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Cool temperatures can limit productivity of temperate grazing systems as poor pasture growth rates in winter create feed shortages for livestock. Ornithopus spp. (serradella) are broadly adapted annual pasture legumes that produce high-quality forage in soil types considered marginal for other temperate legume species. However, serradella establishment is perceived to be difficult in cool-season environments. We used survival analysis to compare germination rate and seedling emergence for two serradella species (yellow serradella and French serradella) against three reference species (Medicago sativa, M. polymorpha and Trifolium subterraneum ) in four temperature treatments (10/5, 15/10, 20/15 and 25/20°C; max/min). We also compared shoot relative growth rate and photosynthetic rate at 15/10°C (cool) and 23/18°C (warm). Cool temperatures (10/5, 15/10°C) did not slow germination rates for serradella relative to the reference species, but warm temperatures (20/15, 25/20°C) delayed emergence and reduced post-emergent shoot growth rates. Once established, Ornithopus spp. had similar mean photosynthetic rates and stomatal conductance at cool temperatures to the reference species. We conclude that, contrary to common perception, cool temperatures did not adversely influence germination, emergence, or early growth of Ornithopus spp. relative to the reference species.

Background and Purpose: The interrelation of cognitive performance, cerebrovascular damage and brain functional connectivity (FC) in advanced arteriosclerosis remains unclear. Our aim was to investigate the associations between FC, white matter damage and cognitive impairment in carotid artery disease., Methods: Seventy-one participants with a recent cerebrovascular event and with written informed consent underwent resting-state functional magnetic resonance imaging and the Addenbrooke's Cognitive Examination - Revised (ACE-R). Network and inter-hemispheric FC metrics were compared between cognitively normal and impaired subjects, and interrelated with cognition. In order to explore the nature of FC changes, their associations with microstructural damage of related white matter tracts and cognitive performance were investigated, followed by mediation analysis., Results: Participants with global cognitive impairment showed reduced FC compared to the cognitively intact subjects within the central executive network (CEN), and between hemispheres. Patients with executive dysfunction had decreased CEN FC whilst patients with memory loss demonstrated low FC in both the CEN and the default mode network (DMN). Global performance correlated with connectivity metrics of the CEN hub with DMN nodes, and between hemispheres. Cingulum mean diffusivity (MD) was negatively correlated with ACE-R and CEN-DMN FC. The cingulum MD-cognition association was partially mediated by CEN-DMN FC., Conclusions: Long-range functional disconnection of the CEN with DMN nodes is the main feature of cognitive impairment in elderly subjects with symptomatic carotid artery disease. Our findings provide further support for the connectional diaschisis concept of vascular cognitive disorder, and highlight a mediation role of functional disconnection to explain associations between microstructural white matter tract damage and cognitive impairment., (© 2019 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Cytomegalovirus (CMV) infection is a potentially fatal complication in patients receiving haematopoietic stem cell transplantation (HSCT), but recent evidence indicates that CMV has strong anti-leukaemia effects due in part to shifts in the composition of natural killer (NK) cell subsets. NK cells are the primary mediators of the anti-leukaemia effect of allogeneic HSCT, and infusion of allogeneic NK cells has shown promise as a means of inducing remission and preventing relapse of several different haematological malignancies. The effectiveness of these treatments is limited, however, when tumours express human leucocyte antigen (HLA)-E, a ligand for the inhibitory receptor NKG2A, which is expressed by the vast majority of post-transplant reconstituted and ex-vivo expanded NK cells. It is possible to enhance NK cell cytotoxicity against HLA-E pos malignancies by increasing the proportion of NK cells expressing NKG2C (the activating receptor for HLA-E) and lacking the corresponding inhibitory receptor NKG2A. The proportion of NKG2C pos /NKG2A neg NK cells is typically low in healthy adults, but it can be increased by CMV infection or ex-vivo expansion of NK cells using HLA-E-transfected feeder cells and interleukin (IL)-15. In this review, we will discuss the role of CMV-driven NKG2C pos /NKG2A neg NK cell expansion on anti-tumour cytotoxicity and disease progression in the context of haematological malignancies, and explore the possibility of harnessing NKG2C pos /NKG2A neg NK cells for cancer immunotherapy., (© 2018 British Society for Immunology.)

Background and Purpose: The role of clinical factors, cerebral infarcts and hippocampal damage in vascular cognitive impairment (VCI) subtypes remains unclear., Methods: Non-demented patients with carotid stenosis and recent transient ischemic attack/stroke had cognitive assessment and brain magnetic resonance imaging (MRI). Amnestic VCI was defined as memory impairment; non-amnestic VCI was any other subdomain impairment. Associations of MRI metrics [log-transformed total ischemic lesion load (log TILL), mesiotemporal atrophy (MTA) score, hippocampal mean diffusivity (hipMD)] with cognitive performance were assessed., Results: A hundred and eight patients, 47 with amnestic VCI and 21 with non-amnestic VCI, were assessed. A higher MTA (odds ratio 12.89, P = 0.001) and left hipMD (odds ratio 4.43, P = 0.003) contributed to amnestic VCI versus normal. Age-adjusted fluency correlated with log TILL (P = 0.002). Age-adjusted memory was associated with left hipMD (P = 0.001), MTA (P < 0.001) but not log TILL (P = 0.14). Left hipMD, MTA and smoking showed classification potential between amnestic VCI versus normal (area 0.859, P < 0.001)., Conclusions: Neuroimaging assists stratification in amnestic VCI characterized by hippocampal changes and in non-amnestic VCI by higher ischemic burden. MTA and hippocampal diffusivity show diagnostic biomarker potential., (© 2017 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

