The p53 Codon 72 PRO/PRO Genotype May Be Associated with Initial Central Visual Field Defects in Caucasians with Primary Open Angle Glaucoma

Background

Loss of vision in glaucoma is due to apoptotic retinal ganglion cell loss. While p53 modulates apoptosis, gene association studies between p53 variants and glaucoma have been inconsistent. In this study we evaluate the association between a p53 variant functionally known to influence apoptosis (codon 72 Pro/Arg) and the subset of primary open angle glaucoma (POAG) patients with early loss of central visual field.

Methods

Genotypes for the p53 codon 72 polymorphism (Pro/Arg) were obtained for 264 POAG patients and 400 controls from the U.S. and in replication studies for 308 POAG patients and 178 controls from Australia (GIST). The glaucoma patients were divided into two groups according to location of initial visual field defect (either paracentral or peripheral). All cases and controls were Caucasian with European ancestry.

Results

The p53-PRO/PRO genotype was more frequent in the U.S. POAG patients with early visual field defects in the paracentral regions compared with those in the peripheral regions or control group (p = 2.7×10⁻⁵). We replicated this finding in the GIST cohort (p  = 7.3×10⁻³, and in the pooled sample (p = 6.6×10⁻⁷) and in a meta-analysis of both the US and GIST datasets (1.3×10⁻⁶, OR 2.17 (1.58–2.98 for the PRO allele).

Conclusions

These results suggest that the p53 codon 72 PRO/PRO genotype is potentially associated with early paracentral visual field defects in primary open-angle glaucoma patients.     

The counter regulatory response induced by CpG oligonucleotides prevents bleomycin induced pneumopathy

Bleomycin (BLM) induces life-threatening pneumonitis and pulmonary fibrosis in 20% of patients, limiting its use as a chemotherapeutic agent. Oligonucleotides expressing immunostimulatory CpG motifs (CpG ODN) stimulate cells that express Toll-like receptor 9 to initiate an inflammatory response. This short-lived inflammation is physiologically suppressed by a counter-regulatory process that peaks five days later. Using a murine model of BLM-induced lung injury, the effect of CpG ODN treatment on pulmonary inflammation, fibrosis and mortality was examined. Administering CpG ODN 5 days before BLM (so that the peak of the counter-regulatory process induced by CpG ODN coincided with BLM delivery) resulted in a dose-dependent reduction in pulmonary toxicity (p < 0.005). Delaying the initiation of therapy until the day of or after BLM administration worsened the inflammatory process, consistent with the counter-regulatory process rather than initial pro-inflammatory response being critical to CpG induced protection. The protection afforded by CpG ODN correlated with reduced leukocyte accumulation and inflammatory cytokine/chemokine production in the lungs. These changes were associated with the increased production of IL-10, a critical element of the counter-regulatory process triggered by CpG ODN, and the concomitant down-regulation of BLM-induced IL-17A and TGF-β1 (which promote pulmonary toxicity). This work represents the first example of the physiologic counter-regulation of TLR induced immune activation being harnessed to block an unrelated inflammatory response.     

Detection of interspecies hybridisation in Chondrichthyes: hybrids and hybrid offspring between Australian (Carcharhinus tilstoni) and common (C. limbatus) blacktip shark found in an Australian fishery

Interspecies hybridisation in nature is a well-studied phenomenon, but it has not been analysed using genetic markers in the class Chondrichthyes (sharks, rays and chimeras). Two black-tip whaler shark species (Australian, Carcharhinus tilstoni; Common, C. limbatus) have overlapping distributions in Australia, distinct mitochondrial DNA sequence (ND4, COI, control region) and distinct morphological features such as length at sexual maturity, length at birth and number of vertebrae. A mismatch was observed between species identification using mtDNA sequence and species identification using morphological characters. To test whether hybridisation between the two species was responsible, a nuclear gene with species-specific mutations was sequenced. Extensive interspecies hybridisation was found to be occurring. Hybrids were found from five locations on the eastern Australian coastline, spanning 2,000 km. If hybrid fitness is low and hybrids are common, then fisheries recruitment may be overestimated and the productivity of the black-tip shark fishery may be well below that required to support commercial exploitation. To guard against identification errors, the likelihood of hybridisation and subsequent introgression should be assessed prior to using mtDNA (e.g. barcoding) to identify shark species. The C. limbatus–C. tilstoni species complex provides a unique opportunity to investigate the ability of sharks to adapt to environmental change, in particular, the impact of hybridization on species distributions which favour C. tilstoni along the north and C. limbatus along the south eastern Australian coastline.     

