reduction in the gain or slope of the VO2 response from 9.4(0.2) ml.min-1.W-1 in normoxia to 9.0(0.1) ml.min-1.W-1 in hypoxia, while total lag time was no different between normoxia and hypoxia, at 19.7(3.3) v 21.4(2.9) s. Both the gain and total lag time of the step tests were significantly different from the ramp test. Kinetics of Q during the step transition did not differ between normoxia and hypoxia, at 26.5(2.3) v 31.0(4.9) s, respectively (NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation of VO2 kinetics in humans with pseudorandom binary sequence work rate change.

The dynamic response of oxygen uptake (VO2) was investigated with two different cycle ergometer tests in which the work rate changed as a pseudorandom binary sequence (PRBS). One sequence had 15 units, each of 30-s duration for a total of 450 s (PRBS1). The second had 63 units, each of 5-s duration for a total of 315 s (PRBS2). The useful range of frequencies available for investigation of the dynamic characteristics of the VO2 response as described by their bandwidth were 0.002-0.013 Hz for PRBS1 and 0.003-0.089 Hz for PRBS2. Eight subjects each completed both PRBS tests. Data from four or five consecutive sequences were ensemble averaged to reduce the biological noise. A Fourier analysis was then conducted, with the range of frequencies investigated spanning those of the bandwidth for PRBS2. This was up to the 28th harmonic. For PRBS1, the VO2 response could be adequately reconstructed by including Fourier coefficients only up to the 5th harmonic. In contrast, for PRBS2, there was still a clear pattern in the residuals at the 5th harmonic. The data were not adequately reconstructed until higher-frequency components up to the 28th harmonic were included. Evidence for this came from analysis of the mean square error. The mean square error at the 28th harmonic was reduced to 83 +/- 8% of the mean square error at the 5th harmonic for PRBS1 and to 31 +/- 3% for PRBS2 (P less than 0.0001). These data obtained by Fourier analysis and reconstructed for comparison with the original VO2 response indicate the presence of a high-frequency component that was not apparent when a test with a smaller bandwidth was used as the work rate forcing.

Time domain analysis of oxygen uptake during pseudorandom binary sequence exercise tests.

Pseudorandom binary sequence (PRBS) exercise tests involve repeated switching between two work rates (WR) according to a computer-generated pattern. This paper presents an approach to analysis of O2 uptake (VO2) in the time domain. First, the autocorrelation function (ACF) of the input WR was recognized to be a triangular-shaped pulse that can be taken to be equivalent to a ramp increase followed by a ramp decrease in WR. Then the cross-correlation function of the input (WR) and the output (VO2) was treated as if it were the response to a triangular-shaped pulse. The cross-correlation function was analyzed by fitting a linear summation of the ramp form of a two-component exponential function to this triangular pulse. VO2 responses of eight subjects were obtained from two different PRBS tests, as well as step changes in WR. The first PRBS test consisted of 15 units, each 30 s in duration. Its ACF had a base width of 60 s. The ramp increase-ramp decrease model fit the data throughout the range of response. The second PRBS test had 63 units, each 5 s in duration; thus its ACF base width was 10 s. Again, the ramp model fit adequately. The data from the second PRBS test could be fit by the impulse form of the two-component exponential equation, although the fit in the first 30 s tended to be poorer. The time constants of VO2 dynamics estimated from step and PRBS tests were not significantly different. PRBS tests can be analyzed in the time domain, and the indicators of system dynamics reflect physiological properties similar to those investigated during step changes in WR.

Pathological and epidemiological aspects of the coexistence of maedi-visna and sheep pulmonary adenomatosis.

Between 1982 and 1991, 159 sheep suffering from chronic respiratory disease were subjected to clinical, pathological, histopathological and serological examination. Maedi was diagnosed in 82 sheep and sheep pulmonary adenomatosis (SPA) in another 59. Forty-one of the latter (69.5 per cent) were seropositive for maedi-visna (MV) virus infection, but only six (10.2 per cent) showed concurrent lung lesions of maedi. Even disregarding the MV seronegative sheep and those younger than two years old, the rate of concurrent maedi lesions did not exceed 18 per cent. During a similar period, 5060 sheep from 161 flocks (86 of which also provided the 159 affected animals) were tested for antibodies to MV virus. The average seroprevalence of MV virus infection among flocks in which SPA was detected was 66.4 per cent, whereas in those in which SPA could not be demonstrated, and in those in which necropsies were not performed, the levels of MV virus infection were 55.1 per cent and 43.6 per cent, respectively. The effect of SPA on the seroprevalence of MV virus infection was independent of other factors, such as breed of sheep or the size of the flocks. These results provide evidence that SPA plays a role in the spread of MV virus infection, although a synergistic effect of the simultaneous infection on the expression of concurrent lesions does not seem to occur.

Bovine abortion associated with Neospora caninum in northern Spain.

During 1996, aborted bovine fetuses from dairy herds in norther Spain were examined by histopathological, immunohistochemical and serological methods for evidence of infection by Neospora caninum. Microscopical brain changes consistent with those of neosporosis were detected in 36 of 81 fetuses and specific antibodies (IFAT titres > or = 1/16) in 32 of 63. Eight fetuses with neurological lesions were seronegative, and eight without lesions were seropositive. In 25 of 34 fetal brains with lesions consistent with published descriptions of neosporosis, immunohistochemical analysis revealed the presence of N caninum antigen. Depending on the diagnostic criteria, the prevalence of neosporosis related to bovine abortion was between 32 and 57 per cent of the fetuses submitted and between 33 and 58 per cent of the herds involved. Data on the geographical, seasonal and fetal age distribution of the infection are given for the fetuses examined and the value of the different diagnostic methods is discussed.

Brucella ovis infection in two flocks of sheep.

Brucella ovis infection could not be eradicated from two flocks of sheep despite serologically testing and culling the infected rams for four years. The hypothesis that the ewes played a major role in the maintenance of the infection in both flocks was investigated by using serological, bacteriological and pathological criteria. Specific antibodies against B ovis were demonstrated in 71 ewes in the two flocks. Forty-four of the seropositive ewes were slaughtered for bacteriological and pathological studies and B ovis was isolated from 16 of them; the uterus was the target organ of the infection, although extrauterine infection was also demonstrated in some of the infected ewes.