Indoleamine 2,3-diooxygenase in periaortic fat: mechanisms of inhibition of contraction

Indoleamine 2,3-dioxygenase (IDO) metabolizes L-tryptophan to L-kynurenine, promotes immunosuppression, and has been described as a consumer of superoxide. We discovered IDO expression in periaortic fat and tested the hypothesis that periarterial IDO functionally reduces agonist-induced contraction. Our model was the thoracic aorta, abdominal aorta, and superior mesenteric artery of the male Sprague-Dawley rat. Periaortic fat from the thoracic aorta stained intensely for IDO, the brown fat marker uncoupling protein-1, and oil red O as a general lipid marker. White fat around the mesenteric artery and abdominal aorta stained less for IDO; brown fat was less abundant. IDO activity (kynurenine-to-tryptophan ratio via HPLC) was detected in visceral and mesenteric artery fat (ratio: ～4) but was highest in perithoracic aortic fat (ratio: 10 ± 1.1). In isometric contractile experiments, periadventitial fat reduced ANG II-induced thoracic aortic (with fat: 34% of without fat) and mesenteric artery (with fat: 63% of without fat) maximal contraction. In contrast, periadventitial fat did not reduce agonist-induced contraction in the abdominal aorta. The IDO inhibitor 1-L-methyltryptophan (1-MT) reversed the fat-induced reduction of ANG II-induced contraction in the thoracic aorta but not in the mesenteric artery. The IDO metabolite kynurenine relaxed the thoracic aorta only at high (9 mM) concentrations, whereas the downstream metabolite quinolinic acid (1 mM) relaxed the contracted thoracic aorta (～80%). 1-MT did not correct the reduction in basal superoxide levels observed in the presence of perithoracic aortic fat. We conclude that IDO is an enzyme active primarily in brown fat surrounding the thoracic aorta and depresses aortic contractility.     

Identification of a sporozoite-specific antigen from Toxoplasma gondii

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii -infected humans. Serum antibody to TgERP was detected in humans within 6-8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti- T. gondii IgM detected in sera, or < 30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti- T. gondii IgM detected in sera, or > 30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.     

Automation of Molecular-Based Analyses: A Primer on Massively Parallel Sequencing

Recent advances in genetics have been enabled by new genetic sequencing techniques called massively parallel sequencing (MPS) or next-generation sequencing. Through the ability to sequence in parallel hundreds of thousands to millions of DNA fragments, the cost and time required for sequencing has dramatically decreased. There are a number of different MPS platforms currently available and being used in Australia. Although they differ in the underlying technology involved, their overall processes are very similar: DNA fragmentation, adaptor ligation, immobilisation, amplification, sequencing reaction and data analysis. MPS is being used in research, translational and increasingly now also in clinical settings. Common applications include sequencing of whole genomes, whole exomes or targeted genes for disease-causing gene discovery, genetic diagnosis and targeted cancer therapy. Even though the revolution that is occurring with MPS is exciting due to its increasing use, improving and emerging technologies and new applications, significant challenges still exist. Particularly challenging issues are the bioinformatics required for data analysis, interpretation of results and the ethical dilemma of ‘incidental findings’.     

Direct measurement of spatial autocorrelation at the community level in four plant communities

Abstract. Four sites were sampled to determine spatial autocorrelation in vegetation at the community level. All were in western New Zealand, but on different substrates and of different physiognomy: a terrace forest, a floodplain forest, a mire and the middle of a logging road. In ‘dissimograms’the four communities all showed steady increases in dissimilarity with distance, but with shoulders in the curve for some sites, which could be related to plant morphology. Most of the increase in dissimilarity occurred over very short distances: less than 0.5 m in the forests, less than 1 m in the mire and less than 2 m in the road. Separate analyses of the woody and herbaceous guilds in the floodplain forest showed that herbaceous dissimilarities remained low at distances up to 20 m, probably because of clonal structure in some species. The mire showed low overall dissimilarity, which is attributed to the uniform substrate and the small species pool. Simulations showed that the approach is capable of indicating structure when it is present. Although the dissimogram was clearest when analysing a simulated grid of patches, other types of simulated patchiness showed dissimograms that were clearly distinguishable from those obtained from the vegetation studied. The almost continuous rise in dissimilarity with distance found in the four sites offers no support to the Hierarchy theory, fitting much more closely the alternative Continuum theory.     

Cloning of a Drosophila melanogaster adenine phosphoribosyltransferase structural gene and deduced amino acid sequence of the enzyme

The Aprt locus of Drosophila melanogaster encodes the structural gene for adenine phosphoribosyltransferase (APRT). DNA cloned from microdissected salivary gland polytene chromosome region 62B7-12 was used in conjunction with chromosome walking and hybrid selection of mRNA to isolate the Aprt gene. Aprt lies at cytogenetic position 62B9 and is closely flanked by other genes of unknown function. Nucleotide sequencing shows that four APRT cDNAs have a common 5' terminus with an apparent cap consensus sequence but two different 3' sites of polyadenylation. The distribution of conserved amino acid sequences in APRT from vertebrates, insects and bacteria suggests that they may have shared a common ancestral gene for this ubiquitous enzyme.     

Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.     

Reduction in number of sarcolemmal KATP channels slows cardiac action potential duration shortening under hypoxia

The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (K_ATP) channels. K_ATP channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative K_ATP channel subunit were compared with littermate controls. Evaluation of K_ATP channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80–85% reduction in cardiac K_ATP channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal K_ATP channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress.     

Radium in the Suwannee River and estuary

A two-year study of radium in the Suwannee River has shown that groundwater discharge, via springs, is a very important source of radium both to the river and to offshore Gulf of Mexico waters. Dissolved radium is maintained within relatively narrow limits in the river by uptake into suspended particles. In the estuary, dissolved radium versus salinity profiles show distinctive nonconservative behavior with radium in significant excess of its linear mixing value at mid-salinities. Unlike the situation in many other estuaries, however, desorption of radium from particles cannot account for most of the observed excess. Thus, the anomalously high radium characteristic of much of the west Florida shelf apparently does not have a riverine source. Direct effusion of high-radium groundwater into these coastal waters is thought to be the major supplier of radium, and perhaps other elements as well.