Effects of the Phosphatase Inhibitors Calyculin A and Okadaic Acid on Acetylcholine Synthesis and Content of Rat Hippocampal Formation

Abstract: The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 n M ) and okadaic acid (50 n M ) reduced significantly ( p < 0.01) the synthesis of [³H]ACh from [³H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration-dependent manner ( p < 0.01). This depletion was not the result of a drug-induced increase of spontaneous ACh release, which was not changed significantly ( p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration-dependent manner ( p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly ( p > 0.4) in the presence of calyculin A or okadaic acid. Both high-affinity and low-affinity [³H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration-dependent manner in the presence of calyculin A or okadaic acid; these agents reduced V _max values for high- and low-affinity choline uptake ( p < 0.01) with no significant change in K _m values ( p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.     

Prevalence of trypanosomes,salivary gland hypertrophy virus and <Emphasis Type="Italic">Wolbachia</Emphasis> in wild populations of tsetse flies from West Africa

Background

Tsetse flies are vectors of African trypanosomes, protozoan parasites that cause sleeping sickness (or human African trypanosomosis) in humans and nagana (or animal African trypanosomosis) in livestock. In addition to trypanosomes, four symbiotic bacteria Wigglesworthia glossinidia, Sodalis glossinidius, Wolbachia, Spiroplasma and one pathogen, the salivary gland hypertrophy virus (SGHV), have been reported in different tsetse species. We evaluated the prevalence and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in four tsetse species (Glossina palpalis gambiensis, G. tachinoides, G. morsitans submorsitans, and G. medicorum) that were collected between 2008 and 2015 from 46 geographical locations in West Africa, i.e. Burkina Faso, Mali, Ghana, Guinea, and Senegal.

Results

The results indicated an overall low prevalence of SGHV and Wolbachia and a high prevalence of trypanosomes in the sampled wild tsetse populations. The prevalence of all three infections varied among tsetse species and sample origin. The highest trypanosome prevalence was found in Glossina tachinoides (61.1%) from Ghana and in Glossina palpalis gambiensis (43.7%) from Senegal. The trypanosome prevalence in the four species from Burkina Faso was lower, i.e. 39.6% in Glossina medicorum, 18.08%; in Glossina morsitans submorsitans, 16.8%; in Glossina tachinoides and 10.5% in Glossina palpalis gambiensis. The trypanosome prevalence in Glossina palpalis gambiensis was lowest in Mali (6.9%) and Guinea (2.2%). The prevalence of SGHV and Wolbachia was very low irrespective of location or tsetse species with an average of 1.7% for SGHV and 1.0% for Wolbachia. In some cases, mixed infections with different trypanosome species were detected. The highest prevalence of coinfection was Trypanosoma vivax and other Trypanosoma species (9.5%) followed by coinfection of T. congolense with other trypanosomes (7.5%). The prevalence of coinfection of T. vivax and T. congolense was (1.0%) and no mixed infection of trypanosomes, SGHV and Wolbachia was detected.

Conclusion

The results indicated a high rate of trypanosome infection in tsetse wild populations in West African countries but lower infection rate of both Wolbachia and SGHV. Double or triple mixed trypanosome infections were found. In addition, mixed trypanosome and SGHV infections existed however no mixed infections of trypanosome and/or SGHV with Wolbachia were found.

     

Molecular identification of Salmonella isolates from poultry and meat productsin Irbid City,Jordan

The sensitivity and accuracy of molecular diagnosis of Salmonella from meat and poultry products using polymerase chain reaction (PCR) was compared with conventional microbiological methods. A total of 212 samples representing the most frequently used fresh and frozen meat and poultry products (whole, cut, ground, and processed) were collected from different locations within the city of Irbid. DNA was extracted directly from each food sample and amplified using Salmonella-specific primers. Samples were also analysed using conventional microbiological methods for the presence of Salmonella spp. Results showed that Salmonella was detected in 185 samples out of 212 (87%) by PCR technique, while 172 (81%) samples were detected Salmonella positive by conventional microbiological methods. On the other hand, 27 (12.7%) samples were negative by PCR and 40 (18.8%) samples were negative by conventional microbiological methods. PCR assay proved to be an effective method for Salmonella detection in meat and poultry products with high specificity and sensitivity and more importantly a less time-consuming procedure. Using PCR, Salmonella spp. detection could be achieved within 24–36 h compared to 3–8 days for the conventional microbiological methods.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.