Home range size of adult Indo‐Pacific bottlenose dolphins (Tursiops aduncus) in a coastal and estuarine system is habitat and sex‐specific

This study examined sex‐specific differences in home range size of adult Indo‐Pacific bottlenose dolphins off Bunbury, Western Australia. We applied a new kernel density estimation approach that accounted for physical barriers to movements. A Bayesian mixture model was developed to estimate a sex effect in home range size with latent group partitioning constrained by association data. A post hoc analysis investigated group partitioning relating to the proportion of time spent in open vs. sheltered waters. From 2007 to 2013, photographic‐identification data were collected along boat‐based systematic transect lines (n = 586). Analyses focused on adult dolphins of known sex (sighted ≥ 30 times; n = 22 males and 34 females). The 95% utilization distributions of males varied between 27 and 187 km² (; 94.8 ± 48.15) and for females between 20 and 133 km² (65.6 ± 30.9). The mixture model indicated a 99% probability that males had larger home ranges than females. Dolphins mostly sighted in open waters had larger home ranges than those in sheltered waters. Home ranges of dolphins sighted in sheltered waters overlapped with areas of highest human activity. We suggest that sex differences in home ranges are driven by male mating strategies, and home range size differences between habitats may be influenced by prey availability and predation risk.     

Proteome map of normal rat retina and comparison with the proteome of diabetic rat retina: new insight in the pathogenesis of diabetic retinopathy

We have employed proteomics to establish a proteome map of the normal rat retina. This baseline map was then used for comparison with the early diabetic rat retinal proteome. Diabetic rat retinae were obtained from Dark Agouti rats after 10 wk of streptozotocin-induced hyperglycaemia. Extracted proteins from normal and diabetic rat retinae were separated and compared using 2-DE. A total of 145 protein spots were identified in the normal rat retina using MALDI-MS and database matching. LC-coupled ESI-MS increased the repertoire of identified proteins by 23 from 145 to 168. Comparison with early diabetic rat retinae revealed 24 proteins unique to the diabetic gels, and 37 proteins absent from diabetic gels. Uniquely expressed proteins identified included the HSPs 70.1A and 8, and platelet activating factor. There were eight spots with increased expression and 27 with decreased expression on diabetic gels. Beta catenin, phosducin and aldehyde reductase were increased in expression in diabetes whilst succinyl coA ligase and dihydropyrimidase-related protein were decreased. Identification of such changes in protein expression has given new insights and a more comprehensive understanding of the pathogenesis of diabetic retinopathy, widening the scope of potential avenues for new therapies for this common cause of blindness.     

Sustained nitric oxide production in macrophages requires the arginine transporter CAT2

The aberrant production of nitric oxide (NO) contributes to the pathogenesis of diseases as diverse as cancer and arthritis. Sustained NO production via the inducible enzyme, nitric-oxide synthase 2 (NOS2), requires extracellular arginine uptake. Three closely related cationic amino acid transporter genes (Cat1-3) encode the transporters that mediate most arginine uptake in mammalian cells. Because CAT2 is induced coordinately with NOS2 in numerous cell types, we investigated a possible role for CAT2-mediated arginine transport in regulating NO production. The complexity of arginine transport systems and their biochemically similar transport properties called for a genetic approach to determine the role of CAT2. CAT2-deficient mice were generated and found to be healthy and fertile in contrast to Cat1(-/-) animals. Analysis of cytokine-activated macrophages from Cat2(-/-) mice revealed a 92% reduction in NO production and a 95% reduction in l-Arg uptake. The reduction in NO production was not due to differences in NOS2 protein expression, NOS2 activity, or intracellular l-arginine content. In conclusion, our results show that sustained abundant NO synthesis by macrophages requires arginine transport via the CAT2 transporter.     

Conserved cysteine and tryptophan residues of the endothelin-converting enzyme-1 CXAW motif are critical for protein maturation and enzyme activity

The neprilysin (NEP)/endothelin-converting enzyme (ECE) family of metalloproteases contains a highly conserved carboxyl-terminal tetrapeptide sequence, CXAW, where "C" is cysteine, "X" is a polar amino acid, "A" is an aliphatic residue, and "W" is tryptophan. Although this sequence strongly resembles a prenylation motif, human ECE-1 did not appear to be prenylated when labeled in vivo using various isoprenoid precursors in cell lines expressing ECE-1. We used site-directed mutagenesis to investigate the role of the CXAW motif and determined that the conserved cysteine residue of the CXAW motif in ECE-1, Cys(755), is critical for proper folding of the enzyme, its export from the endoplasmic reticulum, and its maturation in the secretory pathway. In addition, site-directed mutagenesis revealed that the conserved tryptophan residue of the sequence CEVW appears to be important for endoplasmic reticulum export and is essential for enzyme activity. Deletion of Trp(758) or substitution with alanine greatly slowed maturation of the enzyme, and resulted in more than a 90% loss of enzyme activity relative to the wild type. Conservative substitution of the tryptophan with phenylalanine did not reduce activity, whereas replacement with tyrosine, methionine, or leucine reduced enzyme activity by 50%, 75%, and 85%, respectively. Together, these data indicate that the conserved CEVW sequence does not serve as a prenylation signal and that both the conserved cysteine and tryptophan residues are necessary for proper folding and maturation of the enzyme. Furthermore, the conserved tryptophan appears to be critical for enzyme activity.     

