Thrombospondin-1 expression and modulation of Wnt and hippo signaling pathways during the early phase of Trypanosoma cruzi infection of heart endothelial cells

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/β-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/β-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/β-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3β at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3β was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of β-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of β-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating β-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a β-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the β-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.     

Up-regulation of expression of translation factors – a novel molecular mechanism for cadmium carcinogenesis

The molecular mechanisms potentially responsible for cadmium carcinogenesis were investigated by differential gene expression analysis of Balb/c-3T3 cells morphologically transformed with cadmium chloride. Differential display analysis of gene expression revealed overexpression of mouse Translation Initiation Factor 3 (TIF3; GenBank Accession Number AF 271072) and Translation Elongation Factor-1delta (TEF-1delta; GenBank Accession Number AF 304351) in the transformed cells compared with the control cells. The full length cDNAs for TIF3 and TEF-1delta were cloned and sequenced. Transfection of mammalian cells with an expression vector containing either TIF3 or TEF-1delta cDNA resulted in overexpression of the encoded protein. Overexpression of the cDNA-encoded TIF3 and TEF-1delta proteins in NIH3T3 cells was oncogenic as evidenced by the appearance of transformed foci capable of anchorage-independent growth on soft agar and tumorigenesis in nude mouse. Blocking the translation of TIF3 and TEF-1delta proteins using the corresponding antisense mRNA resulted in a significant reversal of the oncogenic potential of cadmium transformed Balb/c-3T3 cells as evidenced from the suppression of anchorage-independent growth on soft agar and diminished tumorigenesis in nude mouse. These findings demonstrate that the up-regulation of expression of TIF3 and TEF-1delta is a novel molecular mechanism responsible, at least in part, for cadmium carcinogenesis.     

Rho kinase is required for CCR7-mediated polarization and chemotaxis of T lymphocytes

A chemokine receptor, CXCR4, and its endogenous ligand, stromal cell-derived factor-1 (SDF-1), have been recognized to be involved in the metastasis of several types of cancers. T140 analogs are peptidic CXCR4 antagonists composed of 14 amino acid residues that were previously developed as anti-HIV agents having inhibitory activity against HIV-entry through its co-receptor, CXCR4. Herein, we report that these compounds effectively inhibited SDF-1-induced migration of human breast cancer cells (MDA-MB-231), human leukemia T cells (Sup-T1) and human umbilical vein endothelial cells at concentrations of 10–100 nM in vitro. Furthermore, slow release administration by subcutaneous injection using an Alzet osmotic pump of a potent and bio-stable T140 analog, 4F-benzoyl-TN14003, gave a partial, but statistically significant (P≤0.05 (t-test)) reduction in pulmonary metastasis of MDA-MB-231 in SCID mice, even though no attempt was made to inhibit other important targets such as CCR7. These results suggest that T140 analogs have potential use for cancer therapy, and that small molecular CXCR4 antagonists could potentially replace neutralizing antibodies as anti-metastatic agents for breast cancer.     

D-Peptides as inhibitors of the DnaK/DnaJ/GrpE chaperone system

DnaK, a Hsp70 homolog of Escherichia coli, together with its co-chaperones DnaJ and GrpE protects denatured proteins from aggregation and promotes their refolding by an ATP-consuming mechanism. DnaJ not only stimulates the gamma-phosphate cleavage of DnaK-bound ATP but also binds polypeptide substrates on its own. Unfolded polypeptides, such as denatured luciferase, thus form ternary complexes with DnaJ and DnaK. A previous study has shown that d-peptides compete with l-peptides for the same binding site in DnaJ but do not bind to DnaK (Feifel, B., Sch?nfeld, H.-J., and Christen, P. (1998) J. Biol. Chem. 273, 11999-12002). Here we report that d-peptides efficiently inhibit the refolding of denatured luciferase by the DnaK/DnaJ/GrpE chaperone system (EC50 = 1-2 microM). The inhibition of the chaperone action is due to the binding of d-peptide to DnaJ (Kd = 1-2 microM), which seems to preclude DnaJ from forming ternary (ATP.DnaK)m.substrate.DnaJn complexes. Apparently, simultaneous binding of DnaJ and DnaK to one and the same target polypeptide is essential for effective chaperone action.     

Stratification of solids,nitrogen and phosphorus in swine manure in deep pits under slatted floors

Manure slurries stored in pits under slatted floors of both finishing and nursery barns were sampled at four different depths to study stratifications of total solids (TS) and nutrients (nitrogen and phosphorus), and to determine the relationship between the stratification of TS and nutrients. The results obtained can be used to improve the management and handling of swine manure in the under-slat storage pits. A management scheme that can be adopted for both the finishing and the nursery barns' pits is the layer-by-layer harvesting of the manure. The thinner manure, which is lower in nutrients, can be spread on land near the production units in larger volumes or it may pumped to land remote from the production units without causing many clogging problems. The thicker manure, higher in nutrients, can be transported to land further away and spread in smaller volumes. The TS content of each stratum can be used to accurately estimate the nitrogen and phosphorus levels in the respective strata so that application rates can easily be adjusted accordingly during the time of land application.     

