In this study, Bax inhibitor-1 (BI-1release from ER microsomes from BI-1-overexpressing cells and BI-1-reconsituted liposomes. Acidic conditions also induced BI-1 protein oligomerization. Interestingly subjecting BI-1-overexpressing cells to acidic conditions induced more Bax recruitment to mitochondria, more cytochrome c release from mitochondria, and more cell death. These findings suggest that BI-1 increases Ca 2؉ leak rates from the ER through a mechanism that is dependent on pH and on the carboxyl-terminal cytosolic region of the BI-1 protein. The findings also reveal a cell death-promoting phenotype for BI-1 that is manifested under low pH conditions. The endoplasmic reticulum (ER)3 contains the largest calcium reserve in the cell (1, 2). Agonist-induced ER calcium release occurs through Ca 2ϩ channels such as inositol trisphosphate (IP 3 ) and ryanodine receptors (3). Calcium uptake into the ER occurs when the calcium release channels are closed (i.e. negative feedback to the IP 3 receptor) (4) and is performed by sarcoplasmic reticulum/ER-associated calcium-activated ATPase pumps (5). In the resting state, the Ca 2ϩ content of the ER reflects a balance between active uptake by sarcoplasmic reticulum/ER-associated calcium-activated ATPase and passive efflux or basal leakage through other Ca 2ϩ channels. This leakage is revealed when sarcoplasmic reticulum/ER-associated calcium-activated ATPase pumps are inhibited by agents such as thapsigargin (6), causing Ca 2ϩ to leak out of the ER into the cytosol.The Bax inhibitor-1 (BI-1) (also known as "testis enhanced gene transcript" (TEGT)) is an antiapoptotic protein capable of inhibiting Bax activation and translocation to mitochondria (7). This ubiquitously expressed protein contains several transmembrane domains and localizes to the ER. The homology of BI-1 sequences among species is striking, and the characteristic hydrophobicity and ER membrane localization are evolutionarily conserved (8). BI-1 affects calcium leakage from the ER as measured with Ca 2ϩ -sensitive, ER-targeted fluorescent proteins and Ca 2ϩ -sensitive dyes (9). However, the mechanism by which BI-1 regulates ER Ca 2ϩ fluxes remains unclear. Here we have provided additional evidence that BI-1 induces passive Ca 2ϩ leakage from the ER and also show that BI-1 activity is regulated by pH in a manner dependent on the carboxyl-terminal cytosolic domain of this protein.* This work was supported, in whole or in part, by National Institutes of Health Grant AG15393 (to J. C. R.). This work was also supported by Korea Research Foundation Grants KRF-2005-070-C00095, E00021, and 2005-015-E00210 and Korea Science and Engineering Foundation Grants R01-2006-000-10422-0 and R01-2007-000-20275-0. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.

BackgroundThe purpose of this study was to compare the outcome of carbapenem versus non-carbapenem antimicrobial therapy for pediatric urinary tract infections (UTIs) caused by extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae.Materials and MethodsFrom 2006 to 2011, 42 episodes of UTI caused by ESBL-producing Enterobacteriaceae were diagnosed at Seoul National University Children's Hospital. Patients were grouped according to the antimicrobials they received into a carbapenem group and a non-carbapenem group. Medical records were retrospectively reviewed to assess treatment outcome, time to defervescence after initiation of treatment, and relapse rate.ResultsThere were 36 children with 42 episodes of UTI caused by ESBL-producing Enterobacteriaceae. Twenty-seven cases (64%) had an underlying urologic disease, 28 (67%) cases were caused by Escherichia coli, and 14 (33%) cases were caused by Klebsiella pneumoniae. Four (10%) cases were treated with carbapenem, 23 cases (55%) were treated with non-carbapenem, and 15 (36%) cases were treated by switching from a carbapenem to a non-carbapenem and vice versa. There was no treatment failure at the time of antimicrobial discontinuation. Between the carbapenem and the non-carbapenem treatment groups, there were no significant differences in bacterial etiology (P = 0.59), time to defervescence after the initiation of antimicrobials (P = 0.28), and relapse rate (P = 0.50). In vitro susceptibility to non-carbapenem antimicrobials did not affect the time to defervescence after the initiation of antimicrobial treatment, and the relapse rate in the non-carbapenem group.ConclusionsThis study found no significant difference in the treatment outcome between pediatric patients treated with carbapenem and those treated with non-carbapenem antimicrobials for UTI caused by ESBL-producing Enterobacteriaceae. Therefore, the initially administered non-carbapenem can be maintained in UTI patients showing clinical improvement.

Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR), consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

p38, which has been suggested to be a sca¡old protein for the assembly of a macromolecular tRNA synthetase complex, contains a leucine zipper-like motif. To understand the importance of the leucine zipper-like motif of p38 (p38LZ) in macromolecular assembly, the p38LZ solution structure was investigated by circular dichroism and nuclear magnetic resonance spectroscopy. The solution structure of p38LZ showed an amphipathic K K-helical structure and characteristics similar to a coiled-coil motif. The protein^protein interaction mediated by p38LZ was examined by an in vitro binding assay. The p43 protein, another non-synthetase component of the complex, could bind to p38LZ via its N-terminal domain, which is also predicted to have a potential coiled-coil motif. Thus, we propose that the p38^p43 complex would be formed by coiledcoil interactions, and the formation of the binary complex would facilitate the macromolecular assembly of aminoacyl-tRNA synthetases. ß