Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1, a novel neuronal protein that regulates energy balance

To identify genes involved in the central regulation of energy balance, we compared hypothalamic mRNA from lean and obese Psammomys obesus, a polygenic model of obesity, using differential display PCR. One mRNA transcript was observed to be elevated in obese, and obese diabetic, P. obesus compared with lean animals and was subsequently found to be increased 4-fold in the hypothalamus of lethal yellow agouti (Ay/a) mice, a murine model of obesity and diabetes. Intracerebroventricular infusion of antisense oligonucleotide targeted to this transcript selectively suppressed its hypothalamic mRNA levels and resulted in loss of body weight in both P. obesus and Sprague Dawley rats. Reductions in body weight were mediated by profoundly reduced food intake without a concomitant reduction in metabolic rate. Yeast two-hybrid screening, and confirmation in mammalian cells by bioluminescence resonance energy transfer analysis, demonstrated that the protein it encodes interacts with endophilins, mediators of synaptic vesicle recycling and receptor endocytosis in the brain. We therefore named this transcript Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 encodes a large proline-rich protein that is expressed predominantly in the brain and is highly conserved between species. Together these data suggest that SGIP1 is an important and novel member of the group of neuronal molecules required for the regulation of energy homeostasis.

Insulin signaling after exercise in insulin receptor substrate-2-deficient mice

The period immediately after exercise is characterized by enhanced insulin action in skeletal muscle, and on the molecular level, by a marked increase in insulin-stimulated, phosphotyrosine-associated phosphatidylinositol (PI) 3-kinase activity. Because the increase in PI 3-kinase activity cannot be explained by increased insulin receptor substrate (IRS)-1 signaling, the present study examined whether this effect is mediated by enhanced IRS-2 signaling. In wild-type (WT) mice, insulin increased IRS-2 tyrosine phosphorylation (2.5-fold) and IRS-2-associated PI 3-kinase activity (3-fold). Treadmill exercise, per se, had no effect on IRS-2 signaling, but in the period immediately after exercise, there was a further increase in insulin-stimulated IRS-2 tyrosine phosphorylation (3.5-fold) and IRS-2-associated PI 3-kinase activity (5-fold). In IRS-2-deficient (IRS-2-/-) mice, the increase in insulin-stimulated, phosphotyrosine-associated PI 3-kinase activity was attenuated as compared with WT mice. However, in IRS-2-/- mice, the insulin-stimulated, phosphotyrosine-associated PI 3-kinase response after exercise was slightly higher than the insulin-stimulated response alone. In conclusion, IRS-2 tyrosine phosphorylation and associated PI 3-kinase activity are markedly enhanced by insulin in the immediate period after exercise. IRS-2 signaling can partially account for the increase in insulin-stimulated phosphotyrosine-associated PI 3-kinase activity after exercise.

Somatostatin modulates G-CSF-induced but not interleukin-3-induced proliferative responses in myeloid 32D cells via activation of somatostatin receptor subtype 2

Somatostatin, originally identified as a peptide involved in neurotransmission, functions as an inhibitor of multiple cellular responses, including hormonal secretion and proliferation. Somatostatin acts through activation of G-protein-coupled receptors of which five subtypes have been identified. We have recently established that human CD34/c-kit expressing hematopoietic progenitors and acute myeloid leukemia (AML) cells exclusively express SSTR2. A major mechanism implicated in the antiproliferative action of somatostatin involves activation of the SH2 domain-containing protein tyrosine phosphatase SHP-1. While 0.1-1 x 10(-9) M of somatostatin, or its synthetic stable analog octreotide, can inhibit G-CSF-induced proliferation of AML cells, little or no effects are seen on GM-CSF- or IL-3-induced responses.
MATERIALS AND METHODS: To study the mechanisms underlying the antiproliferative responses of myeloblasts to somatostatin, clones of the IL-3-dependent murine cell line 32D that stably express SSTR2 and G-CSF receptors were generated. RESULTS: Similar to AML cells, octreotide inhibited G-CSF-induced but not IL-3-induced proliferative responses of 32D[G-CSF-R/SSTR2] cells. Somatostatin induced SHP-1 activity and inhibited G-CSF-induced, but not IL-3-induced, activation of the signal transducer and activator of transcription proteins STAT3 and STAT5.
CONCLUSION: Based on these data and previous results, we propose a model in which recruitment and activation of the tyrosine phosphatase SHP-1 by SSTR2 is involved in the selective negative action of somatostatin on G-CSF-R signaling.

Differential tumor necrosis factor receptor 2-mediated editing of virus-specific CD8+ effector T cells

Much of the CD8+ T cell response in H2b mice with influenza pneumonia is directed at the nucleoprotein366-374 (NP366) and acid polymerase224-233 (PA224) peptides presented by the H2Db MHC class I glycoprotein. These DbNP366- and DbPA224-specific T cell populations are readily analyzed by staining with tetrameric complexes of MHC+ peptide (tetramers) or by cytokine production subsequent to in vitro stimulation with the cognate peptides. The DbPA224-specific CD8+ effector T cells make more tumor necrosis factor (TNF) α than the comparable CD8+DbNP366+ set, a difference reflected in the greater sensitivity of the CD8+DbPA224+ population to TNF receptor (TNFR) 2-mediated apoptosis under conditions of in vitro culture. Freshly isolated CD8+DbNP366+ and CD8+DbPA224+ T cells from influenza-infected TNFR2-/- mice produce higher levels of IFN-γ and TNF-α after in vitro stimulation with peptide, although the avidity of the T cell receptor-epitope interaction does not change. Increased numbers of both CD8+DbPA224+ and CD8+DbNP366+ T cells were recovered from the lungs (but not the spleens) of secondarily challenged TNFR2-/- mice, a pattern that correlates with the profiles of TNFR expression in the TNFR2+/+ controls. Thus, it seems that TNFR2-mediated editing of influenza-specific CD8+ T cells functions to limit the numbers of effectors that have localized to the site of pathology in the lung but does not modify the size of the less activated responder T cell populations in the spleen. Therefore, the massive difference in magnitude for the secondary, although not the primary, response to these DbNP366 and DbPA224 epitopes cannot be considered to reflect differential TNFR2-mediated T cell editing.

Molecular characterization and analysis of a truncated serotonin receptor gene expressed in neural and reproductive tissues of abalone

In molluscs, the neurotransmitter serotonin (5-HT) has been linked to a variety of biological roles including gamete maturation and spawning. The possible involvement of 5-HT in abalone gamete release was demonstrated by a dose-dependent increase in Haliotis rubra gonad contractile bioactivity following 5-HT stimulation. Physiological functions associated with 5-HT, are mediated through binding to 5-HT receptors. A cDNA encoding a putative 5-HT receptor consisting of 359 amino acids was isolated from the tropical abalone H. asinina, termed 5-HT_{1 ha}. The 5-HT_{1 ha} shares G-protein-coupled receptor motifs with metazoan 5-HT receptors, including predicted transmembrane domains, active sites for protein kinase action, and N-linked glycosylation sites. However, the third intracellular loop of 5-HT_{1 ha} is relatively short, and only six transmembrane domains are predicted, implying a truncated receptor. Phylogenetic analysis with known 5-HT receptor genes suggests that 5-HT_{1 ha} belongs to the type 1 5-HT receptor family. Expression analysis by RT-PCR showed that 5-HT_{1 ha}  mRNA was present in all tissues examined, including the neural ganglia and gonad tissues. Immunocytochemistry revealed the presence of 5-HT_{1 ha} specifically within the soma of neuronal cells located in the outer cortex of both cerebral and pleuropedal ganglia. In ovarian and testicular tissues, 5-HT_{1 ha} immunoreactivity was observed in epithelial cells of the outer capsule and connective tissue of the trabeculae to which the gamete follicles adhere. Whether this receptor transcript is translated to a functional protein needs to be verified, but if so, it could play a role in reproduction.

Insulin-stimulated insulin recetor substrate-2-associated phosphatidylinositol 3-kinase activity is enhanced in human skeletal muscle after exercise.