Conjugated Linoleic Acid (CLA), Body Fat,and Apoptosis*

Objective: The objective of the study was to determine if consumption of conjugated linoleic acid (CLA) by mice could induce apoptosis in adipose tissue. Other objectives were to determine the influence of feeding mice CLA for ≤2 weeks on body fat, energy expenditure, and feed intake. Research Methods and Procedures: A mixture of CLA isomers (predominantly c9,t11 and t10,c12) was included in the AIN‐93G diet at 0, 1, and 2%, and fed to mice for 12 days (Trial 1), or was included at 2% and fed to mice for 0, 5, and 14 days (Trial 2). Feed intake was measured daily and energy expenditure was determined by direct calorimetry on day 9 in Trial 1. Retroperitoneal fat pads were analyzed for apoptosis by determination of DNA fragmentation. Results: Dietary CLA reduced feed intake by 10% to 12% (p < 0.01), but either did not influence or did not increase energy expenditure as indicated by heat loss. Body weight was not influenced by consumption of CLA in Trial 1 but was increased (p < 0.01) by CLA in Trial 2. Weights of retroperitoneal, epididymal, and brown adipose tissues were lower (p < 0.01) in animals fed CLA, although liver weight was increased (p < 0.10; Trial 1) or not changed (Trial 2). Analysis of retroperitoneal fat pad DNA from both trials indicated that apoptosis was increased (p < 0.01) by CLA consumption. Discussion: These results are interpreted to indicate that CLA consumption causes apoptosis in white adipose tissue. This effect occurs within 5 days of consuming a diet that contains CLA.     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

Identical digeneans in coral reef fishes from French Polynesia and the Great Barrier Reef (Australia) demonstrated by morphology and molecules

Three coral reef fish species, Zanclus cornutus, Chaetodon vagabundus and Naso lituratus, were collected in French Polynesia and on the Great Barrier Reef, Queensland. These fish species were each infected by one morphologically similar digenean species in both localities; Schistorchis zancli Hanson, 1953 was found in Zanclus cornutus, Preptetos laguncula Bray and Cribb, 1996 in Naso lituratus and Neohypocreadium dorsoporum Machida and Uchida, 1987 in Chaetodon vagabundus. In addition, on the Great Barrier Reef P. laguncula was also found in Naso unicornis and N. dorsoporum in Chaetodon ephippium and Chaetodon flavirostris. Morphometric differences between the species from the two sites were only slight. Sequences from the second internal transcribed spacer of the ribosomal DNA of each worm revealed total homology or negligible divergence between samples from hosts caught in French Polynesia and on the Great Barrier Reef. These results show that across more than 6000 km these digeneans are similar in morphology and genotype. Some species of fishes and molluscs are considered to have distributions that encompass the entire tropical Indo-West Pacific. These findings suggest that at least some of their parasites have similarly broad distributions.     

Induction of umbelliferone in sweet potato cell suspension culture using mercuric chloride

HgCl₂ was used at up to 10 mg l^–1 as an elicitor of phytoalexins in sweet potato (Ipomoea batatas (L.) Lam. cv Centennial) cell suspension cultures. Maximum stimulation of a coumarin compound was after one day of exposure using 1 mg HgCl₂ l^–1. The compound was identified by HPLC and GC-MS analyses as 7-hydroxycoumarin (umbelliferone).     

Adipose Depletion and Apoptosis Induced by Trans-10, Cis-12 Conjugated Linoleic Acid in Mice*

Objective: To compare the effectiveness of a conjugated linoleic acid (CLA) isomer mixture (mCLA) with each main isomer [trans-10,cis-12 CLA (CLA10,12) and cis-9,trans-11 CLA (CLA9,11)] in causing body lipid loss and adipose tissue apoptosis. Research Methods and Procedures: Mice selected over 16 generations for high (MH) or low (ML) energy expenditure and a control group (MC) were fed diets containing either soy oil or soy oil plus mCLA, CLA10,12, or CLA9,11 for 5 days in one study and 14 days in a second study. Results: Mice fed mCLA or CLA10,12 had less body lipid (p < 0.05), smaller retroperitoneal fat pads (p < 0.05), and ate less (p < 0.01) than mice fed no CLA or CLA9,11 for 5 days. Mice consuming 1% mCLA or 0.5% CLA10,12 gained less weight (p < 0.01) and had less body lipid (p < 0.05) and smaller epididymal (p < 0.05) and retroperitoneal fat pads (p < 0.01) than mice consuming either control or 0.5% CLA9,11-containing diets for 14 days. Only mCLA and CLA10,12 increased apoptosis in retroperitoneal fat pads (p < 0.01). The effects of mCLA and CLA10,12 were independent of genetic line except for the effect on adipocyte apoptosis. Mice of the MH line were slightly less sensitive than MC or ML mice to CLA-induced adipose tissue apoptosis. Discussion: CLA10,12, but not CLA9,11, can induce both body fat loss and adipose apoptosis. Although mice of a genotype with less body fat and greater metabolic rate and feed intake appear less sensitive, these CLA effects are robust for mice of varying metabolic background.     

Microbial Population Analysis as a Measure of Ecosystem Restoration

During a controlled oil spill study in a freshwater wetland, four methods were used to track changes in microbial populations in response to in situ remediation treatments, including nutrient amendments and the removal of surface vegetation. Most probable number (MPN) estimates of alkane and aromatic hydrocarbon degraders showed divergence of the alkane and aromatic degrading populations during the first summer of the experiment. Alkane degraders increased in all plots by 1.5 orders of magnitude and aromatic degraders increased in oiled plots by 3.5 orders of magnitude. Phospholipid fatty acid (PLFA) analysis of biomass and community composition showed no essential differences among treatments. Denaturing gradient gel electrophoresis (DGGE) analysis of the sediment microbial community showed some differences in specific populations of organisms with respect to oiled and unoiled plots. Some organisms were only found in the oiled plots. Sediment toxicity measured against single celled algae showed that the oiled sediments were toxic into the second year of the study, but that nutrient addition relieved the toxicity more rapidly than natural attenuation of the oil.