A novel antagonist to the inhibitors of apoptosis (IAPs) potentiates cell death in EGFR-overexpressing non-small-cell lung cancer cells.

In the effort to develop an efficient chemotherapy drug for the treatment of non-small-cell lung cancer (NSCLC), we analyzed the anti-tumorigenic effects of a novel small molecule targeting the inhibitor of apoptosis (IAPs), HM90822B, on NSCLC cells. HM90822B efficiently decreased IAP expression, especially that of XIAP and survivin, in several NSCLC cells. Interestingly, cells overexpressing epidermal growth factor receptor (EGFR) due to the mutations were more sensitive to HM90822B, undergoing cell cycle arrest and apoptosis when treated. In xenograft experiments, inoculated EGFR-overexpressing NSCLC cells showed tumor regression when treated with the inhibitor, demonstrating the chemotherapeutic potential of this agent. Mechanistically, decreased levels of EGFR, Akt and phospho-MAPKs were observed in inhibitor-treated PC-9 cells on phosphorylation array and western blotting analysis, indicating that the reagent inhibited cell growth by preventing critical cell survival signaling pathways. In addition, gene-specific knockdown studies against XIAP and/or EGFR further uncovered the involvement of Akt and MAPK pathways in HM90822B-mediated downregulation of NSCLC cell growth. Together, these results support that HM90822B is a promising candidate to be developed as lung tumor chemotherapeutics by targeting oncogenic activities of IAP together with inhibiting cell survival signaling pathways.

Proteome analysis of light-induced proteins in Synechocystis sp. PCC 6803: identification of proteins separated by 2D-PAGE using N-terminal sequencing and MALDI-TOF MS.

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.

Cyclic AMP-dependent phosphoprotein components I and II interact with beta gamma subunits of transducin in frog rod outer segments.

Components I and II (CI&II) in frog rod outer segments (ROS) are prominent cAMP-dependent protein kinase (PK-A) substrates. Their phosphorylation level is high in the dark, and illumination causes dephosphorylation. In order to understand their physiological role in phototransduction, biochemical characterization of CI&II phosphorylation was performed. Fractionation of phosphorylated ROS proteins showed that CI&II in the soluble fraction were highly phosphorylated by endogenous PK-A, whereas those in the membrane-associated protein fractions were not. The latter proteins could be phosphorylated by purified catalytic subunit of PK-A (PK-Acat) while the former proteins were not, suggesting that membrane-bound CI&II are normally much less phosphorylated. Treatments that dissociate the alpha subunit (alpha t) of transducin (Gt) from beta gamma subunits (beta gamma t) and thus produce excess free subunits of Gt in the soluble fraction caused inhibition of CI&II phosphorylation in the soluble fraction and enhancement of CI&II phosphorylation in the peripheral membrane fractions containing less Gt. Unphosphorylated CI&II tightly associated with the washed ROS membranes could be extracted after phosphorylation by PK-Acat. Phosphorylation also caused elution of beta gamma t from the membrane under the same conditions. Cross-linking by the maleimidobenzoyl-N-hydroxysuccinimide ester of the peripheral membrane fraction produced a distinct phosphorylated 50 kDa product with concurrent disappearance of the beta subunit of transducin (beta t) and phosphorylated CI&II. This phosphorylated cross-linked product was not recognized by a monoclonal anti-alpha t antibody but was recognized by antiserum against beta t, suggesting that the 50 kDa protein is a complex of beta gamma t and CI&II. Amino terminal sequencing of components I and II suggests that they are identical proteins with a unique sequence unrelated to other proteins in protein data bases. Phosphopeptide mapping of phosphorylated CI&II in the soluble fraction yielded two trypsinized phosphopeptides, while that in the peripheral membrane fractions showed only one phosphopeptide. These data suggest that multiple phosphorylation of CI&II alters their cellular localization. We conclude that phosphorylation of CI&II controls their localization in frog ROS and an interaction of CI&II with subunits of Gt regulates their phosphorylation.

Synthesis and antibacterial activity of new cephalosporins with lactonyloxyimino moiety.

A series of 7-¿2-(2-aminothiazol-4-yl)-2-Z-(gamma-lacton-3-yl)oxyimin oacetamido¿ cephalosporins with various substituents at the 3-position in cephem nucleus were synthesized and evaluated microbiologically. The tested compounds showed potent activities but were somewhat less active than cefotaxime or cefixime against a wide variety of Gram-positive and Gram-negative bacteria.

The molecular basis of GTP-binding protein interaction with receptors.

The molecular basis of the interaction between the visual receptor, rhodopsin, and the rod outer segment GTP-binding protein, transducin or Gt, was studied using a synthetic-peptide-competition approach to elucidate the site(s) on the Gt alpha-subunit (alpha t) involved in high-affinity binding to light-activated rhodopsin (R*). Synthetic peptides based on the amino acid sequence of portions of the molecule that interact with rhodopsin can themselves bind the rhodopsin and thus behave as competitive inhibitors of rhodopsin-G-protein interaction. This blockade was assessed by measuring the ability of peptides to inhibit Gt stabilization of the metarhodopsin II conformation of rhodopsin. Based upon this analysis, two regions near the C-terminal of alpha 1 are important for interaction with R*.

Arm wrestler's injury: report of seven cases.

Seven cases of fractures of the humerus from arm wrestling included 2 types of fractures. Two were spiral fractures of the humeral shaft with large butterfly fragments at the junction of the middle and distal one third caused by a violent uncoordinated muscular action. Five were fractures of the medial epicondyle of the humerus as a result of a sudden blow to the elbow. In all 7 cases, the fractures healed and arm function returned to normal.