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‣ Subunit interactions influence the biochemical and biological properties of Hsp104

Schirmer, Eric C.; Ware, Danielle M.; Queitsch, Christine; Kowal, Anthony S.; Lindquist, Susan L.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
551.23344%
Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vitro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severely impair hexamerization. We report that high protein concentrations overcome the assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold, changing Vmax with little effect on Km. In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits hexamerization of wild-type (WT) Hsp104, lowering Vmax with little effect on Km. ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions. To further analyze the effects of subunit interactions on Hsp104, we assessed the effects of mutant Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that hexamerizes but does not hydrolyze ATP reduces the ATPase activity of WT Hsp104 in vitro. In vivo, this mutant is not toxic but specifically inhibits the thermotolerance function of WT Hsp104. Thus, interactions between subunits influence the ATPase activity of Hsp104...

‣ Expression profiling reveals fundamental biological differences in acute myeloid leukemia with isolated trisomy 8 and normal cytogenetics

Virtaneva, Kimmo; Wright, Fred A.; Tanner, Stephan M.; Yuan, Bo; Lemon, William J.; Caligiuri, Michael A.; Bloomfield, Clara D.; de la Chapelle, Albert; Krahe, Ralf
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 30/01/2001 Português
Relevância na Pesquisa
551.23344%
Acute myeloid leukemia (AML) is a heterogeneous group of diseases. Normal cytogenetics (CN) constitutes the single largest group, while trisomy 8 (+8) as a sole abnormality is the most frequent trisomy. How trisomy contributes to tumorigenesis is unknown. We used oligonucleotide-based DNA microarrays to study global gene expression in AML+8 patients with +8 as the sole chromosomal abnormality and AML-CN patients. CD34+ cells purified from normal bone marrow (BM) were also analyzed as a representative heterogeneous population of stem and progenitor cells. Expression patterns of AML patients were clearly distinct from those of CD34+ cells of normal individuals. We show that AML+8 blasts overexpress genes on chromosome 8, estimated at 32% on average, suggesting gene-dosage effects underlying AML+8. Systematic analysis by cellular function indicated up-regulation of genes involved in cell adhesion in both groups of AML compared with CD34+ blasts from normal individuals. Perhaps most interestingly, apoptosis-regulating genes were significantly down-regulated in AML+8 compared with AML-CN. We conclude that the clinical and cytogenetic heterogeneity of AML is due to fundamental biological differences.

‣ Mechanism of biological synergy between cellular Src and epidermal growth factor receptor

Tice, David A.; Biscardi, Jacqueline S.; Nickles, Amanda L.; Parsons, Sarah J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/02/1999 Português
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551.23344%
Overexpression of both cellular Src (c-Src) and the epidermal growth factor receptor (EGFR) occurs in many of the same human tumors, suggesting that they may functionally interact and contribute to the progression of cancer. Indeed, in murine fibroblasts, overexpression of c-Src has been shown to potentiate the mitogenic and tumorigenic capacity of the overexpressed EGFR. Potentiation correlated with the ability of c-Src to physically associate with the activated EGFR and the appearance of two unique in vivo phosphorylations on the receptor (Tyr-845 and Tyr-1101). Using stable cell lines of C3H10T½ murine fibroblasts that contain kinase-deficient (K−) c-Src and overexpressed wild-type EGFR, we show that the kinase activity of c-Src is required for both the biological synergy with the receptor and the phosphorylations on the receptor, but not for the association of c-Src with the receptor. In transient transfection assays, not only epidermal growth factor but also serum- and lysophosphatidic acid-induced DNA synthesis was ablated in a dominant-negative fashion by a Y845F mutant of the EGFR, indicating that c-Src-induced phosphorylation of Y845 is critical for the mitogenic response to both the EGFR and a G protein-coupled receptor (lysophosphatidic acid receptor). Unexpectedly...

‣ Millimeter-scale positioning of a nerve-growth-factor source and biological activity in the brain

Mahoney, Melissa J.; Saltzman, W. Mark
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 13/04/1999 Português
Relevância na Pesquisa
551.23344%
Toxicity prevents the systemic administration of many therapeutic proteins, and attempts at protein targeting via the circulatory system (i.e., “magic bullets”) have failed in all but a few special cases. Direct administration at the target site is a logical alternative, particularly in the central nervous system, but the limits of direct administration have not been defined clearly. Nerve growth factor (NGF) enhances survival of cholinergic neurons and, therefore, has generated considerable interest for the treatment of Alzheimer’s disease. We tested the effectiveness of local delivery by implanting small polymer pellets that slowly released NGF into the central nervous system of adult rats at controlled distances from a target site containing transplanted fetal cholinergic cells. NGF-releasing implants placed within 1–2 mm of the treatment site enhanced the biological function of cellular targets, whereas identical implants placed ≈3 mm from the target site of treatment produced no beneficial effect. Effective NGF therapy required millimeter-scale positioning of the NGF source, and efficacy correlated with the spatial distribution of NGF concentration in the tissue; this result suggests that NGF must be delivered within several millimeters of the target to be effective in treating Alzheimer’s disease. Because the human brain is divided into functional regions that are typically several centimeters in diameter and often irregular in shape...

‣ Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer

Chen, Liaohai; McBranch, Duncan W.; Wang, Hsing-Lin; Helgeson, Roger; Wudl, Fred; Whitten, David G.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 26/10/1999 Português
Relevância na Pesquisa
551.23344%
The fluorescence of a polyanionic conjugated polymer can be quenched by extremely low concentrations of cationic electron acceptors in aqueous solutions. We report a greater than million-fold amplification of the sensitivity to fluorescence quenching compared with corresponding “molecular excited states.” Using a combination of steady-state and ultrafast spectroscopy, we have established that the dramatic quenching results from weak complex formation [polymer(−)/quencher(+)], followed by ultrafast electron transfer from excitations on the entire polymer chain to the quencher, with a time constant of 650 fs. Because of the weak complex formation, the quenching can be selectively reversed by using a quencher-recognition diad. We have constructed such a diad and demonstrate that the fluorescence is fully recovered on binding between the recognition site and a specific analyte protein. In both solutions and thin films, this reversible fluorescence quenching provides the basis for a new class of highly sensitive biological and chemical sensors.