Increase of 5-HT levels is induced both in mouse brain and HEK-293 cells following their exposure to a non-viral tryptophan hydroxylase construct.

Tryptophan hydroxylase type 2 (Tph2) is the rate-limiting enzyme for serotonin (5-HT) biosynthesis in the brain. Dysfunctional Tph2 alters 5-HT biosynthesis, leading to a deficiency of 5-HT, which could have repercussions on human behavior. In the last decade, several studies have associated polymorphisms of the TPH2 gene with suicidal behavior. Additionally, a 5-HT deficiency has been implicated in various psychiatric pathologies, including alcoholism, impulsive behavior, anxiety, and depression. Therefore, the TPH2 gene could be an ideal target for analyzing the effects of a 5-HT deficiency on brain function. The aim of this study was to use the construct pIRES-hrGFP-1a-Tph2-FLAG to treat CD1-male mice and to transfect HEK-293-cells and then to evaluate whether this treatment increases 5-HT production. 5-HT levels were enhanced 48 h post-transfection, in HEK-293 cells. Three days after the ocular administration of pIRES-hrGFP-1a-Tph2-FLAG to mice, putative 5-HT production was significantly higher than in the control in both hypothalamus and amygdala, but not in the brainstem. Further research will be needed on the possible application of this treatment for psychiatric diseases involving a Tph2 dysfunction or serotonin deficiency.

Parasite protein phosphatases: biological function, virulence, and host immune evasion.

Protein phosphatases are enzymes that dephosphorylate tyrosine and serine/threonine amino acid residues. Although their role in cellular processes has been best characterized in higher eukaryotes, they have also been identified and studied in different pathogenic microorganisms (e.g., parasites) in the last two decades. Whereas some parasite protein phosphatases carry out functions similar to those of their homologs in yeast and mammalian cells, others have unique structural and/or functional characteristics. Thus, the latter unique phosphatases may be instrumental as targets for drug therapy or as markers for diagnosis. It is important to better understand the involvement of protein phosphatases in parasites in relation to their cell cycle, metabolism, virulence, and evasion of the host immune response. The up-to-date information about parasite phosphatases of medical and veterinarian relevance is herein reviewed.

Proteomic characterization of the pellicle of Toxoplasma gondii.

Toxoplasma gondii is one of the most successful intracellular parasites in the world. The dynamic, adhesion, invasion, and even replication capabilities of Toxoplasma are based on dynamic machinery located in the pellicle, a three membrane complex that surrounds the parasite. Among the proteins that carry out these processes are inner membrane complex (IMC) proteins, gliding-associated proteins (GAP), diverse myosins, actin, tubulin, and SRS proteins. Despite the importance of the pellicle, the knowledge of its composition is limited. Broad protein identification from an enriched pellicle fraction was obtained by independent digestion with trypsin and chymotrypsin and quantified by mass spectrometry. By trypsin digestion, 548 proteins were identified, while by chymotrypsin digestion, additional 22 proteins were identified. Besides, a group of "sequences related to SAG1" proteins (SRS) were detected together with unidentified new proteins. From identified SRS proteins, SRS51 was chosen for analysis and modeling as its similarities with crystallized adhesion proteins, exhibiting the presence of a spatial groove that is apparently involved in adhesion and cell invasion. As SRS proteins have been reported to be involved in the activation of the host's immune response, further studies could consider them as targets in the design of vaccines or of drugs against Toxoplasma. SIGNIFICANCE: To date, the proteomic composition of the pellicle of Toxoplasma is unknown. Most proteins reported in Toxoplasma pellicle have been poorly studied, and many others remain unidentified. Herein, a group of new SRS proteins is described. Some SRS proteins previously described from pellicle fraction have adhesion properties to the host cell membrane, so their study would provide data related to invasion mechanism and to open possibilities for considering them as targets in the design of immunoprotective strategies or the design of new pharmacological treatments.

Cellular Identification and In Silico Characterization of Protein Phosphatase 2C (PP2C) of Cryptosporidium parvum.

PURPOSE: Cryptosporidium parvum is an Apicomplexa parasite that causes watery diarrhea (cryptosporidiosis), especially in children and immunocompromised adults (the latter in a very severe form). No effective treatment exists against infection by this parasite. Phosphatases participate in the regulation of various cellular functions and are thus considered potential therapeutic targets in many diseases. The aim of the present study was to indirectly identify and in silico characterize a protein phosphatase 2C of C. parvum. METHODS: Western blot and indirect immunofluorescence microscopy were performed with a polyclonal antibody against Leishmania major PP2C. Possible cross-reactivity with LmPP2C was assessed by in silico sequence homology to analyze phylogenetic relationships between distinct C. parvum PP2Cs. In addition, another bioinformatics approach was used to predict the possible relationship and function of C. parvum PP2C in the regulation of several cellular processes. RESULTS: Western blotting showed a protein of approximately 72 kDa. With immunofluorescence, PP2C was localized in the nucleus of oocysts (with some additional labeling in the cytoplasm) and at the apical region of sporozoites. By aligning C. parvum PP2C with known ortholog sequences and carrying out PPI analysis, a determination could be made of the degree of conservation of these enzymes, their possible relationship, and their function in the regulation of several cellular processes associated with a likely nuclear location. CONCLUSION: Microscopic localization by immunofluorescence identified CpPP2C at the nucleus in oocysts and at the apical end of the sporozoite body. Hence, this enzyme could be associated with proteins that have an important role in the regulation of transcription and other processes orchestrated by MAPK kinases, according to in silico analysis.

Pilot study of whole-blood gamma interferon response to the Vibrio cholerae toxin B subunit and resistance to enterotoxigenic Escherichia coli-associated diarrhea.

Enterotoxigenic Escherichia coli (ETEC), which produces heat-labile toxin (LT), is a common cause of travelers' diarrhea (TD). The B subunit of ETEC LT is immunologically related to the B subunit of Vibrio cholerae toxin (CT). In this pilot study we evaluated the whole-blood gamma interferon response to CT B in 17 U.S. adults traveling to Mexico. Only one of nine subjects who demonstrated a cellular immune response as determined by whole-blood gamma interferon production to CT B on arrival to Mexico developed diarrhea, whereas five of eight without a cellular response developed diarrhea. Markers of the cellular immune response to ETEC LT could help in identifying individuals immune to ETEC LT, and these markers deserve additional study.