Human adenovirus 36 induces adiposity, increases insulin sensitivity, and alters hypothalamic monoamines in rats

Objective: Human adenovirus 36 (Ad‐36) increases adiposity and reduces serum lipids in chicken, mouse, and non‐human primate models, and it is linked to obesity in sero‐epidemiological studies in humans. Involvement of the central nervous system (CNS) or adipose tissue in the mechanism of Ad‐36‐induced adiposity is unknown. The effects of Ad‐36 on adiposity and on the neuroendocrine system were investigated in a rat model. Research Methods and Procedures: Five‐week‐old male Wistar rats were inoculated intraperitoneally with Ad‐36 or medium. Results: Despite similar food intakes, infected rats attained significantly greater body weight and fat pad weight by 30 weeks post‐inoculation. Epididymal‐inguinal, retroperitoneal, and visceral fat pad weights of the infected group were greater by 60%, 46%, and 86%, respectively (p < 0.00001). The fasting serum insulin level and homeostasis model assessment index indicated greater insulin sensitivity in the infected group. Visceral adipose tissue expression of glycerol 3‐phosphate dehydrogenase, peroxisome proliferator‐activated receptor γ, and CCAAT/enhancer‐binding protein α and β was markedly increased in the infected animals compared with controls. Ad‐36 decreased norepinephrine levels significantly in the paraventricular nucleus in infected vs. control rats (mean ± standard error, 8.9 ± 1.1 vs. 12.8 ± 1.2 pg/μg protein; p < 0.05). Ad‐36 markedly decreased serum corticosterone in infected vs. control rats (mean ± standard error, 97 ± 41.0 vs. 221 ± 111 ng/mL; p < 0.005). Discussion: The results suggest that the pro‐adipogenic effect of Ad‐36 may involve peripheral as well as central effects. The male Wistar rat is a good model for the elucidation of metabolic and molecular mechanisms of Ad‐36‐induced adiposity.     

A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.     

Structure–toxicity relationship of phenolic analogs as anti-melanoma agents: An enzyme directed prodrug approach

The aim of this study was to identify a phenolic prodrug compound that is minimally metabolized by rat liver microsomes, but yet could form quinone reactive intermediates in melanoma cells as a result of its bioactivation by tyrosinase. In current work, we investigated 24 phenolic compounds for their metabolism by tyrosinase, rat liver microsomes and their toxicity towards murine B16-F0 and human SK-MEL-28 melanoma cells. A linear correlation was found between toxicities of phenolic analogs towards SK-MEL-28 and B16-F0 melanoma cells, suggesting similar mechanisms of toxicity in both cell lines. 4-HEB was identified as the lead compound. 4-HEB (IC₅₀ 48 h, 75 μM) showed selective toxicity towards five melanocytic melanoma cell lines SK-MEL-28, SK-MEL-5, MeWo, B16-F0 and B16-F10, which express functional tyrosinase, compared to four non-melanoma cells lines SW-620, Saos-2, PC3 and BJ cells and two amelanotic SK-MEL-24, C32 cells, which do not express functional tyrosinase. 4-HEB caused significant intracellular GSH depletion, ROS formation, and showed significantly less toxicity to tyrosinase specific shRNA transfected SK-MEL-28 cells. Our findings suggest that presence of a phenolic group in 4-HEB is critical for its selective toxicity towards melanoma cells.     

Metallopeptide-promoted inactivation of angiotensin-converting enzyme and endothelin-converting enzyme 1: toward dual-action therapeutics

A series of metallopeptides based on the amino terminal copper/nickel (ATCUN) binding motif have been evaluated as classical inhibitors and catalytic inactivators of both rabbit and human angiotensin-converting enzyme (hACE), and human endothelin-converting enzyme 1 (hECE-1). The cobalt complex [KGHK–Co(NH₃)₂]²⁺, where KGHK is lysylglycylhistidyllysine, displayed similar K _I and IC₅₀ values to those found for [KGHK–Cu]⁺, in spite of the enhanced charge, and so either the influence of charge is offset by the steric influence of the axially coordinated ammine ligands, or binding is dominated by contributions from the amino acid side chains, especially the C-terminal lysine that mimics the binding pattern observed for lisinopril. Moreover, the inhibition observed for [KGHK–Co(NH₃)₂]²⁺ contrasts with the activation of hACE by Co²⁺(aq), reflecting the stimulation of enzyme activity following replacement of the catalytic zinc cofactor by cobalt ion at each of the two active sites. Quantitative analysis of the dose-dependent stimulation of activity by Co²⁺(aq) yielded apparent affinities of 1.3 ± 0.2 and 56 ± 8 μM for the two sites in the presence of saturating Zn²⁺ (10 μM). Catalytic inactivation of hACE by [KGHK–Cu] ⁺ at subsaturating concentrations had previously been characterized, with k _obs = 2.9 ± 0.5 × 10⁻² min⁻¹. Under similar conditions, the same complex is found to catalytically inactivate hECE-1, with k _obs = 2.12 ± 0.16 × 10⁻² min⁻¹, demonstrating the potential for dual-action activity against two key drug targets in cardiovascular disease. Irreversible inactivation of a drug target represents a novel mechanism of drug action that complements existing classical inhibitor strategies that underlie current drug discovery efforts.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Biochemical characterization of ribosome assembly GTPase RbgA in Bacillus subtilis