The number of peripheral blood B lymphocytes doubles during acute exercise, but the phenotypic composition of this response remains unknown. In two independent exercise studies, using complimentary phenotyping strategies, we investigated the mobilisation patterns of distinct B cell subsets. In study one, nine healthy males (mean±SD age: 22.1±3.4years) completed a continuous cycling bout at 80% V̇O2MAX for 20min. In study two, seven healthy experienced cyclists (mean±SD age: 29.9±4.7years) completed a 30min cycling trial at a workload corresponding to +5% of the individual blood lactate threshold. In study one, CD3-CD19+ B cell subsets were classified into immature (CD27-CD10+), naïve (CD27-CD10-), memory (CD27+CD38-), plasma cells/plasmablasts (CD27+CD38+) and finally, recently purported 'B1' cells (CD27+ CD43+ CD69-). In study two, CD20+ B cells were classified into immature (CD27-IgD-), naïve (CD27-IgD+), and IgM+/IgG+/IgA+ memory cells (CD27+IgD-). Total B cells exhibited a mean increase of 88% (study one) and 60% (study two) during exercise. In both studies, immature cells displayed the greatest increase, followed by memory cells, then naïve cells (study one: immature 130%>mature 105%>naïve 84%; study two: immature 110%>mature 56%>naïve 38%). Our findings show that, unlike T cells and NK cells, B cell mobilisation is not driven by effector status, and, for the first time, that B cell mobilisation during exercise is comprised of immature CD27- IgD-/CD10+ cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A- NK cells, which are potent killers of infected cells expressing human leucocyte antigen (HLA)-E. As HLA-E is also over-expressed in several haematological malignancies and CMV has been linked to a reduced risk of leukaemic relapse, we determined the impact of latent CMV infection on NK cell cytotoxicity against four tumour target cell lines with varying levels of HLA-E expression. NK cell cytotoxicity against K562 (leukaemia origin) and U266 (multiple myeloma origin) target cells was strikingly greater in healthy CMV-seropositive donors than seronegative donors and was associated strongly with target cell HLA-E and NK cell NKG2C expression. NK cell cytotoxicity against HLA-E transfected lymphoma target cells (221.AEH) was ∼threefold higher with CMV, while NK cell cytotoxicity against non-transfected 721.221 cells was identical between the CMV groups. NK cell degranulation (CD107a(+) ) and interferon (IFN)-γ production to 221.AEH cells was localized almost exclusively to the NKG2C subset, and antibody blocking of NKG2C completely eliminated the effect of CMV on NK cell cytotoxicity against 221.AEH cells. Moreover, 221.AEH feeder cells and interleukin (IL)-15 were found to expand NKG2C(+) /NKG2A(-) NK cells preferentially from CMV-seronegative donors and increase NK cell cytotoxicity against HLA-E(+) tumour cell lines. We conclude that latent CMV infection enhances NK cell cytotoxicity through accumulation of NKG2C(+) NK cells, which may be beneficial in preventing the initiation and progression of haematological malignancies characterized by high HLA-E expression., (© 2016 British Society for Immunology.)

Allogeneic hematopoietic stem cell transplant, to reconstitute the hematopoietic and immune status of patients undergoing myeloablative therapy for hematologic disorders, has been of great benefit in minimizing or eradicating disease and extending survival. Patients who undergo allogeneic hematopoietic stem cell transplant (allo-HSCT) are subject to many comorbidities among which the most significant, affecting quality of life (QoL) and survival, are acute GvHD (aGvHD) and chronic GvHD (cGvHD), resulting from donor lymphocytes reacting to and damaging host tissues. Physical activity and exercise have clearly been shown, in both children and adults, to enhance fitness, improve symptomatology and QoL, reduce disease progression and extend survival for many diseases including malignancies. In some cases, vigorous exercise has been shown to be equal to or more effective than pharmacologic therapy. This review addresses how cGvHD affects patients' physical function and physical domain of QoL, and the potential benefits of exercise interventions along with recommendations for relevant research and evaluation targeted at incorporating this strategy as soon as possible after allo-HSCT and ideally, as soon as possible upon diagnosis of the condition leading to allo-HSCT.

Purpose: To investigate the T-lymphocyte response to a period of increased training volume in trained females compared to habitual activity in female controls., Methods: Thirteen trained female (19.8 ± 1.9 yrs) soccer players were monitored during a two-week long high volume training period (increased by 39%) and thirteen female untrained (20.5 ± 2.2 yrs) controls were monitored during two-weeks of habitual activity. Blood lymphocytes, collected at rest, were isolated before and after the two-week period. Isolated lymphocytes were assessed for the cell surface expression of the co-receptor CD28, a marker of T-lymphocyte naivety, and CD57 a marker used to identify highly-differentiated T-lymphocytes. Co-expression of these markers was identified on helper CD4(+) and cytotoxic CD8(+) T-lymphocytes. In addition a further population of γδ(+) T-lymphocytes were identified. Plasma was used to determine Cytomegalovirus (CMV) serostatus., Results: No difference was observed in the T-lymphocyte populations following the two-week period of increased volume training. At baseline the number of total CD3(+), cytotoxic CD8(+), naïve (CD8(+) CD28(+) CD57(-)), intermediate (CD8(+) CD28(+) CD57(+)) T-lymphocytes and the number and proportion of γδ(+) T-lymphocytes were greater in the trained compared to the untrained females (p<0.05). The proportion of CD4(+)T-lymphocytes was greater in the untrained compared to the trained (p<0.05), in turn the CD4(+):CD8(+) ratio was also greater in the untrained females (p<0.05). Inclusion of percentage body fat as a covariate removed the main effect of training status in all T-lymphocyte sub-populations, with the exception of the γδ(+) T-lymphocyte population. 8% of the untrained group was defined as positive for CMV whereas 23% of the trained group was positive for CMV. However, CMV was not a significant covariate in the analysis of T-lymphocyte proportions., Conclusion: The period of high volume training had no effect on T-lymphocyte populations in trained females. However, baseline training status differences were evident between groups. This indicates that long-term exercise training, as opposed to short-term changes in exercise volume, appears to elicit discernible changes in the composition of the blood T-lymphocyte pool., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Purpose: MR imaging-detected carotid plaque hemorrhage is associated with an increased risk of recurrent ischemic cerebrovascular events and could be an indicator of disease progression; however, there are limited data regarding the dynamics of the MR imaging-detected carotid plaque hemorrhage signal. We assessed the temporal change of this signal and its impact on carotid disease progression., Materials and Methods: Thirty-seven symptomatic patients with 54 carotid stenoses of >30% on sonography underwent serial MR imaging during 24 months. A signal-intensity ratio of >1.5 between the carotid plaque and adjacent muscle was defined as plaque hemorrhage, and a change in signal-intensity ratio of >0.31 between time points was considered significant. Sixteen patients underwent ≥2 carotid sonography scans to determine the peak systolic velocities and degree of stenosis with time., Results: Of the 54 carotids, 28 had the presence of hyperintense signal on an MR imaging sequence (PH+) and 26 had the absence of hyperintense signal on an MR imaging sequence (PH-) at baseline. The signal-intensity ratio was stable in 33/54 carotid plaques, but 39% showed a change. Plaque hemorrhage classification did not change in 87% of carotid plaques, but 4 became PH+, and 3, PH-. As a group, PH+ carotids did not change significantly in signal-intensity ratio (P = .585), whereas PH- showed an increased signal-intensity ratio at 24.5 months (P = .02). In PH+ plaques, peak systolic velocities significantly increased by 22 ± 39.8 cm/s from baseline to last follow-up sonography (Z = 2.427, P = .013)., Conclusions: During 2 years, MR imaging-detected carotid plaque hemorrhage status remained stable in most (87%) cases with 4 (7%) incident plaque hemorrhages. PH+ plaques were associated with increased flow velocity during the follow-up period., (© 2015 by American Journal of Neuroradiology.)