Exercise increases skeletal muscle insulin action but the underlying mechanisms mediating this are equivocal. In mouse skeletal muscle, prior exercise enhances insulin-stimulated insulin receptor substrate-2 (IRS-2) signaling (Diabetes 2002;51:479-83), but it is unknown if this also occurs in humans. Hyperinsulinemic-euglycemic clamps were performed on 7 untrained males at rest and immediately after 60 minutes of cycling exercise at ~75% Vo_2peak. Muscle biopsies were obtained at basal, immediately after exercise, and at 30 and 120 minutes of hyperinsulinemia. Insulin infusion increased (P < .05) insulin receptor tyrosine phosphorylation similarly in both the rest and exercise trials. Under resting conditions, insulin infusion resulted in a small, but non–statistically significant increase in IRS-2–associated phosphatidylinositol 3 (PI 3)–kinase activity over basal levels. Exercise per se decreased (P < .05) IRS-2–associated PI 3–kinase activity. After exercise, insulin-stimulated IRS-2–associated PI 3–kinase activity tended to increase at 30 minutes and further increased (P < .05) at 120 minutes when compared with the resting trial. Insulin increased (P < .05) Akt Ser⁴⁷³ and GSK-3α/β Ser²¹/Ser⁹ phosphorylation in both trials, with the response tending to be higher in the exercise trial. In conclusion, in the immediate period after an acute bout of exercise, insulin-stimulated IRS-2 signaling is enhanced in human skeletal muscle.

Analysis of forest policy using multi-attribute value theory

Contents:
1. Role of multi-criteria decision making in natural resource management /​ Gamini Herath and Tony Prato
2. Analysis of forest policy using multi-attribute value theory /​ Jayanath Ananda and Gamini Herath
3. Comparing Riparian revegetation policy options using the analytic hierarchy process /​ M. E. Qureshi and S. R. Harrison
4. Managing environmental and health risks from a lead and zinc smelter : an application of deliberative multi-criteria evaluation /​ Wendy Proctor, Chris McQuade and Anne Dekker
5. Multiple attribute evaluation of management alternatives for the Missouri River System /​ Tony Prato
6. Multi-criteria decision analysis for integrated watershed management /​ Zeyuan Qiu
7. Fuzzy multiple attribute evaluation of agricultural systems /​ Leonie A. Marks and Elizabeth G. Dunn
8. Multi-criteria decision support for energy supply assessment /​ Bram Noble
9. Seaport development in Vietnam : evaluation using the analytic hierarchy process /​ Tran Phuong Dong and David M. Chapman
10. Valuing wetland aquatic resources using the analytic hierarchy process /​ Premachandra Wattage and Simon Mardle
11. Multiple attribute evaluation for national park management /​ Tony Prato
12. The future of MCDA in natural resource management : some generalizations /​ Gamini Herath and Tony Prato.

Qualitative analysis of the care of children in hospital in four countries : part 1

As part of a large study of the care of children in Australian, British, Indonesian, and Thai hospitals, qualitative methods were used to examine differences influenced by culture. Two groups were surveyed: parents of hospitalized children, and staff caring for them. Vignettes were used to invoke discussion, and content analysis was used to examine the data. Subjects were interviewed singly, or in focus groups. These interviews were audiotaped and transcribed verbatim. The data were explored by using content analysis to extract themes of understanding of cultural experiences. This article is the first of a two-part series, and includes a review of the literature, description of the methods used, and results of the parents' interviews. The staff results and discussion will be published in Part 2. Analysis revealed that parents in all countries were primarily concerned with treating the child's illness and the child's recovery. Parents were concerned with their work (employment), but this was a much larger consideration in Indonesia and Thailand, where no social security systems exist, than in Australia and Britain. Communication with staff was the most commonly mentioned theme for parents, indicating that irrespective of the culture in which the care was given, good communication between parents and staff was of paramount importance.

Differential effects of exercise on insulin-signaling gene expression in human skeletal muscle.

