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The purpose of this study was (a) to assess the intensity and energy cost of a single-set resistance training (RT) protocol as recommended by the recent American College of Sports Medicine (ACSM) guidelines for older adults and (b) to compare obtained values to those recently reported as eliciting health benefits via endurance-based physical activity (PA). Five males and 5 females (73.1 +/- 5.5 years) performed 1 set of 15 repetitions of 8 RT exercises while connected to a portable metabolic unit (CosMed K4b2). The RT intensity (metabolic equivalents [METs]) was 3.3 +/- 0.7 (males) and 3.0 +/- 0.6 (females). Energy cost (kcal) was 84.2 +/- 14.6 (males) and 69.7 +/- 17.4 (females). We conclude that a single-set 8-exercise RT protocol may be a feasible alternative for achieving moderate intensity (3-6 METs) for older adults but that additional sets and/or repetitions appear to be necessary to accumulate moderate amounts (150-200 kcal) of PA.

One potentially important mechanism for regulating class Ia phosphoinositide 3-kinase (PI 3-kinase) activity is autophosphorylation of the p85 alpha adapter subunit on Ser608 by the intrinsic protein kinase activity of the p110 catalytic subunit, as this downregulates the lipid kinase activity in vitro. Here we investigate whether this phosphorylation can occur in vivo. We find that p110 alpha phosphorylates p85 alpha Ser608 in vivo with significant stoichiometry. However, p110 beta is far less efficient at phosphorylating p85 alpha Ser608, identifying a potential difference in the mechanisms by which these two isoforms are regulated. The p85 alpha Ser608 phosphorylation was increased by treatment with insulin, platelet-derived growth factor, and the phosphatase inhibitor okadaic acid. The functional effects of this phosphorylation are highlighted by mutation of Ser608, which results in reduced lipid kinase activity and reduced association of the p110 alpha catalytic subunit with p85 alpha. The importance of this phosphorylation was further highlighted by the finding that autophosphorylation on Ser608 was impaired, while lipid kinase activity was increased, in a p85 alpha mutant recently discovered in human tumors. These results provide the first evidence that phosphorylation of Ser608 plays a role as a shutoff switch in growth factor signaling and contributes to the differences in functional properties of different PI 3-kinase isoforms in vivo.

The purpose of this study was to compare linear periodization (LP), daily undulating periodization (DUP), and reverse linear periodization (RLP) for gains in local muscular endurance and strength. Sixty subjects (30 men, 30 women) were randomly assigned to LP, DUP, or RLP groups. Maximal repetitions at 50% of the subject's body weight were recorded for leg extensions as a pretest, midtest, and posttest. Training involved 3 sets (leg extensions) 2 days per week. The LP group performed sets of 25 repetition maximum (RM), 20RM, and 15RM changing every 5 weeks. The RLP group progressed in reverse order (15RM, 20RM, 25RM), changing every 5 weeks. The DUP group adjusted training variables between each workout (25RM, 20RM, 15RM repeated for the 15 weeks). Volume and intensity were equated for each training program. No significant differences were measured in endurance gains between groups (RLP = 73%, LP = 56%, DUP = 55%; p = 0.58). But effect sizes (ES) demonstrated that the RLP treatment (ES = 0.27) was more effective than the LP treatment (control) and the DUP treatment (ES = -0.02) at increasing muscular endurance. Therefore, it was concluded that making gradual increases in volume and gradual decreases in intensity was the most effective program for increasing muscular endurance.

The purpose of this study was to compare linear periodization (LP) and daily undulating periodization (DUP) for strength gains. Twenty men (age = 21 +/- 2.3 years) were randomly assigned to LP (n = 10) or DUP (n = 10) groups. One repetition maximum (1RM) was recorded for bench press and leg press as a pre-, mid-, and posttest. Training involved 3 sets (bench press and leg press), 3 days per week. The LP group performed sets of 8 RM during weeks 1-4, 6 RM during weeks 4-8, and 4 RM during weeks 9-12. The DUP group altered training on a daily basis (Monday, 8 RM; Wednesday, 6 RM; Friday, 4 RM). Analysis of variance with repeated measures revealed statistically significant differences favoring the DUP group between T1 to T2 and T1 to T3. Making program alterations on a daily basis was more effective in eliciting strength gains than doing so every 4 weeks.

The phosphatidylinositol 3′-kinase (PI3K) signaling pathway is frequently dysregulated in BRAF mutated melanomas and serves to promote the growth of tumors initiated by expression of BRAF. The most prevalent mechanism of PI3K pathway activation occurs via silencing expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, but mutational activation of PIK3CA, encoding the 110kDa catalytic subunit of PI3′-kinase-a, has also been observed at low frequency. However, the PI3K catalytic isoform dependence of BRAF mutated melanoma remains poorly understood, as does the differential isoform dependence imbued by discrete nodes of PI3K pathway dysregulation. To characterize the PI3K isoform dependence of BRAF/PTEN as well as BRAF/PIK3CA melanoma, we administered a panel of isoform-selective PI3K inhibitors to cultured melanoma cells and to genetically engineered mouse (GEM) models of melanoma of both genotypes. These studies revealed that, while BRAF/PIK3CA melanoma was sensitive to p110a-selective inhibition, inhibition of BRAF/PTEN melanoma cell proliferation required combined blockade of p110a (PIK3CA), p110d (PIK3CD) and p110g (PIK3CG). Moreover, in GEM models of BRAF/PIK3CA or BRAF/PTEN melanoma, isoform-selective PI3K inhibition elicited only a cytostatic effect as monotherapy, but enhanced melanoma regression in response to BRAFV600E→MEK→ERK pathway-targeted inhibition. Most interestingly, treatment with GDC0032, a p110b-sparing PI3K inhibitor, forestalled the onset of MEK inhibitor (GDC-973, cobimetinib) resistant disease in the BRAF/PTEN GEM model of melanoma. Together, these data suggest that PIK3CAmutational status may be used to select for melanoma patients that will respond to a p110a-selective inhibitor. By contrast, PTEN melanoma patients will require a p110b-sparing pan-class I PI3K inhibitor. Moreover, although monotherapy PI3K inhibition did not elicit dramatic tumor regression, blockade of this pathway may be a useful strategy to extend the duration of response to BRAF pathway-targeted therapy.

