Diagnostic test for mucopolysaccharidosis. I. Direct method for quantifying excessive urinary glycosaminoglycan excretion.
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This direct method for quantifying excessive urinary glycosaminoglycan excretion exploits the specific binding of 1,9-dimethylmethylene blue (DMB). The procedure obviates cumbersome and labor-intensive procedures for separating glycosaminoglycans from other constituents of urine. Pediatric pharmaceutical formulations (except heparin), in concentrations expected in urine, do not interfere with spectrophotometry, nor does protein. Results can be expressed in terms of urinary creatinine; thus the test is applicable to very small urine specimens (0.1 mL), such as those obtainable from neonates. In a pilot study, results of the direct DMB test for 48 urine specimens agreed with the clinical diagnosis, and quantitative measurements correlated moderately (r = 0.76) with results of a commonly used procedure (carbazole-borate reactivity after precipitation with cetylpyridinium chloride). The present method was also used to assess metabolic correction in a patient with Hurler's syndrome after treatment by bone-marrow transplantation. This quantitative method surmounts the major technical problems of developing mass screening programs for infants, thus offering the potential for earlier diagnosis and treatment of mucopolysaccharidosis diseases. (+info)
Simple bacterial preservation medium and its application to proficiency testing in water bacteriology.
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A medium composed of nutrient broth, 1.8% boric acid, and 1% sodium chloride at pH 7.0 was shown to maintain the stability of Escherichia coli cultures for up to 10 days at room temperature. By using this preservation medium for preparing a simulated sample a successful proficiency test survey in water bacteriology was conducted. (+info)
Simple spectrophotometric quantification of urinary excretion of glycosaminoglycan sulfates.
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We describe a simple, rapid, precise, and sensitive spectrophotometric method for measuring urinary glycosaminoglycan (GAG) sulfate excretion. The GAG sulfates are precipitated with cetylpyridinium chloride, resuspended in water, and mixed with the basic dye 1,9-dimethylmethylene blue to produce a complex with the polyanionic molecule of sulfated GAGs. Absorbance is read at 535 nm. The standard curve for reaction was linear up to 12 micrograms of the different GAGs: dermatan sulfate, heparan sulfate, keratan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate. Within- and between-run precision (CV), measured at three different GAG concentrations (normal and pathological), varied from 1.6% to 2.5% and from 1.8% to 4.5%, respectively. Analytical recovery ranged from 71% to 107%. Urinary GAG excretion, measured by this procedure, correlates (r = 0.837; p less than 0.001) with the values obtained with the borate-carbazole reaction (Anal Biochem 1962;4:330-4). (+info)
Chicken liver TGGCA protein purified by preparative mobility shift electrophoresis (PMSE) shows a 36.8 to 29.8 kd microheterogeneity.
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The TGGCA protein, the chicken homologue of HeLa cell NF-I, was purified to homogeneity from liver tissue by a procedure which includes preparative mobility shift electrophoresis (PMSE) as the final step. PMSE was here adjusted for the isolation of the TGGCA protein, but can be used as a general method to characterize the protein moiety of specific DNA-binding proteins. The TGGCA protein is a family of 6 protein species, which show minor differences in molecular weight from 36.8kd to 29.8kd. This microheterogeneity differs from the size distribution reported for HeLa cell NF-I polypeptides. All species of the TGGCA protein bind identically to a synthetic DNA-binding site and appear to be highly related in primary structure. We discuss the possible functional importance of this microheterogeneity. (+info)
Laboratory assessment of physical and chemical methods of preserving urine specimens.
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Three methods of preserving simulated specimens of urine were studied with six test strains of bacteria. Viable counts were measured by a surface viable count and by the filter-paper-strip method during a holding period of 72 hours. Refrigeration at approximately 4 degrees C was effective and reliable. Boric acid (1-8%) at room temperature was toxic for the strain of Escherichia coli at a density of 10(7) cfu/ml but this may not be significant at the higher concentration of bacterial cells often found in clinical specimens. NaCl-polyvinylpyrrolidone (PVP) solutions containing PVP of mol. wt 44 000 or 700 000 were not effective; they were toxic for the Gram-negative strains and did not retard the growth of Micrococcus subgroup 3. The two methods of measuring viable counts were compared for specimens held under different conditions; the specificity of the filter-paper-strip method was high but the sensitivity was low when many of the specimens contained approximately 10(5) cfu/ml. (+info)
Proteus mirabilis urease. Partial purification and inhibition by boric acid and boronic acids.
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Urease was purified 800-fold and partially characterized from Proteus mirabilis, the predominant microorganism associated with urinary stones. Boric acid is a rapid reversible competitive inhibitor of urease. The pH-dependence of inhibition exhibited pKa values of 6.25 and 9.3, where the latter value is probably due to the inherent pKa of boric acid. Three boronic acids also were shown to inhibit urease competitively. (+info)