Heart rate (HR) is a valuable and widespread measure for physical training programs, although its description of conditioning is limited to the cardiac response to exercise. More comprehensive measures of exercise adaptation include cardiac output (Q̇), stroke volume (SV) and oxygen uptake (V̇O2), but these physiological parameters can be measured only with cumbersome equipment installed in clinical settings. In this work, we explore the ability of pulse transit time (PTT) to represent a valuable pairing with HR for indirectly estimating Q̇, SV and V̇O2 non-invasively. PTT was measured as the time interval between the peak of the electrocardiographic (ECG) R-wave and the onset of the photoplethysmography (PPG) waveform at the periphery (i.e. fingertip) with a portable sensor. Fifteen healthy young subjects underwent a graded incremental cycling protocol after which HR and PTT were correlated with Q̇, SV and V̇O2 using linear mixed models. The addition of PTT significantly improved the modeling of Q̇, SV and V̇O2 at the individual level ([Formula: see text] for SV, 0.548 for Q̇, and 0.771 for V̇O2) compared to predictive models based solely on HR ([Formula: see text] for SV, 0.503 for Q̇, and 0.745 for V̇O2). While challenges in sensitivity and artifact rejection exist, combining PTT with HR holds potential for development of novel wearable sensors that provide exercise assessment largely superior to HR monitors.

Obesity has been associated with accelerated biological ageing and immunosenescence. As the prevalence of childhood obesity is increasing, we wanted to determine if associations between obesity and immunosenescence would manifest in children. We studied 123 Mexican American adolescents aged 10-14 (mean 12·3 ± 0·7) years, with body weights ranging from 30·1 to 115·2 kg (mean 52·5 ± 14·5 kg). Blood samples were obtained to determine proportions of naive, central memory (CM), effector memory (EM), senescent and early, intermediate and highly differentiated subsets of CD4(+) and CD8(+) T cells. Overweight and obese children had significantly lowered proportions of early CD8(+) T cells (B = -11·55 and -5·51%, respectively) compared to healthy weight. Overweight children also had more EM (B = +7·53%), late (B = +8·90%) and senescent (B = +4·86%) CD8(+) T cells than healthy weight children, while obese children had more intermediate CD8(+) (B = +4·59%), EM CD8(+) (B = +5·49%), late CD4(+) (B = +2·01%) and senescent CD4(+) (B = +0·98%) T cells compared to healthy weight children. These findings withstood adjustment for potentially confounding variables, including age, gender and latent cytomegalovirus and Epstein-Barr virus infections. We conclude that excess body mass, even in adolescence, may accelerate immunosenescence and predispose children to increased risks of incurring immune-related health problems in adulthood., (© 2014 British Society for Immunology.)

Success of long duration space missions will depend upon robust immunity. Decreased immunity has been observed in astronauts during short duration missions, as evident by the reactivation of latent herpes viruses. Seventeen astronauts were studied for reactivation and shedding of latent herpes viruses before, during, and after 9-14 days of 8 spaceflights. Blood, urine, and saliva samples were collected 10 days before the flight (L-10), during the flight (saliva only), 2-3h after landing (R+0), 3 days after landing (R+3), and 120 days after landing (R+120). Values at R+120 were used as baseline levels. No shedding of viruses occurred before flight, but 9 of the 17 (designated "virus shedders") shed at least one or more viruses during and after flight. The remaining 8 astronauts did not shed any of the 3 target viruses (non-virus shedders). Virus-shedders showed elevations in 10 plasma cytokines (IL-1α, IL-6, IL-8, IFNγ, IL-4, IL-10, IL-12, IL-13, eotaxin, and IP-10) at R+0 over baseline values. Only IL-4 and IP-10 were elevated in plasma of non-virus shedders. In virus shedders, plasma IL-4 (a Th2 cytokine) was elevated 21-fold at R+0, whereas IFNγ (a Th1 cytokine) was elevated only 2-fold indicating a Th2 shift. The inflammatory cytokine IL-6 was elevated 33-fold at R+0. In non-shedding astronauts at R+0, only IL-4 and IP-10 levels were elevated over baseline values. Elevated cytokines began returning to normal by R+3, and by R+120 all except IL-4 had returned to baseline values. These data show an association between elevated plasma cytokines and increased viral reactivation in astronauts., (Copyright © 2012. Published by Elsevier Ltd.)

The relation between haem biosynthesis and intestinal iron absorption is not well understood, we therefore investigated the effect of compounds that alter haem metabolism on duodenal iron absorption. CD1 mice were treated with either an inhibitor (succinyl acetone (SA)) or stimulator (2-allyl-2-isopropylacetamide (AIA)) of haem biosynthesis. 5-Aminolaevulinic acid (ALA) dehydratase and urinary ALA and porphobilinogen (PBG) levels, were determined. Intestinal iron absorption was assayed with in vivo and in vitro techniques. Liver hepcidin (Hamp1) and duodenal iron transporter mRNA levels were measured using RT-PCR. AIA caused increased hepatic ALA synthase (1.6-fold) and ALA dehydratase (1.4-fold, both p<0.005) activities and increased urinary ALA and PBG excretion (2.1- and 1.4-fold, p<0.005, p<0.05, respectively). In vivo intestinal iron absorption was reduced to 49% of control (p<0.005). Mice treated with SA showed decreased urinary ALA and PBG levels (75 and 55% control, both p<0.005) and reductions in both ALA synthase and ALA dehydratase activities (77 and 56% control, p<0.05, p<0.005, respectively) in the liver. Liver and duodenal haem and cytochrome oxidase levels were not significantly decreased. Iron absorption was enhanced (1.26-fold, p<0.05) and hepatic Hamp1 mRNA was reduced (53% of control, p<0.05). In vitro duodenal iron uptake after mice were injected with SA also demonstrated an increase in Fe(III) reduction and uptake (1.27- and 1.41-fold, p<0.01 respectively). Simultaneous injections of SA and ALA blocked the enhancing effect on iron absorption seen with SA alone. We conclude that alterations in haem biosynthesis can influence iron absorption and in particular, the intermediate ALA seems to be an inhibitor of iron absorption.

Background: The brush border ferric reductase (Dcytb) is critical for the absorption of dietary iron and appears to be expressed on the duodenal enterocyte brush border. The Dcytb expression is increased in severe iron-deficient anaemia, but the situation in a more typical mild iron deficiency is unclear. This study investigated Dcytb expression in patients with normal iron status or mild iron deficiency and its relationships with enterocyte iron status., Materials and Methods: Duodenal biopsy specimens and blood samples were obtained from 32 patients undergoing routine upper gastrointestinal endoscopy. Twenty-three specimens (six iron-deficient and 17 iron-replete) were processed for light-microscopy (LM) and for immunohistochemistry with antibodies against Dcytb and heavy/light chain ferritin subunits. The nine remaining biopsies (three iron-deficient and six iron-replete) were processed for electron microscopy (EM). Immunolocalization of Dcytb and intracellular ferritin was performed with appropriate primary antibodies followed by 10-nm gold conjugate labels., Results: The LM process showed a strong negative correlation between immunolabelling intensity of Dcytb on the enterocyte brush border and serum iron saturation (P < 0.001), but only a weak negative correlation between this antigen and haemoglobin (P = 0.08) or serum ferritin concentrations (P = 0.4). EM confirmed anti-Dcytb preferential labelling of microvilli rather than enterocyte cytoplasm (P = 0.001), but preferential antiferritin labelling of cytoplasm (P < 0.02). There was no correlation with enterocyte cytoplasmic ferritin labelling (i.e. enterocyte iron status and Dcytb expression)., Conclusions: Enterocyte Dcytb brush border expression is increased even in mild iron deficiency and may be related to serum iron saturation. The lack of correlation with enterocyte ferritin expression deserves further study with direct measurement of intracellular iron.