The cyclic-AMP response element binding (CREB) protein has been shown to have a pivotal role in cell survival and cell proliferation. Transgenic rodent models have revealed a role for CREB in higher-order brain functions, such as memory and drug addiction behaviors. CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours. It is the central position of CREB, downstream from key developmental and growth signalling pathways, which gives CREB this ability to influence a spectrum of cellular activities, such as cell survival, growth and differentiation, in both normal and cancer cells. We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade. The mechanism by which CREB regulates glioblastoma (GBM) tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways that then modulate the expression of three key cell cycle factors, cyclin B, D and proliferating cell nuclear antigen (PCNA). Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms. The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself. Given that CREB sits at the hub of key cancer cell signalling pathways, understanding the role of glioma-specific CREB function may lead to improved novel combinatorial anti-tumour therapies, which can complement existing PI3K-specific drugs undergoing early phase clinical trials. ispartof: Oncogenesis vol:3 issue:6 ispartof: location:United States status: published

used to visualize mouse colon with fluorescent. Mice distal colon was sprayed with 200ul of 50pM NPs and allowed to incubate for 10 minutes. Then distal colon was washed with water and imaged with spyglass. Then mice were sacrificed and colon was extracted for Raman spectroscopy. Result : We Successful labeled S440 SERS nanoparticles with 5-ROX fluorophore and an EGFR-affibody. SpyGlass system is able to detect nanoparticles bound to tumors when applied at 50-pM concentration. SpyGlass video and Raman scan suggest polyp specificity of SERS NP, and qPCR results show EGFR up-regulation in tumor compared to normal. Conclusion : Fluorescent Endoscopy with SERS nanoparticle labled with EGFR showed specific binding to mice colon polyp. Raman spectroscopy gave additional corresponding result. These multi-modality imaging with targeted nanoparticle spraying concept could give rise to new diagnostic tool in the near future.

Analysis of the glycogen synthase kinase-3beta (GSK-33) activity in several colon cancer cell lines suggested a correlation between comparatively low enzyme activity and moderate to high differentiation status. Treatment of LIM2537 cells, a poorly differentiated colon cancer cell line, with the potent differentiating agent sodium butyrate resulted in 34% reduction in GSK-3beta activity in the treated cells (P < 0.028, n = 3). Decreases in GSK-3beta activity were paralleled by stabilization of cytoplasmic beta-catenin, a hallmark of Wnt signaling. However, in contrast to Wnt signaling, expression of the beta-catenin/ TCF target genes c-myc and cyclin D1 did not appear to be increased in the sodium butyrate-treated cells. Interestingly, expression of membrane-bound beta-catenin was increased in the sodium butyrate-treated cells. This suggests that, in the context of cellular differentiation, increases in beta-catenin expression may be sequestered at the cell membrane and suggests that a possible role of sodium butyrate in promoting differentiation may be via increasing the levels of beta-catenin available for cell adhesion.

As the colonic epithelium is physiologically exposed to butyrate and to activators of protein kinase C, we examined the effect of the protein kinase C signalling pathway on butyrate-induced expression of markers of differentiation. Activators and inhibitors of protein kinase C were used in combination with butyrate and effects on the expression of markers of differentiation examined in colon cancer cell lines. When the protein kinase C activator phorbol myristate acetate (100 nM) was added for 24 h prior to the addition of 2 mM butyrate, there was a synergistic increase in alkaline phosphatase activity (154 +/- 11% above that for butyrate alone, P = 0.003) in a concentration- and time-dependent manner. Butyrate-induced expression of carcinoembryonic antigen and interleukin-8, dome formation and cell turnover were also markedly augmented by pre-treatment with phorbol myristate acetate. A similar effect was observed with propionate or acetate (but not other differentiating agents), when phorbol myristate acetate and butyrate were added concurrently, or when other protein kinase C activators were used. Pharmacological inhibition of protein kinase C activity did not alter butyrate-induced alkaline phosphatase activity, but abrogated the augmentation induced by phorbol myristate acetate. We conclude that protein kinase C does not mediate the differentiating effects of butyrate on colon cancer cells, but its activation regulates butyrate-induced cellular differentiation.

