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PDF file - 81K, Supplementary data comprising the materials and methods for (i) crystallography (ii) gene expression studies (iii) siRNA studies and Supplementary Figure legends 1-7

Evaluation of FRalpha expression by tumor cell lines

Table of Patient Characteristics

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen–specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcϵ receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127–41. ©2017 AACR.

XLS file - 703K, Gene expression data for CCT128930: Genes that showed at least a 1.5 Fold Change in at least one treated sample compared to vehicle control and were differentially expressed between the treatment groups (Welch ANOVA FDR5%)

Description of additional experiments and procedures carried out in this study

Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component.Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148.Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed.Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial. Clin Cancer Res; 18(14); 3912–23. ©2012 AACR.

XLS file - 66K, Genes that showed at least a 1.5 Fold Change in at least one treated sample compared to vehicle control and were differentially expressed between the treatment groups (Welch ANOVA FDR5%) for both CCT128930 and AT13148

PDF file - 553K, Supplementary Figure 1. Phosphorylation of AKT substrates remains suppressed, despite pSer473 AKT induction. Supplementary Figure 2. The pharmacokinetic-pharmacodynamic profile and antitumor activity of AT13148 in PTEN-deficient PC3 human prostate cancer xenografts. Supplementary Figure 3. Pharmacodynamic biomarker study of AT13148(40mg/kg) in MES-SA human tumor xenografts. Supplementary Figure 4. The antitumour activity of AT13148 in mutant KRAS A549 human lung cancer xenografts. Supplementary Figure 5. Mouse body weight data for the MES-SA (Figure 4B) and BT474 (Figure 4C) efficacy studies. Supplementary Figure 6. The effect of AT13148 versus CCT128930 on apoptosis and cell cycle distribution in U87MG human glioblastoma cells. Supplementary Figure 7. Differential effects of 24h treatment of 10M each of the AGC kinase inhibitor AT13148 and the selective AKT inhibitor CCT128930 on cell cycle effects in PTEN-deficient U87MG human glioblastoma cells. Supplementary Table 1. Summary of the inhibitory activity of 10��M AT13148 against in vitro. Supplementary Table 2. Summary of the inhibitory activity of AT13148 against a panel of protein kinases Supplementary Table 3. Summary of the in vitro cytotoxicity of AT13148 against a panel of human tumor cell lines harboring different defects in the PI3K-AKT signaling pathway. Supplementary Table 4. Summary of the pharmacokinetics of AT13148 in mouse plasma and tissues following iv or oral administration

Beta-glucans are large polysaccharides produced by a range of prokaryotic and eukaryotic organisms. They have potential immunostimulatory properties and have been used with therapeutic intent as anti-microbial and anti-tumour agents. A range of other potentially beneficial effects have been described, and oral forms of beta-glucans are widely available over-the-counter and online. Parenteral formulations are popular in parts of Asia and are the subject of ongoing trials, worldwide. Beta-glucans are also potential contaminants of pharmaceutical products, and high levels have been described in some blood products. However, little is known about the clinical effects of such contamination, considerable uncertainty exists over the level at which immunostimulation may occur, and there are no guidelines available on acceptable levels. We encountered beta-glucan contamination of one of our products, and we suspect that others may encounter similar issues since the origin of beta-glucan contamination includes commonly used filters and solutions applied in the manufacture of biotherapeutic agents. It is likely that regulators will increasingly enquire about beta-glucan levels in pharmaceutical products, especially those with an immunomodulatory mechanism of action. Here, we review the literature on beta-glucans in pharmaceutical products and propose an acceptable level for therapeutic agents for parenteral use.

PurposeThis purpose of the study is to examine the labour supply decisions with respect to earnings and considers whether we are willing or indeed able to work less. The authors specifically focus on the three points of time, i.e. beginning of the sample, pre and post Global Financial Crisis.Design/methodology/approachThe study regression analysis by utilises microdata from the UK Labour Force Survey (LFS) regarding individual hours worked in three separate survey periods: 1994q2, 2007q2 and 2015q2FindingsThe results suggest that we are far from income-satiated. The elasticity of hours worked with respect to earnings is stubbornly inelastic and for some demographic cohorts positive, implying the desire to work more. The authors find that job flexibility matters in facilitating reduced hours of work, but that jobs are not becoming more flexible. The authors also do see a secular reduction in hours worked, accompanied by a shift to working later in life, but these appear to be down to factors other than higher wages.Research limitations/implicationsThe study has important research implications in terms of understanding the dynamics of the labour market on the whole and in the pre and post global financial crisis periods.Practical implicationsThe research has profound policy implication in terms of labour and employment policy.Social implicationsThere are important social implications, particularly in terms of household labour supply decisions and substitution between work and leisure.Originality/valueThe study has significant element of originality in terms of understanding the changing dynamics of labour market. This is the first study which has investigated the labour market in the light of empirical evidence and in the various time periods.

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen–specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcϵ receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127–41. ©2017 AACR.

Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed. Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial. Clin Cancer Res; 18(14); 3912–23. ©2012 AACR.

Recent changes to non-clinical cancer guidelines offer a golden opportunity to expedite the translation of new anticancer drugs into the clinic. In this review we look at how these guidelines can be implemented and how they can be integrated with non-clinical and clinical study design to produce robust and safe clinical trials.

The paper examines changes in UK regional employment during the period of the New Labour administration, 1997–2010, with the Blair and Brown administrations considered separately. The paper employs a shift-share analysis of workplace employment data by industry and subregion, using annual data from the UK Labour Force Survey. The results reveal significant regional shifts, with interesting spatial dynamics in and around the capital and resilient employment growth in the provinces. Keywords: shift-share analysis, workplace employment, labour-force survey, New Labour

Recent interest into the possible benefits of l -theanine found in tea has raised the issue that there are few data available on amounts of l -theanine contained in cups of commercially-available teas, prepared by a standard method. HPLC along with a standard method of preparing tea was employed here to determine amounts of l -theanine in cups of tea and the effects that various preparation factors have on amounts of l -theanine extracted. Brewing time was found to be a major determinant of the amount of l -theanine extracted, while the addition of small amounts of milk and sugar made no significant difference. High levels of milk resulted in a marked lowering of the level of detectable l -theanine. Contrary to previous research, a standard (200 ml) cup of black tea was found to contain the most l -theanine (24.2 ± 5.7 mg) while a cup of green tea contained the least (7.9 ± 3.8 mg).

In debates about employment creation there is increasing concern about the quality as well as the quantity of jobs. The authors examine the regional distribution of workplace employment in the UK using the ‘quality of jobs’ framework. Findings reveal evidence of marked regional differences in average ‘job quality’, with the gap between regions growing over the decade from 1997, and the focus of high-quality job creation biased towards already advantaged regions. There is evidence of increased job polarisation across most UK regions. The analyses suggest an important role of the public sector in providing high-quality jobs, particularly outside London and the South East.

RNA isolated from goldfish retinas before and during optic nerve regeneration, when translated in vitro, directed the synthesis of neurofilament proteins that are normally found in high levels in the optic nerve. The major neurofilament proteins of the goldfish optic nerve comprise a group of four isoelectric variants of molecular weight 58,000 (58K) which we have identified previously as ON1-ON4. The levels of ON1 and ON2 within the optic nerve had been shown to decrease shortly after optic nerve crush and then increase to precrush levels during the regeneration process. Employing two-dimensional electrophoretic analysis of in vitro translation products and immunoprecipitations with antibodies specific for the ON proteins and an anti-intermediate filament monoclonal antibody, we show that ON1 and ON2 are encoded by mRNA synthesized in the retinas. The synthesis of ON3 and ON4 by retina RNA was undetected. This confirms data from previous ex vivo experiments that indicated that ON1 and ON2 are of neuronal origin whereas ON3 and ON4 are nonneuronal. ON1 and ON2 synthesis increases dramatically during optic nerve regeneration to levels 10- and 30-fold over precrush levels, respectively. In addition to ON1 and ON2, the synthesis of a previously unidentified 52K protein is observed at relatively high levels 20 and 32 days after optic nerve crush, but is unobserved before regeneration. Thus, optic nerve regeneration can be correlated with specific changes in intermediate filament gene expression within the retina.

The predominant proteins (58K) of the intermediate filament complex in the goldfish visual pathway consist of a series of isoelectric variants. Previous biochemical studies have shown that proteins ON1 and ON2 are of neuronal origin, whereas ON3 and ON4 are of nonneuronal origin. Polyclonal antibodies, purified by affinity chromatography, that are specific for ON1 and ON2 or ON3 and ON4 have been used to localize histologically the ON proteins within the normal and crushed optic nerve. Anti-ON1/ON2 antiserum presented a pattern consistent with intraaxonal staining. A nonneuronal staining pattern was observed with anti-ON3/ON4 antiserum. The two patterns were distinct from and complementary to each other. The data suggest that ON3 and ON4 represent a novel glial fibrillary acidic protein. The results are discussed in terms of the function of these proteins in development, plasticity, and regeneration.

Enantio- and regioselective syntheses of the pyrrolidine 5,5-trans-lactams benzyl (3aS,6aR)-6,6-dimethyl-5-oxohexahydropyrrolo[3,2-b]pyrrole-1(2H)-carboxylate and benzyl (3aS,6aR)-6S-ethyl-5-oxohexahydropyrrolo[3,2-b]pyrrole-1(2H)-carboxylate from a common intermediate 2-ethoxy-3S-(2,2,2-trifluoro-acetylamino)-pyrrolidine-1-carboxylic acid benzyl ester are reported. The key step in both syntheses is the acyl iminium ion mediated condensation of the pyrrolidine with a ketene acetal.

Hepatitis C virus (HCV) is the cause of the majority of transfusion-associated hepatitis and a significant proportion of community-acquired hepatitis worldwide. Infection by HCV frequently leads to persistent infections that result in a range of clinical conditions including an asymptomatic carrier state, severe chronic active hepatitis, cirrhosis and, in some cases, hepatocel-lular carcinoma. The HCV genome consists of a single-stranded, positive sense RNA containing an open reading frame of approximately 9060 nucleotides. This is translated into a single polyprotein of approximately 3020 amino acids (C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B), which in turn is processed by a series of host and viral proteinases into at least 10 cleavage products. The N-terminal portion of the NS3 protein encodes a serine proteinase that is responsible for the cleavage at the NS3–4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. The 54 amino acid NS4A protein is a cofactor that binds to the NS3 protein and enhances its proteolytic activity. This report describes the expression of a recombinant NS3–4A proteinase fusion protein in Escherichia coli and the in vitro characterization of the enzyme activity using synthetic peptide substrates. It then demonstrates how these results were employed to guide the design of potent inhibitors of this enzyme.

Several racemic bicyclo[3.2.1]octene derivatives have been synthesised and evaluated as inhibitors of influenza virus sialidases. The 5-acetamido-bicyclo[3.2.1]octenol 4 showed modest activity against influenza A and B virus sialidases.

Because induction of the chicken ovalbumin (Ov) gene by steroid hormones requires concomitant protein synthesis, efforts have focused on defining the binding site in the Ov gene for a labile transcription factor. Previous gel mobility shift studies identified one such site in the steroid-dependent regulatory element (SDRE) between -900 and -853. To ascertain whether estrogen and glucocorticoid affect the binding of this labile protein, genomic footprinting of the Ov gene was done by treating primary oviduct cell cultures with dimethyl sulfate. Several alterations that include steroid-dependent protection of guanine residues -889 and -885 and hypersensitivity of adenine residues -892 and -865 were observed. Of particular importance, the in vivo footprinting data are corroborated by two functional studies, one with linker-scanning mutations and the other with point mutations. Ten-base-pair linker-scanning mutations between -900 and -878 severely reduced the induction by estrogen and glucocorticoid. Likewise, point mutations of the protected guanine residues profoundly attenuated the response to these steroid hormones. In addition, in vitro binding activity correlated with in vivo functional activity. For example, mutant A4e shows no transcriptional activity in response to steroid hormones, and a corresponding oligomer does not bind protein in vitro. In contrast, mutant A4c is fully active in both contexts. These data support the contention that the ovalbumin gene is regulated by a steroid hormone-induced transcriptional cascade that culminates in the binding of chicken ovalbumin induced regulatory protein or protein complex (Chirp-I) to a DNA element from -891 to -878 in the SDRE.

Several racemic bicyclo[3.2.1]octene derivatives have been synthesised and evaluated as inhibitors of influenza virus sialidases. The 5-acetamido-bicyclo[3.2.1]octenol 4 showed modest activity against influenza A and B virus sialidases.

2566 Background: AT13148 is a first-in-class, orally bioavailable, dual ROCK I/II inhibitor, which potently inhibits ROCK I/II (6, 4 nM), PKA (3 nM), AKT (38 nM) and p70S6K (8 nM). Broad anti-cance...

Analogues of 4-Guanidino-Neu5Ac2en (Zanamivir) have been prepared containing carbamate substituents at the 7-hydroxy position. (4S,5R,6R)-5-Acetylamino-6-{1R-[(6-aminohexyl)carbamoyloxy]-2R,3-dihydroxypropyl}-4-guanidino-5,6-dihydro-4H-pyran-2carboxylic acid and (4S,5R,6R)-5-Acetylamino-6-{1R-[heptylcarbamoyloxy]-2R,3-dihydroxypropyl}-4-guanidino-5,6-dihydro4H-pyran2-carboxylic acid were the two analogues possessing activity comparable to Zanamivir, showing potent inhibition of influenza virus sialidases and good antiviral activity in vitro.

Hormone-responsive genes rely on complex regulatory elements known as hormone response units to integrate various regulatory signals. Characterization of the steroid-dependent regulatory element (SDRE) in the check ovalbumin gene (--892 to --796) suggests that it functions as a hormone response unit. Previous studies using gel mobility shift assays and several types of footprinting analyses demonstrated that proteins bind to this entire element in vitro even in the absence of steroid hormones. However, the genomic footprinting experiments described herein indicate that the binding of three different proteins or protein complexes to the SDRE requires estrogen and corticosterone, suggesting that the chromatin structure of this site is restricted in vivo. Transfection experiments using linker scanning and point mutations support the contention that the binding of these three complexes is essential for induction of the ovalbumin gene by steroid hormones. In addition, functional analyses suggest that a fourth complex is also necessary for maximal induction. These and other data suggest that the SDRE functions as a hormone response unit to coordinate signals generated by two steroid hormones.

Peptides based upon the non-prime side residues of the NS4A-4B cleavage site of hepatitis C virus (HCV) NS3-4A proteinase containing an alpha-ketoamide moiety in place of the scissile amide bond are potent inhibitors of this enzyme.

Tamoxifen acts as a strong estrogen antagonist in human breast but as an estrogen agonist in the uterus. The action of tamoxifen is mediated through estrogen receptors (ERalpha and ERbeta), which bind to a variety of responsive elements, to activate transcription. To examine the role of these varied elements in the response to antiestrogens, we studied the activation of a panel of differing promoters, by these compounds, in human breast, bone, and endometrial derived cell lines. No agonistic activity was observed in breast cells, whereas all antiestrogens, particularly tamoxifen, exhibited agonistic effects in uterine cell lines. All antiestrogens studied were agonistic in co-transfections of a collagenase reporter gene and ERbeta, but tamoxifen alone was agonistic with ERalpha in (uterine) HEC-1-A cells. The ERalpha mediated, agonism of tamoxifen was not observed in primary cultures of human uterine stromal cells, whereas the ERbeta-mediated agonism of all selective estrogen receptor modulators was present. This suggests that the two receptors operate by distinct pathways and that the response of cells to antiestrogens is dependent on the ER subtypes expressed.

Genomic clones for Cyp4a12 and a novel member of the murine Cyp4a gene family were isolated. The novel gene, designated Cyp4a14, has a GC rich sequence immediately 5′ of the transcription start site, and is similar to the rat CYP4A2 and CYP4A3 genes. The Cyp4a14 gene spans approximately 13 kb, and contains 12 exons; sequence similarity to the rat CYP4A2 gene sequence falls off 300 bp upstream from the start site. In view of the known sex-specific expression of the rat CYP4A2 gene, the expression and inducibility of Cyp4a14 was examined. The gene was highly inducible in the liver when mice were treated with the peroxisome proliferator, methylclofenapate; induction levels were low in control animals and no sex differences in expression were observed. By contrast, the Cyp4a12 RNA was highly expressed in liver and kidney of control male mice but was expressed at very low levels in liver and kidney of female mice. Testosterone treatment increased the level of this RNA in female liver slightly, and to a greater extent in the kidney of female mice. In agreement with studies on the cognate RNA, expression of Cyp4a12 protein was male-specific in the liver of control mice and extremely high inducibility of Cyp4a10 protein, with no sex differences, was also demonstrated. In view of the overlapping patterns of inducibility of the three Cyp4a genes, we investigated whether the three genes were co-localized in the genome. Two overlapping yeast artificial chromosome (YAC) clones were isolated, and the three Cyp4a genes were shown to be present on a single YAC of 220 kb. The Cyp4a genes are adjacent to the Cyp4b1 gene, with Cyp4a12 most distant from Cyp4b1. The clustering of these two gene subfamilies in the mouse was replicated in the human, where the CYPA411 and CYP4B1 genes were present in a single YAC clone of 440 kb. However, the human CYP4F2 gene was mapped to chromosome 19. Phylogenetic analysis of the CYP4 gene families demonstrated that CYP4A and CYP4B are more closely related than CYP4F.

Alex R. Bell ‘, Paul S. Joys ’, Richard Savory ’, Paula Barratt Tim J.B. Gray ’, Nancy A. Jenkins , Debra J. Gilbert ‘, Neal G. Copeland ’ and David R. Bell a ”Department of Life Science, University of Nottingham, NG7 2RD.; bSanofi Winthrop Pharmaceuticals Research, Northumberland, England.; Mammalian Genetics Laboratory, ABL-Basic Research Program, NCIFrederick Cancer Research and Development Centre, Frederick, USA.

Three murine peroxisome-proliferator-activated-receptor (PPAR) genes were localised to chromosome 15 (PPAR alpha), chromosome 17 (PPAR beta) and chromosome 6 (PPAR gamma). The expression of the three PPAR RNAs was determined using a specific RNase protection assay. In liver RNA, PPAR alpha was expressed at the highest level, with 20-fold lower levels of PPAR beta, and very low levels of PPAR gamma. The three PPAR RNAs showed no sex-specific differences in expression, and the levels of these transcripts were unaffected by treatment of mice with testosterone or the potent peroxisome proliferator, methylclofenapate. In agreement with this data, the level of PPAR alpha protein in liver was unchanged after treatment of mice with methylclofenapate. Investigation of the tissue-specific distribution revealed that the PPAR alpha RNA was expressed at highest levels in liver, to moderate levels in kidney and brown adipose tissue, and at low levels elsewhere. PPAR beta was expressed at moderate levels in liver, and lower levels in other tissues, including brown adipose tissue. In contrast, PPAR gamma RNA was expressed at low levels in liver or epididymal white adipose tissue and at very low levels elsewhere, but was expressed at high levels in brown adipose tissue. The tissue distribution of these receptors suggests an important role in lipid metabolism and toxicity for individual members of the PPAR family. The expression of PPAR alpha and PPAR beta RNAs was examined in 13 strains of mice, and the levels of expression varied within a fourfold range. Polymorphism in the size of PPAR alpha RNA from Swiss-Webster mice was detected, and shown to be due to a 2-bp mutation in the 3' non-coding region of PPAR alpha in Swiss Webster mice.

The AGC kinase AKT is a key component of the phosphatidylinositol 3-kinase (PI3K) pathway, which is frequently deregulated in cancer, making AKT a target of major therapeutic interest. However, PI3K signaling through both AKT-dependent and AKT-independent mechanisms involving other AGC kinases, such as p70S6K, PKA, SGK and ROCK, is important in a range of cancers. Hence, the pharmacological inhibition of these multiple AGC kinases may increase response rates and minimize clinical resistance compared with targeting AKT alone. The clinical drug candidate AT13148 is a multi-AGC kinase, ATP-competitive inhibitor, identified utilizing high-throughput X-ray crystallography and fragment-based lead discovery techniques. Screening of this oral small molecule against a panel of kinases at 10μM revealed >80% inhibition of the structurally related AGC kinases AKT, PKA, ROCK2, p70S6K, MSK, RSK1/2, and SGK. We demonstrate that AT13148 has antiproliferative activity in a range of in vitro models harboring relevant genetic abnormalities, including PTEN, KRAS, PIK3CA and HER2 aberrations. AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK and SGK substrate phosphorylation and induction of apoptosis in both a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects both in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer and PTEN-deficient MES-SA uterine tumor xenografts was demonstrated. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP competitive inhibitors of AKT, is not a therapeutically relevant reactivation step for this compound. We used gene expression microarray studies to characterize the underlying molecular mechanisms of action of AT13148 and the selective AKT inhibitor CCT128930, and observed the induction of upstream regulators including insulin receptor substrate-2 and PIK3IP1 due to compensatory feedback loops, consistent with blockade of AKT signaling. These studies also showed that AT13148 and CCT128930 have distinct molecular effects in cancer cells: AT13148 had a predominant effect on apoptosis genes and caused a greater apoptotic phenotype, while CCT128930 modulated genes in the network regulating cell cycle. This finding emphasizes the functional differences of AT13148 as a multi-AGC kinase inhibitor in contrast to a more AKT-selective inhibitor. In view of the potential mechanistic advantages detailed above, and the potent antitumor activity observed at well tolerated doses against established human tumor xenografts with clinically relevant genetic drivers, the clinical utility of such an AGC kinase inhibitor strategy will now be assessed in a first-in-human Phase I trial of AT13148. 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 928. doi:1538-7445.AM2012-928

We report the cDNA sequence and predicted amino acid sequence of a novel type I keratin, designated as GK50, and show that keratin expression in the goldfish optic nerve is highly complex. The GK50 protein is one of at least three type I keratins expressed in goldfish optic nerve based on both antibody reactivity and blot-binding to the type II keratin ON3. After optic nerve crush in situ hybridization shows a localized increase in GK50 mRNA expression in the crush zone. This is in contrast to ON3 mRNA which shows a localized increase that is limited to the proximal and distal margins of the crush zone, suggesting a diversity of keratin expression in different cell types of the goldfish optic nerve.

The distribution of intermediate filament proteins in optic nerve and spinal cord from rat, hamster, goldfish, frog, and newt were analyzed by two-dimensional gel electrophoresis. General as well as specific monoclonal and polyclonal antibodies were reacted against putative intermediate filament proteins. In vitro incubations of excised optic nerve in the presence of [35S]methionine distinguished between neuronal and nonneuronal intermediate filament proteins. The proteins of the intermediate filament complex in the two tissues for rat and hamster were similar. The typical neurofilament triplet and glial fibrillary acidic protein (GFAP) were observed. Vimentin was more concentrated in the optic nerve than in the spinal cord. The goldfish, newt, and frog contained neurofilament proteins in the 145–150K range and in the 70–85K range. In addition, predominant neurofilament proteins in the 58–62K molecular-weight range were found in all three species. In contrast to mammalian species, the goldfish, newt, and frog displayed extensive heterogeneity between optic nerve and spinal cord in the expression of both neuronal and nonneuronal intermediate filament proteins. The distinctive presence of low-molecular-weight intermediate filament proteins and their high concentration in the optic nerve and spinal cord of these nonmammalian vertebrates is discussed in terms of neuronal development and regeneration.

The four diastereomeric 3,16-diacetates 6, 9, 10 and 11 were prepared from gitoxin 1 by the routes shown in Schemes 1 and 2 and tested for inotropic activity in the isolated guinea-pig atrial preparation. In line with earlier findings in the digitoxigenin series, derivatives with a 3 alpha-acetoxy function, viz 9 and 10, possessed high biological activity.

The intermediate-filament proteins expressed in the goldfish retina were investigated by immunohistochemistry and by immunoblotting. Polyclonal antibodies that previously had been raised against the goldfish optic nerve neurofilament (ON1 and ON2) and glial filament (ON3 and ON4) proteins were used in this study. Anti-ON1/ON2 antiserum reacted on a retinal immunoblot with two proteins having molecular weights and isoelectric points corresponding to those of ON1 and ON2. Histologically, the most pronounced anti-ON1/ON2 reactivity was observed in the ganglion cell layer of the goldfish retina. The anti-ON3/ON4 antiserum reacted with a single protein on a retinal immunoblot. This protein had a molecular weight and isoelectric point which corresponded to the goldfish optic nerve glial filament proteins. This anti-serum labeled horizontal cells in retina sections. Three previously unidentified goldfish visual-pathway intermediate-filament proteins sharing a molecular weight of 60K were observed on two-dimensional gels of retinal cytoskeletal proteins and on retinal immunoblots which were probed with a monoclonal antibody which recognizes an epitope common to all intermediate Filament proteins. The possible existence of homologs of mammalian GFAP and vimentin in the goldfish retina was also explored. Antibodies directed against mammalian GFAP and vimentin labeled the Muller fibers and the cone horizontal cells, respectively. However, immunoblot analysis and a comparison of the two-dimensional gel electrophoresis patterns of goldfish retinal and rat spinal cord cytoskeletal proteins demonstrated a lack of goldfish proteins identical to the mammalian intermediate-filament proteins.

The work reported is the result of an analysis of post-nuclear-attack railroad operation in 12 rail activity centers. The analysis has been based upon the transportation of a single commodity--food. The selected nuclear attack involved 375 4-megaton weapons directed against retaliatory bases and industrial and population centers. The determination of post-attack food transportation requirements was based on the number and location of survivors by county, or similar divisions, and the geographical configuration of surviving yards and rail lines. Interstate food shipments were traced from the point where each rail activity center would be entered to points within 5 or 10 miles of survivors. Supply routes were selected by which food deliveries could be economically made to all survivors and empty freight cars removed. Traffic patterns were established together with yard assignments and requirements for rolling stock. The capability of the rail facilities to accommodate additional traffic was also determined.

The report describes the procedures that were used to evaluate the preliminary designs of advanced landing craft as part of the Navy's Advanced Amphibious Landing Craft Program. The procedures were applied initially to more than 30 different existing and proposed landing craft, and complete analysis was made for five advanced craft after screening out marginal craft at each stage of the analysis. Measures of effectiveness used were Force-Time Effectiveness, Time to Deliver 200,000 sq ft of Vehicles Ashore, Marine Forces or Cargo Lost, Response Time, Mean Productivity per Craft by Type, and Mean Cargo Transfer Rates. Standoff distances of 5 and 25 nautical miles were used. The computer programs used in the analysis were provided by SRI and the Naval Weapons Laboratory. Data are presented bearing on the effectiveness of each type of craft and each craft mix. A description also is given of the SRI program GAMUT, which is a simulation covering much the same ground as the STS-2 package but with a great reduction in the level of detail that is considered. It provides the means of rapidly and cheaply changing the input conditions and operating procedures used in the simulation. Selected preliminary results of the GAMUT model are given.