Home About us Contact
|
Home About us Contact | ||
|
|
||
Mutant Form (mutant + form)
Selected AbstractsT lymphocyte rolling and recruitment into peripheral lymph nodes is regulated by a saturable density of L-selectin (CD62L)EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2007Elena Galkina Dr. Abstract L-selectin mediates tethering and rolling of lymphocytes in high endothelial venules (HEV) of lymph nodes (LN) and of leukocytes at inflammatory sites. We used transgenic mice expressing varying levels of wild-type or a non-cleavable mutant form of L-selectin on T cells to determine the relationship between L-selectin density, tethering and rolling, and migration into LN. T cells expressing supraphysiological levels of either wild-type or non-cleavable L-selectin showed rolling parameters similar to C57BL/6 T cells in hydrodynamic flow assays and during rolling in Peyer's patch HEV. In contrast, PMA- or antigen-activated T cells and L-selectin+/, T cells expressing subphysiological levels of L-selectin showed reduced numbers of rolling cells with increased rolling velocity. Short-term homing studies showed that elevated expression of L-selectin above physiological levels had no effect on T cell migration to LN; however, low L-selectin expression resulted in reduced T cell homing to LN. Thus, T lymphocyte migration into LN is regulated by the density of cell surface L-selectin. In addition, there is a saturable density of L-selectin required for optimal homing to PLN in C57BL/6 mice, the L-selectin level on circulating naive T cells promotes optimal homing, and increased expression above saturating levels promotes no further increase in T cell recruitment. [source] Changes in adult olfactory bulb neurogenesis in mice expressing the A30P mutant form of alpha-synucleinEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2009Franz Marxreiter Abstract In familial and sporadic forms of Parkinson's disease (PD), alpha-synuclein pathology is present in the brain stem nuclei and olfactory bulb (OB) long before Lewy bodies are detected in the substantia nigra. The OB is an active region of adult neurogenesis, where newly generated neurons physiologically integrate. While accumulation of wild-type alpha-synuclein is one of the pathogenic hallmarks of non-genetic forms of PD, the A30P alpha-synuclein mutation results in an earlier disease onset and a severe clinical phenotype. Here, we study the regulation of adult neurogenesis in the subventricular zone (SVZ)/OB system in a tetracycline-suppressive (tet-off) transgenic model of synucleinopathies, expressing human mutant A30P alpha-synuclein under the control of the calcium/calmodulin-dependent protein kinase II alpha (CaMK) promoter. In A30P transgenic mice alpha-synuclein was abundant at the site of integration in the glomerular cell layer of the OB. Without changes in proliferation in the SVZ, significantly fewer newly generated neurons were observed in the OB granule cell and glomerular layers of A30P transgenic mice than in controls, most probably due to increased cell death. By tetracycline-dependent abrogation of A30P alpha-synuclein expression, OB neurogenesis and programmed cell death was restored to control levels. Our results indicate that, using A30P conditional (tet-off) mice, A30P alpha-synuclein has a negative impact on olfactory neurogenesis and suppression of A30P alpha-synuclein enhances survival of newly generated neurons. This finding suggests that interfering with alpha-synuclein pathology can rescue newly generated neurons, possibly leading to new targets for therapeutic interventions in synucleinopathies. [source] Hypothalamic,endocrine aspects in Huntington's diseaseEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2006Åsa Petersén Abstract Huntington's disease (HD) is a hereditary and fatal disorder caused by an expanded CAG triplet repeat in the HD gene, resulting in a mutant form of the protein huntingtin. Wild-type and mutant huntingtin are expressed in most tissues of the body but the normal function of huntingtin is not fully known. In HD, the neuropathology is characterized by intranuclear and cytoplasmic inclusions of huntingtin aggregates, and cell death primarily in striatum and cerebral cortex. However, hypothalamic atrophy occurs at early stages of HD with loss of orexin- and somatostatin-containing cell populations. Several symptoms of HD such as sleep disturbances, alterations in circadian rhythm, and weight loss may be due to hypothalamic dysfunction. Endocrine changes including increased cortisol levels, reduced testosterone levels and increased prevalence of diabetes are found in HD patients. In HD mice, alterations in the hypothalamic,pituitary,adrenal axis occurs as well as pancreatic ,-cell and adipocyte dysfunction. Increasing evidence points towards important pathology of the hypothalamus and the endocrine system in HD. As many neuroendocrine factors are secreted into the cerebrospinal fluid, blood and urine, it is possible that their levels may reflect the disease state in the central nervous system. Investigating neuroendocrine changes in HD opens up the possibility of finding biomarkers to evaluate future therapies for HD, as well as of identifying novel targets for therapeutic interventions. [source] Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylaseFEBS JOURNAL, Issue 5 2003Structural, functional implications Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302,6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19,33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15,K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein- l -isoaspartic acid O -methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 °C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR,,34 min to ,,31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR,,34 min species decreased with a biphasic time-course with t0.5 -values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of ,,1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 °C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial ,-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32,Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition. [source] Cytoplasmic splicing of tRNA in Saccharomyces cerevisiaeGENES TO CELLS, Issue 3 2007Tohru Yoshihisa The splicing of nuclear encoded RNAs, including tRNAs, has been widely believed to occur in the nucleus. However, we recently found that one of the tRNA splicing enzymes, splicing endonuclease, is localized to the outer surface of mitochondria in Saccharomyces cerevisiae. These results suggested the unexpected possibility of tRNA splicing in the cytoplasm. To investigate this possibility, we examined whether cytoplasmic pre-tRNAs are bona fide intermediates for tRNA maturation in vivo. We isolated a new reversible allele of temperature-sensitive (ts) sen2 (HA-sen2-42), which encodes a mutant form of one of the catalytic subunits of yeast splicing endonuclease. The HA-sen2-42 cells accumulated large amounts of pre-tRNAs in the cytoplasm at a restrictive temperature, but the pre-tRNAs were diminished when the cells were transferred to a permissive temperature. Using pulse-chase/hybrid-precipitation techniques, we showed that the pre-tRNAs were not degraded but rather converted into mature tRNAs during incubation at the permissive temperature. These and other results indicate that, in S. cerevisiae, pre-tRNAs in the cytoplasm are genuine substrates for splicing, and that the splicing is indeed carried out in the cytoplasm. [source] Point mutations of 3BP2 identified in human-inherited disease cherubism result in the loss of functionGENES TO CELLS, Issue 11 2004S. M. Shahjahan Miah Adaptor protein 3BP2 positively regulates the high affinity IgE receptor (Fc,RI)-mediated activation of degranulation in mast cells. Genetic study identified the point mutations of 3BP2 gene in human-inherited disease cherubism. The multiple cysts in cherubism lesion of jaw bones are filled with the activated osteoclasts and stromal cells, including mast cells. By over-expression study using rat basophilic leukaemia RBL-2H3 mast cells, we have analysed the effect of the point mutations on the function of 3BP2 protein, which plays a positive regulatory role on Fc,RI-mediated mast cell activation. Over-expression of 3BP2 mutants suppressed the antigen-induced degranulation and cytokine gene transcription. Antigen-induced phosphorylation of Vav1, activation of Rac1, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK), inhibitor of nuclear factor ,B kinase (IKK) and nuclear factor of activated T cells (NFAT) were all impaired in the cells over-expressing the cherubism mutants of 3BP2. Furthermore, cherubism mutations of 3BP2 may abrogate the binding ability to interact with chaperone protein 14-3-3. These results demonstrate that over-expression of the mutant form of 3BP2 inhibits the antigen-induced mast cell activation. It suggests that point mutations of 3BP2 gene cause the dysfunction of 3BP2 in vivo. [source] Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5GENES TO CELLS, Issue 10 2001Kyoko Shirakabe Background Runx2, formerly called PEBP2,A or Cbfa1, is a transcription factor whose deletion causes a complete lack of ossification. It directly regulates the expression of osteoblast-specific genes through the osteoblast-specific cis -acting element found in the promoter region of these genes. Results In this study, we have found conditions in which induction of the expression of Runx2 is not accompanied by expression of an osteoblast-specific gene, osteocalcin in C2C12 cells. This finding suggests the existence of a repressor of the activity of Runx2. We have then found that the homeobox protein Msx2 is able to repress the transcription activity of Runx2 by interacting with it. Furthermore, our results have shown that the other homeobox protein Dlx5 has an activity which interferes with both abilities of Msx2 to interact with Runx2 and repress its transcription activity. It has previously been shown that a missense mutation of Msx2 (P148H) causes Boston-type craniosynostosis in humans. Interestingly, while this mutant form of Msx2 was able to bind to Runx2 and repress its activity, these abilities of Msx2 (P148H) were not subject to regulation by Dlx5. Conclusion These results suggest that regulation of the activity of Runx2 by Msx2 and Dlx5 plays an important role in the mammalian skull development. [source] The melanoma-associated 24 base pair duplication in p16INK4a is functionally impairedINTERNATIONAL JOURNAL OF CANCER, Issue 4 2005Therese M. Becker Abstract Melanoma-associated germline mutations affecting the tumor suppressor and cyclin-dependent kinase (CDK) inhibitor, CDKN2A/p16INK4a, have been identified in over 100 melanoma-prone families worldwide. To predict the melanoma risk for carriers of specific mutations, mutant p16INK4a can be tested in biochemical and cellular assays. In most cases, p16INK4a mutations with predicted disease relation, due to segregation with melanoma, are functionally impaired in such assays. The N-terminal 24 base pair duplication of CDKN2A, however, encodes a p16INK4a variant previously shown to have wild-type function, despite segregating with melanoma in at least 5 melanoma families. To clarify whether the duplication mutation has a cell cycle regulatory defect or behaves like wild-type p16INK4a, we reanalyzed the cell cycle-inhibitory activity of this mutation. Stable cell clones of the p16-null WMM1175 melanoma cell line inducible for ectopic p16INK4a were used in this study. In these cells, p16INK4a expression can be controlled at physiologic levels. Our results show that in comparison to wild-type p16INK4a, the duplication mutant induced weaker S-phase inhibition and cells expressing this mutant form