The PI3K regulatory subunits p55α and p50α regulate cell death in vivo.

The phosphatidylinositol 3-kinase (PI3K) regulatory subunits p55α and p50α are coordinately transcriptionally upregulated by signal transducer and activator of transcription 3 (Stat3) at the onset of mammary gland involution, a process that requires Stat3. Deletion of both p55α and p50α subunits in vivo abrogated mammary epithelial cell death during involution. This was associated also with reduced cytosolic levels and activity of the cysteine protease cathepsin L, which is implicated in lysosomal-mediated programmed cell death (LM-PCD) and is upregulated in involution. Furthermore, involution is delayed in cathepsin L-deficient mice suggesting that the p55α/p50α subunits mediate cell death in part by elevating the level of cathepsin L resulting in increased cytosolic activity. Surprisingly, we found that p55α/p50α localize to the nucleus where they bind to chromatin and regulate transcription of a subset of inflammatory/acute phase genes that are also Stat3 targets. Our findings reveal a novel role for these PI3K regulatory subunits as regulators of LM-PCD in vivo.

Analysis of in vivo-derived amyloid-beta polypeptides by on-line two-dimensional chromatography-mass spectrometry.

The presence of senile plaques composed of amyloid-beta (Abeta) polypeptides within brain tissue is normally used as a definitive postmortem diagnosis for Alzheimer's Disease (AD). Therefore, these polypeptides have been investigated as potential biomarkers of the disease state. However, at present, there is a lack of a robust assay for the detection of such polypeptides derived from in vivo sources. Such an assay is essential for analysis of biological samples from model AD systems. To overcome this problem we have developed a new single-step assay utilizing two dimensional-chromatography in conjunction with mass spectrometry. The method consists of on-line size-exclusion chromatography (SEC) to provide initial separation of analytes from the sample (based on their molecular weight) coupled with sample preconcentration prior to analysis by microbore high-performance liquid chromatography-mass spectrometry (HPLC-MS). This provides an extremely versatile and powerful assay which can separate specific analytes from cell lysate in a single step without further sample handling. The use of mass spectrometry as the detection system yields much more structural information than can be obtained from traditional ELISA and sandwich ELISA antibody assays. Furthermore, the on-line sample cleanup protocol minimizes sample handling and facilitates assay automation. Utilizing this new assay we have been able to detect Abeta 1-40 and Abeta 1-42 at cellular concentration levels directly from cell lysates. Moreover, we have detected multiple peptide responses within the same analysis, some of which have been tentatively identified as other ragged C-termini Abeta polypeptides derived from Abeta 1-42, based on their molecular weight, as well as oxidized Abeta polypeptides.

Selective increase in cellular A beta 42 is related to apoptosis but not necrosis.

Amyloid beta protein ending at 42 (A beta 42) plays an important role in the pathology of Alzheimer's disease (AD). Here we show an increase in cellular A beta 42 in damaged neurons, with both ELISA and immunocytochemistry. The cellular A beta 42 increase was caused by 3-day treatments with H2O2, etoposide or melphalan, all of which induce genotoxic apoptosis, but not by treatment with sodium azide, which causes necrosis. Secreted A beta was similarly decreased with all these treatments. The cellular A beta 42 increase appeared even with minimal damage (ELISA) and A beta 42-positive cells were TUNEL negative (double staining), indicating that any early apoptosis mechanism may induce the cellular A beta 42 increase. Thus, neuronal apoptosis and cellular A beta 42 increase may be linked in a way that contributes importantly to AD pathology.

Detection and quantitation of cellularly derived amyloid beta peptides by immunoprecipitation-HPLC-MS.

A quantitative method for detection of amyloid beta peptides using immunoprecipitation-HPLC-mass spectrometry (IP-LC-MS) is described. Comparison of IP-LC-MS with sandwich ELISA revealed comparable results in the analysis of A beta 1-40 and A beta 1-42 derived from fetal guinea pig cell media and cell lysates. The use of IP-LC-MS not only allows a quantitative method for A beta 1-40 and A beta 1-42 peptides present in Alzheimer's disease (AD), but allows detection of other A beta peptide species that may also play a role in the onset of AD in humans.

pPV: a novel IRES-containing vector to facilitate plasmid immunization and antibody response characterization.

BACKGROUND: The ability to derive immunological reagents for basic and applied research in a timely fashion is a basic requirement of many research projects and is becoming increasingly important as the number of novel gene products of potential interest continues to evolve rapidly. DNA immunization provides a means of facilitating the production of antibody reagents by circumventing the need to derive either purified protein or define peptides before initiating an in vivo immunization protocol. OBJECTIVES: The DNA construct pPV, for plasmid vaccination, has been designed to facilitate the generation and characterization of antibody reagents against either random or defined molecular targets. STUDY DESIGN: pPV incorporates mammalian regulatory and structural features that promote expression of a bifunctional messenger RNA (mRNA) from a single promoter within mammalian cells both in vitro and in vivo. The bifunctional mRNA encodes a control epitope (human IL5), and the 'test' epitope expressed as a tagged recombinant polypeptide in either a random 'shot-gun' mode or a predetermined fashion. In addition, to aid subsequent characterization of antibody responses elicited in vivo, a T7 promoter is included to enable in vitro expression of tagged recombinant polypeptides. RESULTS: The utility and functionality of pPV for the in vitro expression of recombinant protein and the in vivo elicitation of antibody responses is illustrated using a defined 'test' epitope, human proIL1 beta. CONCLUSION: It is anticipated pPV will find particular utility in the future rapid generation and characterization of antibody reagents against the plethora of novel genes emerging from ongoing genomics activity in a directed or genome wide fashion.

Initial culture behaviour of rat blastocysts on selected feeder cell lines.

To increase our understanding of rat embryos in culture and to attempt the isolation of blastocyst-derived cell lines, we examined the initial growth behaviour of rat blastocysts from four strains of rat on four different feeder cell layers. The feeders used were a continuous cell line of murine embryonic fibroblasts (STO), primary mouse (MEF) or primary rat (REF) embryonic fibroblasts, and a continuous cell line of rat uterine epithelial cells (RUCs). A medium that gave optimum plating efficiencies for murine ES cells was used in the rat embryo culture. Each culture system allowed hatching and attachment of the blastocysts, that is, the behaviour was similar on each feeder and each strain for the first 2 days in culture. Subsequently, there was a rapid differentiation of the Inner Cell Mass (ICM) cells on fibroblastic feeder cell layers (STO > MEF > REF), and this was generally complete after 3-6 days in primary culture. On RUCs, the ICM was found to increase in size without differentiation up to and including day 4 and in some cases longer. Embryo-derived cells were obtained by disaggregating and passaging ICMs on REF and RUC feeders. Rounded, refractile, and epithelial-like cells were isolated on REF and colonies of ES-like cells on the RUCs. The ES-like cells were positive for expression of alkaline phosphatase and stage-specific embryonic-antigen 1. This is an important first step towards the derivation and culture of pluripotent ES cells from the rat.