The use of various FES protocols to encourage increases in physical activity and to augment physical fitness and reduce heart disease risk is a relatively new, but growing field of investigation. The evidence so far supports its use in improving potential health benefits for patients with SCI. Such benefits may include more efficient and safer cardiac function; greater stimulus for metabolic, cardiovascular, and pulmonary training adaptations; and greater stimulus for skeletal muscle training adaptations. In addition, the availability of relatively inexpensive commercial FES units to elicit muscular contractions, the ease of use of gel-less, reusable electrodes, and the increasing popularity of home and commercial upper body exercise equipment mean that such benefits are likely to be more accessible to the SCI population through increased convenience and decreased cost. The US Department of Health and Human Services has identified those with SCI as a "special population" whose health problems are accentuated, and so need to be specifically addressed. FES presents "a clear opportunity.... For health promotion and disease prevention efforts to improve the health prospects and functional independence of people with disabilities." As a corollary to this, the Centers for Disease Control and Prevention have recommended the development of techniques to prevent or ameliorate secondary disabilities in persons with a SCI. Patients with SCI have an increased susceptibility to cardiac morbidity and mortality in the acute and early stages of their injury. Most of these patients make an excellent adaptation except when confronted with infection or hypoxia. SCI by itself does not promote atherosclerosis; however, in association with multiple secondary conditions related to SCI, along with advancing age, patients with SCI are predisposed to relatively greater risk of heart disease. The epidemiologic significance of this is reflected in demographic studies that indicate an increasing number of SCI patients becoming aged. Currently 71,000 (40%) of the total 179,000 patients with SCI living in the United States are older than 40 years, and 45,000 have injuries sustained more than 20 years earlier. In addition, new injuries in the older population are increasing (currently 11% of all injuries), and some of these new patients with SCI already have pre-existing cardiac disease. Studies have demonstrated that improved lifestyle, physical activity, lipid management, and dietary restrictions can affect major risk factors for coronary artery disease. Therefore an aggressive cardiac prevention program is appropriate for patients with SCI as part of their rehabilitation. At a given submaximal workload, arm exercise is performed at a greater physiologic cost than is leg exercise. At maximal effort, however, physiologic responses are generally greater in leg exercise than arm exercise. Arm exercise is less efficient and less effective than lower body exercise in developing and maintaining both central and peripheral aspects of cardiovascular fitness. The situation is further compounded in SCI because of poor venous return as a result of lower-limb blood pooling, as a result of lack of sympathetic tone, and a diminished or absent venous "muscle pump" in the legs. This latter mechanism perhaps contributes the greatest diminution in the potential for aerobic performance in the SCI population. Obtaining a cardiopulmonary training effect in individuals with SCI is quite possible. Current studies indicate decreases in submaximal HR, respiratory quotient, minute ventilation, and oxygen uptake, with increases in maximal power output, oxygen uptake, minute ventilation, and lactic acid. Individuals with SCI have been shown to benefit from lower limb functional electrical stimulation (FES)-induced exercise. Studies have consistently reported increases in lower limb strength and cycle endurance performance with these protocols, as well as improvements in metabolic and

In order to model the lifetime of the electrochemical cell in an Alkali Metal Thermal to Electric Converter (AMTEC), studies of TiN electrodes on β″-alumina solid electrolytes (BASE) have been made to determine the performance parameters over time. The first of a series of experiments in which TiN electrodes are studied in a Vapor Exposure Test Cell is described here, with preliminary results from electrochemical measurements made on the cell. The TiN electrodes tested here perform well, with minimal degradation over 1000 hours of operation at 850 °C.

Tyrosine phosphorylation is now recognised as a key event in the activation of the macrophage respiratory burst. Since vanadate, a phosphotyrosine phosphatase (PTP) inhibitor is able to enhance the respiratory burst, we proposed that agents which prime the macrophage for enhance respiratory burst activity may do so by suppressing cellular PTP activity. The level of PTP activity in murine bone marrow-derived macrophages (BMM) was assessed by the ability of cell lysates to dephosphorylate 32 P-labelled RR- src peptide. In contrast to our hypothesis, pretreatment of BMM with bacterial lipopolysaccharide (LPS), tumor necrosis factor α (TNF α ) or granulocyte/macrophage-colony stimulating factor (GMCSF), agents which prime for enhanced respiratory burst activity, was found to dramatically increase the level of cellular PTP activity. The time-course for this increase correlated well with the time course of priming by these agents. In addition, colony stimulating factor-1, a cytokine which does not prime the macrophage respiratory burst, did not enhance PTP levels. The physiological relevance of the increased PTP activity was further supported by confirming it was active against endogenous tyrosine phosphorylated substrates. Interestingly, phorbol myristate acetate and zymosan, agents which trigger the macrophage respiratory burst, were found to inhibit the PTP activity of BMM. Our results demonstrate the regulation of cellular PTP activity by priming agents and further highlight the importance of tyrosine phosphorylation and dephosphorylation events in the regulation of macrophage function.

To the Editor: In the April 15 issue of Clinical Cancer Research, Levine et al. ([1][1]) reported the frequent occurrence of somatic mutations in the phosphatidylinositol 3-kinase p110α catalytic subunit gene PIK3CA in breast and ovarian cancer. While their study broadly supports the conclusions of

Loss-of-function mutations in the gene coding for perforin (PRF1) markedly reduce the ability of cytotoxic T lymphocytes and natural killer cells to kill target cells, causing immunosuppression and impairing immune regulation. In humans, nearly half of the cases of type 2 familial hemophagocytic lymphohistiocytosis are due to bi-allelic PRF1 mutations. The partial inactivation of PRF1 due to mutations that promote protein misfolding or the common hypomorphic allele coding for the A91V substitution have been associated with lymphoid malignancies in childhood and adolescence. To investigate whether PRF1 mutations also predispose adults to cancer, we genotyped 566 individuals diagnosed with melanoma (101), lymphoma (65), colorectal carcinoma (30) or ovarian cancer (370). The frequency of PRF1 genotypes was similar in all disease groups and 424 matched controls, indicating that the PRF1 status is not associated with an increased susceptibility to these malignancies. However, four out of 15 additional individuals diagnosed with melanoma and B-cell lymphoma during their lifetime expressed either PRF1A91V or the rare pathogenic PRF1R28C variant (p = 0.04), and developed melanoma relatively early in life. Both PRF1A91V- and PRF1R28C-expressing lymphocytes exhibited severely impaired but measurable cytotoxic function. Our results suggest that defects in human PRF1 predispose individuals to develop both melanoma and lymphoma. However, these findings require validation in larger patient cohorts.

Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with greater than 50% of tumors expressing the BRAF(V600E) oncoprotein. BRAF(V600E) is a constitutively active protein kinase that promotes sustained activation of BRAF→MEK→ERK signaling in the melanoma cell. Importantly, the marked tumor regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF(V600E) in melanoma maintenance. However, since most patients relapse with lethal vemurafenib resistant disease, understanding and preventing mechanism(s) of drug resistance will be critical to extending the durability of patients' responses. Although mutational activation of BRAF is often the initiating event, progression of BRAF mutated melanomas is frequently associated with silencing of the PTEN tumor suppressor, a negative regulator of PI3′-kinase signaling. Whilst PTEN silencing is common in human melanoma, mutational activation of PIK3CA (encoding the alpha catalytic subunit of PI3′-kinase) is extremely rare, even though PIK3CA mutations are common in other human cancers. Since PTEN is reported to have tumor suppressor functions independent of its PI3′-lipid phosphatase activity, PTEN silencing may have more profound tumor promoting activity than mutational activation of PIK3CA. To test this, we generated mice carrying a conditional knock-in allele of Pik3ca that permits Cre-induced expression of PIK3CA(H1047R), a mutationally activated form of PIK3CA detected in many cancer types. Expression of PIK3CA(H0147R) in melanocytes elicited no obvious phenotype, consistent with the absence of phenotype in PTEN(Null) melanocytes. As observed previously, melanocyte-specific expression of BRAF(V600E) combined with PTEN silencing led to metastatic melanoma. Interestingly, metastatic melanoma also arose from melanocytes expressing both BRAF(V600E) and PIK3CA(H1047R), although these tumors grew more slowly than their BRAF(V600E)/PTEN(Null) counterparts. Both BRAF(V600E)/PTEN(Null) and BRAF(V600E)/PIK3CA(H1047R) melanomas were sensitive to the tumor prevention effects of pharmacological blockade of class 1 PI3′-kinases. However, the clinically relevant subset of BRAF(V600E)/PTEN(Null) melanoma was resistant to pharmacological blockade of AKT. Using genetically engineered mouse models and melanoma-derived cell lines, we demonstrate cooperative anti-tumor effects of combining RAF and PI3′-kinase inhibitors, suggesting that combined pathway-targeted chemotherapy may maximize melanoma patients' therapeutic response while delaying the onset of lethal drug resistant disease. Using patient derived xenografts propagated in immunocompromised mice we explored the cause and consequences of drug resistance, which was selected by continuous vemurafenib administration. As observed previously, resistant tumors displayed continued dependence on BRAF(V600E)→MEK→ERK signaling due to elevated BRAF(V600E) expression. However, remarkably, we observed that vemurafenib-resistant melanomas displayed a striking drug dependency for their continued proliferation, such that cessation of drug administration led to regression of established drug-resistant tumors. We further demonstrated that a discontinuous dosing strategy, which exploits the fitness deficit displayed by drug-resistant cells in the absence of the vemurafenib, forestalls the onset of lethal drug resistant disease. These data highlight the concept that drug-resistant cells may also show dependency, such that altered drug dosing may prevent the emergence of lethal drug resistance. Citation Format: Martin McMahon, Meghna Das Thakur, Victoria Marsh, Jillian Silva, Allison Landman, Marian Deuker, Fernando Salangsang, Nancy Pryer, Wayne Phillips, Mitchell Levesque, Reinhard Dummer, Marcus Bosenberg, William R. Sellers, Darrin Stuart. Targeting BRAF and PI3′-kinase signaling for therapy of melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY17-03. doi:10.1158/1538-7445.AM2013-SY17-03

An accumulation of international scientific evidence indicates that physical inactivity is detrimental to health and that moderate levels of physical activity confer significant health benefits. Unfortunately, in countries where major surveys of physical activity have been conducted, the prevalence of sedentary behaviour has been found to be as high as 40%. In the US, where approximately 30% of adults report little or no physical activity, the Centers for Disease Control and the American College of Sports Medicine recently issued guidelines and recommendations on the amount and frequency of moderate levels of physical activity necessary to elicit health benefits in predominantly sedentary adults. These guidelines utilise a physical activity-health paradigm and, uniquely, recommend the potential effectiveness of activities of daily living or "lifestyle activity' for achieving health benefits. This article briefly reviews the rationale behind these guidelines and, in view of the historical association of the exercise training-fitness model to health, highlights some challenges and potential problems in applying these new guidelines to the general population.

The purpose of this study was to determine the effects of a maximal isokinetic training program on strength and muscle mass in a group of untrained men 73 +/- 7.3 (range 68-82) years of age. Testing and training were conducted on an isokinetic dynamometer. Training consisted of one set of six maximal knee flexion and extension movements at 60 deg*s(-1) performed three times per week for 12 weeks. The subjects trained the right leg only, the left leg serving as a within-subject control. Trained leg peak torque increased by 18.1% for knee extension (from 107.2 +/- 42.9 to 126.6 +/- 44.6 Nm; p0.05) and by 18.2% for knee flexion (from 85.1 +/- 21.1 to 100.4 + or - 25.5 Nm; p0.05). Urinary creatinine levels increased by 24.3% (NS). We conclude that maximal isokinetic training at 60 deg*s(-1) increases strength and a urinary index of muscle mass in a group of aged untrained men.

Mutations in the p110α subunit of phosphoinositide 3-kinase (PI3K) (encoded by the gene PIK3CA) are frequent in human cancers. To better understand the role of mutant PIK3CA in the initiation or progression of tumorigenesis in vivo, we generated a conditional (Cre recombinase-inducible) knock-in mouse model of the common activating mutation, Pik3caH1047R, expressed from the endogenous locus. We have then targeted the mutation to specific tissues by crossing our mouse to mice expressing Cre under the control of tissue-specific promoters. The mouse mammary gland was targeted using a transgenic mouse expressing Cre under the control of the mouse mammary tumor virus promoter (MMTV-Cre). To target the gastrointestinal tract, we used the A33CrePR2 mouse, expressing Ru486-inducible version of Cre under the control of the gpa33 promoter. Tumors were formed in both these models but only following a long latency (>15 months) suggesting that a ‘second-hit’ may be necessary for tumor development. Similarly, activation of this mutation in the mouse ovary, by injection of an adenovirus expressing Cre into the ovarian bursa, revealed that Pik3caH1047R alone is a weak activator of AKT capable of inducing premalignant hyperplasia in ovarian surface epithelium. Interestingly, we found that combining Pik3caH1047R mutation with deletion of Pten in the mouse ovary resulted in robust epithelial AKT signaling and progression to ovarian serous adenocarcinomas and granulosa cell tumors within 6 months. Therapeutic inhibition of PI3K/mTOR in these mice delayed tumor growth and prolonged survival. This novel mouse is proving to be a fascinating model with which to explore the biological consequences and clinical significance of Pik3caH1047R mutation in diverse tissue types and a valuable tool for the preclinical evaluation of PI3K pathway inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3289. doi:1538-7445.AM2012-3289

