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691.
692.
693.
Damásio AR Ribeiro LF Ribeiro LF Furtado GP Segato F Almeida FB Crivellari AC Buckeridge MS Souza TA Murakami MT Ward RJ Prade RA Polizeli ML 《Biochimica et biophysica acta》2012,1824(3):461-467
Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan β-1,3 or β-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in β-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state. 相似文献
694.
Mucins are linear O-glycosylated glycoproteins involved in inflammation, cell adhesion, and tumorigenesis. Cancer-associated mucins often possess increased expression of the T (Galβ1,3GalNAcαThr/Ser) and Tn (GalNAcαThr/Ser) cancer antigens, which are diagnostic markers for several cancers, including colon cancer. We have used AFM based single-molecule forced unbinding under near physiological conditions to investigate the self-interactions between porcine submaxillary mucin (PSM) as well as between PSM analogs possessing various carbohydrates including the T- and Tn-antigen. Distributions of unbinding forces and corresponding force loading rates were determined for force loading rates from 0.18 nN/s to 39 nN/s, and processed to yield most probable unbinding forces f* and lifetimes of the interactions. Parameter f* varied in the range 27 to 50 pN at force loading rates of about 2 nN/s among the various mucins. All mucin samples investigated showed self-interaction, but the tendency was greatest for PSM displaying only the Tn-antigen (Tn-PSM) or a mixture of Tn-, T-antigen, and the trisaccharide Fucα1,2Galβ1,3GalNAc (Tri-PSM). Weaker self-interactions were observed for native PSM (Fd-PSM), which consists of a nearly equal mixture of the longer core 1 blood group A tetrasaccharide (GalNAcα1,3(Fucα1,2)Galβ1,3GalNAcαSer/Thr) and Tn-antigen. The data are consistent with the truncated Tn and T glycans enhancing self-interaction of the mucins. These carbohydrate cancer antigens may, thus, play an active role in the disease by constitutively activating mucin and mucin-type receptors by self-association on cells. 相似文献
695.
Suijkerbuijk SJ van Dam TJ Karagöz GE von Castelmur E Hubner NC Duarte AM Vleugel M Perrakis A Rüdiger SG Snel B Kops GJ 《Developmental cell》2012,22(6):1321-1329
Chromosomal stability is safeguarded by a mitotic checkpoint, of which BUB1 and Mad3/BUBR1 are core components. These paralogs have similar, but not identical, domain organization. We show that Mad3/BUBR1 and BUB1 paralogous pairs arose by nine independent gene duplications throughout evolution, followed by parallel subfunctionalization in which preservation of the ancestral, amino-terminal KEN box or kinase domain was mutually exclusive. In one exception, vertebrate BUBR1-defined by the KEN box-preserved the kinase domain but allowed nonconserved degeneration of catalytic motifs. Although BUBR1 evolved to a typical pseudokinase in some vertebrates, it retained the catalytic triad in humans. However, we show that putative catalysis by human BUBR1 is dispensable for error-free chromosome segregation. Instead, residues that interact with ATP in conventional kinases are essential for conformational stability in BUBR1. We propose that parallel evolution of BUBR1 orthologs rendered its kinase function dispensable in vertebrates, producing an unusual, triad-containing pseudokinase. 相似文献
696.
André Ricardo de Lima Damásio Benevides C. Pessela Fernando Segato Rolf Alexander Prade Jose Manuel Guisan Maria de Lourdes T.M. Polizeli 《Process Biochemistry》2012,47(12):2411-2417
A gene encoding α-l-arabinofuranosidase (abfA) from Aspergillus niveus was identified, cloned, and successfully expressed in Aspergillus nidulans. Based on amino acid sequence comparison, the 88.6 kDa enzyme could be assigned to the GH family 51. The characterization of the purified recombinant AbfA revealed that the enzyme was active at a limited pH range (pH 4.0–5.0) and an optimum temperature of 70 °C. The AbfA was able to hydrolyze arabinoxylan, xylan from birchwood, debranched arabinan, and 4-nitrophenyl arabinofuranoside. Synergistic reactions using both AbfA and endoxylanase were also assessed. The highest degree of synergy was obtained after the sequential treatment of the substrate with endoxylanase, followed by AbfA, which was observed to release noticeably more reducing sugars than that of either enzyme acting individually. The immobilization of AbfA was performed via ionic adsorption onto various supports: agarose activated by polyethyleneimine polymers, cyanogen bromide activated Sepharose, DEAE-Sepharose, and Sepharose-Q. The Sepharose-Q derivative remained fully active at pH 5 after 360 min at 60 °C, whereas the free AbfA was inactivated after 60 min. A synergistic effect of arabinoxylan hydrolysis by AbfA immobilized in Sepharose-Q and endoxylanase immobilized in glyoxyl agarose was also observed. The stabilization of arabinofuranosidases using immobilization tools is a novel and interesting topic. 相似文献
697.
Dam AD Mitchell AS Rush JW Quadrilatero J 《Apoptosis : an international journal on programmed cell death》2012,17(1):48-60
Oxidative stress has a well-established role in numerous intracellular signaling pathways, including apoptosis. Glutathione
is an important cellular antioxidant and is the most abundant low molecular weight thiol in the cell. Although previous work
has shown a link between glutathione and apoptosis, this relationship has not been defined in skeletal muscle. The present
investigation examined the effect of glutathione depletion on skeletal muscle apoptotic signaling, and mitochondrial apoptotic-susceptibility.
Administration of l-buthionine-[S,R]-sulfoximine (BSO; 30 mM in drinking water for 10 days) caused glutathione depletion in whole muscle and
isolated mitochondria, as well as elevated muscle catalase protein content and reactive oxygen species (ROS) generation. Glutathione
depletion was associated with elevated DNA fragmentation, mitochondrial Bax levels, Poly(ADP-ribose) polymerase (PARP) cleavage,
and calpain activity; however, caspase-3, -8, and -9 activity were not altered. BSO administration was also associated with
higher cytosolic and nuclear protein levels of apoptosis-inducing factor (AIF), but not cytochrome c, second mitochondria-derived
activator of caspase (Smac), or endonuclease G (EndoG). In addition, isolated mitochondria from BSO animals demonstrated significantly
lower membrane potential, increased Ca2+-induced permeability transition pore opening, and greater basal and ROS-induced AIF and cytochrome c release. These results
demonstrate that glutathione depletion in skeletal muscle increases caspase-independent signaling, as well as augments mitochondrial-associated
apoptotic events to subsequent cell death stimuli. 相似文献
698.
Zhang L Huang H Zhou F Schimmel J Pardo CG Zhang T Barakat TS Sheppard KA Mickanin C Porter JA Vertegaal AC van Dam H Gribnau J Lu CX ten Dijke P 《Molecular cell》2012,46(5):650-661
TGF-β members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-β family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-β signaling. Depletion of RNF12 dramatically reduced TGF-β/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-β family signaling. 相似文献
699.
Dam Anh Tran Anthony Shakeshaft Anh Duc Ngo John Rule David P. Wilson Lei Zhang Christopher Doran 《PloS one》2012,7(12)
In Vietnam, premature mortality due to AIDS-related conditions is commonly associated with late initiation to antiretroviral therapy (ART). This study explores reasons for late ART initiation among people living with HIV (PLHIV) from the perspectives of health care providers and PLHIV. The study was undertaken in six clinics from five provinces in Vietnam. Baseline CD4 counts were collected from patient records and grouped into three categories: very late initiators (≤100 cells/mm3 CD4), late initiators (100–200 cells/mm3) and timely initiators (200–350 cells/mm3). Thirty in-depth interviews with patients who started ART and 15 focus group discussions with HIV service providers were conducted and thematic analysis of the content performed. Of 934 patients, 62% started ART very late and 11% initiated timely treatment. The proportion of patients for whom a CD4 count was obtained within six months of their HIV diagnosis ranged from 22% to 72%. The proportion of patients referred to ART clinics by voluntary testing and counselling centres ranged from 1% to 35%. Structural barriers to timely ART initiation were poor linkage between HIV testing and HIV care and treatment services, lack of patient confidentiality and a shortage of HIV/AIDS specialists. If Vietnam’s treatment practice is to align with WHO recommendations then the connection between voluntary counselling and testing service and ART clinics must be improved. Expansion and decentralization of HIV/AIDS services to allow implementation at the community level increased task sharing between doctors and nurses to overcome limited human resources, and improved patient confidentiality are likely to increase timely access to HIV treatment services for more patients. 相似文献
700.
Hansen HV Christensen EM Dam H Gluud C Wetterslev J Kessing LV;Early Intervention Affective Disorders 《PloS one》2012,7(3):e32950