Skeletal muscle insulin sensitivity is enhanced after acute exercise and short-term endurance training. We investigated the impact of exercise on the gene expression of key insulin-signaling proteins in humans. Seven untrained subjects (4 women and 3 men) completed 9 days of cycling at 63 ± 2% of peak O₂ uptake for 60 min/day. Muscle biopsies were taken before, immediately after, and 3 h after the exercise bouts (on days 1 and 9). The gene expression of insulin receptor substrate-2 and the p85α subunit of phosphatidylinositol 3-kinase was significantly higher 3 h after a single exercise bout, although short-term training ameliorated this effect. Gene expression of insulin receptor and insulin receptor substrate-1 was not significantly altered at any time point. These results suggest that exercise may have a transitory impact on the expression of insulin receptor substrate-2 and phosphatidylinositol 3-kinase; however, the predominant actions of exercise on insulin sensitivity appear not to reside in the transcriptional activation of the genes encoding major insulin-signaling proteins.

Provision of meals on wheels to the elderly : case studies in the era of local government reform

Australia’s ageing population is on the increase. It is predicted that by 2021 one quarter of Victoria’s elderly population will be aged 60 and over. Not only are people living longer, but they also wish to remain living in the community. The Home and Community Care (HACC) Program was established in 1985 to facilitate and assist the elderly and disabled to remain in their home. In Victoria around 51% of HACC funds are managed by local governments, a level of government that has recently undergone major reforms, which ultimately impacted on the manner in which services were provided. The HACC program funds Meals on Wheels, a service that provides meals for those elderly who no longer can prepare their own meals. The aim of this study was to assess the Meals on Wheels service provided by two Melbourne councils with different service philosophies. The study has four main components: (1) Menu analysis by food and variety; (2) Analysis of actual meals; (3) Clients assessment of Food Services; and (4) Client assessment of the organoleptic qualities of Meals on Wheels. Two Melbourne councils were chosen for their different approaches to service delivery. The City of South tendered out both meal production and delivery, while the City of North maintained its MOW service in-house. The case study method of research allowed for each council’s service to be assessed objectively and without comparison. Several methodologies were used for collecting data in this study. Menu analysis was carried out by comparing the MOW menus with the HACC menu planning guidelines together with general menu planning principles. Analysis of actual meals was in two stages. The weights of the meals were recorded and compared with the HACC recommended food serving portions and meal combinations over a five-day period were analysed for their nutrient contents. Face to face interviews were conducted with clients for their assessment of MOW and the assessment of the organoleptic qualities of the meals was carried out over a five-day period. The results concluded that both councils menus were based on sound menu planning principles, but did not conform with Home and Community Care menu planning guidelines fully and did not include a serve of bread, fruit and milk. The weight analysis of the meal combinations revealed some discrepancies between actual meals and Home and Community Care guidelines by not meeting the recommended serving sizes. Meal combinations generally met Home and Community Care standard for kilojoule and protein, but other nutrients, such as thiamin, riboflavin, magnesium, calcium and zinc were generally below the recommended levels for Meals On Wheels. The majority of study group lived alone and received four to five meals per week. Delivery times of meals, selective menus and food quality were issues raised by clients. Whilst the quality and variety of vegetables was raised by clients they generally rated the organoleptic qualities of the meals as satisfactory. This study examined the four components of the service. A simple method of evaluation the service was developed, which highlight discrepancies with HACC standards and encouraged the councils to set a customer satisfaction standard. A number of recommendations are made to ensure that meals are aesthetically pleasing, including a list of different methods for preparing vegetables. The provision of additional foods, such as a “snack pack” is recommended to improve the supply of essential nutrients that were below the Home and Community Care standards. Meals on Wheels is a vital support service for the elderly living in the community and as such should aim to provide a high quality service that meets the needs of its clients.

Location rather than CD62L phenotype is critical in the early establishment of influenza-specific T cell memory

The rapid recall of influenza virus-specific CD8+ T cell effector function is protective, although our understanding of T cell memory remains incomplete. Recent debate has focused particularly on the CD62L lymph node homing receptor. The present analysis shows that although functional memory can be established from both CD62Lhi and CD62Llo CD8+ T cell subsets soon after initial encounter between naive precursors and antigen, the optimal precursors are CD8+CD44hiCD25lo immune lymphocytes isolated from draining lymph nodes on day 3.5 after influenza virus infection. Analysis of primed T cells at different times after challenge indicates that the capacity to transfer memory is diminished at the peak of the primary cytotoxic T lymphocyte response, challenging speculations that the transition to memory first requires full differentiation to effector status. It seems that location rather than CD62Lhi/lo phenotype may be the more profitable focus for further dissection of the early establishment of T cell memory.