The respiratory burst is a key element of the bactericidal armamentarium of phagocytes. In this study we have shown that a virulent strain of Yersinia enterocolitica serogroup O:9 completely inhibited the ability of murine bone marrow-derived macrophages to mount a respiratory burst in response to stimulation by zymosan. This property of the bacterium was abrogated by curing the strain of its 71.5-kb virulence plasmid and by transposon mutagenesis of the plasmid-borne yopD gene. Derivatives of the bacterium which were unable to inhibit the respiratory burst were also less able to disrupt cytoskeletal actin and to resist phagocytosis. yopD mutants also showed an impaired ability to dephosphorylate phosphotyrosine residues in macrophage proteins and were completely avirulent for mice. All of these defects were fully or partly restored by trans-complementation of a yopD mutant with a cloned yopD gene. The results of this study and those of previous work with YopD (R. Rosqvist, A. Forsberg, and H. Wolf-Watz, Infect. Immun. 59:4562-4569, 1991) suggest that YopD functions chiefly by facilitating the transport of virulence plasmid-encoded proteins, such as YopE, a cytotoxin, and YopH, a protein tyrosine phosphatase, across the cytoplasmic membrane to their targets within host cells. The combined action of these Yops on cytoplasmic proteins, especially actin, could explain the effects of virulent Y. enterocolitica on macrophage morphology, phagocytic capacity, and respiratory burst activity, all of which rely on cytoskeletal integrity to function normally.

We have investigated the relationship between the expression of the p47-phox and p67-phox cytosolic components of the NADPH oxidase and priming of the macrophage respiratory burst. Western blot analysis revealed that murine bone marrow-derived macrophages (BMM) contain immunoreactive proteins detected by antisera raised against recombinant human p47-phox and p67-phox. Priming BMM by exposure to tumor necrosis factor or (TNF-α) or lipopolysaccharide (LPS) increased the levels of p47-phox and p67-phox. Colony-stimulating factor 1 (CSF-1), which we previously found to have a negative effect on the priming of murine macrophages, had no effect on the level of p47-phox but down-regulated that of p67-phox. Our results suggest that the regulatory effects of LPS, TNF-α, and CSF-1 on the respiratory burst of BMM may be due to modulation of the expression of the p47-phox and p67-phox cytosolic components of the NADPH oxidase. J. Leukoc. Biol. 55: 530–535; 1994.

The activity of phosphatidylinositol (PI) 3-kinase was examined in murine bone marrow-derived macrophages (BMM) stimulated with the haematopoietic growth factors colony stimulating factor-1 (CSF-1) and granulocyte/macrophage-CSF (GM-CSF). PI 3-kinase was immunoprecipitated from cell lysates using anti-phosphotyrosine antibody or an antibody directed against the 85K subunit of PI 3-kinase, and the activity assayed by the phosphorylation of PI in the presence of [gamma 32P]-ATP. The results demonstrate that CSF-1 increases the activity of PI 3-kinase, as compared to the non-stimulated control, in murine macrophages. Maximum activity was seen after 10 min of stimulation with CSF-1 at 3000-5000 U/ml. The dose-response of CSF-1 is consistent with other biochemical effects of CSF-1 seen in the BMM. GM-CSF also stimulated PI 3-kinase activity although to a lesser extent than CSF-1, correlating well with their degree of mitogenic activity on the BMM. Non-mitogenic macrophage activating agents, such as the phorbol myristate acetate, lipopolysaccharide, concanavalin A and formyl-methionyl-leucyl-phenylalanine, did not significantly increase the PI 3-kinase activity. Furthermore, CSF-1 failed to stimulate PI 3-kinase activity in resident peritoneal macrophages, a population of macrophages with poor proliferative capacity. These results suggest that the PI 3-kinase activity may be involved in the haemopoietic growth factor signalling pathways regulating macrophage growth.

3045 Background: Chemotherapy-induced hepatic injury (CIHI) represents a major cause of morbidity post-resection of colorectal liver metastases in patients treated with neoadjuvant chemotherapy. Prediction of patients at risk of developing CIHI may be possible by analysis of genes associated with forms of toxicity and chemotherapy drug metabolism. Methods: Two hundred twenty-two patients provided 234 tissue bank or pathology archive samples following hepatic resection, which were examined for CIHI and had DNA and RNA extracted for genetic analyses. CIHI subtypes were scored according to previously validated systems on haematoxylin eosin, and Masson trichrome slides of non-tumourous liver. Variants within genes relevant to damage neutralisation from oxaliplatin (nucleotide excision repair enzymes ERCC1 and ERCC2, and glutathione-S-transferase enzymes GSTM1 and GSTT1), and enzymes involved in metabolism of 5FU [thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD)...

Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention.

