Quantitation of autoradiographic grains in different zones of articular cartilage with image analyzer

Summary A novel method is introduced for the estimation of grain numbers in autoradiographic sections of articular cartilage with an image analyzer. It is based on separation of grains from the underlying structures by gray level thresholding and determination of the percentage of total area occupied by grains in a relatively large measuring field. The mean grain size is used as a reference to calculate grain numbers per cell profile and per unit area of tissue in various zones of bovine articular cartilage labelled with ³⁵S-sulphate in tissue culture. The results demonstrate considerable zonal differences as well as site related topographic variation in the rate of ³⁵S-sulphate incorporation. The largest site-related variation in the grain counts was observed in the superficial zone, suggesting a delicate control of proteoglycan synthesis in this zone.The IBAS program used in this work is available from Dr. J.J. Parkkinen or through Bitnet or EARN mail: MLAMMI at FINKUO     

Demonstration of chondroitin sulphate and glycoproteins in articular cartilage matrix using periodic acid-Schiff (PAS) method

Summary Staining of articular cartilage by the periodic acid-Schiff (PAS) method was measured using microspectrophotometry. Standard PAS technique with 2 h oxidation produced a distinct Schiff reaction in the cartilage sections. The staining increased with depth of the articular cartilage demonstrating distribution of the glycoproteins. The modified PAS method included a second, longer periodic acid treatment, which made the uronic acid of glycosaminoglycans PAS-positive. The modified PAS method proved to be highly specific for chondroitin sulphate, which was determined from the samples with gas chromatography. A statistically significant correlation between the Schiff reactivity and galactosamine content of the sections was observed. It is concluded that for articular cartilage standard and modified PAS methods are useful procedures for demonstrating local changes of glycoproteins and chondroitin sulphate, respectively.     

Indentation stiffness of young canine knee articular cartilage—Influence of strenuous joint loading

The indentation stiffness of knee articular cartilage subjected to strenuous physical training (SPT: treadmill running 20 km day⁻¹ for 15 weeks, n = 6) of young Beagles was tested and compared to that obtained from age-matched (55 weeks, n = 9) controls. The mathematical solution for the shear modulus, as determined from indentation of an elastic layer bonded to a rigid half space, was extended to small Poisson's ratios and applied to the analysis of cartilage response after a step stress (0.39 MPa) application. In these measurements with an impervious, plane-ended indenter, the equilibrium deformation was systematically greater than values predicted from the instant response by the linear biphasic theory. Therefore, the accurate determination of Poisson's ratio from the creep curves was not possible. The mean shear modulus (calculated by using the deformation at 900 s after load application and assuming a constant Poisson's ratio of 0.40 for the matrix) of canine knee articular cartilage was 0.37 MPa. While the cartilage thickness was not affected by SPT, the cartilage of the lateral tibial plateau was stiffer (13.3%, p<0.05) than that in controls. However, in the femoral condyles, the stiffness was at the control level or even below. Our results on cartilage structure and properties suggest that SPT, in contrast to our previous findings with moderate training, does not necessarily improve the biological properties of articular cartilage in young animals.     

Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry

Summary Selected commonly used cationic dyes, viz. Thionin, Safranin O, Toluidine Blue O, Dimethylmethylene Blue, Cuprolinic Blue, Cupromeronic Blue,N, N-Diethylpseudoisocyanine, and a modified PAS-method, and staining method, with a variety of alternative procedures, e.g., variation of pH, use of the critical electrolyte concentration method, and blocking reactions (methylation-saponification, carboxymethylation), were tested to select optimal staining procedures for the semiquantitative histochemical estimation of glycosaminoglycans by microspectrophotometry in sections of articular cartilage. The methods were carried out on 3 m-thick paraffin and 1 m-thick glycolmethacrylate sections of bovine articular cartilage. The staining intensity of the sections was measured from spots 25 m apart using a leitz MPV 3 microspectrophotometer, starting at the surface of the cartilage and ending up at the tidemark. The result was compared with the fixed-charge density graph determined from the adjacent articular cartilage.Of the dyes tested, Thionin and Safranin O proved to be excellent cationic dyes for the histochemical quantification of cartilage matrix proteoglycans, since the staining intensity curves showed a linear correlation (r=0.900–0.995) with the fixed charge density curves from the adjacent cartilage. Also, the stain distribution was consistently uniform across the sections. In 1 m-thick glycolmethacrylate sections, the Safranin O staining gradient showed almost perfect identity with the fixed-charge density curve. Cuprolinic Blue and Cupromeronic Blue combined with the critical electrolyte concentration technique were also useful for the microspectrophotometric assays of glycosaminoglycans, but the presence of metachromasia should be checked prior to the measurements. The reliability of blocking procedures for quantitative histochemical work was not convincing.     

