In an investigation of heat shock proteins (HSPs) in the brains of Alzheimer's disease (AD) patients and cognitively intact control subjects, we found that 2 HSPs, termed "HSP72" and "GRP78," ...underwent major changes in expression in AD. HSP72, which was present at very low levels in control brains, increased dramatically in AD patients, and was localized exclusively in neuritic plaques and neurofibrillary tangles. We hypothesize that HSP72 is induced as an early response to the formation of abnormal proteins, perhaps targeting them for proteolysis. In contrast, GRP78 increased in AD only in neurons that remained cytologically normal, especially in the CA3 subfield of the hippocampus and the deep layers of the entorhinal cortex. The increased expression of GRP78 within successfully surviving neurons suggests that this protein may protect such cells from AD-specific damage.
DAMIC (Dark Matter in CCDs) is an experiment searching for dark matter particles employing fully-depleted charge-coupled devices. Using the bulk silicon which composes the detector as target, we ...expect to observe coherent WIMP-nucleus elastic scattering. Although located in the SNOLAB laboratory, 2 km below the surface, the CCDs are not completely free of radioactive contamination, in particular coming from radon daughters or from the detector itself. We present novel techniques for the measurement of the radioactive contamination in the bulk silicon and on the surface of DAMIC CCDs. Limits on the Uranium and Thorium contamination as well as on the cosmogenic isotope 32 Si, intrinsically present on the detector, were performed. We have obtained upper limits on the 238 TJ (232 Th) decay rate of 5 (15) kg_1 d_1 at 95% CL. Pairs of spatially correlated electron tracks expected from 32 Si-32 P and 210 Pb-210 Bi beta decays were also measured. We have found a decay rate of 80+l10 -65 kg_1 d_1 for 32 Si and an upper limit of - 35 kg-1 d-1 for 210 Pb, both at 95% CL.
A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B ...lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (μ+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.
We have characterized the association between the binding protein, BiP (also known as GRP 78), and misfolded forms of the influenza virus hemagglutinin precursor, HA0. BiP is a heat-shock-related ...protein that binds to unassembled immunoglobulin heavy chain and to a variety of misfolded proteins in the lumen of the ER. A small fraction (5-10%) of newly synthesized HA0 in CV-1 cells was found to be misfolded and retained in the ER. When glycosylation was blocked with tunicamycin, all of the HA0 produced was similarly misfolded. The misfolded HA0 was retained as relatively small (9-25-S) complexes associated with BiP. In these complexes the top domains of HA0 were correctly folded judging by their reactivity with minoclonal antibodies, but the polypeptides were cross-linked via anomalous interchain disulfides. The association with BiP was noncovalent and easily broken by warming to 37°C or by adding ATP to the lysate. Pulse-chase experiments showed that HA0's self-association into complexes occurred immediately after synthesis and was followed rapidly by BiP association. The misfolded, BiP-associated HA0 was not transported to the plasma membrane but persisted as complexes in the ER for a long period of time before degradation (t1/2 = 6 h). The results suggested that BiP may be part of a quality control system in the ER and that one of its functions is to detect and retain misfolded proteins.
The relationship of N-linked glycosylation and association with heavy chain binding protein (BiP) to the secretion of Factor VIII (FVIII), von Willebrand Factor (vWF), and tissue plasminogen ...activator (tPA) was studied in Chinese hamster ovary (CHO) cells. FVIII has a heavily glycosylated region containing 20 clustered potential N-linked glycosylation sites. A significant proportion of FVIII was detected in a stable complex with BiP and not secreted. Deletion of the heavily glycosylated region resulted in reduced association with BiP and more efficient secretion. Tunicamycin treatment of cells producing this deleted form of FVIII resulted in stable association of unglycosylated FVIII with BiP and inhibition of efficient secretion. vWF contains 17 potential N-linked glycosylation sites scattered throughout the molecule. vWF was transiently associated with BiP and efficiently secreted demonstrating that CHO cells are competent to secrete a highly glycosylated protein. tPA, which has three utilized N-linked glycosylation sites, exhibited low level association with BiP and was efficiently secreted. Disruption of N-linked glycosylation of tPA by either site-directed mutagenesis or tunicamycin treatment resulted in reduced levels of secretion and increased association with BiP. This effect was enhanced by high levels of tPA expression. The glycosylation state and extent of association with BiP could be correlated with secretion efficiency.
Glucose is the major source of brain energy and is essential for maintaining normal brain and neuronal function. Hypoglycemia causes impaired synaptic transmission. This occurs even before ...significant reduction in global cellular ATP concentration, and relationships among glycolysis, ATP supply, and synaptic transmission are not well understood. We demonstrate that the glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (3-PGK) are enriched in synaptic vesicles, forming a functional complex, and that synaptic vesicles are capable of accumulating the excitatory neurotransmitter glutamate by harnessing ATP produced by vesicle-bound GAPDH/3-PGK at the expense of their substrates. The GAPDH inhibitor iodoacetate suppressed GAPDH/3-PGK-dependent, but not exogenous ATP-dependent, (3)Hglutamate uptake into isolated synaptic vesicles. It also decreased vesicular (3)Hglutamate content in the nerve ending preparation synaptosome; this decrease was reflected in reduction of depolarization-induced (3)Hglutamate release. In contrast, oligomycin, a mitochondrial ATP synthase inhibitor, had minimal effect on any of these parameters. ADP at concentrations above 0.1 mm inhibited vesicular glutamate and dissipated membrane potential. This suggests that the coupled GAPDH/3-PGK system, which converts ADP to ATP, ensures maximal glutamate accumulation into presynaptic vesicles. Together, these observations provide insight into the essential nature of glycolysis in sustaining normal synaptic transmission.
To investigate the function of heavy chain binding protein (BiP, GRP 78) in the endoplasmic reticulum, we have characterized
its interaction with a model plasma membrane glycoprotein, the G protein ...of vesicular stomatitis virus. We used a panel of
well characterized mutant G proteins and immunoprecipitation with anti-BiP antibodies to determine if BiP interacted with
newly synthesized G protein and/or mutant G proteins retained in the endoplasmic reticulum. We made three major observations:
1) BiP bound transiently to folding intermediates of wild-type G protein which were incompletely disulfide-bonded; 2) BiP
did not bind stably to all mutant G proteins which remain in the endoplasmic reticulum; and 3) BiP bound stably only to mutant
G proteins which do not form correct intrachain disulfide bonds.
This paper discusses changes in employee commuting in the ten largest Slovenian employment centers from 2000 to 2009. Based on analysis of the SRDAP database, changes are presented in employee ...commuter routes between source and target municipalities. The results show a significant increase in both the scope of employee commuting and number of routes. Reasons for these changes are explained by the construction of freeway infrastructure, which has made it possible to travel faster from one municipality to another and to commute to employment centers. The scope and direction of commuting also depend on changes in the socioeconomic structure of the urban system, especially suburbanization, the economic crisis affecting local employment centers, and changing job locations within the regions themselves.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with ...light chains. The heavy chain-BiP complex can be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the CH1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line ( CH1) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.
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