Simple sequence repeat oligonucleotides were used to probe the tomato genome for elements displaying variability amongst commercial cultivars. The oligonucleotide (GACA)4 was found to be particularly informative on genotype screening blots, hybridising to a highly polymorphic family of elements, and was used to clone one such member from a lambda library. The GACA-hybridisation was localised to a 1.3-kbHinfI fragment within the original 15-kb lambda insert. This 1,349-bp subclone (pT-GACA-2:1.3) was used to probe 27 Californian processing varieties and found to be capable of distinguishing all from each other, thus demonstrating its utility as a genetic fingerprinting probe for cultivar identification. Hybridisation occurred to approximately 10 major high molecular weight (> 4-kb) bands, most of which segregated independently in F2 populations, as well as a large number of less clearly resolvable smaller fragments. Sequence analysis of the cloned element reveals that it is almost entirely composed of GACA or GATA repeats. These tetranucleotides are organised into distinct repetitive domains, consisting either of tandem arrays of each tetranucleotide or interspersions of GACA and GATA to form dodecanucleotides that are then further repeated. The boundaries between domains contain sufficient departures from the concensus repeat to allow construction of unique polymerase chain reaction (PCR) primers. Amplification from two such contiguous regions identifies length variation in both, thus yielding a genotype screen appropriate for high-throughput applications, such as assessment of purity in F1 hybrid seed lots.

10610 Background: The PI3K/AKT/mTOR signaling pathway is a central network in a number of cancers, including EOC. Identifying associations between key molecules and clinical outcomes may help expos...

The PI3kinase/AKT/mTOR pathway plays key roles in cell growth, proliferation and survival with mTOR providing a hub for coordinating signaling in response to growth factors, hormones and nutrients. This pathway is one of the most frequently mutated or abnormally activated pathways in ovarian cancer, thus making it an excellent therapeutic target for this disease. The aims of this study were: 1. To investigate the potency of PF-4691502 (PF502) a dual PI3 kinase and mTOR inhibitor to inhibit ovarian tumor cell proliferation and induce cell death. 2. To profile exponentially growing ovarian tumor cells using gene expression, copy number variations and activation of the PI3 kinase pathway. 3. To define a predictive signature or biomarker that defines ovarian tumor cell sensitivity to PF502. A panel of 39 ovarian tumor cells were treated with PF502 (1nM-30µM) for 72h and the dose at which proliferation was inhibited by 50% (GI50) determined. Cells showed a 50-fold difference in PF502 sensitivity with GI50 values ranging from 15nM to 700nM, with the majority (85%) of cells having a GI50 of less than 200nM, these cells were classified as sensitive and the remaining 6 cell lines with a GI50 greater than 350nM were classified as resistant. PF502 also caused cell death in a subset of sensitive ovarian tumor cells. Increase in PIK3CA gene copy number was observed in 40% of the cell lines but this did not correlate with PF502 sensitivity nor did activating mutations in PIK3CA or inactivation of PTEN. Gene expression data was generated from exponentially growing cells using high-density oligonucleotide microarrays. A predictive model of response to PF502 was generated using the gene expression data. Exhaustive 10-fold cross validation of the model showed that tumour cell sensitivity could be predicted with high (96%) accuracy using approximately 20-30 genes. A differential gene expression analysis was performed using all data and 89 genes were found significantly different between sensitive and resistant cell lines. Pathway analysis revealed an over expression of inflammatory markers within the resistant class. Gene set enrichment analysis further supported an increased inflammatory signal within resistant cells, and revealed that both MYC and RAS signaling were also elevated providing a clear indication of potential targets for combination therapies in PF502 resistant disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4130.

We have investigated the relationship between tyrosine phosphorylation and respiratory-burst activity in mouse bone-marrow-derived macrophages (BMM). We demonstrate that zymosan, an agent known to trigger the macrophage respiratory burst, also triggers the activation of tyrosine kinase activity, resulting in rapid tyrosine phosphorylation on numerous proteins, and provide evidence for the role of tyrosine phosphorylation in the triggering of the BMM respiratory burst. Agents, such as tumour necrosis factor alpha (TNF alpha), interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS), which prime the macrophage for an enhanced zymosan-triggered respiratory burst, increase tyrosine phosphorylation triggered by zymosan. The zymosan-triggered tyrosine phosphorylation and respiratory-burst activity were partially suppressed by the tyrosine kinase inhibitors alpha-cyano-3-ethoxy-4-hydroxy-5-phenylmethylcinnamide (ST638) and herbimycin A. In addition, pre-exposure of BMM to vanadate, a phosphotyrosine phosphatase inhibitor, greatly enhanced the ability of zymosan to induce tyrosine phosphorylation and trigger the respiratory burst. These data highlight the importance of the balance between tyrosine kinase and phosphotyrosine phosphatase activity in determining the ultimate level of tyrosine phosphorylation in BMM and suggest that zymosan-triggered tyrosine phosphorylation is an important biochemical signal for triggering of the respiratory burst.

Preexposure of human monocytes to recombinant human interleukin-4 (IL-4) suppressed the respiratory burst response to a number of different stimuli, including phorbol myristate acetate, zymosan, platelet-activating factor and the chemotactic peptide, f-met-leuphe. Under similar conditions, the respiratory burst of murine macrophages was enhanced by preexposure to recombinant murine IL-4. By conducting our studies on cells from different species under similar conditions we have demonstrated that there is a significant disparity in the effects of IL-4 on human and murine macrophages thus providing an explanation for some apparent inconsistencies in the literature and highlighting the need for caution when extrapolating data across species barriers.

