Interaction of a novel GDP exchange inhibitor with the Ras protein

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.     

Monitoring protein refolding induced by disulfide formation using capillary isoelectric focusing-electrospray ionization mass spectrometry

Rapid growth in the biotechnology industry has led to a dramatic increase in attention to the protein folding problem. Understanding protein-folding pathways is essential to the production of biopharmaceuticals since commercial production of recombinant proteins often requires a protein-refolding process for recovery of high yields. Protein folding coupled to the formation of disulfide bonds presents one of the simplest approaches to studying folding intermediates. On-line capillary isoelectric focusing-electrospray ionization mass spectrometry (CIEF-ESIMS) is demonstrated for kinetic studies of disulfide bond-induced protein refolding. Refolding intermediates of bovine pancreatic ribonuclease A, a model system for this study, are blocked at different stages by alkylating free thiols with iodoacetate. The alkylation reaction results in the introduction of charge (-1) and mass (59) differences for each alkylation site, providing the means for predictable separation and direct identification of refolding intermediates using CIEF-ESIMS. Besides the observation of refolding intermediates containing different numbers of disulfide bonds and even mixed disulfides, the two-dimensional resolving power of CIEF-ESIMS allows the determination of conformational heterogeneity among groups of refolding intermediates.     

Intracellular retention of recombinant GABAB receptors

gamma-Aminobutyric acid type B (GABAB) receptors mediate the transmission of slow and prolonged inhibitory signals in the central nervous system. Two splice variants of GABAB receptors, GABABR1a and GABABR1b, were recently cloned from a mouse cortical and cerebellar cDNA library. As predicted, these receptors belong to the G protein-coupled receptor superfamily. We have used epitope-tagged versions of GABABR1a receptors to study the cellular distribution of these proteins in a variety of non-neuronal and neuronal cell types. Here we report that recombinant GABAB receptors fail to reach the cell surface when expressed in heterologous systems and are retained in the endoplasmic reticulum when introduced into COS cells. In addition, we prove that recombinant GABAB receptors are excluded from the cell surface when overexpressed in ganglion neurons and we further demonstrate that they fail to activate in superior cervical ganglion neurons. Together our observations suggest that recombinant GABAB receptors require additional information for functional targeting to the plasma membrane.     

Sentinel Chicken Surveillance Program in Australia, July 2002 to June 2003

Detection of flavivirus seroconversions in sentinel chicken flocks located throughout Australia is used to provide an early warning of increased levels of Murray Valley encephalitis (MVE) and Kunjin (KUN) virus activity in the region. During the 2002-2003 season low levels of flavivirus activity were detected in northern Australia compared to previous years. MVE and KUN virus activity was detected in the Kimberley and Pilbara regions of Western Australia and the Northern Territory but not in north Queensland, New South Wales or Victoria. This is similar to the previous season. There were no reported cases of disease caused by either virus.     

Role of mu heavy chain in B cell development. I. Blocked B cell maturation but complete allelic exclusion in the absence of Ig alpha/beta

There is good evidence for a signaling role played by Ig heavy chain in the developmental transition through the pre-B cell stage. We have previously described signal-capable or signal-incapable mutants of mu heavy chain in which a signaling defect is caused by failure to associate with the Ig alpha/beta heterodimer. To further characterize the role of Ig heavy chain-mediated signaling in vivo, as well as in B cell development and allelic exclusion, we have created transgenic mice in which the B cells express these signal-capable and signal-incapable mutant mu chains. Failure of mu to signal via Ig alpha/beta results in a block in B cell development in mice expressing the signal-incapable mu. A small number of B cells in these animals do escape the developmental block and are expressed in the spleen and the periphery as B220+ transgenic IgM+ cells. These cells respond to LPS by proliferating but show no response to T-independent-specific Ag. In contrast, B cells expressing the signal-capable B cell receptor show a strong signaling response to Ag-specific stimulus. There is no Ig alpha seen in association with signal-deficient IgM. Thus, the B cell receptor complex is not assembled, and no signal can be delivered. Despite the block in developmental signaling, allelic exclusion is complete. There is no detectable coexpression of transgenic IgM and endogenous murine IgM, nor is there rearrangement of the endogenous heavy chain genes. This suggests that differing signaling mechanisms are responsible for the developmental transition and allelic exclusion and thus allows for separate examination of these signaling mechanisms.     

Chlorinated hydrocarbon pesticides in blood of newborn babies in India

Umbilical cord blood collected during labor of 100 Indian women was analyzed for organochlorine pesticides by gas-liquid chromatography with electron-capture detection. Significant levels of p,p'-DDT and its metabolites, p,p'-TDE and p,p'-DDE, as well as alpha-, beta-, and gamma-isomers of BHC were estimated. Residues in the neonatal blood were related to age, dietetic habits, and area of residence of the mothers. The study highlights the extent of placental transfer of the body burden of toxic chemicals from the mother to the fetus.     

A levels-of-processing analysis of Bloom's taxonomy.

Tested the cumulative hierarchial assumption of B. S. Bloom's (1956) taxonomy by orienting 80 American and 52 Australian undergraduates at 4 taxonomic levels (Knowledge, Application, Synthesis, Evaluation) to the same study material and subsequently administering an unexpected memory test. With the exception of the Evaluation category, recall generally increased, as predicted, as taxonomic level increased. Bloom's taxonomy appears to possess some cross-national validity, at least for the 2 nationalities sampled, since the overall recall performance of the groups was quite similar. In general, moderate support was obtained for the cumulative hierarchical assumption, but it is concluded that the Evaluation category is misplaced as the apex level of the taxonomy. (27 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)