Undifferentiated F9 embryonal carcinoma cells produce a short-chain collagen molecule.

The undifferentiated F9 embryonal carcinoma cells produce a unique collagen that decreases in amount during retinoic acid-induced differentiation of F9 cells into basement-membrane parietal endoderm. A bacterial-collagenase-sensitive protein of approx. 60,000 Da was resolved on polyacrylamide-gel electrophoresis. After pepsin digestion, two pepsin-resistant fragments containing hydroxyproline were demonstrated, suggesting that a portion of the molecule has a stable triple helix. The mRNA from the undifferentiated F9 cells translates a collagenase-sensitive protein with a molecular mass consistent with the 60,000 Da collagenous protein produced by undifferentiated F8 cells.     

Nature of the fast and slow refolding reactions of iron(III) cytochrome c

The fast and slow refolding reactions of iron(III) cytochrome c (Fe(III) cyt c), previously studied by Ikai et al. (Ikai, A., Fish, W. W., & Tanford, C. (1973) J. Mol. Biol. 73, 165--184), have been reinvestigated. The fast reaction has the major amplitude (78%) and is 100-fold faster than the slow reaction in these conditions (pH 7.2, 25 degrees C, 1.75 M guanidine hydrochloride). We show here that native cyt c is the product formed in the fast reaction as well as in the slow reaction. Two probes have been used to test for formation of native cyt c. absorbance in the 695-nm band and rate of reduction of by L-ascorbate. Different unfolded species (UF, US) give rise to the fast and slow refolding reactions, as shown both by refolding assays at different times after unfolding ("double-jump" experiments) and by the formation of native cyt c in each of the fast and slow refolding reactions. Thus the fast refolding reaction is UF leads to N and the slow refolding reaction is Us leads to N, where N is native cyt c, and there is a US in equilibrium UF equilibrium in unfolded cyt c. The results are consistent with the UF in equilibrium US reaction being proline isomerization, but this has not yet been tested in detail. Folding intermediates have been detected in both reactions. In the UF leads to N reaction, the Soret absorbance change precedes the recovery of the native 695-nm band spectrum, showing that Soret absorbance monitors the formation of a folding intermediate. In the US leads to N reaction an ascorbate-reducible intermediate has been found at an early stage in folding and the Soret absorbance change occurs together with the change at 695 nm as N is formed in the final stage of folding.     

Multicompartmental distribution of the tuberous sclerosis gene products,hamartin and tuberin

Mutations of the TSC1 and TSC2 genes give rise to the clinical disorder of tuberous sclerosis characterized by the development of hamartomas predominantly affecting the central nervous system, kidney, skin, lung, and heart. The function of the gene products, hamartin and tuberin, is not well understood but we have previously suggested a role in vesicular transport. To define the subcellular compartment(s) involved with these two proteins, biochemical characterization of hamartin and tuberin was performed in primary tissues and cell lines. Fractionation of cell lysates identified both proteins in the cytosolic, microsomal, and cytoskeletal compartments. In each of these fractions, hamartin and tuberin formed a stable complex in coimmunoprecipitation analyses. Further, they colocalized extensively in discrete, vesicular structures in the cytoplasm. Within the microsomal compartment, hamartin and tuberin behaved as peripheral membrane proteins that associate with the cytosolic leaflet of membranous domains. Immunoisolation of tuberin-bound vesicles using magnetic beads showed an enrichment of rap1, rab5, and caveolin-1, all of which have been found in specialized lipid microdomains, caveolae. Our data suggest that hamartin and tuberin are multicompartmental proteins that partially reside in caveolin-1-enriched structures and potentially affect their signaling.     