To investigate the functional significance of mutations in Ferroportin that cause hereditary iron overload, we directly measured the iron efflux activity of the proteins expressed in Xenopus oocytes. We found that wild type and mutant Ferroportin molecules (A77D, N144H, Q248H and V162Delta) were all expressed at the plasma membrane at similar levels. All mutations caused significant reductions in (59)Fe efflux compared to wild type but all retained some residual transport activity. A77D had the strongest effect on (59)Fe efflux (remaining activity 9% of wild-type control), whereas the N144H mutation retained the highest efflux activity (42% of control). The Q248H and V162Delta mutations were intermediate between these values. Co-injection of mutant and wild-type mRNAs revealed that the A77D and N144H mutations had a dominant negative effect on the function of the WT protein.

Intron 3 and the flanking exons of the calmodulin gene have been amplified, cloned, and sequenced from 18 members of the gastropod genus Littorina. From the 48 sequences, at least five different gene copies have been identified and their functionality characterized using a strategy based upon the potential protein product predicted from flanking exon data. The functionality analyses suggest that four of the genes code for functional copies of calmodulin. All five copies have been identified across a wide range of littorinid species although not ubiquitously. Using this novel approach based on intron sequences, we have identified an unprecedented number of potential calmodulin copies in Littorina, exceeding that reported for any other invertebrate. This suggests a higher number of, and more ancient, gene duplications than previously detected in a single genus.

In this study, well-characterised animal models of altered iron metabolism were used to investigate link(s) between haem biosynthesis and intestinal iron absorption. Mice rendered iron deficient by feeding a low-iron diet for 3-4 weeks showed low levels of hepatic non-haem iron and hepcidin mRNA, with reduced urinary 5-aminolaevulinic acid (ALA) excretion and enhanced intestinal iron absorption. Hepatic ALA synthase activity was reduced while ALA dehydratase activity was increased. Iron-loaded mice had markedly increased liver non-haem iron and hepcidin mRNA, with increased urinary ALA excretion. Intestinal iron absorption was decreased mainly due to a reduction in transfer of absorbed iron from mucosa to the carcass. Hepatic ALA synthase activity was increased and ALA dehydratase activity moderately reduced. Mice exposed to hypoxia (0.5 atm) for 1-3 days had reduced hepatic hepcidin mRNA and urinary ALA excretion, while intestinal iron absorption was increased. Hepatic ALA synthase activity was reduced. The ALA dehydratase activity in liver and spleen was markedly enhanced. Injection of ALA to iron-deficient mice or hypoxic mice reduced their intestinal iron absorption to normal levels. This study further supports the hypothesis that alterations in haem biosynthesis influence duodenal iron absorption. ALA in particular appears to function as a modulator in controlling intestinal iron absorption.

Iron metabolism in animals is altered by haemolytic anaemia induced by phenylhydrazine (PHZ). In common with a number of other modulators of iron metabolism, the mode and the mechanisms of this response are yet to be determined. However, recent studies have shown increased expression of the ferrous transporter DMT1 in the duodenum and other tissues of mice administered PHZ. We examined the expression of the ferric reductase Dcytb, DMT1 and some other genes involved in Fe metabolism in tissues of mice dosed with PHZ. The expression of iron-related genes in the duodenum, liver, and spleen of the mice were evaluated using Northern blot analyses, RT-PCR and immunocytochemistry. Dcytb, and DMT1 mRNA and protein increased markedly in the duodenum of mice given PHZ. The efflux protein Ireg1 also increased in the duodenum of the treated mice. These changes correlated with a decrease in hepatic hepcidin expression. Dcytb, DMT1, Ireg1 and transferrin receptor 1 mRNA expression in the spleen and liver of mice treated with PHZ responded to the enhanced iron demand associated with the resulting stimulation of erythropoiesis. Enhanced iron absorption observed in PHZ-treated animals is facilitated by the up-regulation of the genes involved in iron transport and recycling. The probable association of the erythroid and the store regulators of iron homeostasis and absorption in the mice is discussed.

Background: Haem biosynthesis may regulate intestinal iron absorption through changes in cellular levels of delta-aminolaevulinic acid (ALA), haem and perhaps other intermediates. CoCl2 and NiCl2 are activators of haem oxygenase, the rate-limiting enzyme in haem catabolism. Co2+ and Ni2+ may also regulate and increase iron absorption through a mechanism that simulates hypoxic conditions in the tissues., Design: We assayed intestinal iron absorption in mice dosed with CoCl2 or NiCl2. The effects of these metal ions on splenic and hepatic levels of ALA synthase and dehydratase as well as urinary levels of ALA and phosphobilinogen were also assayed., Results: While Co2+ enhanced iron absorption when administered to mice at doses of 65, 125 and 250 micromoles kg(-1) body weight, Ni2+ was effective only at the highest dose. Ni2+ but not Co2+ at the highest dose reduced urinary ALA in the treated mice. Both metals ions increased splenic expression of haem oxygenase 1 and iron regulated protein 1, proteins involved, respectively, in haem degradation and iron efflux. Co2+ induced erythropoietin expression., Conclusions: The data suggest that while the effect of Ni2+ on iron absorption could be explained by effects on ALA, the effect of Co2+ may not be explained simply by changes in haem metabolism; therefore, effects mediated by alterations of specific haemoproteins by mechanisms that simulate tissue hypoxia could be important.

Improved search algorithms and scoring functions are required before the identification of peptide tandem MS data can be considered to be fully reliable and automatable. The development of models that can accurately predict product ion spectra from a peptide sequence would certainly help achieve this goal, but this firstly requires a better understanding of the process of fragmentation of peptides in the gas-phase. We summarize recent developments in this area and show that the prediction of product ion spectra is feasible and should improve the identification of peptide tandem MS data, especially for peptides that currently give low or insignificant scores with current search algorithms.

Background: Genetic haemochromatosis is a common hereditary iron loading disorder in humans. The disease is associated with loss of function mutations in the HFE gene. This is thought to change iron stores via increased iron absorption., Aims: In this study we investigated how adaptation of mucosal reductase activity is engaged in this process and how the changes compare with adaptation seen when an iron deficient diet is fed., Methods: Duodenal mucosal surface reductase was measured with nitroblue tetrazolium in age matched groups of male Hfe knockout mice (Hfe) and wild- type mice fed a purified diet containing normal (iron adequate), high (iron rich), or low (iron deficient) iron concentrations., Results: Reductase activity increased when mice were fed an iron deficient diet and decreased when they were fed an iron rich diet. Total villus activity, as measured by the average area under the activity curve along the crypt-villus axis, was increased 2.8-2.9-fold by iron deficiency in both genotypes. Approximately half of this difference was attributable to the significantly increased length of the villi in mice on an iron deficient diet (p<0.05). Hfe knockout did not affect villus length but increased mucosal reductase activity near the villus tips. Similar increases (1.3-1.6-fold) were seen on all diets but the increase was significant for iron deficient and iron loaded diets only (p<0.05)., Conclusion: Hfe gene product and dietary iron downregulate villus reductase activity in mice.

Dysregulated production of IL-6 and its receptor (IL-6R) are implicated in the pathogenesis of multiple myeloma, autoimmune diseases and prostate cancer. The IL-6R complex comprises two molecules each of IL-6, IL-6R, and the signaling molecule, gp130. Here, we report the x-ray structure (2.4 A) of the IL-6R ectodomains. The N-terminal strand of the Ig-like domain (D(1)) is disulfide-bonded to domain D(2), and domains D(2) and D(3), the cytokine-binding domain, are structurally similar to known cytokine-binding domains. The head-to-tail packing of two closely associated IL-6R molecules observed in the crystal may be representative of the configuration of the physiological dimer of IL-6R and provides new insight into the architecture of the IL-6R complex.

Dcytb has been identified as the mammalian transplasma ferric reductase that catalyzes the reduction of ferric to ferrous iron in the process of iron absorption. Its mRNA and protein levels are up-regulated by several independent stimulators of iron absorption. Furthermore, its cDNA encodes putative binding sites for heme and ascorbic acid. Using Northern and Western blots, RT-PCR and confocal microscopy, we studied the expression and localisation of Dcytb in cell lines and tissues of CD1 mice. Dcytb expression and function were modulated by iron. Dcytb and DMT1, both predominantly localised in the apical region of the duodenum were up-regulated in iron deficiency. Dcytb, the iron regulated ferric reductase may also utilize cytoplasmic ascorbate as electron donor for transmembrane reduction of iron. Dcytb expression was found in other tissues apart from the duodenum and its regulation and functions at these other sites are of interest in iron metabolism.

Duodenal cytochrome b (Dcytb) is a haem protein similar to the cytochrome b561 protein family. Dcytb is highly expressed in duodenal brush-border membrane and is implicated in dietary iron absorption by reducing dietary ferric iron to the ferrous form for transport via Nramp2/DCT1 (divalent-cation transporter 1)/DMT1 (divalent metal-transporter 1). The protein is expressed in other tissues and may account for ferric reductase activity at other sites in the body.

Background: HFE knockout mice (C57BL/6 x 129/Ola strain) mimic the functional aberrations of human hereditary haemochromatosis (HH) in short-term experiments. The present study investigates functional and morphological long-term changes., Methods: HFE(o/o), HFE(+/o) and HFE(+/+) mice were maintained on iron-rich and control diets for 2 weeks, 3, 12 and 18 months. Light microscopic tissue iron distribution, pathomorphological alterations, tissue iron content and oxidative stress were analysed in liver, pancreas, spleen, gastrointestinal tract, kidneys and myocardium. Additionally, duodenal 59Fe absorption and 59Fe whole body loss were measured., Results: Iron distribution between organs and microscopic iron deposition in the tissues resembled the patterns described in HH. After 3 months of iron-rich feeding duodenal 59Fe absorption decreased to approximately 15% of iron-adequate controls but remained about twice as high in HFE(o/o) as in HFE(+/+) mice. Hepatic iron concentrations reached only half the values known to induce hepatic fibrosis in rats and humans, while whole body 59Fe loss was about twice as high. Consequently no hepatic fibrosis developed, although massive hepatocellular iron deposition and indication for oxidative stress were observed., Conclusion: C57BL/6 x 129/O1a HFE(o/o) mice mimic HH iron distribution and the regulation of intestinal iron absorption after long-term feeding. However, characteristic morphological late changes in untreated HH are not modelled.

Central infusion of angiotensin IV or its more stable analogues facilitates memory retention and retrieval in normal animals and reverses amnesia induced by scopolamine or by bilateral perforant pathway lesions. These peptides bind with high affinity and specificity to a novel binding site designated the angiotensin AT(4) receptor. Until now, the AT(4) receptor has eluded molecular characterization. Here we identify the AT(4) receptor, by protein purification and peptide sequencing, to be insulin-regulated aminopeptidase (IRAP). HEK 293T cells transfected with IRAP exhibit typical AT(4) receptor binding characteristics; the AT(4) receptor ligands, angiotensin IV and LVV-hemorphin 7, compete for the binding of [(125)I]Nle(1)-angiotensin IV with IC(50) values of 32 and 140 nm, respectively. The distribution of IRAP and its mRNA in the brain, determined by immunohistochemistry and hybridization histochemistry, parallels that of the AT(4) receptor determined by radioligand binding. We also show that AT(4) receptor ligands dose-dependently inhibit the catalytic activity of IRAP. We have therefore demonstrated that the AT(4) receptor is IRAP and propose that AT(4) receptor ligands may exert their effects by inhibiting the catalytic activity of IRAP thereby extending the half-life of its neuropeptide substrates.

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to cause enteropathy, but the mechanism by which this toxicity occurs is less well established. This paper sets out to test the hypothesis that these drugs affect oxidative phosphorylation in jejunal tissue, thereby interfering with energy metabolism and rendering the tissue vulnerable to damage. Jejunal tissue obtained from rats and humans was used for in vitro determinations of oxygen uptake, lactate production and energy charge levels in the presence of indomethacin, a commonly used NSAID. In the rat jejunal tissue, drug concentrations of 0.5 mM and 2.5 mM produced significant decreases in oxygen uptake (P<0.01) and energy charge levels in the tissue (P<0.05). There was a corresponding increase in lactate production by the tissue at these indomethacin concentrations (P<0.05). Rat jejunum examined by electron microscopy after incubation with various concentrations of indomethacin showed ultrastructural effects of the drug on mitochondrial morphology. In human tissue, an inhibitory effect of indomethacin on oxygen uptake was seen, but the effects on lactate production and energy charge were less conclusive. These findings suggest that indomethacin affects mitochondria and thereby impairs energy metabolism in jejunal tissue.

Background: A part of the mechanism of the gastrointestinal toxicity exhibited by non-steroidal anti-inflammatory drugs is believed to involve the uncoupling of mitochondrial oxidative phosphorylation. Most previous uncoupling studies have used rat liver mitochondria. There is little information on the effects of the drugs on mitochondria from other species., Aim: To study the effect of indometacin on isolated liver mitochondria from rats, mice and humans., Methods: We studied the effects of indometacin on respiration and adenosine triphosphate synthesis by isolated liver mitochondria from rats, mice and humans. Its effects were compared with those of dinitrophenol, a classical uncoupler., Results: Indometacin uncoupled oxidative phosphorylation at low concentrations (P < 0.05) and inhibited respiration at high concentrations (P < 0.01) in all three species. Adenosine triphosphate synthesis was, however, more sensitive to dinitrophenol or indometacin at lower concentrations in mouse and human compared to rat liver mitochondria (P < 0.05)., Conclusions: The current study shows that indometacin acts as an inhibitory uncoupler in human mitochondria. It also demonstrates that the responses of rat, mouse and human mitochondria to indometacin are broadly similar.

