Evaluation of a rapid diagnostic test for bacterial vaginosis.

OBJECTIVE: To evaluate a new rapid diagnostic test for bacterial vaginosis. DESIGN: Comparison of a new biochemical diamine test with low technology tests and microbiological culture. SETTING: General practice, family planning clinic. MAIN OUTCOME MEASURES: Comparison of the new diamine test with microbiological culture for Gardnerella vaginalis, with clue cells, and with the amine test. RESULTS: Two hundred and twenty-nine vaginal swabs were assayed quantitatively by the new diamine test. When compared with microbiological culture of Gardnerella vaginalis, the sensitivities and specificities were 86% and 81%, respectively. When compared with clue cell findings, the sensitivities and specificities were 97% and 83%, respectively. In the third comparison with the amine test the sensitivity was 94% and the specificity was 84%. Since microbiological diagnosis of organisms related to bacterial vaginosis is difficult, the new test and existing sideroom tests may be more sensitive to the condition and the true frequency of false positives may be less than the specificity in this study suggests. CONCLUSIONS: The new diamine test is accurate, sensitive and specific, and provides the basis for the rapid diagnosis of bacterial vaginosis. Such a test is needed if bacterial vaginosis is to be diagnosed and managed effectively in both general and specialist practice.

Degradation of [3H]chondroitin 4-sulphate and re-utilization of the [3H]hexosamine component by the isolated perfused rat liver.

Radiolabelled chondroitin 4-sulphate was isolated after incubation of rat rib cartilage with N-acetyl-D-[6-3H]galactosamine. After proteolytic digestion of the tissue with either papain or trypsin the released [3H]chondroitin 4-sulphate was added to an isolated perfused rat liver system. Analysis of perfusate after several hours perfusion showed that radiolabelled amino sugars were secreted by the liver in a low-molecular-weight form and as components of glycoproteins.

Purification and some kinetic properties of rat liver glucosamine synthetase.

1. Glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was purified about 300-fold from rat liver by two techniques. One procedure utilized the protective action of fructose 6-phosphate and gave a relatively stable preparation, the other yielded an unstable enzyme (half-life of about 20h), free of contaminant activities, on which kinetic experiments were performed. Although the properties of the two preparations showed slight differences, the unstabilized form could be converted into the stabilized form. 2. During preparation the enzyme retained its sensitivity to the feedback inhibitor, UDP-N-acetylglucosamine. 3. The reversibility of the enzyme-catalysed reaction could not be demonstrated. There was no apparent requirement for a cofactor. 4. The pH optimum was at 7.5, at which pH the reaction obeyed a Ping Pong Bi Bi rate equation. At pH values outside the range 6.9-7.6 and at temperatures below 29 degrees C the velocity was described by an ordered Bi Bi rate equation. 5. The molecular weight of the enzyme, determined by two procedures, was 360000-400000. 6. The aminotransferase was unable to utilize ammonia as a substrate.

Studies on the control of hexosamine biosynthesis by glucosamine synthetase.

1. The nature of the feedback inhibition of hexosamine biosynthesis on rat liver glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) by UDP-N-acetylglucosamine was investigated in detail. 2. Further modifiers of physiological importance are described. Glucose 6-phosphate and AMP potentiated the UDP-N-acetylglucosamine inhibition, and UTP behaved as an activator. These three compounds only exerted their action when the feedback inhibitor was bound to the enzyme. 3. ATP also inhibited the enzyme. 4. The actions of these various effectors are discussed in kinetic terms. 5. An interpretation of these findings with reference to the regulation of hexosamine biosynthesis is presented.

Binding of substrates and modifiers to glucosamine synthetase.

1. The binding of substrates and effectors to glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was studied by using the ligand to alter the denaturation rate of the enzyme. The free enzyme bound fructose 6-phosphate, glucose 6-phosphate and UDP-N-acetylglucosamine, but not glutamine, AMP or UTP. Glucose 6-phosphate and AMP increased the binding of UDP-N-acetylglucosamine whereas UTP decreased the interaction between the enzyme and the feedback inhibitor. UDP-N-acetylglucosamine induced a glutamine-binding site on the enzyme. 2. Selective thermal or chemical denaturation revealed that the UDP-N-acetylglucosamine-binding site was not located at the catalytic site. The UTP site could not be distinguished from that for the nucleotide sugar. The AMP- and glucose 6-phosphate-binding sites were distinct from the catalytic and feedback-inhibitor-binding sites. 3. The specificity of the glutamine-binding site was investigated by using a series of potential analogues. 4. A model is proposed for the action of the effectors and the mechanism of the reaction discussed in kinetic and chemical terms.