Prolin-rich tyrosine kinase 2 (PYK2) expression and localization in mouse testis

Prolin-rich kinase 2 (PYK2) is a nonreceptor tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). PYK2 is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha (TNF-alpha), changes in osmolarity, elevation in intracellular calcium concentration, angiotensin, and UV irradiation. PYK2 has ligand sequences for Src homology 2 and 3 (SH-2 and SH-3), and has binding sites for paxillin and p130(cas). Activation of PYK2 leads to modulation of ion channel function, phosphorylation of tyrosine residues, and activation of the MAP kinase signaling pathways. Immunocytochemistry shows that PYK2 is present in mouse germinal and Sertoli cells (ser). Northern blot and immunoprecipitation analysis demonstrate that, among germinal cells, PYK2 is more abundant in spermatocytes (spc) and spermatids (spt); in addition, immunofluorescence analysis clearly shows that the diffuse cytoplasmic localization of PYK2 changes in a specific cellular compartment in spt and spermatozoa.     

Atypical Human Infections by Animal Trypanosomes

The two classical forms of human trypanosomoses are sleeping sickness due to Trypanosoma brucei gambiense or T. brucei rhodesiense, and Chagas disease due to T. cruzi. However, a number of atypical human infections caused by other T. species (or sub-species) have been reported, namely due to T. brucei brucei, T. vivax, T. congolense, T. evansi, T. lewisi, and T. lewisi-like. These cases are reviewed here. Some infections were transient in nature, while others required treatments that were successful in most cases, although two cases were fatal. A recent case of infection due to T. evansi was related to a lack of apolipoprotein L-I, but T. lewisi infections were not related to immunosuppression or specific human genetic profiles. Out of 19 patients, eight were confirmed between 1974 and 2010, thanks to improved molecular techniques. However, the number of cases of atypical human trypanosomoses might be underestimated. Thus, improvement, evaluation of new diagnostic tests, and field investigations are required for detection and confirmation of these atypical cases.

Key Learning Points

• The classical human trypanosomoses are human African trypanosomosis (HAT) or sleeping sickness, and Chagas disease, the Latin American human trypanosomosis.
• Atypical human infections caused by Trypanosoma species that normally are restricted to animals have been reported. These cases of atypical human trypanosomoses (a-HT) are mostly transient, but some require treatment and can be fatal.
• Only a few cases of a-HT have been fully confirmed, especially in Asia, leading to the hypothesis that the actual prevalence is probably underestimated.
• The detection of a case of a-HT should be based on observation of the parasite by direct microscopy. Evaluating/improving the diagnoses through serological and PCR assays would help in detecting and identifying atypical trypanosomosis infections in humans. These laboratory research and field activities are needed to evaluate the actual occurrence of atypical cases.

Top Five Papers

1. Verma A, Manchanda S, Kumar N, Sharma A, Goel M, et al. (2011) Trypanosoma lewisi or Trypanosoma lewisi-like infection in a 37-day-old infant. Am J Trop Med Hyg 85: 221–224.
2. Deborggraeve S, Koffi M, Jamonneau V, Bonsu FA, Queyson R, et al. (2008) Molecular analysis of archived blood slides reveals an atypical human Trypanosoma infection. Diagn Microbiol Infect Dis 61: 428–433.
3. Vanhollebeke B, Truc P, Poelvoorde P, Pays A, Joshi PP, et al. (2006) Human Trypanosoma evansi infection linked to a lack of apolipoprotein L-I. N Engl J Med 355: 2752–2756.
4. Joshi PP, Shegokar V, Powar S, Herder S, Katti R, et al. (2005) Human trypanosomiasis caused by Trypanosoma evansi in India: the first case report. Am J Trop Med Hyg 73: 491–495.
5. Howie S, Guy M, Fleming L, Bailey W, Noyes H, et al. (2006) A Gambian infant with fever and an unexpected blood film. PLoS Med 3: e355. doi:10.1371/journal.pmed.0030355.

     

Hyaluronan-CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9

Osteoclast (OC) precursors migrate to putative sites of bone resorption to form functionally active, multinucleated cells. The preOC FLG 29.1 cells, known to be capable of irreversibly differentiating into multinucleated OC-like cells, displayed several features of primary OCs, including expression of specific integrins and the hyaluronan (HA) receptor CD44. OC-like FLG 29.1 cells adhered to and extensively migrated through membranes coated with fibronectin, vitronectin, and laminins, but, although strongly binding to HA, totally failed to move on this substrate. Moreover, soluble HA strongly inhibited OC-like FLG 29.1 cell migration on the permissive matrix substrates, and this behavior was dependent on its engagement with CD44, as it was fully restored by function-blocking anti-CD44 antibodies. HA did not modulate the cell-substrate binding affinity/avidity nor the expression levels of the corresponding integrins. MMP-9 was the major secreted metalloproteinase used by OC-like FLG 29.1 cells for migration, because this process was strongly inhibited by both TIMP-1 and GM6001, as well as by MMP-9-specific antisense oligonucleotides. After HA binding to CD44, a strong down-regulation of MMP-9 mRNA and protein was detected. These findings highlight a novel role of the HA-CD44 interaction in the context of OC-like cell motility, suggesting that it may act as a stop signal for bone-resorbing cells.     