Along with the great strides that have been made towards understanding cancer, has come a realization of the complexity of molecular events that lead to malignancy. Proteomics-based approaches, which enable the quantitative investigation of both cellular protein expression levels and protein-protein interactions involved in signaling networks, promise to define the molecules controlling the processes involved in cancer.

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

gp130 is the common signal transducing receptor subunit for the interleukin-6-type family of cytokines. Its extracellular region (sgp130) is predicted to consist of five fibronectin type III-like domains and an NH2-terminal Ig-like domain. Domains 2 and 3 constitute the cytokine-binding region defined by a set of four conserved cysteines and a WSXWS motif, respectively. Here we determine the disulfide structure of human sgp130 by peptide mapping, in the absence and presence of reducing agent, in combination with Edman degradation and mass spectrometry. Of the 13 cysteines present, 10 form disulfide bonds, two are present as free cysteines (Cys(279) and Cys(469)), and one (Cys(397)) is modified by S-cysteinylation. Of the 11 potential N-glycosylation sites, Asn(21), Asn(61), Asn(109), Asn(135), Asn(205), Asn(357), Asn(361), Asn(531), and Asn(542) are glycosylated but not Asn(224) and Asn(368). The disulfide bonds, Cys(112)-Cys(122) and Cys(150)-Cys(160), are consistent with known cytokine-binding region motifs. Unlike granulocyte colony-stimulating factor receptor, the connectivities of the four cysteines in the NH2-terminal domain of gp130 (Cys(6)-Cys(32) and Cys(26)-Cys(81)) are consistent with known superfamily of Ig-like domains. An eight-residue loop in domain 5 is tethered by Cys(436)-Cys(444). We have created a model predicting that this loop maintains Cys(469) in a reduced form, available for ligand-induced intramolecular disulfide bond formation. Furthermore, we postulate that domain 5 may play a role in the disulfide-linked homodimerization and activation process of gp130.

The ability of intestinal mucosa to absorb dietary ferric iron is attributed to the presence of a brush-border membrane reductase activity that displays adaptive responses to iron status. We have isolated a complementary DNA, Dcytb (for duodenal cytochrome b), which encoded a putative plasma membrane di-heme protein in mouse duodenal mucosa. Dcytb shared between 45 and 50% similarity to the cytochrome b561 family of plasma membrane reductases, was highly expressed in the brush-border membrane of duodenal enterocytes, and induced ferric reductase activity when expressed in Xenopus oocytes and cultured cells. Duodenal expression levels of Dcytb messenger RNA and protein were regulated by changes in physiological modulators of iron absorption. Thus, Dcytb provides an important element in the iron absorption pathway.

Aggressive lipid management is likely beneficial for coronary artery disease patients with diabetes mellitus or of advanced age. Nevertheless, a study of a large national sample of patients seen in ambulatory medical practices suggests pharmacologic undertreatment in these high-risk groups.

The gas-phase ion-molecule reactions of neutral alanylglycine have been examined with various mass-selected acylium ions RCO(+) (R= CH(3), CD(3), C(6)H(5), C(6)F(5) and (CH(3))( 2)N), as well as the transacylation reagent O-benzoylbenzophenone in a Fourier transform ion cyclotron resonance mass spectrometer. Reactions of the gaseous dipeptide with acylium ions trapped in the ICR cell result in the formation of energized [M + RCO](+) adduct ions that fragment to yield N-terminal b-type and C-terminal y-type product ions, including a modified b(1) ion which is typically not observed in the fragmentation of protonated peptides. Judicious choice of the acylium ion employed allows some control over the product ion types that are observed (i.e., b versus y ions). The product ion distributions from these ion--molecule reactions are similar to those obtained by collision-activated dissociation in a triple quadrupole mass spectrometer of the authentic N-acylated alanylglycine derivatives. These data indicate that derivatization of the peptide in the gas-phase occurs at the N-terminal amine. Ab initio molecular orbital calculations, performed to estimate the thermochemistry of the steps associated with adduct formation as well as product ion formation, indicate that (i) the initially formed adduct is energized and hence likely to rapidly undergo fragmentation, and (ii) the likelihood for the formation of modified b(1) ions in preference to y(1) ions is dependent on the R substituent of the acylium ion. The reaction of the tetrapeptide valine--alanine--alanine--phenylalanine with the benzoyl cation was also found to yield a number of product ions, including a modified b(1) ion. This result suggests that the new experimental approach described here may provide a tool to address one of the major limitations associated with traditional mass spectrometric peptide sequencing approaches, that is, determination of the identity and order of the two N-terminal amino acids. Analogies are made between the reactions observed here and the derivatization and N-terminal cleavage reactions employed in the condensed-phase Edman degradation method.

Increased left ventricular (LV) mass is often found in adults and is a powerful predictor of cardiovascular mortality. To test the hypothesis that an electrocardiographic estimate of LV mass--the Cornell voltage--is associated with ventricular premature complexes (VPCs) in free-living adults, a cross-sectional analysis of the predictors of VPCs on a 2-minute rhythm strip in a population-based sample of 13,606 middle-aged, African-American and white men and women from 4 US communities in the Atherosclerosis Risk in Communities Study baseline examinations was performed. In adults without known coronary artery disease, the prevalence of VPCs increases monotonically with increasd Cornell voltages within ethnicity and gender groups. Independent of systemic hypertension, serum electrolytes, age, heart rate, educational attainment, gender, and ethnicity, a millivolt increase in Cornell voltage was associated with a 20% to 30% increase in the prevalence odds ratio of VPCs on the 2-minute electrocardiogram. Thus, Cornell voltage is associated with VPCs on a 2-minute electrocardiogram. The association is consistent in African-Americans, whites, men, and women.

Epidermal growth factor (EGF) receptor ligands such as EGF and transforming growth factor-alpha (TGF-alpha) play an important role in controlling the proliferation, survival, morphology, and motility of colonic epithelial cells. There is also increasing evidence that growth factors and extracellular matrix (ECM) proteins cooperate to regulate these cellular processes. We have reported previously that autocrine TGF-alpha and an unidentified ECM protein in the serum-free conditioned medium of the human colon carcinoma cell line LIM1215 synergize to induce spreading of these cells in low-density cultures. We have now purified the ECM protein secreted by LIM1215 cells and show that it synergizes with EGF to induce spreading of LIM1215 cells and other human cell lines from the colon and other tissues. The purified ECM migrated as a single protein band with an apparent molecular mass of approximately 800 kDa on SDS-PAGE under nonreducing conditions and, under reducing conditions, as three protein bands of approximately 360, 210, and 200 kDa. Immunoblotting experiments and mass spectrometry analysis of tryptic digests on the purified protein identified the 360-, 210-, and 200-kDa protein bands as laminin alpha5, beta1, and gamma1 chains, respectively, indicating that LIM1215 cells secrete laminin-10 (alpha5 beta1 gamma1). In serum-free medium, LIM1215 cells adhere to laminin-10 primarily via alpha2 beta1 and alpha3 beta1 integrin receptors. EGF-induced spreading of LIM1215 cells on laminin-10 is partially inhibited by pretreatment of the cells with blocking antibodies directed against integrin alpha3 or beta1 but not alpha2, alpha6, or beta4 subunits. Spreading is almost completely inhibited by blocking alpha3 + alpha2, alpha3 + alpha6, or beta1 + beta4 integrin chains and results in cell death. Increased spreading in the presence of EGF correlates with up-regulation of alpha6 beta4 integrins in these cells after exposure to EGF. These results indicate that colon cancer cells attach and spread on laminin-10 via multiple integrin receptors and suggest a critical role for alpha3 beta1 integrins in the spreading response. Together, our results support the concept that the adhesive properties of colon cancer cells are modulated by autocrine production of TGF-alpha and laminin-10 and autocrine induction of appropriate integrins.