Effect of vascular endothelial growth factor upregulation on retinal gene expression in the Kimba mouse

Background:  The Kimba mouse carries a human vascular endothelial growth factor transgene causing retinal neovascularisation similar to that seen in diabetic retinopathy. Here, we examine the relationship between differential gene expression induced by vascular endothelial growth factor overexpression and the architectural changes that occur in the retinae of these mice.

Methods:  Retinal gene expression changes in juvenile and adult Kimba mice were assayed by microarray and compared with age-matched wild-type littermates. Transcription of selected genes was validated by quantitative real-time polymerase chain reaction. Protein translation was determined using immunohistochemistry and enzyme-linked immunosorbent assay.

Results:  Semaphorin 3C was upregulated, and nuclear receptor subfamily 2, group 3, member 3 (Nr2e3) was downregulated in juvenile Kimba mice. Betacellulin and endothelin 2 were upregulated in adults. Semaphorin 3C colocalized with glial fibrillary acidic protein in Müller cells of Kimba retinae at greater signal intensities than in wild type. Endothelin 2 colocalised to Müller cell end feet and extended into the outer limiting membrane. Endothelin receptor type B staining was most pronounced in the inner nuclear layer, the region containing Müller cell somata.

Conclusions:  An early spike in vascular endothelial growth factor induced significant long-term retinal neovascularisation associated with changes to the retinal ganglion, photoreceptor and Müller cells. Overexpression of vascular endothelial growth factor led to dysregulation of photoreceptor metabolism through differential expression of Nr2e3, endothelin 2, betacellulin and semaphorin 3C. Alterations in the expression of these genes may therefore play key roles in the pathological mechanisms that result from retinal neovascularisation.

Impact of obesity and leptin treatment on adipocyte gene expression in Psammomys obesus

We examined the effects of leptin treatment on the expression of key genes in adipocyte metabolism in Psammomys obesus (P. obesus), a polygenic rodent model of obesity. Lean and obese P. obesus were given three daily intraperitoneal injections of either saline or leptin (total of 45 mg/kg per day) for 7 days. In lean animals, leptin treatment led to reductions in food intake, body weight and fat mass. Pair-fed animals matched for the reduction in food intake of the lean leptin-treated animals demonstrated similar reductions in body weight and fat mass. In obese P. obesus, leptin treatment failed to have any effect on body weight or body fat mass, indicating leptin resistance. Lipoprotein lipase, hormone-sensitive lipase and peroxisome proliferator activated receptor gamma 2 mRNA levels were significantly reduced in lean leptin-treated animals, whereas pair-fed animals were similar to lean controls. Uncoupling protein 2 and glycerol phosphate acyltransferase were also reduced in the lean leptin-treated animals, but not significantly so. Obese animals did not show any gene expression changes after leptin treatment. In conclusion, high circulating concentrations of leptin in lean P. obesus resulted in decreased gene expression of a number of key lipid enzymes, independent of changes in food intake, body weight and fat mass. These effects of leptin were not found in obese P. obesus.

Determination of sodium oxalate in Bayer liquor using flow-analysis incorporating an anion exchange column and tris(2,2`-bipyridyl)ruthenium(II) chemiluminescence detection

Semi-automated flow injection instrumentation, incorporating a small anion exchange column coupled with tris(2,2′-bipyridyl)ruthenium(II) (Ru(bipy)₃²⁺) chemiluminescence detection, was configured and utilised to develop rapid methodology for the determination of sodium oxalate in Bayer liquors. The elimination of both negative and positive interferences from aluminium(III) and, as yet, unknown concomitant organic species, respectively are discussed. The robustness of the methodology was considerably enhanced by using the temporally stable form of the chemiluminescence reagent, tris(2,2′-bipyridyl)ruthenium(III) perchlorate in dry acetonitrile. Real Bayer process samples were analysed and the results obtained compared well with those performed using standard methods within industrial laboratories.