533 Background: The phosphathidylinositol-3-kinase (PI3K) signaling pathway is frequently deregulated in tumor biology and is an attractive target for cancer therapy. Our aim was to characterize the molecular and clinical outcome effects of PIK3CA mutations in breast cancer (BC). Methods: We analyzed 173 BC samples for PIK3CA mutations. Corresponding gene expression profiles were used to understand its effects on the PI3K pathway. We validated a PIK3CA-GS in 2 independent BC cohorts (n = 183) with known PIK3CA mutation status and evaluated its correlation with clinical outcome in 1748 BC samples stratified by treatment and subtype. Results: 26% of BCs had a PIK3CA mutation. Tumors with PIK3CA mutation demonstrated a distinct gene expression signature (p = 0.03 after 1000 perm). In 2 datasets it could discriminate PIK3CA mutation carriers from wild-type (ROC 0.68, 0.71, p = 0.001for both). However, the PIK3CA-GS was correlated with deactivation of the PI3K pathway probably through a negative feedback loop. This observation was supported by: 1) the PIK3CA-GS was significantly correlated with gene expression changes induced by PI3K inhibitors (Connectivity Map, Gene set enrichment analyses) and 2) the PIK3CA-GS was anti-correlated with a GS of PTEN loss (R = -0.3; Saal et al, 2007). Higher levels of the PIK3CA signature were observed in HER-2+ and estrogen receptor positive (ER+), luminal BC subtypes. Whilst there was no association with mutation status alone and prognosis, increasing expression of the PIK3CA-GS (suggesting deactivation) was significantly associated with better clinical outcome in both untreated (p = 0.04) and particularly ER+, luminal-B, tamoxifen only-treated (p = 0.004) BC. Multivariate analysis (HR: 0.4; 95%CI: 0.3–0.7; p = 0.002) confirmed that the PI3KCA-GS provided independent prognostic information. Conclusions: Paradoxically, the PIK3CA-GS correlates with inhibition of the PI3K pathway in ER+ BC and identifies a subgroup of luminal B BCs with a favorable outcome. The PIK3CA-GS may be a better indicator of PI3K pathway dysfunction than mutation status, potentially indicating patients who may benefit from combined endocrine therapy and PI3K inhibition. No significant financial relationships to disclose.

Figures of merit and data reporting for single-element infrared detectors are well established and easily incorporated in a contractual framework. Measured characteristics which predict system performance regardless of detector type, and which provide useful process feedback have been debated and optimized over the last 50 years. In the recent past these measurements have been extended to small arrays of detectors, which have generally been treated as lists of signal detectors. Arrays of detectors are now routinely tested which have 16,000 elements and proposed production arrays have 256,000 elements. This has rendered traditional methods clumsy and incomplete. Recently these issues have been investigated in order to design a software system to accompany a new IRFPA test system product. The authors describe their view of the requirements, and the approaches that were examined and discarded or incorporated during this effort. Different methods of displaying and reporting data, as well as a calculation engine developed as part of the effort, are discussed.© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The purpose of this study was to determine the effectiveness of specific and nonspecific warm-ups on the vertical jump test performed by athletic men. Twenty-nine men (18-23 years) in athletics (speed positions in football) performed vertical jump tests on 4 separate days after completing 4 different warm-up protocols. The 4 warm-up protocols were (a) submaximal jump warm-up, (b) weighted jump warm-up, (c) stretching warm-up, and (d) no warm-up. The weighted jump warm-up protocol required 5 countermovement jumps onto a box, with the athletes holding dumbbells equaling 10% of their body weight. The submaximal jump warm-up protocol required the athletes to perform 5 countermovement jumps at 75% intensity of their past maximum vertical jump score. The stretching warm-up protocol required the athletes to perform 14 different stretches, each held for 20 seconds. The no warm-up protocol required the athletes to perform no activity prior to being tested. Three vertical jumps were measured following each warm-up; the score for analysis was the best jump. The data were analyzed with a repeated measures analysis of variance and Bonferroni post hoc tests. The Bonferroni post hoc tests showed a significant difference (p < 0.001) between the weighted jump warm-up and all other warm-ups. The effect size was 0.380 and the power was 1.00 for the statistical analyses. We concluded that utilizing a weighted resistance warm-up would produce the greatest benefit when performing the vertical jump test.

The purpose of this study was (a) to assess the intensity and energy cost of a single-set resistance training (RT) protocol as recommended by the recent American College of Sports Medicine (ACSM) guidelines for older adults and (b) to compare obtained values to those recently reported as eliciting health benefits via endurance-based physical activity (PA). Five males and 5 females (73.1 +/- 5.5 years) performed 1 set of 15 repetitions of 8 RT exercises while connected to a portable metabolic unit (CosMed K4b2). The RT intensity (metabolic equivalents [METs]) was 3.3 +/- 0.7 (males) and 3.0 +/- 0.6 (females). Energy cost (kcal) was 84.2 +/- 14.6 (males) and 69.7 +/- 17.4 (females). We conclude that a single-set 8-exercise RT protocol may be a feasible alternative for achieving moderate intensity (3-6 METs) for older adults but that additional sets and/or repetitions appear to be necessary to accumulate moderate amounts (150-200 kcal) of PA.

The purpose of this study was to compare linear periodization (LP), daily undulating periodization (DUP), and reverse linear periodization (RLP) for gains in local muscular endurance and strength. Sixty subjects (30 men, 30 women) were randomly assigned to LP, DUP, or RLP groups. Maximal repetitions at 50% of the subject’s body weight were recorded for leg extensions as a pretest, midtest, and posttest. Training involved 3 sets (leg extensions) 2 days per week. The LP group performed sets of 25 repetition maximum (RM), 20RM, and 15RM changing every 5 weeks. The RLP group progressed in reverse order (15RM, 20RM, 25RM), changing every 5 weeks. The DUP group adjusted training variables between each workout (25RM, 20RM, 15RM repeated for the 15 weeks). Volume and intensity were equated for each training program. No significant differences were measured in endurance gains between groups (RLP 5 73%, LP 5 56%, DUP 5 55%; p 5 0.58). But effect sizes (ES) demonstrated that the RLP treatment (ES 5 0.27) was more effective than the LP treatment (control) and the DUP treatment (ES 52 0.02) at increasing muscular endurance. Therefore, it was concluded that making gradual increases in volume and gradual decreases in intensity was the most effective program for increasing muscular endurance.