Changes in Saccharide Metabolism Induced by Infection of Camellia Sinensis by Exobasidium Vexans

Changes in saccharide contents of tea leaves during infection with blister blight fungus Exobasidium vexans Masse was studied. Saccharose and glucose contents decreased in the blistered portions when compared to the normal regions until sporulation and remained constant during the entire period of sporulation. Fructose content increased abruptly during the initiation of sporulation and remained constant up to the end of sporulation in both blistered and non-blistered regions. Starch content continuously decreased in the blistered region. Peroxidase activity was highly enhanced during the final stages of leaf senescence. The activity of acid invertase was inversely proportional to the starch content and closely related to the changes in the saccharose and glucose contents. Protein and chlorophyll contents gradually decreased in the blistered regions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modeling effects of granules on the start-up of anaerobic digestion of dairy wastewater with Langmuir and extended Freundlich equations

The effects of granules-inocula on the start-up of anaerobic reactors treating dairy manure were studied in a batch-fed reactor. The effects of start-up period and ratio of granules to feed were analyzed. Results indicated that the effects of start-up period could be described by Langmuir model, while the Extended Freundlich model could be used to model the effects of ratio of granules to feed on cumulative biogas production. In addition, transmission electron microscopes (TEM) and scanning electron microscope analysis were conducted to elucidate the distribution of microbial population and micro-colonies in granules and manure. From the TEM micrographs analyses, the ratios the Syntrophobacter and methanogens in granule and manure were shown to be 1.57 ± 0.42 and 0.22 ± 0.20, respectively. These results demonstrated that granules-inocula could reduce the period required for onset of biogas by 25%.     

Genetic diversity of UPASI tea clones (Camellia sinensis (L.) O. Kuntze) on the basis of total catechins and their fractions

Tea leaf catechins and the ratio of dihydroxylated to trihydroxylated catechin fractions were analysed to identify the genetic diversity of 26 UPASI released tea clones. Principal component analysis (PCA) based on regression factor separated tea clones into five groups according to their jats (Jats are region based rays for e.g., Assam, China and Cambod origin) as well as their quality constituents (such as total polyphenols, total catechins, amino acids in the green leaves and liquor characteristics of black tea), particularly the catechins. Group 1 represented medium quality (quality of the final produce) clones, such as UPASI-10, UPASI-12 and UPASI-15 and drought tolerant clones like UPASI-1, UPASI-2, UPASI-9 and UPASI-10. Group 2 contained purely "China" cultivars while group 3 possessed high quality tea cultivars. "Assam" (group 5) teas had the lowest ratio of dihydroxylated to trihydroxylated catechin fractions (1:4) than the "Chinery" (group 2) teas (1:5). This biochemical differentiation indicated that there is a vast genetic diversity in UPASI released tea clones in terms of catechin fractions, even though the majority of them were selected from one tea estate located in the Nilgiris.     

Tazobactam forms a stoichiometric trans-enamine intermediate in the E166A variant of SHV-1 beta-lactamase: 1.63 A crystal structure

Many pathogenic bacteria develop antibiotic resistance by utilizing beta-lactamases to degrade penicillin-like antibiotics. A commonly prescribed mechanism-based inhibitor of beta-lactamases is tazobactam, which can function either irreversibly or in a transient manner. We have demonstrated previously that the reaction between tazobactam and a deacylation deficient variant of SHV-1 beta-lactamase, E166A, could be followed in single crystals using Raman microscopy [Helfand, M. S., et al. (2003) Biochemistry 42, 13386-13392]. The Raman data show that maximal populations of an enamine-like intermediate occur 20-30 min after "soaking in" has commenced. By flash-freezing crystals in this time frame, we were able to trap the enamine species. The resulting 1.63 A resolution crystal structure revealed tazobactam covalently bound in the trans-enamine intermediate state with close to 100% occupancy in the active site. The Raman data also indicated that tazobactam forms a larger population of enamine than sulbactam or clavulanic acid does and that tazobactam's intermediate is also the most long-lived. The crystal structure provides a rationale for this finding since only tazobactam is able to form favorable intra- and intermolecular interactions in the active site that stabilize this trans-enamine intermediate. These interactions involve both the sulfone and triazolyl groups that distinguish tazobactam from clavulanic acid and sulbactam, respectively. The observed stabilization of the transient intermediate of tazobactam is thought to contribute to tazobactam's superior in vitro and in vivo clinical efficacy. Understanding the structural details of differing inhibitor effectiveness can aid the design of improved mechanism-based beta-lactamase inhibitors.