Tetrameric mouse acetylcholinesterase: continuum diffusion rate calculations by solving the steady-state Smoluchowski equation using finite element methods

The tetramer is the most important form for acetylcholinesterase in physiological conditions, i.e., in the neuromuscular junction and the nervous system. It is important to study the diffusion of acetylcholine to the active sites of the tetrameric enzyme to understand the overall signal transduction process in these cellular components. Crystallographic studies revealed two different forms of tetramers, suggesting a flexible tetramer model for acetylcholinesterase. Using a recently developed finite element solver for the steady-state Smoluchowski equation, we have calculated the reaction rate for three mouse acetylcholinesterase tetramers using these two crystal structures and an intermediate structure as templates. Our results show that the reaction rates differ for different individual active sites in the compact tetramer crystal structure, and the rates are similar for different individual active sites in the other crystal structure and the intermediate structure. In the limit of zero salt, the reaction rates per active site for the tetramers are the same as that for the monomer, whereas at higher ionic strength, the rates per active site for the tetramers are approximately 67%-75% of the rate for the monomer. By analyzing the effect of electrostatic forces on ACh diffusion, we find that electrostatic forces play an even more important role for the tetramers than for the monomer. This study also shows that the finite element solver is well suited for solving the diffusion problem within complicated geometries.     

Inhibition of erythrocyte acetylcholinesterase by n-butanol at high concentrations

Erythrocyte acetylcholinesterase (AChE) is bound to the membrane by a complex glycosylphosphatidylinositol anchor, so the effect of alcohol on AChE activity may reflect direct and/or membrane-mediated effects. The indication of a direct interaction between n-butanol and AChE molecules is the activation/inhibition of AChE by occupation of the enzyme's active and/or regulatory sites by alcohol. The activation of AChE can occur only at low concentrations of alcohols, while at high concentrations AChE is inhibited. In this work the mechanism of inhibition of erythrocyte AChE by n-butanol at high concentrations was studied. The values of activity, calculated assuming parabolic competitive inhibition, which implies that one or two molecules of inhibitor bind to the enzyme, fit well to the experimental values. From the values of the inhibition constants it was concluded that at high n-butanol concentrations two alcohol molecules usually interact with AChE.     

Genital human papillomavirus testing by in situ hybridization in liquid atypical cytologic materials and follow-up biopsies

OBJECTIVE: To describe cases of HPV testing by DNA in situ hybridization performed on atypical cervicovaginal samples collected by a liquidsed method that were negative for HPV DNA on cytology but revealed cervical intraepithelial neoplasia on follow-up biopsies. STUDY DESIGN: Three hundred ninety-five consecutive SurePath atypical squamous cells of undetermined significance (ASC-US) cytologic samples from asymptomatic, reproductive-age women were tested for human papillomaviruses (HPVs) by the in situ hybridization (ISH) method (Ventana Inform HPV Test, Tucson, Arizona, U.S.A). One hundred (25%) cases underwent follow-up colposcopic biopsy within 3 months of cytology. All the tests (cytology, ISH, histology) were independently evaluated without knowledge of the other tests. RESULTS: One hundred twenty-two (33%) cytologic samples were positive for HPVs. Of a total of 100 (HPV positive and negative) follow-up biopsies, 55 were positive for cervical intraepithelial neoplasia (CIN). Fourteen cases of biopsy-proven CIN tested negative for all HPV types in the prior cytologic samples. Retesting of the 14 CIN tissues by ISH was negative in 10, positive for HPV in 2 and inconclusive in 2. CONCLUSION: There is a small but significant (14%) false negative rate with HPV testing by the Ventana ISH method. Clinically suspicious cases should be followed even if an HPV test is negative.     

HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells

Malignant glioma patients have a dismal prognosis with an urgent need of new treatment modalities. Previously developed gene therapies for brain tumors showed promising results in experimental animal models, but failed in clinical trials due to low transfection rates and insufficient expression of the transgene in tumor cells, as well as low bystander killing effects. We have previously shown that the histone deacetylase inhibitor 4-phenylbutyrate (4-PB) enhances gap junction communication between glioma cells in culture. In this study, we demonstrate an activation of recombinant HSV-tk gene expression, and a dramatic enhancement of gap junction-mediated bystander killing effect by administration of the HSV-tk prodrug ganciclovir together with 4-PB. These findings that 4-PB potentiates "suicide gene" expression as well as enhances gap junctional communication and bystander killing of tumor cells justify further testing of this paradigm as an adjunct to suicide gene therapy of malignant gliomas.     

Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.     

Adaptation of organisms to extreme conditions of deep-sea hydrothermal vents

The deep-sea hydrothermal vents are located along the volcanic ridges and are characterized by extreme conditions such as unique physical properties (temperature, pression), chemical toxicity, and absence of photosynthesis. However, life exists in these particular environments. The primary producers of energy and organic molecules in these biotopes are chimiolithoautotrophic bacteria. Many animals species live in intimate and complex symbiosis with these sulfo-oxidizing and methanogene bacteria. These symbioses imply a strategy of nutrition and a specific metabolic organization involving numerous interactions and metabolic exchanges, between partners. The organisms of these ecosystems have developed different adaptive strategies. In these environments many microorganisms are adapted to high temperatures. Moreover to survive in these environments, living organisms have developed various strategies to protect themselves against toxic molecules such as H2S and heavy metals.     

Substring selection for biomedical document classification

MOTIVATION: Attribute selection is a critical step in development of document classification systems. As a standard practice, words are stemmed and the most informative ones are used as attributes in classification. Owing to high complexity of biomedical terminology, general-purpose stemming algorithms are often conservative and could also remove informative stems. This can lead to accuracy reduction, especially when the number of labeled documents is small. To address this issue, we propose an algorithm that omits stemming and, instead, uses the most discriminative substrings as attributes. RESULTS: The approach was tested on five annotated sets of abstracts from iProLINK that report on the experimental evidence about five types of protein post-translational modifications. The experiments showed that Naive Bayes and support vector machine classifiers perform consistently better [with area under the ROC curve (AUC) accuracy in range 0.92-0.97] when using the proposed attribute selection than when using attributes obtained by the Porter stemmer algorithm (AUC in 0.86-0.93 range). The proposed approach is particularly useful when labeled datasets are small.