Requirement of UNC93B1 reveals a critical role for TLR7 in host resistance to primary infection with Trypanosoma cruzi

UNC93B1 associates with TLR3, 7, and 9, mediating their translocation from the endoplasmic reticulum to the endolysosome, thus allowing proper activation by microbial nucleic acids. We found that the triple-deficient 3d mice, which lack functional UNC93B1 as well as functional endosomal TLRs, are highly susceptible to infection with Trypanosoma cruzi. The enhanced parasitemia and mortality in 3d animals were associated with impaired proinflammatory response, including reduced levels of IL-12p40 and IFN-γ. Importantly, the phenotype of 3d mice was intermediary between MyD88(-/-) (highly susceptible) and TLR9(-/-) (moderately susceptible), indicating the involvement of an additional UN93B1-dependent TLR(s) on host resistance to T. cruzi. Hence, our experiments also revealed that TLR7 is a critical innate immune receptor involved in recognition of parasite RNA, induction of IL-12p40 by dendritic cells, and consequent IFN-γ by T lymphocytes. Furthermore, we show that upon T. cruzi infection, triple TLR3/7/9(-/-) mice had similar phenotype than 3d mice. These data imply that the nucleic acid-sensing TLRs are critical determinants of host resistance to primary infection with T. cruzi.     

Is volumetric modulated arc therapy with constant dose rate a valid option in radiation therapy for head and neck cancer patients?

Background

Intensity-modulated radiotherapy (IMRT) improves dose distribution in head and neck (HN) radiation therapy. Volumetric-modulated arc therapy (VMAT), a new form of IMRT, delivers radiation in single or multiple arcs, varying dose rates (VDR-VMAT) and gantry speeds, has gained considerable attention. Constant dose rate VMAT (CDR-VMAT) associated with a fixed gantry speed does not require a dedicated linear accelerator like VDR-VMAT. The present study explored the feasibility, efficiency and delivery accuracy of CDR-VMAT, by comparing it with IMRT and VDR-VMAT in treatment planning for HN cancer.

Effects of water deprivation on the water content of cattle skin

In cattle the water content of the skin was determined (1) in the normal animals; (2) after a 3-day period of water deprivation (dehydration); (3) one hour after the water deprived animals had resumed drinking (rehydration)and (4) one hour after the beginning of infusion of water into the rumen of normal animals (overhydration). Dehydration reduced the water content of the skin from 70.6 to 65.8% on average. Rehydration led to a partial restoration of the water content of the skin. Overhydration did not have a measurable effect on the water content of the skin. A rough estimation of the total amount of water lost during dehydration from the total skin of each animal indicated that on average the calves lost 315 ml,the oxen 1,336 ml of water from their skins.

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Zusammenfassung Beim Rind wurde der Wassergehalt der Haut bestimmt (1)in normalem Zustand der Tiere; (2) nach einer dreitägigen Wasserenthaltung der Tiere (Dehydration); (3) eine Stunde nachdem die dehydrierten Tiere wieder zu trinken begannen(Rehydration) und (4) eine Stunde nach begonnener Infusion von Wasser in den Pansen von normalen Tieren (Überhydration). Dehydration verursachte einen mittleren Abfall des Wassergehaltes der Haut von 70.6 auf 65.8%. Rehydration führte zu einer teilweisen Wiederherstellung des normalen Wassergehaltes der Haut, während Überhydration ohne messbaren Einfluss blieb. Der berechnete Wasserverlust von der gesamten Haut als Folge der Dehydration war 315 ml bei den Kälbern und 1.336 ml bei den Ochsen.

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Resume On a déterminé la teneur en eau de la peau des bovidés sous quatre conditions: (1) chez des bêtes à l'état normal; (2) par déshydratation (après 3 jours sans abreuvage); (3) une heure après que les bêtes déshydratées aient reçu à boire (réhydratation); (4) une heure après le début d'une infusion d'eau dans la panse de bêtes à l'état normal (surhydratation). L'état de déshydratation a provoqué une baisse moyenne du taux de l'eau cutanée de 70,6% à 65,8%. La réhydratation a eu pour conséquence la reconstitution partielle de la teneur en eau de la peau alors que la surhydratation ne semble pas avoir eu d'effets. La perte en eau calculée par suite de déshydratation fut de 315 ml pour les veaux, de 1,336 ml pour les boeufs.

     

Potentiation of NMDA Receptor-Dependent Cell Responses by Extracellular High Mobility Group Box 1 Protein

Background

Extracellular high mobility group box 1 (HMGB1) protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca²⁺ influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown.

Principal Findings

Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca²⁺-dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130–139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1_(130–139) peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex.

Conclusion

We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1.     

Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.     

Novel surface expression of reticulocalbin 1 on bone endothelial cells and human prostate cancer cells is regulated by TNF-alpha

An unbiased cDNA expression phage library derived from bone-marrow endothelial cells was used to identify novel surface adhesion molecules that might participate in metastasis. Herein we report that reticulocalbin 1 (RCN1) is a cell surface-associated protein on both endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 is an H/KDEL protein with six EF-hand, calcium-binding motifs, found in the endoplasmic reticulum. Our data indicate that RCN1 also is expressed on the cell surface of several endothelial cell lines, including human dermal microvascular endothelial cells (HDMVECs), bone marrow endothelial cells (BMEC), and transformed human bone marrow endothelial cells (TrHBMEC). While RCN1 protein levels were highest in lysates from HDMVEC, this difference was not statistically significant compared BMEC and TrHBMEC. Given preferential adhesion of PCa to bone-marrow EC, these data suggest that RCN1 is unlikely to account for the preferential metastasis of PCa to bone. In addition, there was not a statistically significant difference in total RCN1 protein expression among the PCa cell lines. RCN1 also was expressed on the surface of several PCa cell lines, including those of the LNCaP human PCa progression model and the highly metastatic PC-3 cell line. Interestingly, RCN1 expression on the cell surface was upregulated by tumor necrosis factor alpha treatment of bone-marrow endothelial cells. Taken together, we show cell surface localization of RCN1 that has not been described previously for either PCa or BMEC and that the surface expression on BMEC is regulated by pro-inflammatory TNF-alpha.     

Kinesin II mediates Vg1 mRNA transport in Xenopus oocytes

The subcellular localization of specific mRNAs is a widespread mechanism for regulating gene expression. In Xenopus oocytes microtubules are required for localization of Vg1 mRNA to the vegetal cortex during the late RNA localization pathway. The factors that mediate microtubule-based RNA transport during the late pathway have been elusive. Here we show that heterotrimeric kinesin II becomes enriched at the vegetal cortex of stage III/IV Xenopus oocytes concomitant with the localization of endogenous Vg1 mRNA. In addition, expression of a dominant negative mutant peptide fragment or injection of a function-blocking antibody, both of which impair the function of heterotrimeric kinesin II, block localization of Vg1 mRNA. We also show that exogenous Vg1 RNA or Xcat-2, another RNA that can use the late pathway, recruits endogenous kinesin II to the vegetal pole and colocalizes with it at the cortex. These data support a model in which kinesin II mediates the transport of specific RNA complexes destined for the vegetal cortex.     

Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at -20 degrees C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).