Heme Amplifies the Innate Immune Response to Microbial Molecules through Spleen Tyrosine Kinase (Syk)-dependent Reactive Oxygen Species Generation

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.     

TLR4 recognizes Pseudallescheria boydii conidia and purified rhamnomannans

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.     

Free-energy profiles for catalysis by dual-specificity phosphatases

PTPs (protein tyrosine phosphatases) are fundamental enzymes for cell signalling and have been linked to the pathogenesis of several diseases, including cancer. Hence, PTPs are potential drug targets and inhibitors have been designed as possible therapeutic agents for Type II diabetes and obesity. However, a complete understanding of the detailed catalytic mechanism in PTPs is still lacking. Free-energy profiles, obtained by computer simulations of catalysis by a dual-specificity PTP, are shown in the present study and are used to shed light on the catalytic mechanism. A highly accurate hybrid potential of quantum mechanics/molecular mechanics calibrated specifically for PTP reactions was used. Reactions of alkyl and aryl substrates, with different protonation states and PTP active-site mutations, were simulated. Calculated reaction barriers agree well with experimental rate measurements. Results show the PTP substrate reacts as a bi-anion, with an ionized nucleophile. This protonation state has been a matter of debate in the literature. The inactivity of Cys-->Ser active-site mutants is also not fully understood. It is shown that mutants are inactive because the serine nucleophile is protonated. Results also clarify the interpretation of experimental data, particularly kinetic isotope effects. The simulated mechanisms presented here are better examples of the catalysis carried out by PTPs.     

Increased cerebral oxidative damage and decreased antioxidant defenses in ovariectomized and sham-operated rats supplemented with vitamin A

Previous studies have linked oxidative stress with aging and aging-related processes, including menopause. Abnormalities in the redox state similar to those observed in menopausal women can be modeled experimentally with rat ovariectomy. The aim of the present study was to investigate the effects of vitamin A (retinol palmitate) supplementation (500 or 1,500?IU?kg(-1)?day(-1) for 30?days) on behavioral parameters and brain redox profile in ovariectomized (OVX) and sham-operated rats. Ovariectomy caused pronounced uterine atrophy and decreased locomotor/exploratory activity. Moreover, we found increased hypothalamic and frontal cortex superoxide dismutase/catalase (SOD/CAT) ratio and decreased hippocampal thiol content, accompanied by increased frontal cortex lipid oxidative damage (TBARS) in OVX rats. Vitamin A at 1,500?IUkg(-1)?day(-1) decreased exploratory behavior and decreased total hippocampal thiol content in sham-operated rats, increased hippocampal SOD/CAT ratio and decreased total antioxidant potential in the hippocampus of both sham and OVX groups, and increased cortical TBARS levels in OVX rats. Thus, vitamin A may induce a pro-oxidant state in discrete brain regions of sham-operated and OVX rats. These results suggest some caution regarding the use of high doses of vitamin A supplementation during menopause.     

The spermatogonial stem cell niche in the collared peccary (Tayassu tajacu)

In the seminiferous epithelium, spermatogonial stem cells (SSCs) are located in a particular environment called the "niche" that is controlled by the basement membrane, key testis somatic cells, and factors originating from the vascular network. However, the role of Leydig cells (LCs) as a niche component is not yet clearly elucidated. Recent studies showed that peccaries (Tayassu tajacu) present a peculiar LC cytoarchitecture in which these cells are located around the seminiferous tubule lobes, making the peccary a unique model for investigating the SSC niche. This peculiarity allowed us to subdivide the seminiferous tubule cross-sections in three different testis parenchyma regions (tubule-tubule, tubule-interstitium, and tubule-LC contact). Our aims were to characterize the different spermatogonial cell types and to determine the location and/or distribution of the SSCs along the seminiferous tubules. Compared to differentiating spermatogonia, undifferentiated spermatogonia (A(und)) presented a noticeably higher nuclear volume (P < 0.05), allowing an accurate evaluation of their distribution. Immunostaining analysis demonstrated that approximately 93% of A(und) were GDNF receptor alpha 1 positive (GFRA1(+)), and these cells were preferentially located adjacent to the interstitial compartment without LCs (P < 0.05). The expression of colony-stimulating factor 1 was observed in LCs and peritubular myoid cells (PMCs), whereas its receptor was present in LCs and in GFRA1(+) A(und). Taken together, our findings strongly suggest that LCs, different from PMCs, might play a minor role in the SSC niche and physiology and that these steroidogenic cells are probably involved in the differentiation of A(und) toward type A(1) spermatogonia.     

Sensitivity of double centrifugation sugar fecal flotation for detecting intestinal helminths in coyotes (Canis latrans)

Fecal analysis is commonly used to estimate prevalence and intensity of intestinal helminths in wild carnivores, but few studies have assessed the reliability of fecal flotation compared to analysis of intestinal tracts. We investigated sensitivity of the double centrifugation sugar fecal flotation and kappa agreement between fecal flotation and postmortem examination of intestines for helminths of coyotes (Canis latrans). We analyzed 57 coyote carcasses that were collected between October 2010 and March 2011 in the metropolitan area of Calgary and Edmonton, Alberta, Canada. Before analyses, intestines and feces were frozen at -80 C for 72 hr to inactivate Echinococcus eggs, protecting operators from potential exposure. Five species of helminths were found by postmortem examination, including Toxascaris leonina, Uncinaria stenocephala, Ancylostoma caninum, Taenia sp., and Echinococcus multilocularis. Sensitivity of fecal flotation was high (0.84) for detection of T. leonina but low for Taenia sp. (0.27), E. multilocularis (0.46), and U. stenocephala (0.00). Good kappa agreement between techniques was observed only for T. leonina (0.64), for which we detected also a significant correlation between adult female parasite intensity and fecal egg counts (R(s)=0.53, P=0.01). Differences in sensitivity may be related to parasite characteristics that affect recovery of eggs on flotation. Fecal parasitologic analyses are highly applicable to study the disease ecology of urban carnivores, and they often provide important information on environmental contamination and potential of zoonotic risks. However, fecal-based parasitologic surveys should first assess the sensitivity of the techniques to understand their biases and limitations.     

Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonhereditary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoinositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.     

Mutant p53 Aggregates into Prion-like Amyloid Oligomers and Fibrils: IMPLICATIONS FOR CANCER

Over 50% of all human cancers lose p53 function. To evaluate the role of aggregation in cancer, we asked whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form. The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was demonstrated using x-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, and anti-amyloid immunoassay. The x-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross β-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils accelerated the aggregation of WT p53C, a behavior typical of a prion. The R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect and may serve as a potential target for cancer therapy.     

Reduced muscle carnosine content in type 2, but not in type 1 diabetic patients

Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n=58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (-45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle.