PubMed · 15658463
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Tomoyuki Yamasaki, Hiromu Nakajima. 2004. [Phosphofructokinase (PFK)].. https://pubmed.ncbi.nlm.nih.gov/15658463/
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Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is characterized by varying degrees of developmental delay, motor and gait abnormalities, seizures, and thrombosis. Biochemical abnormalities include homocystinuria and hyperhomocysteinemia. Clinical severity correlates with MTHFR activity in cultured fibroblasts; activity can also be assayed in cultured amniocytes and chorionic villus cells (CVC). Forty-four private mutations have been identified, limiting the use of direct mutation detection for prenatal diagnosis. However, intragenic polymorphisms have been identified, making prenatal diagnosis by linkage analysis a possible option, even without knowledge of deleterious mutations. Prenatal diagnosis for severe MTHFR deficiency has been available by biochemical methodologies, but molecular genetic approaches have not yet been reported. We performed prenatal diagnosis for severe MTHFR deficiency in 11 at-risk pregnancies in seven families. A combined approach of linkage analysis and enzymatic assays was used in six pregnancies; linkage analysis alone was performed in one pregnancy. Linkage analysis for the 677C > T or 1298A > C polymorphisms predicted that all seven fetuses were unaffected. For six of these seven fetuses, enzymatic activities were also measured and demonstrated concordant results. Of the 10 pregnancies in which enzymatic assays were performed, activities in cultured amniocytes predicted six unaffected fetuses (1.4-7.1 nmol CHO/mg prot/h (U)) and one affected fetus (0.24 U [control 3.1-9.6 U]). Three pregnancies assessed via CVCs demonstrated two unaffected fetuses (3.6 and 7.7 U) and 1 affected fetus (0 U [control 4.5-7.8 U]). These values were compared to those of the probands (range = 0.02-0.7 U (control 2.4-11.7 U)) in cultured fibroblasts. Our findings suggest that linkage analysis for severe MTHFR deficiency can be a practical approach for prenatal diagnosis.
AIM: The diagnosis of Helicobacter pylori infection can be made easily by the rapid urease test during endoscopy. The mainly commercial rapid urease test available in routine practice, is in liquid phase, need to be stored at 4 degrees C and generally they are not ready to use. Recently a new rapid urease test, the Pronto Dry, has been reported to be faster in the final reading, ready to use, and it can be stored at room temperature. Aim of the study was to evaluate the diagnostic accuracy and the reaction time of Pronto Dry vs liquid phase-rapid urease test, before and after treatment of Helicobacter pylori infections. METHODS: A total of 315 untreated dyspeptic patients and 323 post-treatment patients, were enrolled in this study. At endoscopy, 5 biopsy samples were obtained from the antrum and from the corpus for histology; culture and rapid urease tests (liquid phase and Dry test). Helicobacter pylori status was defined according to European guidelines. Sensitivity and specificity of both rapid urease test were assessed at 5, 15, 30 minutes, and 3 and 24 hours after the endoscopy. RESULTS: One hundred and eleven out of 315 untreated dyspeptic patients were found to be positive for Helicobacter pylori infection, and 56/323 patients were found still positive after treatment. Sensitivity at 5, 15, 30 minutes, and 3 and 24 hours in untreated patients were 45%, 71.2%, 81.1%, 90.1% and 91.9% respectively for the Pronto Dry vs 6.3%, 31.5%, 51.3%, 78.4% and 90.1% for liquid phase rapid urease test. Sensitivity at the same times in not eradicated patients were 33.9%, 66.1%, 85.7%, 92.8 and 92.8% respectively for the Pronto Dry vs 3.6%, 37.5%, 55.3%73.2%, 92.8% for liquid phase rapid urease test. CONCLUSIONS: Pronto Dry showed to have higher sensitivity in pre and post treatment setting compared to liquid phase-rapid urease test within 3 hours of incubation time.
We report here a new pH-indicator-based assay for galactosyltransferase. The method is simple and fast, requires no specialized equipment, labeled substrate, or other expensive materials, and is thus expected to have broad applications including automated high-throughput screening. The method is based upon the detection of absorbance change of a pH indicator, phenol red, in response to proton release that accompanies the galactosyltransferase-catalyzed galactose transfer. The assay was used to compare three galactosyltransferases in our collection. As demonstrated here, subtle differences in substrate specificity were readily discerned with this sensitive method. All three enzymes accept both N-acetylglucosamine and glucose as acceptor but the relative activity varies with the origin of the enzyme. The method was demonstrated to be useful in the initial characterization of recombinant galactosyltransferase from crude cell extract. Optimal metal cofactor Mn(2+) concentration and temperature were determined with the method. Overall, the method offers a great improvement over current methods in reducing time and material consumption. It is the first pH-sensitive method for galactosyltransferase. The principles of using pH indicator in galactosyltransferase assay should be applicable to other glycosyltransferase enzymes.