of p16INK4a retained colony formation ability. We also show that the cell cycle-regulatory defect of the p16INK4a duplication mutant was associated with decreased inhibition of pRb phosphorylation even though it retained significant binding to CDK4. © 2005 Wiley-Liss, Inc. [source] Rap1 and p38 MAPK mediate 8-chloro-cAMP-induced growth inhibition in mouse fibroblast DT cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2006Young-Ho Ahn 8-Cl-cAMP, which is known to induce differentiation, growth inhibition, and apoptosis in various cancer cells, has been investigated as a putative anti-cancer drug. Previously, we reported that 8-Cl-cAMP and its metabolite 8-Cl-adenosine induce growth inhibition and apoptosis through p38 mitogen-activated protein kinase (MAPK) activation. To further investigate the signal mechanisms that regulate the cellular effects of 8-Cl-cAMP, we focused on a small GTPase Rap1 that is known to be involved in growth inhibition and reverse-transformation. 8-Cl-cAMP and 8-Cl-adenosine could increase Rap1 activity, which was blocked by ABT702,an adenosine kinase inhibitor. This suggests that 8-Cl-cAMP-induced Rap1 activation is also dependent on the metabolic degradation of 8-Cl-cAMP. Overexpression of a constitutively active mutant form of Rap1 (Rap1V12) attenuated cellular growth and soft-agar colony formation, which was basically the same effect as that observed with the 8-Cl-cAMP treatment. Furthermore, the Rap1V12 transfectant showed a high level of p38 MAPK activation. However, 8-Cl-cAMP-induced Rap1 activation was not diminished by SB203580, a p38 MAPK inhibitor, suggesting that Rap1 activation might act upstream of p38 MAPK activation during 8-Cl-cAMP-induced growth inhibition. J. Cell. Physiol. 209: 1039,1045, 2006. © 2006 Wiley-Liss, Inc. [source] Hepatitis B virus markers in anti-HBc only positive individuals,JOURNAL OF MEDICAL VIROLOGY, Issue 3 2001Bernard Weber Abstract Isolated reactivity to hepatitis B virus (HBV) core antigen (anti-HBc) is observed relatively frequently in immunocompromised individuals, intravenous drug abusers (IVDA), and in the presence of HCV infection. The reason for the lack of HBsAg is not clear. The aim of the present study was to investigate which factors (genetic variability of S gene, low-level HBsAg, and immune complexes may be responsible for the failure of HBsAg detection with commercial HBsAg screening assays. Dilution series of two recombinant HBsAg escape mutants and dilutions of serum samples from chronic HBV carriers with multiple insertions in the a determinant and different HBsAg subtypes were tested with a highly sensitive assay that detects wild-type HBsAg (Elecsys HBsAg, Roche Diagnostics, Penzberg, Germany) and two assays that detect HBV wild-type and escape mutants (Murex HBsAg Version 3, Murex and Enzygnost HBsAg 5.0, Dade Behring, Marburg, Germany). Elecsys HBsAg showed in comparison to Murex HBsAg Version 3 and Enzygnost HBsAg 5.0 a reduced sensitivity for escape mutant detection. On the other hand, the best performance for HBsAg subtype detection was obtained with Elecsys HBsAg. In the second part of the study, a selected panel of isolated anti-HBc reactive (n,=,104) serum samples (AxSYM Core) was submitted to testing by Elecsys HBsAg, Murex HBsAg Version 3, Enzygnost HBsAg 5.0, and HBsAg detection after immune complex dissociation (ICD) and anti-HBs determination with two different assays (AxSYM Ausab and Elecsys Anti-HBs). To assess the specificity of anti-HBc test results, all the samples were tested by a second anti-HBc assay (Elecsys Anti-HBc). Quantitative HBV DNA detection was undertaken with a commercially available HBV PCR assay (Amplicor HBV Monitor). HCV infection was present in 65.4% of anti-HBc only reactive individuals. Five AxSYM Core positive samples were negative by Elecsys Anti-HBc. Overall, 15 (14.4%) AxSYM Ausab negative samples gave positive results with Elecsys Anti-HBs (median value: 21 IU/ml). No low-level HBsAg carrier was detected among the isolated anti-HBc reactive individuals with Elecsys HBsAg. There was no evidence for the presence of immune complexes. Only one sample was repeatedly reactive by the Murex HBsAg, suggesting that the a mutant form of HBsAg was responsible for the isolated anti-HBc reactivity, however neutralisation assay was not interpretable and HBV DNA PCR was negative. Fifteen (14.4%) anti-HBc only positive individuals were HBV DNA carriers with concentrations ranging from 800 to more than >4,000,000 copies of viral DNA/ml. In conclusion, the most probable explanations for isolated anti-HBc reactivity in our study group are a possible interference of HBsAg synthesis by HCV infection (65.4%) and divergence of results of anti-HBs assays (14.4%). There is no evidence for the presence of low-level HBsAg carriers and immune complexes. HBsAg mutants cannot be excluded definitively by the test strategy used in the present evaluation. J. Med. Virol. 64:312,319, 2001. © 2001 Wiley-Liss, Inc. [source] ,-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2009Nishani T. Hettiarachchi Abstract Parkinson's disease (PD) is characterized in part by the presence of ,-synuclein (,-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the ,-synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type ,-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of ,-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the ,-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+]i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of ,-synuclein. However, only WT ,-syn transfected cells displayed significantly impaired viability. Our findings suggest that ,-syn regulates Ca2+ entry pathways and, consequently, that abnormal ,-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis. [source] The catalytic domain of human neuropathy target esterase mediates an organophosphate-sensitive ionic conductance across