NSAIDs counteract H. pylori VacA toxin-induced cell vacuolation in MKN 28 gastric mucosal cells

The relationship between nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori-induced gastric mucosal injury is still under debate. VacA toxin is an important H. pylori virulence factor that causes cytoplasmic vacuolation in cultured cells. Whether and how NSAIDs affect VacA-induced cytotoxicity is unclear. This study was designed to evaluate the effect of NSAIDs on H. pylori VacA toxin-induced cell vacuolation in human gastric mucosal cells in culture (MKN 28 cell line). Our data show that 1) NSAIDs (indomethacin, aspirin, and NS-398) inhibit VacA-induced cell vacuolation independently of inhibition of cell proliferation and prostaglandin synthesis; 2) NSAIDs impair vacuole development/maintenance without affecting cell binding and internalization of VacA; and 3) NSAIDs, as well as the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid, also inhibit cell vacuolation induced by ammonia. We thus hypothesize that NSAIDs might protect MKN 28 cells against VacA-induced cytotoxicity by inhibiting VacA channel activity required for vacuole genesis.     

Layer-by-Layer coated tyrosinase: An efficient and selective synthesis of catechols

Agaricus bisporous tyrosinase was immobilized on commercial available epoxy-resin Eupergit®C250L and then coated by the Layer-by-Layer method (LbL). The two novel heterogeneous biocatalysts were characterized for their morphology, pH and storage stability, kinetic properties (K_m, V_max, V_max/K_m) and reusability. These biocatalysts were used for the efficient and selective synthesis of bioactive catechols under mild and environmental friendly experimental conditions. Ascorbic acid was added in the reaction medium to inhibit the formation of ortho-quinones, thus avoiding the known enzyme suicide inactivation process. Catechols were obtained mostly in quantitative yields and conversion of substrate. Tyrosinase immobilized on Eupergit®C250L and coated by the LbL method showed better catalytic activities, higher pH and storage stability, and reusability with respect to immobilized uncoated tyrosinase. Since chemical procedures to synthesize catechols are often expensive and with high environmental impact, the use of immobilized tyrosinase represents an efficient alternative for the preparation of this family of bioactive compounds.     

Oxysterol-induced up-regulation of MCP-1 expression and synthesis in macrophage cells

To investigate the proinflammatory potential of cholesterol and cholesterol oxidation products (oxysterols), which are present in oxidized low-density lipoproteins, foam cells, and fibrotic plaque, we used an in vitro model mimicking the challenge of macrophage cells by the cholesterol accumulating within the central core of atheroma. A biologically representative oxysterol mixture was shown to be potentially able to sustain a chronic inflammatory process within the vascular wall by up-regulating the expression of defined proinflammatory genes. In particular, expression and synthesis of the major chemokine for monocytes/macrophages, namely monocyte chemotactic protein-1 (MCP-1), were consistently increased when cells of the macrophage lineage (U937 cell line) were incubated with this mixture. On the contrary, an identical concentration of unoxidized cholesterol in no case modified expression or synthesis of the chemokine. Up-regulated expression and synthesis of MCP-1 by the oxysterol mixture was clearly dependent on a net increment of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor kappaB (NF-kappaB) nuclear binding. The results indicate that cholesterol may contribute to the progression of atherosclerotic lesions by strongly up-regulating crucial proinflammatory factors like MCP-1, but only after having been oxidized to oxysterols.     

Identification, cloning, nucleotide sequence and chromosomal map location of hns, the structural gene for Escherichia coli DNA-binding protein H-NS

Summary Beginning with a synthetic oligonucleotide probe derived from its amino acid sequence, we have identified, cloned and sequenced the hns gene encoding H-NS, an abundant Escherichia coli 15 kDa DNA-binding protein with a possible histone-like function. The amino acid sequence of the protein deduced from the nucleotide sequence is in full agreement with that determined for H-NS. By comparison of the restriction map of the cloned gene and of its neighboring regions with the physical map of E. coli K12 as well as by hybridization of the hns gene with restriction fragments derived from the total chromosome, we have located the hns gene oriented counterclockwise at 6.1 min on the E. coli chromosome, just before an IS30 insertion element.     

Flexible nonlinear blind signal separation in the complex domain

This paper introduces an Independent Component Analysis (ICA) approach to the separation of nonlinear mixtures in the complex domain. Source separation is performed by a complex INFOMAX approach. The neural network which realizes the separation employs the so called "Mirror Model" and is based on adaptive activation functions, whose shape is properly modified during learning. Nonlinear functions involved in the processing of complex signals are realized by pairs of spline neurons called "splitting functions", working on the real and the imaginary part of the signal respectively. Theoretical proof of existence and uniqueness of the solution under proper assumptions is also provided. In particular a simple adaptation algorithm is derived and some experimental results that demonstrate the effectiveness of the proposed solution are shown.     

Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo.