TEACHING MARKETING AND MANAGEMENT TO AN EXTENSION AUDIENCE IN AN INTER-DISCIPLINARY SETTING

This paper discusses how economists utilize an inter-disciplinary workshop to teach marketing and management concepts to beef cattle producers and beef industry advisors. Range and animal scientists along with economists teach concepts in the classroom and then demonstrate these concepts with hands-on field activities in an 8-day Ranch Practicum, spread over an 8-month period.Teaching/Communication/Extension/Profession,

Ultraviolet radiation triggers apoptosis of fibroblasts and skin keratinocytes mainly via the BH3-only protein Noxa

To identify the mechanisms of ultraviolet radiation (UVR)–induced cell death, for which the tumor suppressor p53 is essential, we have analyzed mouse embryonic fibroblasts (MEFs) and keratinocytes in mouse skin that have specific apoptotic pathways blocked genetically. Blocking the death receptor pathway provided no protection to MEFs, whereas UVR-induced apoptosis was potently inhibited by Bcl-2 overexpression, implicating the mitochondrial pathway. Indeed, Bcl-2 overexpression boosted cell survival more than p53 loss, revealing a p53-independent pathway controlled by the Bcl-2 family. Analysis of primary MEFs lacking individual members of its BH3-only subfamily identified major initiating roles for the p53 targets Noxa and Puma. In the transformed derivatives, where Puma, unexpectedly, was not induced by UVR, Noxa had the dominant role and Bim a minor role. Furthermore, loss of Noxa suppressed the formation of apoptotic keratinocytes in the skin of UV-irradiated mice. Collectively, these results demonstrate that UVR activates the Bcl-2–regulated apoptotic pathway predominantly through activation of Noxa and, depending on cellular context, Puma

Bcl-2–regulated apoptosis and cytochrome c release can occur independently of both caspase-2 and caspase-9

Apoptosis in response to developmental cues and stress stimuli is mediated by caspases that are regulated by the Bcl-2 protein family. Although caspases 2 and 9 have each been proposed as the apical caspase in that pathway, neither is indispensable for the apoptosis of leukocytes or fibroblasts. To investigate whether these caspases share a redundant role in apoptosis initiation, we generated caspase-2−/−9−/− mice. Their overt phenotype, embryonic brain malformation and perinatal lethality mirrored that of caspase-9−/− mice but were not exacerbated. Analysis of adult mice reconstituted with caspase-2−/−9−/− hematopoietic cells revealed that the absence of both caspases did not influence hematopoietic development. Furthermore, lymphocytes and fibroblasts lacking both remained sensitive to diverse apoptotic stimuli. Dying caspase-2−/−9−/− lymphocytes displayed multiple hallmarks of caspase-dependent apoptosis, including the release of cytochrome c from mitochondria, and their demise was antagonized by several caspase inhibitors. These findings suggest that caspases other than caspases 2 and 9 can promote cytochrome c release and initiate Bcl-2–regulated apoptosis

Pro-Apoptotic Apoptosis Protease–Activating Factor 1 (Apaf-1) Has a Cytoplasmic Localization Distinct from Bcl-2 or Bcl-XL

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease–activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2–like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-xL were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-xL precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-xL could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2–like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation

Transcriptomic classification of genetically engineered mouse models of breast cancer identifies human subtype counterparts

Background: Human breast cancer is a heterogeneous disease consisting of multiple molecular subtypes. Genetically engineered mouse models are a useful resource for studying mammary cancers in vivo under genetically controlled and immune competent conditions. Identifying murine models with conserved human tumor features will facilitate etiology determinations, highlight the effects of mutations on pathway activation, and should improve preclinical drug testing. Results: Transcriptomic profiles of 27 murine models of mammary carcinoma and normal mammary tissue were determined using gene expression microarrays. Hierarchical clustering analysis identified 17 distinct murine subtypes. Cross-species analyses using three independent human breast cancer datasets identified eight murine classes that resemble specific human breast cancer subtypes. Multiple models were associated with human basal-like tumors including TgC3(1)-Tag, TgWAP-Myc and Trp53-/-. Interestingly, the TgWAPCre-Etv6 model mimicked the HER2-enriched subtype, a group of human tumors without a murine counterpart in previous comparative studies. Gene signature analysis identified hundreds of commonly expressed pathway signatures between linked mouse and human subtypes, highlighting potentially common genetic drivers of tumorigenesis. Conclusions: This study of murine models of breast carcinoma encompasses the largest comprehensive genomic dataset to date to identify human-to-mouse disease subtype counterparts. Our approach illustrates the value of comparisons between species to identify murine models that faithfully mimic the human condition and indicates that multiple genetically engineered mouse models are needed to represent the diversity of human breast cancers. The reported trans-species associations should guide model selection during preclinical study design to ensure appropriate representatives of human disease subtypes are used

Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis

SummaryIn stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2–α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other’s surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis

Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity

Prosurvival Bcl-2–like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function

Strong signature of natural selection within an FHIT intron implicated in prostate cancer risk

Previously, a candidate gene linkage approach on brother pairs affected with prostate cancer identified a locus of prostate cancer susceptibility at D3S1234 within the fragile histidine triad gene (FHIT), a tumor suppressor that induces apoptosis. Subsequent association tests on 16 SNPs spanning approximately 381 kb surrounding D3S1234 in Americans of European descent revealed significant evidence of association for a single SNP within intron 5 of FHIT. In the current study, resequencing and genotyping within a 28.5 kb region surrounding this SNP further delineated the association with prostate cancer risk to a 15 kb region. Multiple SNPs in sequences under evolutionary constraint within intron 5 of FHIT defined several related haplotypes with an increased risk of prostate cancer in European-Americans. Strong associations were detected for a risk haplotype defined by SNPs 138543, 142413, and 152494 in all cases (Pearson's χ2 = 12.34, df 1, P = 0.00045) and for the homozygous risk haplotype defined by SNPs 144716, 142413, and 148444 in cases that shared 2 alleles identical by descent with their affected brothers (Pearson's χ2 = 11.50, df 1, P = 0.00070). In addition to highly conserved sequences encompassing SNPs 148444 and 152413, population studies revealed strong signatures of natural selection for a 1 kb window covering the SNP 144716 in two human populations, the European American (π = 0.0072, Tajima's D= 3.31, 14 SNPs) and the Japanese (π = 0.0049, Fay & Wu's H = 8.05, 14 SNPs), as well as in chimpanzees (Fay & Wu's H = 8.62, 12 SNPs). These results strongly support the involvement of the FHIT intronic region in an increased risk of prostate cancer. © 2008 Ding et al

Correlation of omega-3 levels in serum phospholipid from 2053 human blood samples with key fatty acid ratios

<p>Abstract</p> <p>Background</p> <p>This research was conducted to explore the relationships between the levels of omega-3 fatty acids in serum phospholipid and key fatty acid ratios including potential cut-offs for risk factor assessment with respect to coronary heart disease and fatal ischemic heart disease.</p> <p>Methods</p> <p>Blood samples (n = 2053) were obtained from free-living subjects in North America and processed for determining the levels of total fatty acids in serum phospholipid as omega-3 fatty acids including EPA (eicosapentaenoic acid, 20:5 n-3) and DHA (docosahexaenoic acid, 22:6 n-3) by combined thin-layer and gas-liquid chromatographic analyses. The omega-3 levels were correlated with selected omega-6: omega-3 ratios including AA (arachidonic acid, 20:4n-6): EPA and AA:(EPA+DHA). Based on previously-published levels of omega-3 fatty acids considered to be in a 'lower risk' category for heart disease and related fatality, 'lower risk' categories for selected fatty acid ratios were estimated.</p> <p>Results</p> <p>Strong inverse correlations between the summed total of omega-3 fatty acids in serum phospholipid and all four ratios (omega-6:omega-3 (n-6:n-3), AA:EPA, AA:DHA, and AA:(EPA+DHA)) were found with the most potent correlation being with the omega-6:omega-3 ratio (R²= 0.96). The strongest inverse relation for the EPA+DHA levels in serum phospholipid was found with the omega-6: omega-3 ratio (R²= 0.94) followed closely by the AA:(EPA+DHA) ratio at R²= 0.88. It was estimated that 95% of the subjects would be in the 'lower risk' category for coronary heart disease (based on total omega-3 ≥ 7.2%) with omega-6:omega-3 ratios <4.5 and AA:(EPA+DHA) ratios <1.4. The corresponding ratio cut-offs for a 'lower risk' category for fatal ischemic heart disease (EPA+DHA ≥ 4.6%) were estimated at < 5.8 and < 2.1, respectively.</p> <p>Conclusions</p> <p>Strong inverse correlations between the levels of omega-3 fatty acids in serum (or plasma) phospholipid and omega-6: omega-3 ratios are apparent based on this large database of 2053 samples. Certain fatty acid ratios may aid in cardiovascular disease-related risk assessment if/when complete profiles are not available.</p

Destruction of tumor vasculature and abated tumor growth upon VEGF blockade is driven by proapoptotic protein Bim in endothelial cells

VEGF deprivation induces Bim expression in tumor endothelial cells, and Bim is needed for anti-VEGF–driven endothelial cell death and tumor shrinkage