liposome membranesJOURNAL OF NEUROCHEMISTRY, Issue 2 2001Philip J. Forshaw In humans and other vertebrates, reaction of organophosphates with a neuronal membrane protein, neuropathy target esterase (NTE), initiates events which culminate in axonal degeneration. The initiation process appears to involve modification of a property of the protein distinct from its esterase activity, subsequent to formation of a negatively charged adduct with the active site serine residue. Here, we show that membrane patches from liposomes containing NEST, a recombinant hydrophobic polypeptide comprising the esterase domain of human NTE, display a transmembrane ionic conductance with both stable and high-frequency flickering components. An asymmetric current,voltage relationship suggested that ion flow was favoured in one direction relative to the membrane and its associated NEST molecules. Flow of anions was slightly favoured compared with cations. The flickering current formed a much larger proportion of the overall conductance in patches containing wild-type NEST compared with the catalytically inactive S966A mutant form of the protein. The conductance across patches containing NEST, but not those with the S966A mutant, was significantly reduced after adding neuropathic organophosphates to the bathing medium. By contrast, non-neuropathic covalent inhibitors of the catalytic activity of NEST did not reduce NEST-mediated conductance. Future work may establish whether NTE itself mediates an organophosphate-sensitive ion flux across intracellular membranes within intact cells. [source] Failure to farnesylate Rheb protein contributes to the enrichment of G0/G1 phase cells in the Schizosaccharomyces pombe farnesyltransferase mutantMOLECULAR MICROBIOLOGY, Issue 6 2001Wenli Yang Protein farnesylation is important for a number of physiological processes, including proliferation and cell morphology. The Schizosaccharomyces pombe mutant, cpp1,, defective in farnesylation, exhibits distinct phenotypes, including morphological changes and sensitivity to the arginine analogue, canavanine. In this work, we report a novel phenotype of this mutant, enrichment of G0/G1 phase cells. This phenotype results mainly from the inability to farnesylate the Rheb G-protein, as normal cell cycle progression can be restored to the mutant by expressing a mutant form of SpRheb (SpRheb-CVIL) that can bypass farnesylation. In contrast, a farnesylation-defective mutant of SpRheb (SpRheb-SVIA) is incapable of restoring the normal cell cycle profile to the cpp1, mutant. Inhibition of SpRheb expression leads to the accumulation of cells at the G0/G1 phase of the cell cycle. This growth arrest phenotype of the sprheb, disruption can be complemented by the introduction of wild-type sprheb+. The complementation is dependent on farnesylation, as the farnesylation-defective SpRheb-SVIA mutant is incapable of complementing the sprheb, disruption. Other mutants of SpRheb, E40K and S20N, are also incapable of complementing the sprheb, disruption. Furthermore, efficient complementation can be obtained by the expression of human Rheb but not Saccharomyces cerevisiae Rheb. Our findings suggest that protein farnesylation is important for cell cycle progression of S. pombe cells and that farnesylated SpRheb is critical in this process. [source] Pathological biochemistry of ,-synucleinopathyNEUROPATHOLOGY, Issue 5 2007Takeshi Iwatsubo Lewy bodies (LBs) are hallmark lesions in the brains of patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB). We raised a monoclonal antibody LB509 against purified LBs from the brains of patients with DLB that strongly immuolabled LBs, and found that ,-synuclein is one of the major components of LBs. Thus, the deposition of ,-synuclein, an abundant presynaptic brain protein, as fibrillary aggregates in affected neurons or glial cells, was highlighted as a hallmark lesion of a subset of neurodegenerative disorders, including PD, DLB and multiple system atrophy collectively referred to as synucleinopathies. Importantly, the identification of missense mutations in and multiplication of ,-synuclein gene in some pedigrees of familial PD has strongly implicated ,-synuclein in the pathogenesis of PD and other synucleinopathies. We then examined the specific post-translational modifications that characterize and underlie the aggregation of ,-synuclein in synucleinopathy brains by mass spectrometry and using a specific antibody, and found that serine 129 of ,-synuclein deposited in synucleinopathy lesions is selectively and extensively phosphorylated. Furthermore we generated transgenic C. elegans overexpressing ,-synuclein in neurons, and found that overexpression of familial PD-linked mutant form of ,-synuclein impairs functions of dopamine neurons. These findings collectively underscore the importance of deposition of ,-synuclein as well as its phosphorylation in the pathogenesis of ,-synucleinopathies. [source] A mutant form of PTEN linked to autismPROTEIN SCIENCE, Issue 10 2010Roberta E. Redfern Abstract The tumor suppressor, phosphatase, and tensin homologue deleted on chromosome 10 (PTEN), is a phosphoinositide (PI) phosphatase specific for the 3-position of the inositol ring. PTEN has been implicated in autism for a subset of patients with macrocephaly. Various studies identified patients in this subclass with one normal and one mutated PTEN gene. We characterize the binding, structural properties, activity, and subcellular localization of one of these autism-related mutants, H93R PTEN. Even though this mutation is located at the phosphatase active site, we find that it affects the functions of neighboring domains. H93R PTEN binding to phosphatidylserine-bearing model membranes is 5.6-fold enhanced in comparison to wild-type PTEN. In contrast, we find that binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) model membranes is 2.5-fold decreased