Pseudomonas exotoxin A. Membrane binding, insertion, and traversal

Using vesicle targets composed of phosphatidylcholine and cholesterol (1:1 molar ratio), we found that Pseudomonas aeruginosa exotoxin A (PTx) binding and insertion are not only dependent on pH (Zalman, L.S., and Wisnieski, B.J. (1985) Infect. Immun. 50, 630-635) but also on ionic strength, reaching a maximum in pH 4 buffer that contains 150-200 mM NaCl. Insertion was monitored by photolabeling with an intramembranous probe. Higher levels of binding and insertion were attained with vesicles that contained 2.5 mol% dicetylphosphate than with neutral vesicles. Positively charged vesicles (2.7 mol% stearylamine) were the least effective targets. At pH 7.4, all binding levels were depressed. While PTx binding increased with increasing temperature, the relative proportion of the vesicle-associated toxin that was photolabeled decreased. The most likely explanation for the decrease is that the bilayer translocation rates increased with increasing temperature, and hence fewer PTx molecules were accessible at the time of photolabeling. At 37 degrees C, binding and insertion both plateaued within 10 min of lowering the pH to 4. After 10 min, the amount of bound toxin decreased slightly with time but there was a dramatic decrease in photolabeling, indicating that inserted PTx had begun to cross the bilayer. This was verified by the finding that when PTx was incubated with vesicles that contained trypsin, cleavage occurred only in those samples in which the pH was shifted down to pH 4. Entry is triggered by an acid-induced conformational change that promotes productive binding and insertion. After insertion, the kinetics of membrane traversal appear to be regulated by the physical properties of the bilayer.     

Assessing the significance of conserved genomic aberrations using high resolution genomic microarrays

Genomic aberrations recurrent in a particular cancer type can be important prognostic markers for tumor progression. Typically in early tumorigenesis, cells incur a breakdown of the DNA replication machinery that results in an accumulation of genomic aberrations in the form of duplications, deletions, translocations, and other genomic alterations. Microarray methods allow for finer mapping of these aberrations than has previously been possible; however, data processing and analysis methods have not taken full advantage of this higher resolution. Attention has primarily been given to analysis on the single sample level, where multiple adjacent probes are necessarily used as replicates for the local region containing their target sequences. However, regions of concordant aberration can be short enough to be detected by only one, or very few, array elements. We describe a method called Multiple Sample Analysis for assessing the significance of concordant genomic aberrations across multiple experiments that does not require a-priori definition of aberration calls for each sample. If there are multiple samples, representing a class, then by exploiting the replication across samples our method can detect concordant aberrations at much higher resolution than can be derived from current single sample approaches. Additionally, this method provides a meaningful approach to addressing population-based questions such as determining important regions for a cancer subtype of interest or determining regions of copy number variation in a population. Multiple Sample Analysis also provides single sample aberration calls in the locations of significant concordance, producing high resolution calls per sample, in concordant regions. The approach is demonstrated on a dataset representing a challenging but important resource: breast tumors that have been formalin-fixed, paraffin-embedded, archived, and subsequently UV-laser capture microdissected and hybridized to two-channel BAC arrays using an amplification protocol. We demonstrate the accurate detection on simulated data, and on real datasets involving known regions of aberration within subtypes of breast cancer at a resolution consistent with that of the array. Similarly, we apply our method to previously published datasets, including a 250K SNP array, and verify known results as well as detect novel regions of concordant aberration. The algorithm has been fully implemented and tested and is freely available as a Java application at http://www.cbil.upenn.edu/MSA.     

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization,cDNA cloning,and expressed sequence tag analysis

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.     

RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain

microRNAs (miRNAs) are small (approximately 22 nucleotide) regulatory RNAs which play fundamental roles in many biological processes. Recent studies have shown that the expression of many miRNAs is altered in various human tumors and some miRNAs may function as oncogenes or tumor suppressor genes. However, with the exception of glioblastoma multiforme, the expression of miRNAs in brain tumors is unknown. Furthermore, methods to profile miRNAs from formalin-fixed, paraffin-embedded (FFPE) archival tissues or to study their cellular and subcellular localization in FFPE tissues have been lacking. Here we report the coordinated miRNA expression analysis from the tissue level to the subcellular level, using the RAKE (RNA-primed, array-based, Klenow Enzyme) miRNA microarray platform in conjunction with Locked Nucleic Acid (LNA)-based in situ hybridization (LNA-ISH) on archival FFPE human brains and oligodendroglial tumors. The ability to profile miRNAs from archival tissues at the tissue level, by RAKE microarrays, and at the cellular level by LNA-ISH, will accelerate studies of miRNAs in human diseases.     

Application of Multiplexed Kinase Inhibitor Beads to Study Kinome Adaptations in Drug-Resistant Leukemia

Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.