Wayne A. Wilson

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of α1,4-linked glucose residues with branches introduced through the addition of α1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Background The treatment options for trichomoniasis are largely limited to nitroimidazole compounds (metronidazole and tinidazole). Few alternatives exist in cases of recalcitrant infections or in cases of nitroimidazole hypersensitivity. Recently, the intravaginal administration of boric acid has been advocated as an alternative treatment of trichomoniasis. However, no in vitro studies are available that directly assess the sensitivity of Trichomonas vaginalis to boric acid. Methods We examined the sensitivity of common laboratory strains and recent clinical isolates of T. vaginalis to boric acid. The effect of increasing concentrations of boric acid on parasite growth and viability was determined, and a minimal lethal concentration was reported. The effect of pH on boric acid toxicity was assessed and compared with that of lactic and acetic acid. Results Boric acid is microbicidal to T. vaginalis, and its antitrichomonal activity is independent of environmental acidification. Unlike acetic acid and lactic acid, boric acid exposure results in growth suppression and lethality over a wide range of pH (5–7) and under conditions that are normally permissible for growth in vitro. Conclusions The microbicidal effect of boric acid on T. vaginalis, coupled with its previous clinical use in treating vaginal candidiasis, supports the continued inclusion of boric acid in the therapeutic arsenal for treating trichomoniasis.

Trichomonas vaginalis, a flagellated protozoan, is the agent responsible for trichomoniasis, the most common nonviral sexually transmitted infection worldwide. A reported 200 million cases are documented each year with far more cases going unreported. However, T. vaginalis is disproportionality under studied, especially considering its basic metabolism. It has been reported that T. vaginalis does not grow on sucrose. Nevertheless, the T. vaginalis genome contains some 11 putative sucrose transporters and a putative β-fructofuranosidase (invertase). Thus, the machinery for both uptake and cleavage of sucrose appears to be present.

Several species of trichomonad have been shown to accumulate significant quantities of glycogen during growth, suggesting an important role for this compound in cell physiology. We provide the first analysis of the changes in glycogen content and glycogen phosphorylase activity that occur during in vitro growth of two trichomonad species: Trichomonas vaginalis and Trichomonas tenax. Both species accumulated glycogen following inoculation into fresh medium and utilized this compound during logarithmic growth. Glycogen phosphorylase activity also varied during growth in a species-specific manner. The expression of phosphorylase genes in T. vaginalis remained constant during growth and thus transcriptional control did not explain the observed fluctuations in phosphorylase activity. After cloning, expression, and purification, two recombinant glycogen phosphorylases from T. vaginalis and one recombinant glycogen phosphorylase from T. tenax had robust activity and, in contrast to many other eukaryotic glycogen phosphorylases, did not appear to be regulated by reversible protein phosphorylation. Furthermore, allosteric regulation, if present, was not mediated by compounds known to impact the activity of better characterized phosphorylases.

Giardia intestinalis is the causative agent of human giardiasis, a common diarrheal illness worldwide. Despite its global distribution and prevalence, many questions regarding its basic biology and metabolism remain unanswered. In this study, we examine the accumulation and degradation of glycogen, an important source of stored carbon and energy, during the in vitro growth and differentiation of G. intestinalis . We report that, as G. intestinalis progresses through its growth cycle, cultures of trophozoites accumulate glycogen during the lag and early logarithmic phases of growth and then utilize this compound during their remaining logarithmic growth. As cultures enter the stationary phase of growth, they re-accumulate glycogen stores. The activity of glycogen phosphorylase, an enzyme involved in glycogen metabolism, also varied throughout in vitro trophozoite growth. During the in vitro induction of trophozoite differentiation into water-resistant cyst forms, the cultures initially accumulated stores of glycogen which diminished throughout transition to the cyst form. This observation is suggestive of a role for glycogen in the differentiation process. These studies represent the first thorough analysis of changes in glycogen content and glycogen phosphorylase activity during G. intestinalis growth and differentiation.