for the mutant PTEN in comparison to wild-type PTEN. The structural change previously found for wild-type PTEN upon interaction with PI(4,5)P2, is absent for H93R PTEN. Consistent with the increased binding to phosphatidylserine, we find enhanced plasma membrane association of PTEN-GFP in U87MG cells. However, this enhanced plasma membrane association does not translate into increased PI(3,4,5)P3 turnover, since in vivo studies show a reduced activity of the H93R PTEN-GFP mutant. Because the interaction of PI(4,5)P2 with PTEN's N-terminal domain is diminished by this mutation, we hypothesize that the interaction of PTEN's N-terminal domain with the phosphatase domain is impacted by the H93R mutation, preventing PI(4,5)P2 from inducing the conformational change that activates phosphatase activity. [source] A non-invasive method based on saliva to characterize transthyretin in familial amyloidotic polyneuropathy patients using FT-ICR high-resolution MSPROTEOMICS - CLINICAL APPLICATIONS, Issue 6-7 2010Gonçalo da Costa Abstract Purpose: To identify, characterize and perform a relative quantification of human transthyretin (TTR) variants in human saliva. Experimental design: Serum and saliva samples were collected from healthy and familial amyloidotic polyneuropathy (FAP) patients, proteins separated by SDS-PAGE, TTR bands excised, in-gel digested and analyzed by MALDI-FTICR. Results: We identified and performed a relative quantification of mutated and native TTR forms in human saliva, based on FTICR-MS. The results are quantitatively identical to the ones obtained with human serum. In FAP patients subjected to cadaveric liver transplant, the TTR mutant form is no longer detected in saliva, while in patients receiving a domino liver from a FAP donor the mutant form of TTR becomes detectable in saliva, thus demonstrating the serum origin of TTR in saliva. Conclusions and clinical relevance: Saliva TTR originates in serum and the ratio of mutant to native TTR is preserved. The method provides a non-invasive detection of mutated TTR and a relative quantification of TTR forms. Diagnostic and disease prognosis of FAP is crucial at early stages of the disease and after liver transplantation, the only curative therapy. A suitable non-invasive method was developed for monitoring the most important FAP biomarker in human saliva. [source] Identification of a novel germline MET mutation in dogsANIMAL GENETICS, Issue 3 2006A.T. Liao Summary The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor that mediates multiple functions such as migration, cycling and survival by binding to hepatocyte growth factor (HGF). Dysregulation of MET through inappropriate expression or mutation has been shown to play an important role in human cancers. Furthermore, inherited mutations in MET are known to contribute to the development of gastric and renal cancer in humans. Lastly, mouse models of MET mutations lead to the development of a wide variety of cancers including lymphomas, sarcomas and some forms of carcinoma. In the process of cloning canine MET, a novel germline point mutation was found in the juxtamembrane domain (G966S) in two of the templates used for cloning, both of which were derived from Rottweiler dogs, a breed believed to be at high risk for the development of several cancers. Screening of germline DNA from a variety of breeds revealed that this mutation was present in approximately 70% of Rottweiler dogs and <5% of all other breeds examined, suggesting a breed-specific heritable mutation. Stable transfection of the G966S mutant form of MET into NIH3T3 cells resulted in enhanced baseline scattering and migration of the cells, which was further increased in the presence of HGF. This study supports the notion that particular dog breeds may carry germline mutations that contribute to high rates of cancer in a manner similar to heritable, cancer-associated mutations in humans. [source] Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substratesACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2009Narayanasami Sukumar (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed ,100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2,Å resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30,mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state. [source] A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactionsACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2009Bret D. Freudenthal Eukaryotic proliferating cell nuclear antigen (PCNA) is an essential replication accessory factor that interacts with a variety of proteins involved in DNA replication and repair. Each monomer of PCNA has an N-terminal domain A and a C-terminal domain B. In the structure of the wild-type PCNA protein, domain A of one monomer interacts with domain B of a neighboring monomer to form a ring-shaped trimer. Glu113 is a conserved residue at the subunit interface in domain A. Two distinct X-ray crystal structures have been determined of a mutant form of PCNA with a substitution at this position (E113G) that has previously been studied because of its effect on translesion synthesis. The first structure was the expected ring-shaped trimer. The second structure was an unanticipated nontrimeric form of the protein. In this nontrimeric form, domain A of one PCNA monomer interacts with domain A of a neighboring monomer, while domain B of this monomer interacts with domain B of a different neighboring monomer. The B,B interface is stabilized by an antiparallel ,-sheet and appears to be structurally similar to the A,B interface observed in the trimeric form of PCNA. The A,A interface, in contrast, is primarily stabilized by hydrophobic interactions. Because the E113G substitution is located on this hydrophobic surface, the A,A interface should be less favorable in the case of the wild-type protein. This suggests that the side chain of Glu113 promotes trimer formation by destabilizing these possible alternate subunit interactions. [source] Escherichia coli MltA: MAD phasing and refinement of a tetartohedrally twinned protein crystal structureACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2005Thomas R. M. Barends Crystals were grown of a mutant form of the bacterial cell-wall maintenance protein MltA that diffracted to 2.15,Å resolution. When phasing with molecular replacement using the native structure failed, selenium MAD was used to obtain initial phases. However, after MAD phasing the crystals were found to be tetartohedrally twinned, hampering correct space-group determination and refinement. A refinement protocol was designed to take tetartohedral twinning into account and was successfully applied to refine the structure. The refinement protocol is described and the reasons for the failure of molecular replacement and the success of MAD are discussed in terms of the effects of the tetartohedral twinning. [source] Crystallization and preliminary X-ray diffraction analysis of a protease-resistant mutant form of human galectin-8ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Hiromi Yoshida A crystal of a protease-resistant mutant form of human galectin-8, a tandem-repeat-type galectin with two carbohydrate-recognition domains, was obtained using the hanging-drop method and was found to belong to the tetragonal space group P43212, with unit-cell parameters a = 78.93, b = 78.93, c = 132.05,Å. Diffraction data were collected to a resolution of 3.4,Å. [source] Small cell astrocytoma: An aggressive variant that is clinicopathologically and genetically distinct from anaplastic oligodendrogliomaCANCER, Issue 10 2004Arie Perry M.D. Abstract BACKGROUND Small cell glioblastoma (GBM) is a variant with monomorphous, deceptively bland nuclei that often is misdiagnosed as anaplastic oligodendroglioma. METHODS To elucidate its clinicopathologic and genetic features, the authors studied 71 adult patients (median age, 57 years), including 22 patients who were identified from a set of 229 GBMs (10%) that had been characterized previously by epidermal growth factor receptor (EGFR)/EGFR-vIII variant immunohistochemistry. Tumors also were analyzed by fluorescence in situ hybridization for 1p, 19q, 10q, and EGFR copy numbers. RESULTS Radiologically, 37% of tumors that were not selected for grade showed minimal to no enhancement. Similarly, 33% of tumors had no endothelial hyperplasia or necrosis histologically, qualifying only as anaplastic astrocytoma (Grade III) using World Health Organization criteria. Nevertheless, such tumors progressed rapidly, with mortality rates that were indistinguishable from their Grade IV counterparts. The median survival for 37 patients who were followed until death was 11 months. Oligodendroglioma-like histology included chicken-wire vasculature (86%), haloes (73%), perineuronal satellitosis (58%), and microcalcifications (45%), although mucin-filled microcystic spaces were lacking. No small cell astrocytomas had 1p/19q codeletions, whereas EGFR amplification and 10q deletions were present in 69% and 97% of small cell astrocytomas, respectively. The tumors expressed EGFR and EGFR-vIII more commonly than nonsmall cell GBMs (83% vs. 35% [P < 0.001]; 50% vs. 21% [P < 0.001] respectively). CONCLUSIONS Small cell astrocytoma is an aggressive histologic variant that behaved like primary GBM, even in the absence of endothelial hyperplasia and necrosis. Despite considerable morphologic overlap with anaplastic oligodendroglioma, clinicopathologic and genetic features were distinct. Fifty percent of small cell astrocytomas expressed the constitutively activated vIII mutant form of EGFR, and molecular testing for 10q deletion improved the diagnostic sensitivity over EGFR alone. Cancer 2004. © 2004 American Cancer Society. [source] Gene therapy mediates cone rescue and rejuvenation in the R91W mutant form of Rpe65-deficiency miceACTA OPHTHALMOLOGICA, Issue 2009Y ARSENIJEVIC Purpose Given the advances of gene therapy studies to cure RPE65-derived Leber Congenital Amaurosis (LCA) (clinical trials phase I), it is of prime importance to examine how cones can be rescued in different mutant contexts. Consequently, we evaluated the effect on retinal activity and cone survival of lentivirus-mediated gene therapy in the R91W knock-in mouse model expressing the mutant Rpe65R91W gene. Methods An HIV-1-derived lentiviral vector (LV) expressing either the GFP or the mouse Rpe65 cDNA under the control of a 0.8 kb fragment of the human Rpe65 promoter (R0.8) was produced. LV-R0.8-RPE65 or GFP was injected into 5-days-old (P5) or 1 month-old R91W mice. Functional and morphological retinal rescues were investigated at 4 months of age. Results Increased light sensitivity was detected by ERG and pupillary light responses in animals injected with LV-R0.8-RPE65 at both P5 and 1 month compared to controls. Histological analysis showed improved expression of cone markers and cone outersegment morphology. Furthermore, the density of cones in the region of RPE65 delivery after treatment at P5 reached the wild type level. However, before injection at 1 month of age, only a fraction of the cones (40% of the number found in WT animals) in the Rpe65R91W/R91W mice expressed cone transducin, this fraction increased to 64% after treatment. Moreover, these cones appeared normal. Conclusion We show that lentivirus-mediated Rpe65 gene transfer is very efficacious in early treatments and still efficient during the course of cone degeneration. Moreover, the treatment at 1 month shows a rejuvenation process of the diseased cones. Thus patient suffering from R91W mutation might benefit from a prolonged therapeutic window. [source] Analysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro,DEVELOPMENTAL DYNAMICS, Issue 2 2002Anthony M. Delise Abstract During embryonic limb development, cartilage formation is presaged by a crucial mesenchymal cell condensation phase. N-Cadherin, a Ca2+ -dependent cell,cell adhesion molecule, is expressed in embryonic chick limb buds in a spatiotemporal pattern suggestive of its involvement during cellular condensation; functional blocking of N-cadherin homotypic binding, by using a neutralizing monoclonal antibody, results in perturbed chondrogenesis in vitro and in vivo. In high-density micromass cultures of embryonic limb mesenchymal cells, N-cadherin expression level is high during days 1 and 2, coincident with active cellular condensation, and decreases upon overt chondrogenic differentiation from day 3 on. In this study, we have used a transfection approach to evaluate the effects of gain- and loss-of-function expression of N-cadherin constructs on mesenchymal condensation and chondrogenesis in vitro. Chick limb mesenchymal cells were transfected by electroporation with recombinant expression plasmids encoding wild-type or two mutant extracellular/cytoplasmic deletion forms of N-cadherin. Expression of the transfected N-cadherin forms showed a transient profile, being high on days 1,2 of culture, and decreasing by day 3, fortuitously coincident with the temporal profile of endogenous N-cadherin gene expression. Examined by means of peanut agglutinin (PNA) staining for condensing precartilage mesenchymal cells, cultures overexpressing wild-type N-cadherin showed enhanced cellular condensation on culture days 2 and 3, whereas expression of the deletion mutant forms (extracellular/cytoplasmic) of N-cadherin resulted in a decrease in PNA staining, suggesting that a complete N-cadherin protein is required for normal cellular condensation to occur. Subsequent chondrogenesis was also affected. Cultures overexpressing the wild-type N-cadherin protein showed enhanced chondrogenesis, indicated by increased production of cartilage matrix (sulfated proteoglycans, collagen type II, and cartilage proteoglycan link protein), as well as increased cartilage nodule number and size of individual nodules, compared with control cultures and cultures transfected with either of the two mutant N-cadherin constructs. These results demonstrate that complete N-cadherin function, at the levels of both extracellular homotypic binding and cytoplasmic linkage to the cytoskeleton by means of the catenin complex, is required for chondrogenesis by mediating functional mesenchymal cell condensation. © 2002 Wiley-Liss, Inc. [source] Neurotoxicity of channel mutations in heterologously expressed ,7-nicotinic acetylcholine receptorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2001Ronald J. Lukas Abstract Nicotinic acetylcholine receptors (nAChR) composed of chick ,7 subunits mutated to threonine at amino acid valine-251 in the putative channel-lining M2 domain were expressed heterologously in several neuron-like and non-neuronal mammalian cell lines. Expression of mutant ,7-nAChR is toxic to neuron-like cells of the human neuroblastoma cell lines SH-SY5Y and IMR-32, but not to several other cell types. Growth in the presence of the ,7-nAChR antagonist methyllycaconitine (MLA) protects against neurotoxicity, as does gradual downregulation of functional, mutant ,7-nAChR in surviving transfected SH-SY5Y cells. Relative to wild-type ,7-nAChR, functional ,7-nAChR mutants show a higher affinity for agonists, slower rates of desensitization, and sensitivity to dihydro-,-erythroidine (DH,E) as an agonist, but they retain sensitivity to MLA as a competitive antagonist. These findings demonstrate that expression of hyperfunctional, mutant forms of Ca2+ -permeable ,7-nAChR is toxic to neuron-like cells. [source] Mutagenic probes of the role of Ser209 on the cavity shaping loop of human monoamine oxidase AFEBS JOURNAL, Issue 16 2009Jin Wang The available literature implicating human monoamine oxidase A (MAO A) in apoptotic processes reports levels of MAO A protein that do not correlate with activity, suggesting that unknown mechanisms may be involved in the regulation of catalytic function. Bioinformatic analysis suggests Ser209 as a possible phosphorylation site that may be relevant to catalytic function because it is adjacent to a six-residue loop termed the ,cavity shaping loop' from structural data. To probe the functional role of this site, MAO A Ser209Ala and Ser209Glu mutants were created and investigated. In its membrane-bound form, the MAO A Ser209Glu phosphorylation mimic exhibits catalytic and inhibitor binding properties similar to those of wild-type MAO A. Solubilization in detergent solution and purification of the Ser209Glu mutant results in considerable decreases in these functional parameters. By contrast, the MAO A Ser209Ala mutant exhibits similar catalytic properties to those of wild-type enzyme when purified. Compared to purified wild-type and Ser209Ala MAO A proteins, the Ser209Glu MAO A mutant shows significant differences in covalent flavin fluorescence yield, CD spectra and thermal stability. These structural differences in the purified MAO A Ser209Glu mutant are not exhibited in quantitative structure,activity relationship patterns using a series of para -substituted benzylamine analogs similar to the wild-type enzyme. These data suggest that Ser209 in MAO A does not appear to be the putative phosphorylation site for regulation of MAO A activity and demonstrate that the membrane environment plays a significant role in stabilizing the structure of MAO A and its mutant forms. [source] Role of the surface charges D72 and K8 in the function and structural stability of the cytochrome c6 from Nostoc sp.FEBS JOURNAL, Issue 13 2005PCC 711 We investigated the role of electrostatic charges at positions D72 and K8 in the function and structural stability of cytochrome c6 from Nostoc sp. PCC 7119 (cyt c6). A series of mutant forms was generated to span the possible combinations of charge neutralization (by mutation to alanine) and charge inversion (by mutation to lysine and aspartate, respectively) in these positions. All forms of cyt c6 were functionally characterized by laser flash absorption spectroscopy, and their stability was probed by urea-induced folding equilibrium relaxation experiments and differential scanning calorimetry. Neutralization or inversion of the positive charge at position K8 reduced the efficiency of electron transfer to photosystem I. This effect could not be reversed by compensating for the change in global charge that had been introduced by the mutation, indicating a specific role for K8 in the formation of the electron transfer complex between cyt c6 and photosystem I. Replacement of D72 by asparagine or lysine increased the efficiency of electron transfer to photosystem I, but destabilized the protein. D72 apparently participates in electrostatic interactions that stabilize the structure of cyt c6. The destabilizing effect was reduced when aspartate was replaced by the small amino acid alanine. Complementing the mutation D72A with a charge neutralization or inversion at position K8 led to mutant forms of cyt c6 that were more stable than the wild-type under all tested conditions. [source] Assembly of the Rieske iron,sulphur protein into the cytochrome bf complex in thylakoid membranes of isolated pea chloroplastsFEBS JOURNAL, Issue 2 2000Aliki Kapazoglou The assembly of the Rieske iron,sulphur protein into the cytochrome bf complex was examined following import of 35S-labeled precursor protein by isolated pea chloroplasts. Rieske protein assembled into the cytochrome bf complex was resolved from unassembled Rieske protein and from other membrane complexes by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. Four mutant forms of the Rieske protein were able to assemble into the cytochrome bf complex in isolated chloroplasts. These were a triple substitution mutant, C107S/H109R/C112S, replacing conserved residues involved in the ligation of the [2Fe-2S] centre; the mutant ,45,52 which removed a glycine-rich region predicted to form a flexible hinge between the hydrophobic membrane-associated region and the hydrophilic lumenal domain; and mutants ,168,173 and ,177,179 which removed two C-terminal regions, which are highly conserved in chloroplast and cyanobacterial Rieske proteins. This indicates that the [2Fe,2S] cluster, the glycine-rich region and the C-terminal region are not essential for stable assembly of the Rieske protein into the cytochrome bf complex in isolated chloroplasts. [source] Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-,1,induced apoptosis in rat hepatocytes,HEPATOLOGY, Issue 4 2005Susana Solá Ursodeoxycholic acid (UDCA) inhibits classical mitochondrial pathways of apoptosis by either directly stabilizing mitochondrial membranes or modulating specific upstream targets. Furthermore, UDCA regulates apoptosis-related genes from transforming growth factor ,1 (TGF-,1),induced hepatocyte apoptosis by a nuclear steroid receptor (NSR),dependent mechanism. In this study, we further investigated the potential role of the glucocorticoid receptor (GR) in the antiapoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF-,1,induced apoptosis of primary rat hepatocytes through a GR-dependent effect. Immunoprecipitation assays and confocal microscopy showed that UDCA enhanced free GR levels with subsequent GR nuclear translocation. Interestingly, when a carboxy-terminus deleted form of GR was used, UDCA no longer increased free GR and/or GR translocation, nor did it protect against TGF-,1,induced apoptosis. In co-transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF-,1. Finally, using a flourescently labeled UDCA molecule, the bile acid appeared diffuse in the cytosol but was aggregated in the nucleus of hepatocytes. Both siRNA assays and transfection experiments with either wild-type or mutant forms of GR showed that nuclear trafficking occurs through a GR-dependent mechanism. In conclusion, these results further clarify the antiapoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis. (HEPATOLOGY 2005;42:925,934.) [source] NF-,B inhibition triggers death of imatinib-sensitive and imatinib-resistant chronic myeloid leukemia cells including T315I Bcr-Abl mutantsINTERNATIONAL JOURNAL OF CANCER, Issue 2 2009Nadia Lounnas Abstract The Bcr-Abl inhibitor imatinib is the current first-line therapy for all newly diagnosed chronic myeloid leukemia (CML). Nevertheless, resistance to imatinib emerges as CML progresses to an acute deadly phase implying that physiopathologically relevant cellular targets should be validated to develop alternative therapeutic strategies. The NF-,B transcription factor that exerts pro-survival actions is found abnormally active in numerous hematologic malignancies. In the present study, using Bcr-Abl-transfected BaF murine cells, LAMA84 human CML cell line and primary CML, we show that NF-,B is active downstream of Bcr-Abl. Pharmacological blockade of NF-,B by the IKK2 inhibitor AS602868 prevented survival of BaF cells expressing either wild-type, M351T or T315I imatinib-resistant mutant forms of Bcr-Abl both in vitro and in vivo using a mouse xenograft model. AS602868 also affected the survival of LAMA84 cells and of an imatinib-resistant variant. Importantly, the IKK2 inhibitor strongly decreased in vitro survival and ability to form hematopoietic colonies of primary imatinib resistant CML cells including T315I cells. Our data strongly support the targeting of NF-,B as a promising new therapeutic opportunity for the treatment of imatinib resistant CML patients in particular in the case of T315I patients. The T315I mutation escapes all currently used Bcr-Abl inhibitors and is likely to become a major clinical problem as it is associated with a poor clinical outcome. © 2009 UICC [source] | ||||||||||||||||||||||||||||