p125 is localized in endoplasmic reticulum exit sites and involved in their organization

Transport vesicles coated with the COPII complex, which is assembled from Sar1p, Sec23p-Sec24p, and Sec13p-Sec31p, are involved in protein export from the endoplasmic reticulum (ER). We previously identified and characterized a novel Sec23p-interacting protein, p125, that is only expressed in mammals and exhibits sequence homology with phosphatidic acid-preferring phospholipase A(1) (PA-PLA(1)). In this study, we examined the localization and function of p125 in detail. By using immunofluorescence and electron microscopy, we found that p125 is principally localized in ER exit sites where COPII-coated vesicles are produced. Analyses of chimeric proteins comprising p125 and two other members of the mammalian PA-PLA(1) family (PA-PLA(1) and KIAA0725p) showed that, for localization to ER exit sites, the p125-specific N-terminal region is critical, and the putative lipase domain is interchangeable with KIAA0725p but not with PA-PLA(1). RNA interference-mediated depletion of p125 affected the organization of ER exit sites. The structure of the cis-Golgi compartment was also substantially disturbed, whereas the medial-Golgi was not. Protein export from the ER occurred without a significant delay in p125-depleted cells. Our study suggests that p125 is a mammalian-specific component of ER exit sites and participates in the organization of this compartment.     

The influence of terrestrial ecosystems on climate

Terrestrial ecosystems influence climate by affecting how much solar energy is absorbed by the land surface and by exchanging climatically important gases with the atmosphere. Recent model analyses show widespread qualitative agreement that terrestrial ecological processes will have a net positive feedback effect on 21st-century global warming, and, therefore, cannot be ignored in climate-change projections. However, the quantitative uncertainty in the net feedback is large. The uncertainty in 21st-century carbon dioxide emissions resulting from terrestrial carbon cycle-climate feedbacks is second in magnitude only to the uncertainty in anthropogenic emissions. We estimate that this translates into an uncertainty in global warming owing to the land surface of 1.5 degrees C by 2100. We also emphasise the need to improve our understanding of terrestrial ecological processes that influence land-atmosphere interactions at relatively long timescales (decadal-century) as well as at shorter intervals (e.g. hourly).     

T-Cell activation by t(9;22) acute lymphoblastic leukemia-derived dendritic-like cells is associated with increased tapasin expression

Dendritic-like cells from t(9;22) acute lymphoblastic leukemia (ALL) blasts can activate T cells, while the original unmodified leukemic blasts cannot. To determine whether these functional differences were associated with differences in antigen-processing machinery (APM) component expression, we have measured the level of APM component expression in unmodified blasts and ALL-derived dendritic-like cells. Seven t(9;22) ALL patient samples and one cell line were studied for APM component expression utilizing a unique panel of recently developed monoclonal antibodies and a recently developed intracellular staining technique. In addition, the HLA class I antigen cell surface expression was measured. HLA class I antigens were similarly expressed on the unmodified blasts and on the autologous dendritic-like cells. Intracellular HLA class I antigen and tapasin expression (P=0.03 for both) were upregulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. These results provide a potential mechanism for the ability of t(9;22) ALL-derived dendritic-like cells to induce T-cell activation and, suggest that tapasin upregulation may serve as a marker to standardize and monitor the quality of the dendritic-like cells used in immunotherapy. Supported partially by The Heidi Leukemia Research Fund, Buffalo, NY, and by PHS grants CA 67108 and CA 16056 awarded by the National Cancer Institute, DHHS.     

KCN1, a Novel Synthetic Sulfonamide Anticancer Agent: In Vitro and In Vivo Anti-Pancreatic Cancer Activities and Preclinical Pharmacology

The purpose of the present study was to determine the in vitro and in vivo anti-cancer activity and pharmacological properties of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1. In the present study, we investigated the in vitro activity of KCN1 on cell proliferation and cell cycle distribution of pancreatic cancer cells, using the MTT and BrdUrd assays, and flow cytometry. The in vivo anti-cancer effects of KCN1 were evaluated in two distinct xenograft models of pancreatic cancer. We also developed an HPLC method for the quantitation of the compound, and examined its stability in mouse plasma, plasma protein binding, and degradation by mouse S9 microsomal enzymes. Furthermore, we examined the pharmacokinetics of KCN1 following intravenous or intraperitoneal injection in mice. Results showed that, in a dose-dependent manner, KCN1 inhibited cell growth and induced cell cycle arrest in human pancreatic cancer cells in vitro, and showed in vivo anticancer efficacy in mice bearing Panc-1 or Mia Paca-2 tumor xenografts. The HPLC method provided linear detection of KCN1 in all of the matrices in the range from 0.1 to 100 µM, and had a lower limit of detection of 0.085 µM in mouse plasma. KCN1 was very stable in mouse plasma, extensively plasma bound, and metabolized by S9 microsomal enzymes. The pharmacokinetic studies indicated that KCN1 could be detected in all of the tissues examined, most for at least 24 h. In conclusion, our preclinical data indicate that KCN1 is a potential therapeutic agent for pancreatic cancer, providing a basis for its future development.     

Inside the Mind of a Medicinal Chemist: The Role of Human Bias in Compound Prioritization during Drug Discovery

Medicinal chemists’ “intuition” is critical for success in modern drug discovery. Early in the discovery process, chemists select a subset of compounds for further research, often from many viable candidates. These decisions determine the success of a discovery campaign, and ultimately what kind of drugs are developed and marketed to the public. Surprisingly little is known about the cognitive aspects of chemists’ decision-making when they prioritize compounds. We investigate 1) how and to what extent chemists simplify the problem of identifying promising compounds, 2) whether chemists agree with each other about the criteria used for such decisions, and 3) how accurately chemists report the criteria they use for these decisions. Chemists were surveyed and asked to select chemical fragments that they would be willing to develop into a lead compound from a set of ∼4,000 available fragments. Based on each chemist’s selections, computational classifiers were built to model each chemist’s selection strategy. Results suggest that chemists greatly simplified the problem, typically using only 1–2 of many possible parameters when making their selections. Although chemists tended to use the same parameters to select compounds, differing value preferences for these parameters led to an overall lack of consensus in compound selections. Moreover, what little agreement there was among the chemists was largely in what fragments were undesirable. Furthermore, chemists were often unaware of the parameters (such as compound size) which were statistically significant in their selections, and overestimated the number of parameters they employed. A critical evaluation of the problem space faced by medicinal chemists and cognitive models of categorization were especially useful in understanding the low consensus between chemists.     

Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 microM) was approximately 80% compared to allicin treatment alone (approximately 50%). Also N-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 microM, 30 min) the redox state for 2GSH/oxidized glutathione shifted from EGSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 microM, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/10(6) cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status.     

The role of a spiny plant refuge in structuring grazed shortgrass steppe plant communities

This study evaluates the hypothesis that biological grazing refuges have an important role in plant-grazer interactions of grasslands with a long history of grazing. We assessed the hypothesis that clumps of the spiny cactus Opuntia polyacantha provide biological refuges from cattle grazing, affecting cover and seedhead production of associated vascular plants in the shortgrass steppe of the North America. The study was based on sampling inside and outside Opuntia clumps in eight long-term moderately grazed pastures established 60 yr ago and their respective ungrazed controls. Opuntia clumps provided a refuge for seedhead production of the dominant grass ( Bouteloua gracilis ) and for cover and seedhead production of many plant groups. Clumps were also a refuge for species sensitive to grazing (species that decrease with grazing) and barrel-cacti, but not for species preferred by cattle (species with greater proportion in the diet than in the field), exotics or weeds. Our results suggest that these effects were mainly through changes in the microenvironmental conditions resulting from protection effects, even though all potential microenvironmental effects could not be measured. Cacti promoted some negative effects on other plant groups, probably due to the space occupied by cladodes inside cactus clumps. The refuge effects observed at the group level did not translate into strong community level effects. Species diversity ( H' ) was greater in cactus clumps due to lower dominance rather than greater richness. The presence of Opuntia clumps increased landscape-scale diversity. This ecological role of Opuntia clumps as refuge from cattle grazing should be taken into consideration in management practices aimed at cactus eradication in order to increase forage availability for livestock. We discuss the potential role of plant community productivity and grazing history with regard to the importance of natural refuges in structuring grassland communities.