Persons with spinal cord injury experience considerable difficulty, during aerobic exercise, in achieving even moderate levels of oxygen uptake. Therefore, the effect of static functional neuromuscular stimulation (FNS) on this parameter during arm crank exercise (ACE) was investigated. Eight subjects with spinal cord injury, lesion levels range C6-T12, performed ACE at 60% and 80% of VO 2peak , both with and without concurrent FNS of the lower limbs. ACE was performed with no FNS, with FNS at 40 milliamperes (mA), and with FNS at 80 mA. Significant differences in oxygen uptake were found between ACE without FNS and ACE with FNS-80 mA at 60% VO 2peak (10% increase, 12.5 ml.kg -1 .min -1 to 13.7 ml.kg -1 .min -1 , P

1. The role of intracellular platelet-activating factor (Paf) in arachidonic acid (AA) mobilization from guinea-pig peritoneal macrophages has been investigated by use of the potent and selective Paf receptor antagonists, WEB 2086 and CV 6209. 2. Adherent macrophages contained cell-associated Paf which was increased by exposure to formyl-methionyl-leucyl-phenylalanine (fMLP), endotoxin and the ionophore, A23187. However, only endotoxin and A23187 caused release of detectable amounts of Paf into the extracellular medium. 3. Exogenous Paf and each of the above stimuli mobilized previously incorporated [14C]-AA and increased the generation of prostacyclin (PGI2) in resident macrophages. 4. WEB 2086 (10-100 microM) and CV 6209 (0.1-10 microM) reduced both basal and stimulated PGI2 generation and WEB 2086 inhibited the mobilization of [14C]-AA. In addition, WEB 2086 (10 microM) inhibited fMLP-and Paf-induced superoxide anion generation. Responses to A23187 were not inhibited by either antagonist. 5. Activation of macrophages by fMLP caused a short burst of intracellular Paf generation but none was detected in the supernatants. The time-course of PGI2 synthesis followed closely that of Paf. 6. These data suggest that intracellular Paf generation is important for subsequent AA mobilization and may have a wider role in signal transduction processes.

Murine bone marrow-derived macrophages (BMM) undergo DNA synthesis in response to growth factors such as colony stimulating factor-1 (CSF-1) and granulocyte-macrophage CSF (GM-CSF). These macrophages can also be "activated," but without subsequent DNA synthesis, by a number of other agents, including lipopolysaccharide (LPS), concanavalin A, zymosan, formyl-methionyl-leucyl-phenylalanine (FMLP), and the Ca2+ ionophore, A23187. When BMM are treated with a range of stimuli, there is some, although not perfect, correlation between transient elevations in both c-myc mRNA and c-fos mRNA levels and increases in DNA synthesis. However, enhanced DNA synthesis and oncogene expression are readily dissociated from rises in inositol phosphates and, by implication, phospholipase C-mediated hydrolysis of phosphatidyl inositol 4,5-bisphosphate. Superoxide formation in BMM can also be dissociated from the other responses and does not necessarily depend on protein kinase C activation.

The electrical conductivity structure of a section of the San Andreas fault is investigated by means of an electromagnetic induction experiment that utilizes a grounded electric dipole source. Location of the study area is 18 km southeast of Hollister, where the fault runs adjacent to the Gabilan Mountains. The rerults indicate a sharp contrast in conductivity across the fault zone. The resistivity of the sediments on the northeast side of the fault is ≈ 20 Ω m compared to ≈ 1000 Ω m for the Gabilan granites on the southwest side. There is evidence for a zone of high conductivity beneath the Gabilans. The high conductivities beneath the Gabilans correlate with the results of earlier seismic and gravity studies. A gravity survey in the area indicates the presence of a pocket of low-density rocks located ≈ 1–9 km beneath the Gabilans and ≈ 2 km southwest of the surface trace of the fault. The depth to the conducting layer there correlates reasonably well with the upper bound of these rocks. Farther away from the fault there is a discontinuity in seismic velocity at a depth of 12 km. The depth to the conducting layer in this area 13 km. The low-density, highly conducting rocks adjacent to the fault zone are interpreted as sedimentary rocks like those on the northeast side of the fault, or fault gouge. Farther from the fault zone the seismic velocities and electrical conductivity at depth are consistent with the hypothesis that the Gabilans are underlain by a slab of serpentinized oceanic crust.

Phagocytic cells can be primed for enhanced stimulated release of superoxide anion (O2-) by exposure to a variety of biologic agents, including gamma-interferon and lipopolysaccharide. We examined the role of calcium ion in this priming, using the calcium ionophore ionomycin. Preincubation with ionomycin, 1 to 10 nM, primed human neutrophils to release up to 7-fold more O2- during stimulation with 1 microM formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). With 160 nM phorbol myristate acetate as stimulus, ionomycin caused a doubling of O2- production in mouse peritoneal macrophages. Incubation of phagocytes with ionomycin at priming concentrations did not directly stimulate O2- release. Priming of neutrophils occurred in 1-2 min and was associated with a marked reduction in the lag time for O2- release after f-Met-Leu-Phe stimulation and with an increase in the rate of O2- production. Kinetic analysis of NADPH-dependent O2(-)-producing activity in sonicates of resting human neutrophils incubated with sodium dodecyl sulfate suggested that modification of the enzyme responsible for the respiratory burst was not responsible for priming. Priming of neutrophils with ionomycin had no apparent effect on either the activity or subcellular distribution of protein kinase C. The effect of ionomycin on the cytosolic free calcium concentration ([Ca2+]c) was assessed in neutrophils using the calcium-sensitive fluorescent dye fura-2. Ionomycin at priming concentrations caused an approximate doubling of the base-line [Ca2+]c. When neutrophils were exposed to various concentrations of ionomycin, a parallel rise in [Ca2+]c and priming was observed. A rise in [Ca2+]c of approximately 0.8 microM caused half-maximal priming. These results suggest that an increase in [Ca2+]c is not sufficient to initiate release of O2-, but they support the concept that Ca2+ can serve as a second messenger in this event.