The gas phase fragmentation reactions of protonated serine and its YNHCH(CH2X)CO2H derivatives, beta-chloroalanine, S-methyl cysteine, O-methyl serine, and O-phosphoserine, as well as the corresponding N-acetyl model peptides have been examined via electrospray ionization tandem mass spectrometry (MS/MS). In particular, the competition between losses from the side chain and the combined loss of H2O and CO from the C-terminal carboxyl group of the amino acids or H2O or CH2CO from the N-acetyl model peptides are compared. In this manner the effect of the leaving group (Y = H or CH3CO, vary X) or of the neighboring group can be examined. It was found that the amount of HX lost from the side chain increases with the proton affinity of X [OP(O)(OH)2 > OCH3 approximately equals OH > Cl]. The ion due to the side chain loss of H2O from the model peptide N-acetyl serine is more abundant than that from protonated serine, suggesting that the N-acetyl group is a better neighboring group than the amino group. Ab initio calculations at the MP2(FC)/6-31G*//HF/6-31G* level of theory suggest that this effect is due to the transition state barrier for water loss from protonated N-acetyl serine being lower than that for protonated serine. The mechanism for side chain loss has been examined using MS3 tandem mass spectrometry, independent synthesis of proposed product ion structures combined with MS/MS, and hydrogen/deuterium exchange. Neighboring group rather than cis 1,2 elimination processes dominate in all cases. In particular, the loss of H3PO4 from O-phosphoserine and N-acetyl O-phosphoserine is shown to yield a 3-membered aziridine ring and 5-membered oxazoline ring, respectively, and not the dehydroalanine moiety. This is in contrast to results presented by DeGnore and Qin (J. Am. Soc. Mass Spectrom. 1998, 9, 1175-1188) for the loss of H3PO4 from larger peptides, where dehydroalanine was observed. Alternate mechanisms to cis 1,2 elimination, for the formation of dehydroalanine in larger phosphoserine or phosphothreonine containing peptides, are proposed.

Patients discharged from the hospital on an angiotensin-converting enzyme inhibitor had a significant decrease in 30-day death or readmission (by 30% in 1,195 unselected Medicare patients hospitalized with congestive heart failure). Other independent predictors of poor outcome determined by multivariate analysis included history of congestive heart failure, male gender, increased number of comorbidities, higher serum blood urea nitrogen, and lower serum sodium.

Iron protein succinylate is a non-toxic therapeutic iron compound. We set out to characterise the structure of this compound and investigate the importance of digestion and intestinal reduction in determining absorption of the compound. The structure of the compound was investigated by variable temperature Mössbauer spectroscopy, molecular size determinations and kinetics of iron release by chelators. Intestinal uptake was determined with radioactive compound force fed to mice. Reduction of the compound was determined by in vitro incubation with intestinal fragments. The compound was found to contain only ferric iron, present as small particles including sizes below 10 nm. The iron was released rapidly to chelators. Digestion with trypsin reduced the molecular size of the compound. Intestinal absorption of the compound was inhibited by a ferrous chelator (ferrozine), indicating that reduction to ferrous iron may be important for absorption. The native compound was a poor substrate for duodenal reduction activity, but digestion with pepsin, followed by pancreatin, released soluble iron complexes with an increased reduction rate. We conclude that iron protein succinylate is absorbed by a mechanism involving digestion to release soluble, available ferric species which may be reduced at the mucosal surface to provide ferrous iron for membrane transport into enterocytes.

Insulin regulates glucose metabolism in adipocytes via a phosphatidylinositide 3-kinase (PI3K)-dependent pathway that appears to involve protein phosphorylation. However, the generation of phosphoinositides is not sufficient for insulin action, and it has been suggested that insulin regulation of glucose metabolism may involve both PI3K-dependent and -independent pathways, the latter being insulin specific. To test this hypothesis, we have designed a phosphoprotein screen to study insulin-specific phosphoproteins that may be either downstream or in parallel to PI3K. Nineteen insulin-regulated phosphospots were detected in the cytosol and high speed pellet fractions, only six of which were significantly regulated by platelet-derived growth factor. Importantly, almost all (92%) of the insulin-specific phosphoproteins identified using this approach were sensitive to the PI3K inhibitor wortmannin. Thus, we obtained no evidence for an insulin-specific, PI3K-independent signaling pathway. A large proportion (62%) of the insulin-specific phosphoproteins were enriched in the same high speed pellet fraction to which PI3K was recruited in response to insulin. Thus, our data suggest that insulin specifically stimulates the phosphorylation of a novel subset of downstream targets and this may in part be because of the unique localization of PI3K in response to insulin in adipocytes.

To identify proteins that bind mammalian IAP homolog A (MIHA, also known as XIAP), we used coimmuno-precipitation and 2D immobilized pH gradient/SDS PAGE, followed by electrospray ionization tandem mass spectrometry. DIABLO (direct IAP binding protein with low pI) is a novel protein that can bind MIHA and can also interact with MIHB and MIHC and the baculoviral IAP, OpIAP. The N-terminally processed, IAP-interacting form of DIABLO is concentrated in membrane fractions in healthy cells but released into the MIHA-containing cytosolic fractions upon UV irradiation. As transfection of cells with DIABLO was able to counter the protection afforded by MIHA against UV irradiation, DIABLO may promote apoptosis by binding to IAPs and preventing them from inhibiting caspases.

A rhodacyanine dye called MKT-077 has shown a highly selective toxicity toward several distinct human malignant cell lines, including bladder carcinoma EJ, and has been subjected to clinical trials for cancer therapy. In the pancreatic carcinoma cell line CRL-1420, but not in normal African green monkey kidney cell line CV-1, it is selectively accumulated in mitochondria. However, both the specific oncogenes responsible for its selective toxicity toward cancer cells, and its target proteins in these cancer cells, still remain to be determined. This study was conducted using normal and ras-transformed NIH 3T3 fibroblasts to determine whether oncogenic ras mutants such as v-Ha-ras are responsible for the selective toxicity of MKT-077 and also to identify its targets, using its derivative called "compound 1" as a specific ligand. We have found that v-Ha-ras is responsible for the selective toxicity of MKT-077 in both in vitro and in vivo. Furthermore, we have identified and affinity purified at least two distinct proteins of 45 kD (p45) and 75 kD (p75), which bind MKT-077 in v-Ha-ras-transformed cells but not in parental normal cells. Microsequencing analysis has revealed that the p45 is a mixture of beta- and gamma-actin, whereas the p75 is HSC70, a constitutive member of the Hsp70 heat shock adenosine triphosphatase family, which inactivates the tumor suppressor p53. MKT-077 binds actin directly, bundles actin filaments by cross-linking, and blocks membrane ruffling. Like a few F-actin-bundling proteins such as HS1, alpha-actinin, and vinculin as well as F-actin cappers such as tensin and chaetoglobosin K (CK), the F-actin-bundling drug MKT-077 suppresses ras transformation by blocking membrane ruffling. These findings suggest that other selective F-actin-bundling/capping compounds are also potentially useful for the chemotherapy of ras-associated cancers.