The ribosome biogenesis GTPase A protein RbgA is involved in the assembly of the large ribosomal subunit in Bacillus subtilis, and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45 S in sucrose density gradients. Here, we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow k(cat) (14 h(-1)) and has a high K(m) (90 μM). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K(+) ions for GTPase activity, which was confirmed experimentally. Interaction with 50 S subunits, but not 45 S intermediates, increased GTPase activity by ～55-fold. Stable association with 50 S subunits and 45 S intermediates was nucleotide-dependent, and GDP did not support strong interaction with either of the subunits. GTP and guanosine 5'-(β,γ-imido)triphosphate (GMPPNP) were sufficient to promote association with the 45 S intermediate, whereas only GMPPNP was able to support binding to the 50 S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.     

Lack of organ specific commitment of vagal neural crest cell derivatives as shown by back-transplantation of GFP chicken tissues

Neural crest cells (NCC) are multipotent progenitors that migrate extensively throughout the developing embryo and generate a diverse range of cell types. Vagal NCC migrate from the hindbrain into the foregut and from there along the gastrointestinal tract to form the enteric nervous system (ENS), the intrinsic innervation of the gut, and into the developing lung buds to form the intrinsic innervation of the lungs. The aim of this study was to determine the developmental potential of vagal NCC that had already colonised the gut or the lungs. We used transgenic chicken embryos that ubiquitously express green fluorescent protein (GFP) to permanently mark and fate-map vagal NCC using intraspecies grafting. This was combined with back-transplantation of gut and lung segments, containing GFP-positive NCC, into the vagal region of a second recipient embryo to determine, using immunohistochemical staining, whether gut or lung NCC are competent of re-colonising both these organs, or whether their fate is restricted. Chick(GFP)-chick intraspecies grafting efficiently labelled NCC within the gut and lung of chick embryos. When segments of embryonic day (E)5.5 pre-umbilical midgut containing GFP-positive NCC were back-transplanted into the vagal region of E1.5 host embryos, the GFP-positive NCC remigrated to colonise both the gut and lungs and differentiated into neurons in stereotypical locations. However, GFP-positive lung NCC did not remigrate when back-transplanted. Our studies suggest that gut NCC are not restricted to colonising only this organ, since upon back-transplantation GFP-positive gut NCC colonised both the gut and the lung.     

Airway epithelial versus immune cell Stat1 function for innate defense against respiratory viral infection

The epithelial surface is often proposed to actively participate in host defense, but evidence that this is the case remains circumstantial. Similarly, respiratory paramyxoviral infections are a leading cause of serious respiratory disease, but the basis for host defense against severe illness is uncertain. Here we use a common mouse paramyxovirus (Sendai virus) to show that a prominent early event in respiratory paramyxoviral infection is activation of the IFN-signaling protein Stat1 in airway epithelial cells. Furthermore, Stat1-/- mice developed illness that resembled severe paramyxoviral respiratory infection in humans and was characterized by increased viral replication and neutrophilic inflammation in concert with overproduction of TNF-alpha and neutrophil chemokine CXCL2. Poor control of viral replication as well as TNF-alpha and CXCL2 overproduction were both mimicked by infection of Stat1-/- airway epithelial cells in culture. TNF-alpha drives the CXCL2 response, because it can be reversed by TNF-alpha blockade in vitro and in vivo. These findings pointed to an epithelial defect in Stat1-/- mice. Indeed, we next demonstrated that Stat1-/- mice that were reconstituted with wild-type bone marrow were still susceptible to infection with Sendai virus, whereas wild-type mice that received Stat1-/- bone marrow retained resistance to infection. The susceptible epithelial Stat1-/- chimeric mice also exhibited increased viral replication as well as excessive neutrophils, CXCL2, and TNF-alpha in the airspace. These findings provide some of the most definitive evidence to date for the critical role of barrier epithelial cells in innate immunity to common pathogens, particularly in controlling viral replication.