Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.     

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.     

Microvascular dysfunction after transient high glucose is caused by superoxide-dependent reduction in the bioavailability of NO and BH(4)

We hypothesized that transient high-glucose concentration interferes with mediation by nitric oxide (NO) of flow-induced dilation (FID) of arterioles due to enhanced production of superoxide. In isolated, pressurized (80 mmHg) rat gracilis muscle arterioles ( approximately 130 microm) after transient high-glucose treatment (tHG; incubation with 30 mM glucose for 1 h), FID was reduced (maximum: control, 38 +/- 4%; after tHG, 17 +/- 3%), which was not further diminished by the NO synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME; 18 +/- 2%). Correspondingly, an enhanced polyethylene-glycol-SOD (PEG-SOD)-sensitive superoxide production was detected after tHG in carotid arteries by dihydroethydine (DHE) staining. Presence of PEG-SOD during tHG prevented the reduction of FID (41 +/- 3%), which could be inhibited by l-NAME (20 +/- 4%). Administration of PEG-SOD after tHG did not prevent the reduction of FID (22 +/- 3%). Sepiapterin, a precursor of the NO synthase cofactor tetrahydrobiopterin (BH(4)), administered during tHG did not prevent the reduction of FID (maximum, 15 +/- 5%); however, it restored FID when administered after tHG (32 +/- 4%). Furthermore, inhibition of either glycolysis by 2-deoxyglucose or mitochondrial complex II by 2-thenoyltrifluoroacetone reduced the tHG-induced DHE-detectable enhanced superoxide production in carotid arteries and prevented FID reduction in arterioles (39 +/- 5 and 35 +/- 2%). Collectively, these findings suggest that in skeletal muscle arterioles, a transient elevation of glucose via its increased metabolism, elicits enhanced production of superoxide, which decreases the bioavailability of NO and the level of the NOS cofactor BH(4), resulting in a reduction of FID mediated by NO.     

The discovery of 3-(N-alkyl)aminopropylphosphonic acids as potent S1P receptor agonists

3-(N-Alkyl)aminopropylphosphonic acids are potent agonists of four of the five known sphingosine-1-phosphate (S1P) receptor subtypes.     

Quantitative microinjection of trehalose into mouse oocytes and zygotes,and its effect on development

Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10mM Tris and 15mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes.     

Microarray Analysis of Gene Expression in Rat Hippocampus After Chronic Ethanol Treatment

It is thought that changes in gene expression in the brain mediate chronic ethanol-induced complex behaviors such as tolerance, dependence, and sensitization, and also relate to ethanol-induced brain toxicity. Using high-density filter-based cDNA microarrays (GeneFilters), we analyzed the expression of over 5000 genes in the dorsal hippocampus of rats treated with 12% ethanol or tap water for 15 months. Ethanol-induced changes in gene expression were particularly prominent in two groups of genes. One group consisted of oxidoreductases, including ceruloplasmin, uricase, branched-chain alpha-keto acid dehydrogenase, NADH ubiquinone oxidoreductase, P450, NAD⁺-isocitrate dehydrogenase, and cytochrome c oxidase, which may be related to ethanol-induced oxidative stress. The other group of genes included ADP-ribosylation factor, RAS related protein rab10, phosphatidylinositol 4-kinase, dynein-associated polypeptides, and dynamin-1, which seem to be involved in membrane trafficking. The results may reveal some of the pathways involved in ethanol-induced pathophysiological changes.     

Role of thromboxane in producing portal hypertension following trauma-hemorrhage

Thromboxane A2 (TXA2) and endothelin-1 (ET-1) have been proposed as the important vasoconstrictors that increase portal venous resistance in paracrine or autocrine fashion. We hypothesized that the hepatic damage following trauma-hemorrhage (T-H) is induced by the impaired hepatic circulation due to the increased production of vasoconstrictors such as ET-1 and TXA2 by the liver. To test this, male Sprague-Dawley rats (n = 6/group) were subjected to trauma (i.e., midline laparotomy) and hemorrhage (35-40 mmHg for 90 min followed by fluid resuscitation) or sham operation. At 2 or 5 h after the end of resuscitation, the liver was isolated and perfused and portal inflow pressure, bile flow, and release of ET-1 and thromboxane B2 (TXB2; a stable metabolite of TXA2) into the perfusate were measured. The level of portal pressure was higher at 5 h following T-H compared with 2 h after T-H and sham. The portal pressure was inversely correlated to the amount of bile production. Furthermore, the bile flow was significantly correlated to the hepatic damage as evidenced by release of lactate dehydrogenase into the perfusate. The level of ET-1 at 5 h following T-H in the perfusate after 30 min of recirculation did not show any difference from sham. However, the levels of TXB2 in the T-H group were significantly higher than those in sham at that interval. These results indicate that the increased release of TXA2 but not ET-1 following T-H might be responsible for producing the increased portal resistance, decreased bile production, and hepatic damage.     

Systemic bacterial invasion induced by sleep deprivation

Profound sleep disruption in humans is generally believed to cause health impairments. Through comparative research, specific physical effects and underlying mechanisms altered by sleep deprivation are being elucidated. Studies of sleep-deprived animals previously have shown a progressive, chronic negative energy balance and gradual deterioration of health, which culminate in fatal bloodstream infection without an infectious focus. The present study investigated the conditions antecedent to advanced morbidity in sleep-deprived rats by determining the time course and distribution of live microorganisms in body tissues that are normally sterile. The tissues cultured for microbial growth included the blood, four major organs, six regional lymph nodes, the intestine, and the skin. The principal finding was early infection of the mesenteric lymph nodes by bacteria presumably translocated from the intestine and bacterial migration to and transient infection of extraintestinal sites. Presence of pathogenic microorganisms and their toxins in tissues constitutes a septic burden and chronic antigenic challenge for the host. Bacterial translocation and pathogenic sequelae provide mechanisms by which sleep deprivation appears to adversely affect health.     

Brief communication: cutmarks on a plio-pleistocene hominid from Sterkfontein, South Africa

Cutmarks inflicted by a stone tool were observed on the right maxilla of Stw 53, an early hominid partial skull from Sterkfontein "Member 5" (South Africa). The morphology of the marks, their anatomical placement, and the lack of random striae on the specimen all support an interpretation of this linear damage as cutmarks. The location of the marks on the lateral aspect of the zygomatic process of the maxilla is consistent with that expected from slicing through the masseter muscle, presumably to remove the mandible from the cranium. Although radioisotopic dates are not available and relative faunal dating of the deposit from which Stw 53 derives is problematic, the morphology of the hominid skull suggests a Plio-Pleistocene age for the specimen. This therefore constitutes the earliest unambiguous evidence that hominids disarticulated the remains of one another.