Elevation of glutathione in phenylalanine mustard-resistant murine L1210 leukemia cells

Murine L1210 leukemia cells resistant to the antineoplastic agent L-phenylalanine mustard have a 1.5-2.0-fold elevation in their cellular GSH and GSSG content as compared to drug-sensitive cells. Cellular uptake of L-[U-14C]cystine and its incorporation into GSH of the resistant tumor are correspondingly elevated. Synthesis of gamma-glutamylcysteine, GSH, and GSSG is elevated 1.5-2.0-fold in cell-free preparations of the resistant tumor. This increased synthesis of GSH is attributed to increased cellular content (1.6-fold) of gamma-glutamylcysteine synthetase. GSH synthetase activity is equivalent in both drug-sensitive and -resistant cells. Investigation into the hydrolysis of selected peptides by cell-free preparations of both sensitive and resistant tumors suggest that aminopeptidase M participates in the formation of L-cysteine from L-Cys-Gly. This is supported by the observation that these preparations readily degrade L-Leu-p-nitroanilide and L-Ala-L-Ala-L-Ala, known substrates for aminopeptidase M, but not dipeptidase. The failure of the tumors to degrade Gly-D-Ala, a dipeptidase substrate, and the marked inhibition of L-Ala-Gly, L-Cys-Gly, and L-Ala-L-Ala-L-Ala hydrolysis by Bestatin further support a role for aminopeptidase M in the generation of L-cysteine from L-Cys-Gly. These results suggest that the drug-resistant tumor cell has developed an efficient mechanism for maintenance of elevated GSH which involves both gamma-glutamyl transpeptidase-initiated catabolism of GSH to cysteine and its reutilization by gamma-glutamylcysteine synthetase.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Selected anthropometric variables and aerobic fitness as predictors of cardiovascular disease risk in children

The aim of this study was to assess the suitability of body mass index, waist circumference, waist-to-height ratio and aerobic fitness as predictors of cardiovascular risk factor clustering in children. A cross-sectional study was conducted with 290 school boys and girls from 6 to 10 years old, randomly selected. Blood was collected after a 12-hour fasting period. Blood pressure, waist circumference (WC), height and weight were evaluated according to international standards. Aerobic fitness (AF) was assessed by the 20-metre shuttle-run test. Clustering was considered when three of these factors were present: high systolic or diastolic blood pressure, high low-density lipoprotein (LDL) cholesterol, high triglycerides, high plasma glucose, high insulin concentrations and low high-density lipoprotein (HDL) cholesterol. A ROC curve identified the cut-off points of body mass index (BMI), WC, waist-to-height ratio (WHtR) and AF as predictors of risk factor clustering. BMI, WC and WHR resulted in significant areas under the ROC curves, which was not observed for AF. The anthropometric variables were good predictors of cardiovascular risk factor clustering in both sexes, whereas aerobic fitness should not be used to identify cardiovascular risk factor clustering in these children.     

Cell proliferation and STAT6 pathways are negatively regulated in T cells by STAT1 and suppressors of cytokine signaling

Suppressor of cytokine signaling (SOCS) proteins have emerged as important regulators of cytokine signals in lymphocytes. In this study, we have investigated regulation of SOCS expression and their role in Th cell growth and differentiation. We show that SOCS genes are constitutively expressed in naive Th cells, albeit at low levels, and are differentially induced by Ag and Th-polarizing cytokines. Whereas cytokines up-regulate expression of SOCS1, SOCS2, SOCS3, and cytokine-induced Src homology 2 protein, Ags induce down-regulation of SOCS3 within 48 h of Th cell activation and concomitantly up-regulate SOCS1, SOCS2, and cytokine-induced Src homology 2 protein expression. We further show that STAT1 signals play major roles in inducing SOCS expression in Th cells and that induction of SOCS expression by IL-4, IL-12, or IFN-gamma is compromised in STAT1-deficient primary Th cells. Surprisingly, IL-4 is a potent inducer of STAT1 activation in Th2 but not Th1 cells, and SOCS1 or SOCS3 expression is dramatically reduced in STAT1(-/-) Th2 cells. To our knowledge, this is the first report of IL-4-induced STAT1 activation in Th cells, and suggests that its induction of SOCS, may in part, regulate IL-4 functions in Th2 cells. In fact, overexpression of SOCS1 in Th2 cells represses STAT6 activation and profoundly inhibits IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 cDNA construct enhances cell proliferation and induces constitutive activation of STAT6 in Th2 cells. These results are consistent with a model where IL-4 has dual effects on differentiating T cells: it simulates proliferation/differentiation through STAT6 and autoregulates its effects on Th2 growth and effector functions via STAT1-dependent up-regulation of SOCS proteins.     

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

Meta-analysis of Correlated Traits via Summary Statistics from GWASs with an Application in Hypertension

Genome-wide association studies (GWASs) have identified many genetic variants underlying complex traits. Many detected genetic loci harbor variants that associate with multiple—even distinct—traits. Most current analysis approaches focus on single traits, even though the final results from multiple traits are evaluated together. Such approaches miss the opportunity to systemically integrate the phenome-wide data available for genetic association analysis. In this study, we propose a general approach that can integrate association evidence from summary statistics of multiple traits, either correlated, independent, continuous, or binary traits, which might come from the same or different studies. We allow for trait heterogeneity effects. Population structure and cryptic relatedness can also be controlled. Our simulations suggest that the proposed method has improved statistical power over single-trait analysis in most of the cases we studied. We applied our method to the Continental Origins and Genetic Epidemiology Network (COGENT) African ancestry samples for three blood pressure traits and identified four loci (CHIC2, HOXA-EVX1, IGFBP1/IGFBP3, and CDH17; p < 5.0 × 10⁻⁸) associated with hypertension-related traits that were missed by a single-trait analysis in the original report. Six additional loci with suggestive association evidence (p < 5.0 × 10⁻⁷) were also observed, including CACNA1D and WNT3. Our study strongly suggests that analyzing multiple phenotypes can improve statistical power and that such analysis can be executed with the summary statistics from GWASs. Our method also provides a way to study a cross phenotype (CP) association by using summary statistics from GWASs of multiple phenotypes.     

Breakdown of tolerance to a neo-self antigen in double transgenic mice in which B cells present the antigen

Transgenic (Tg) mice expressing a foreign Ag, hen egg lysozyme (HEL), under control of the alphaA-crystallin promoter ("HEL-Tg" mice) develop immunotolerance to HEL attributed to the expression of HEL in their thymus. In this paper we analyzed the immune response in double (Dbl)-Tg mice generated by mating the HEL-Tg mice with Tg mice that express HEL Abs on their B cells ("Ig-Tg" mice). The B cell compartment of the Dbl-Tg mice was unaffected by the HEL presence and was essentially identical to that of the Ig-Tg mice. A partial breakdown of tolerance was seen in the T cell response to HEL of the Dbl-Tg mice, i.e., their lymphocyte proliferative response against HEL was remarkably higher than that of the HEL-Tg mice. T-lymphocytes of both Dbl-Tg and Ig-Tg mice responded to HEL at concentrations drastically lower than those found stimulatory to lymphocytes of the wild-type controls. Cell mixing experiments demonstrated that 1) the lymphocyte response against low concentrations of HEL is due to the exceedingly efficient Ag presenting capacity of the Ab expressing B cells and 2) breakdown of tolerance in Dbl-Tg mice can also be attributed to the APC capacity of B cells, that sensitize in vivo and stimulate in vitro populations of T cells with low affinity toward HEL, assumed to be escapees of thymic deletion. These results thus indicate that T cell tolerance can be partially overcome by the highly potent Ag presenting capacity of Ab expressing B cells.     