Premature ventricular contractions (PVCs) are associated with an increased risk of cardiovascular disease and mortality. Many epidemiologic studies measure a continuous short rhythm strip to ascertain PVCs as a screening tool to identify persons at highest risk. Despite its widespread use in epidemiologic studies, the rhythm strip has not been completely validated. Therefore, a continuous 2-min rhythm strip was measured on 242 consecutive individuals referred for ambulatory ECG monitoring. Prevalence of at least one PVC on the 2-min rhythm strip was compared to a gold standard, the average number of PVCs per hr on ambulatory recording. The prevalence of any PVCs on the 2-min rhythm strip was 19%. As average PVCs per hr increased on the ambulatory ECG recording, sensitivity increased while specificity slowly decreased. Sensitivity ranged from 26-100% and specificity ranged from 81-100% across the distribution of average PVCs per hr on ambulatory monitoring. Area under the receiver operator characteristic (ROC) curve of the 2-min rhythm strip compared to 24-hr results was 0.943. Area under ROC curves were not statistically different (P > 0.05) by age, gender, hypertension status, or history of myocardial infarction. In this clinical population, utilizing the 2-min rhythm strip as an indicator of average PVCs per hr had excellent specificity and moderate to low sensitivity across most of the distribution of average PVCs per hr. The use of a short rhythm strip to detect PVCs may be considered useful in epidemiologic investigations of cardiovascular disease and mortality for detecting high frequency PVCs in populations. The use of a short rhythm strip as a screening tool to detect PVCs in clinical practice is not warranted, based on our findings and the existing literature. However, an awareness that PVCs on a 2-min rhythm strip consistently identify high frequency PVCs on 24-hr recordings should be helpful to clinicians.

The proteomic definition of plasma membrane proteins is an important initial step in searching for novel tumor marker proteins expressed during the different stages of cancer progression. However, due to the charge heterogeneity and poor solubility of membrane-associated proteins this subsection of the cell's proteome is often refractory to two-dimensional electrophoresis (2-DE), the current paradigm technology for studying protein expression profiles. Here, we describe a non-2-DE method for identifying membrane proteins. Proteins from an enriched membrane preparation of the human colorectal carcinoma cell line LIM1215 were initially fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 4-20%). The unstained gel was cut into 16 x 3 mm slices, and peptide mixtures resulting from in-gel tryptic digestion of each slice were individually subjected to capillary-column reversed phase-high performance liquid chromatography (RP-HPLC) coupled with electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS). Interrogation of genomic databases with the resulting collision-induced dissociation (CID) generated peptide ion fragment data was used to identify the proteins in each gel slice. Over 284 proteins (including 92 membrane proteins) were identified, including many integral membrane proteins not previously identified by 2-DE, many proteins seen at the genomic level only, as well as several proteins identified by expressed sequence tags (ESTs) only. Additionally, a number of peptides, identified by de novo MS sequence analysis, have not been described in the databases. Further, a "targeted" ion approach was used to unambiguously identify known low-abundance plasma membrane proteins, using the membrane-associated A33 antigen, a gastrointestinal-specific epithelial cell protein, as an example. Following localization of the A33 antigen in the gel by immunoblotting, ions corresponding to the theoretical A33 antigen tryptic peptide masses were selected using an "inclusion" mass list for automated sequence analysis. Six peptides corresponding to the A33 antigen, present at levels well below those accessible using the standard automated "nontargeted" approach, were identified. The membrane protein database may be accessed via the World Wide Web (WWW) at http://www.ludwig. edu.au/jpsl/jpslhome.html.

Background: The pathogenesis of NSAID-induced gastrointestinal damage is believed to involve a nonprostaglandin dependent effect as well as prostaglandin dependent effects. One suggestion is that the nonprostaglandin mechanism involves uncoupling of mitochondrial oxidative phosphorylation., Aims: To assess the role of uncoupling of mitochondrial oxidative phosphorylation in the pathogenesis of small intestinal damage in the rat., Methods: We compared key pathophysiologic events in the small bowel following (i) dinitrophenol, an uncoupling agent (ii) parenteral aspirin, to inhibit cyclooxygenase without causing a 'topical' effect and (iii) the two together, using (iv) indomethacin as a positive control., Results: Dinitrophenol altered intestinal mitochondrial morphology, increased intestinal permeability and caused inflammation without affecting gastric permeability or intestinal prostanoid levels. Parenteral aspirin decreased mucosal prostanoids without affecting intestinal mitochondria in vivo, gastric or intestinal permeability. Aspirin caused no inflammation or ulcers. When dinitrophenol and aspirin were given together the changes in intestinal mitochondrial morphology, permeability, inflammation and prostanoid levels and the macro- and microscopic appearances of intestinal ulcers were similar to indomethacin., Conclusions: These studies allow dissociation of the contribution and consequences of uncoupling of mitochondrial oxidative phosphorylation and cyclooxygenase inhibition in the pathophysiology of NSAID enteropathy. While uncoupling of enterocyte mitochondrial oxidative phosphorylation leads to increased intestinal permeability and low grade inflammation, concurrent decreases in mucosal prostanoids appear to be important in the development of ulcers.

In order to observe cellular changes caused by mutation of the tumor suppressors, APC and p53, we have generated protein expression profiles of mouse colon epithelial cells using two-dimensional electrophoresis (2-DE). Crypts, polyps and stroma were isolated from normal, multiple intestinal neoplasia (MIN) and p53-null mice, each with a C57Black/6J background, and subjected to 2-DE in two separate pH ranges (pH 3-10 and pH 6-11). No significant differences in protein expression patterns were observed between the normal, MIN and p53-null colon epithelial crypts. However, 64 proteins from the MIN polyps showed a 2-fold or greater difference in intensity that was statistically significant as assessed by the Wilcoxon rank-sum test (p < or = 0.05). Of these, calreticulin, carbonic anhydrase I and a new member of the glutathione-S-transferase theta family of proteins have so far been identified using an in-gel digestion protocol coupled with reversed-phase high performance liquid chromatography (RP-HPLC) ion-trap mass spectrometry. In addition, 38 marker proteins have been identified in a continuing effort to generate a comprehensive 2-DE database of proteins expressed by mouse colon epithelial cells (these databases are available at http://www.ludwig.edu.au/jpsl/jpslhome. html).