ADAM17 but not ADAM10 mediates tumor necrosis factor-alpha and L-selectin shedding from leukocyte membranes

The release of tumor necrosis factor-alpha (TNF-alpha) from cellular membranes has been shown by different laboratories to be controlled by a disintegrin and metalloprotease, ADAM10 or ADAM17. In contrast, only ADAM17 has shown to be involved in L-selectin shedding. To determine the specific roles of ADAM10 and ADAM17 in the processing of TNF-alpha and L-selectin shedding, antisense oligonucleotides (ASO) targeting both ADAM10 and ADAM17 were identified. We show that ISIS 16337 reduces ADAM17 mRNA and ISIS 100750 reduces ADAM10 mRNA in a sequence-specific and dose-dependent manner in both Jurkat and THP-1 cells. The ADAM17 ASO (ISIS 16337) inhibited both TNF-alpha secretion in THP-1 cells and L-selectin shedding in Jurkat cells, whereas the ADAM10 ASO (ISIS 100750) did not significantly inhibit release of either protein. These results suggest that ADAM17 is one of the major metalloproteases involved in L-selectin shedding as well as TNF-alpha processing. The biologic substrates for ADAM10 in Jurkat and THP-1 cells remain to be elucidated.     

Cell-cycle arrest in TrkA-expressing NIH3T3 cells involves nitric oxide synthase

We have examined nerve growth factor (NGF)-triggered signaling in two NIH3T3 cell lines exogenously expressing the NGF receptor, TrkA. TRK1 cells cease to proliferate and extend long processes in response to NGF, while E25 cells continue to proliferate in the presence of NGF. These two cell lines express similar levels of TrkA and respond to NGF with rapid elevation of mitogen-activated protein kinase (MAPK) activity. MAPK activation is slightly more sustained for E25 cells than for TRK1 cells, although sustained activation of MAPK has been suggested to cause cell-cycle arrest. As judged by NADPH-diaphorase staining, nitric oxide synthase (NOS) activity is increased in TRK1 cells upon exposure to NGF. In contrast, diaphorase staining in E25 cells is unaffected by NGF treatment. Immunocytochemistry shows that levels of the brain NOS (bNOS) isoform are increased in TRK1, but not E25, cells exposed to NGF. Furthermore, Western blots show that NGF elevated cyclin-dependent kinase inhibitor, p21(WAF1), in TRK1 cells only. NGF-induced p21(WAF1) expression, cell-cycle arrest and process extension are abolished by N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS. The inactive enantiomer, D-NAME, did not inhibit these responses. Furthermore, even though E25 cells do not respond to NGF or nitric oxide donors, they do undergo a morphological change in response to NGF plus a nitric oxide donor. Therefore, NOS and p21(WAF1) are induced only in the TrkA-expressing NIH3T3 cell line that undergoes cell-cycle arrest and morphological changes in response to NGF. These results demonstrate that sustained activation of MAPK is not the sole determining factor for NGF-induced cell-cycle arrest and implicate NO in the cascade of events leading to NGF-induced morphological changes and cell-cycle arrest.     

Biomass and aggregation analysis of human embryonic kidney 293 suspension cell cultures by particle size measurement

A method has been developed to monitor the cell growth of aggregated human embryonic kidney 293 (HEK293) suspension cultures by measuring cumulative particle volume and the particle size distribution. This method employs a particle size analyzer that determines the size of individual particles by detecting their light obscuration (blockage) or scattering. Cell counts derived from the cumulative volume of the cell particle correlate well with manual cell counts from a hemacytometer at different stages of growth. This correlation was further confirmed by quantifying total cellular protein of the samples. Simultaneously, the aggregation state of the samples can also be monitored and mathematically described. Results from this study demonstrate that this simple and reproducible method allows the direct measurement of cumulative cell volume and the degree of cell aggregation, as well as an indirect assessment of cell counts.     

Indirect selection of thermal tolerance during experimental evolution of Drosophila melanogaster

Natural selection alters the distribution of a trait in a population and indirectly alters the distribution of genetically correlated traits. Long‐standing models of thermal adaptation assume that trade‐offs exist between fitness at different temperatures; however, experimental evolution often fails to reveal such trade‐offs. Here, we show that adaptation to benign temperatures in experimental populations of Drosophila melanogaster resulted in correlated responses at the boundaries of the thermal niche. Specifically, adaptation to fluctuating temperatures (16–25°C) decreased tolerance of extreme heat. Surprisingly, flies adapted to a constant temperature of 25°C had greater cold tolerance than did flies adapted to other thermal conditions, including a constant temperature of 16°C. As our populations were never exposed to extreme temperatures during selection, divergence of thermal tolerance likely reflects indirect selection of standing genetic variation via linkage or pleiotropy. We found no relationship between heat and cold tolerances in these populations. Our results show that the thermal niche evolves by direct and indirect selection, in ways that are more complicated than assumed by theoretical models.     

Novel pyrrolyl 2-aminopyridines as potent and selective human β-secretase (BACE1) inhibitors

The proteolytic enzyme β-secretase (BACE1) plays a central role in the synthesis of the pathogenic β-amyloid in Alzheimer’s disease. Recently, we reported small molecule acylguanidines as potent BACE1 inhibitors. However, many of these acylguanidines have a high polar surface area (e.g. as measured by the topological polar surface area or TPSA), which is unfavorable for crossing the blood–brain barrier. Herein, we describe the identification of the 2-aminopyridine moiety as a bioisosteric replacement of the acylguanidine moiety, which resulted in inhibitors with lower TPSA values and superior brain penetration. X-ray crystallographic studies indicated that the 2-aminopyridine moiety interacts directly with the catalytic aspartic acids Asp32 and Asp228 via a hydrogen-bonding network.     

5'-to-3' exoribonuclease activity in bacteria: role of RNase J1 in rRNA maturation and 5' stability of mRNA

Although the primary mechanism of eukaryotic messenger RNA decay is exoribonucleolytic degradation in the 5'-to-3' orientation, it has been widely accepted that Bacteria can only degrade RNAs with the opposite polarity, i.e. 3' to 5'. Here we show that maturation of the 5' side of Bacillus subtilis 16S ribosomal RNA occurs via a 5'-to-3' exonucleolytic pathway, catalyzed by the widely distributed essential ribonuclease RNase J1. The presence of a 5'-to-3' exoribonuclease activity in B. subtilis suggested an explanation for the phenomenon whereby mRNAs in this organism are stabilized for great distances downstream of "roadblocks" such as stalled ribosomes or stable secondary structures, whereas upstream sequences are never detected. We show that a 30S ribosomal subunit bound to a Shine Dalgarno-like element (Stab-SD) in the cryIIIA mRNA blocks exonucleolytic progression of RNase J1, accounting for the stabilizing effect of this element in vivo.     

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.     

Thermodynamic benchmark study using Biacore technology

A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor-enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 degrees C. van't Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.     

Identification of potent and selective TACE inhibitors via the S1 pocket

By focusing on the P1 portion of the piperidine beta-sulfone ligands we identified a motif that induces selectivity and resulted in a series of TACE inhibitors that demonstrated excellent in vitro potency against isolated TACE enzyme and excellent selectivity over MMPs 1, 2, 9, 13, and 14.