Variability of nutrient limitation in the Archipelago Sea,SW Finland

Over a two year study period, zooplankton was sampledin Gazi Bay, Kenya, using a 335 μm mesh size Bongonet. Two Way Indicator Species Analysis (TWINSPAN)classification technique demonstrated that rainfalland tidal regime had substantial influence on thezooplankton community structure. Samples collectedduring the rainy season months clustered together whentreated with TWINSPAN. Furthermore, theclustering was more pronounced for neap tidesamples than for spring tide ones. Samples obtainedduring spring tide did not give a clear cut pattern. Canonical Correspondence Analysis (C.C.A.) confirmedthese findings, a clustering together of rainy/neaptide samples; and little separation (based onenvironmental variables) between samplingstations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of some natural pesticides on entomogenous muscardine fungi.

Five commercial preparations of natural pesticides were tested for in vitro compatibility with muscardine fungi, Beauveria brongniartii and Metarhizium anisopliae. Neemark (azadirachtin) was found compatible with both the fungi. Phytoallexin, the natural fungicide, significantly inhibited the growth of both the fungi, while other natural pesticides showed moderate to severe inhibition.     

Conditions affecting direct gene transfer into rodent muscle in vivo.

This report extends our previous findings that mouse muscle cells in situ can take up naked DNA injected intramuscularly in vivo. Various conditions such as needle type, speed of injection, volume of injection fluid, tonicity of injection fluid, type of solute, type of muscle, physiologic condition of the muscle and age of the animals were appraised for their effect on the levels of luciferase activity expressed from the pRSVL plasmid. Specific conditions such as the use of normal saline as an injection fluid increased the efficiency of expression. The implantation of DNA pellets was an effective way to deliver DNA to muscle, especially for smaller muscle groups. Also, newborn and adult rat muscles expressed plasmid DNA delivered intramuscularly.     

Chemical regulators of carotenogenesis by Blakeslea trispora

The activation of the carotene biosynthetic pathway in Blakeslea trispora was found to occur by trisporic acid and many other compounds such as abscisic acid, β-ionone, α-ionone and vitamin A which share significant structural similarity with trisporic acid. The magnitude of stimulatory activities of these effectors was in the order trisporic acid > abscisic acid > β-ionone > α-ionone > vitamin A. Comparison of structures and stimulatory activities of all the effectors indicated that the short length of the side chain and the presence of a keto group in the ring structure of the trisporic acid molecule contributed significantly to the biological activity towards carotenogenesis.     

The rate of calcium extraction during EDTA decalcification from thin bone slices as assessed with atomic absorption spectrophotometry

Summary The rate of calcium extraction with EDTA (ethylenediamine tetraacetic acid) from thin bone slices (300 m-2mm thick) was determined by aid of an atomic absorption spectrophotometer. A 0.5 mm thick bone slice was completely decalcified with 15% (0.40 M), 8% (0.22 M), and 4% (0.11 M) EDTA in 24 h, 3 days, and 5 days, respectively (vol. 15 ml, temp. 4° C, pH 7.4). At 37 and 60° C the speed of demineralization was slightly increased as compared with that at 20° C, while no difference was observed between 4 and 20° C. Bone slices with a thickness of 0.3, 0.5, 1 and 2 mm were decalcified-in the same order-in 24 h, 2, 3, and 5 days (8% EDTA, 4° C, pH 7.4). At pH 7.4, the decalcification rate was a little slower than at pH 5.0 and 8.5. Agitation did not affect the decalcifying velocity, nor did the volume of the agent, except when the volume was very small. The demineralization of ordinary bone, containing both compact and spongy bone, was found to be more rapid than that of homogeneous bone reported earlier. The acidic buffers and New Decalc^®, which served as reference substances, exerted a more vigorous decalcifying effect than EDTA. K formate/formic acid buffer, pH 3.15, demineralized a 1 mm thick bone slice in 24 h, and 2 days was needed with Na lactate/lactic acid buffer, pH 3.70. With New Decalc^®, pH 0.9, the corresponding demineralization was accomplished in 1.5 h. Atomic absorption spectrophotometer proved to be a useful tool in the evaluation of calcium extraction velocity from bone slices.     

The relative thickness of intracellular membranes in epithelial cells of the ventral lobe of the rat prostate

Synopsis The relative thickness of intracellular membranes of epithelial cells in the ventral lobe of the rat prostrate was measured by a densitometric method. Glutaraldehyde perfusion followed by ruthenium tetroxide immersion fixation appeared to be the most suitable method for membrane thickness measurements. By thickness, the membranes could be roughly subdivided into three groups. The inner and outer membranes of the mitochondrion made up the thinnest membranes of the cell. The second group of membranes consisted of the membranes of the rough-surfaced endoplasmic reticulum and the Golgi apparatus, the different faces of the latter organelle, and the Golgi vesicles. The thickest group of membranes included those of the cell membrane, secretory granules, condensing vacuoles, lysosomes, autophagic vacuoles and multivesicular bodies. The differences in thickness of the membranes are probably due to the varying protein/lipid ratio, and the qualities and proportions of the different lipids in the membranes.