The role of human glutathione S-transferases (hGSTs) in the detoxification of the food-derived carcinogen metabolite N-acetoxy-PhIP, and the effect of a polymorphism in hGSTA1 on colorectal cancer risk

Food-derived heterocyclic amines (HCAs), particularly 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are implicated in the etiology of human colorectal cancer (CRC) via a process of N-oxidation followed by O-acetylation or O-sulfation to form electrophilic metabolites that react with DNA. Glutathione S-transferases (GSTs) detoxify activated carcinogen metabolites by catalysis of their reaction with GSH. However, among HCAs, only N-acetoxy-PhIP has been shown to be a substrate for the GSTs. By using a competitive DNA-binding assay, we confirm that hGSTA1-1 is an efficient catalyst of the detoxification of N-acetoxy-PhIP. Further, we show that hGSTs A2-2, P1-1, M1-1, T1-1 and T2-2 appear to have low activity towards N-acetoxy-PhIP, and that hGSTs A4-4, M2-2, M4-4 and Z1-1 appear to have no activity towards N-acetoxy-PhIP. A genetic polymorphism in the 5'-regulatory sequence of hGSTA1 has been shown to correlate with the relative and absolute levels of expression of GSTA1/GSTA2 in human liver. Examination of hGSTA1 allele frequency in 100 Caucasian CRC patients and 226 Caucasian controls demonstrated a significant over-representation of the homozygous hGSTA1*B genotype among cases compared to controls (24.0 and 13.7%, respectively, P=0.04). This corresponds to an odds ratio for risk of CRC of 2.0 (95% CI 1.0-3.7) when comparing homozygous hGSTA1*B individuals with all other genotypes. Thus, individuals who are homozygous hGSTA1*B, and who would be predicted to have the lowest levels of hGSTA1 expression in their livers, appear to be at risk of developing CRC, possibly as a result of inefficient hepatic detoxification of N-acetoxy-PhIP.     

Construction of phosphorylatable chimeric monoclonal antibody CC49 with a casein kinase I recognition site

Phosphorylation sites for casein kinase I were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (CK1) encoding two casein kinase I phosphorylation sites into an expression vector. The phosphorylation sites were created by incorporating the predicted consensus sequences for phosphorylation by the casein kinase I at the carboxyl terminus of the heavy-chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49CK1) was expressed and purified. The MAb-chCC49CK1 protein can be phosphorylated by the casein kinase I with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49CK1 protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into MAb provides new reagents for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, the casein kinase I recognition site can also be used to introduce phosphorylation sites into proteins.     

Th2-induced airway mucus production is dependent on IL-4Ralpha, but not on eosinophils

Mucus hyperproduction in asthma results from airway inflammation and contributes to clinical symptoms, airway obstruction, and mortality. In human asthmatics and in animal models, excess mucus production correlates with airway eosinophilia. We previously described a system in which TCR transgenic CD4 Th2 cells generated in vitro were transferred into recipient mice and activated in the respiratory tract with inhaled Ag. Th2 cells stimulated airway eosinophilia and a marked increase in mucus production, while mice that received Th1 cells exhibited airway inflammation without eosinophilia or mucus. Mucus could be induced by IL-4-/- Th2 cells at comparable levels to mucus induced by IL-4+/+ Th2 cells. In the current studies we dissect further the mechanisms of Th2-induced mucus production. When IL-4-/- Th2 cells are transferred into IL-4Ralpha-/- mice, mucus is not induced, and BAL eosinophilia is absent. These data suggest that in the absence of IL-4, IL-13 may be critical for Th2-induced mucus production and eosinophilia. To determine whether eosinophils are important in mucus production, IL-5-/- Th2 cells were transferred into IL-5-/- recipients. Eosinophilia was abolished, yet mucus staining in the epithelium persisted. These studies show definitively that IL-5, eosinophils, or mast cells are not essential, but signaling through IL-4Ralpha is critically important in Th2 cell stimulation of mucus production.     

(2-amino-phenyl)-amides of omega-substituted alkanoic acids as new histone deacetylase inhibitors

A variety of omega-substituted alkanoic acid (2-amino-phenyl)-amides were designed and synthesized. These compounds were shown to inhibit recombinant human histone deacetylases (HDACs) with IC(50) values in the low micromolar range and induce hyperacetylation of histones in whole cells. They induced expression of p21WAF1/Cip1 and caused cell-cycle arrest in human cancer cells. Compounds in this class showed efficacy in human tumor xenograft models.