Cell-cell interaction with APC, not IL-23, is required for naive CD4 cells to acquire pathogenicity during Th17 lineage commitment

Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when presensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-β, respectively. In this study, we generated two Th17 subpopulations by using a system in which naive CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-β and, concurrently, are activated either with HEL presented by APCs, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL. Naive CD4 cells activated by the anti-CD3/CD28 Abs acquired pathogenicity, however, when cocultured with HEL/APC. Importantly, the naive CD4 cells did not acquire pathogenicity when cocultured with APCs stimulated with LPS or when separated from the HEL-presenting cells by a semipermeable membrane. Unlike with presensitized Th17, soluble IL-23 does not participate in pathogenicity acquisition by naive CD4 cells; no pathogenicity was induced by adding IL-23 to cultures activated with anti-CD3/CD28 Abs. Furthermore, Abs against IL-23 or IL-23R did not inhibit acquisition of pathogenicity in cultures of naive CD4 cells activated by HEL/APC. Our data thus show that, unlike presensitized CD4 cells, naive CD4 cells polarized toward Th17 phenotype acquire pathogenicity only by direct interaction with APCs presenting the Ag, with no apparent involvement of soluble IL-23. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in pathogenic and other immune processes, along with the IL-23-dependent Th17 cells.     

Both Th1 and Th17 are immunopathogenic but differ in other key biological activities

The role of Th17 lymphocytes in immunopathogenic processes has been well established, but little is known about their basic cell features. In this study, we compared polarized Th1 and Th17 for key biological activities related to pathogenicity and trafficking. Th1 and Th17 lineages were derived from TCR-transgenic CD4 murine cells specific against hen egg lysozyme. When adoptively transferred into mice expressing hen egg lysozyme in their eyes, both Th1 and Th17 induced ocular inflammation but with slight differences in histological pathology. PCR analysis revealed selective expression of IFN-gamma or IL-17 in eyes of Th1 or Th17 recipients, respectively. Additionally, Th1 and Th17 were found to differ in three other key activities: 1) Th17 cells were inferior to Th1 cells in their capacity to trigger massive lymphoid expansion and splenomegaly; 2) the proportion of Th1 cells among infiltrating cells in inflamed recipient eyes declined rapidly, becoming a minority by day 7, whereas Th17 cells remained in the majority throughout this period; and 3) remarkable differences were noted between Th1 and Th17 cells in their expression of certain surface markers. In particular, reactivated Th1 expressed higher levels of CD49d and alpha(4)beta(7) (mucosal homing) in vitro and higher levels of CXCR3 (Th1 trafficking) in vivo. Reactivated Th17, however, expressed higher levels of alpha(E)beta(7) (epithelial tissue homing) and CD38 (activation, maturation and trafficking) in vitro, but in vivo Th17 expressed higher levels of alpha(4)beta(7) and CCR6 (lymphocyte trafficking). These data reveal that Th1 and Th17 cells differ in several key biological activities influencing migration and pathogenic behavior during inflammatory disease.     

Intense photon emission induced by fracture of γ–irradiated Dy‐, Tm‐, Sm‐ and Mn‐doped CaSO4 single crystals

When an γ‐irradiated Dy‐, Tm‐, Sm‐ or Mn‐doped CaSO₄ crystal is impulsively deformed, two peaks appear in the ML intensity versus time curve, whereby the first ML peak is found in the deformation region and the second in the post‐deformation region of the crystals. In this study, intensities I_m1 and I_m2 corresponding to first and second ML peaks, respectively, increased linearly with an impact velocity v₀ of the piston used to deform the crystals, and times t_m1 and t_m2 corresponding to the first and second ML peaks, respectively, decreased with impact velocity. Total ML intensity initially increased with impact velocity and then reached a saturation value for higher values of impact velocity. ML intensity increased with increasing γ‐doses and size of crystals. Results showed that the electric field produced as a result of charging of newly‐created surfaces caused tunneling of electrons to the valence band of the hole‐trapping centres. The free holes generated moved in the valence band and their subsequent recombination with electron trapping centres released energy, thereby resulting in excitation of luminescent centres. Copyright © 2012 John Wiley & Sons, Ltd.