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Reduced Activity (reduced + activity)
Selected AbstractsReduced Activity of CD13/Aminopeptidase N (APN) in Aggressive Meningiomas Is Associated with Increased Levels of SPARCBRAIN PATHOLOGY, Issue 1 2010Christian Mawrin Abstract Meningiomas are the second most common brain tumors in adults, and meningiomas exhibit a tendency to invade adjacent structures. Compared with high-grade gliomas, little is known about the molecular changes that potentially underlie the invasive behavior of meningiomas. In this study, we examined the expression and function of the membrane alanyl-aminopeptidase [mAAP, aminopeptidase N (APN), CD13, EC3.4.11.2] zinc-dependent ectopeptidase in meningiomas and meningioma cell lines, based on its prior association with tumor invasion in colorectal and renal carcinomas. We found a significant reduction of APNmRNA and protein expression, as well as enzymatic activity, in high-grade meningiomas. While meningioma tumor cell proliferation was not affected by either pharmacologic APN inhibition or siRNA-mediated APN silencing, APN pharmacologic and siRNA knockdown significantly reduced meningioma cell invasion in vitro. Next, we employed pathway-specific cDNA microarray analyses to identify extracellular matrix and adhesion molecules regulated by APN, and found that APN-siRNA knockdown substantially increased the expression of secreted protein, acidic and rich in cysteine (SPARC)/osteonectin. Finally, we demonstrated that SPARC, which has been previously associated with meningioma invasiveness, was increased in aggressive meningiomas. Collectively, these results suggest that APN expression and enzymatic function is reduced in aggressive meningiomas, and that alterations in the balance between APN and SPARC might favor meningioma invasion. [source] Scared fish get lazy, and lazy fish get fatJOURNAL OF ANIMAL ECOLOGY, Issue 4 2009Frank Johansson Summary 1Many biological textbooks present predator-induced morphological changes in prey species as an example of an adaptive response, because the morphological change is associated with lower predation risk. Here we show that the adaptive morphological response observed in many systems may actually be an indirect effect of decreased activity , which reduces the predation risk , rather than a direct adaptive response. 2One of the classical examples comes from crucian carp, where the presence of pike leads to a deeper body. We manipulated pike cues (presence and absence) and water current (standing and running water) and found that both standing water and pike cues similarly and independently induced a deeper body. 3Since the presence of pike cues as well as standing water might be associated with low swimming activity, we suggest that the presence of pike causes a reduction in activity (antipredator behaviour). Reduced activity subsequently induces a deeper body, possibly because the energy saved is allocated to a higher growth rate. 4Our result suggests that even if morphological change is adaptive, it might be induced indirectly via activity. This important conceptual difference may be similar in many other systems. [source] In vitro new dialysis protocol to assay the antiseptic properties of a quaternary ammonium compound polymerized with denture acrylic resinLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2004C. Pesci-Bardon Abstract Aims:, To develop an in vitro protocol in order to assess the antiseptic properties of a quaternary ammonium compound polymerized with acrylic denture resin base, using experimental resin discs and dialysis membranes. Methods and Results:, Experimental acrylic resin discs were polymerized with Poly 202063A, an ammonium compound (2,50%). Antiseptic properties were assayed against two reference strains (Escherichia coli, Staphylococcus aureus) and a laboratory strain (Candida albicans), using three different conditions (test A, B and C). In test A, according to classical protocols the resin discs were first soaked in large volumes of microbial inoculum (45 ml). An original dialysis protocol was then designed to recreate the small biofilm volume on the prosthetic surface. In test B, discs and bacterial inoculum (600 ,l) were introduced in a dialysis bag and dialysed against a sterile buffer. A bactericidal effect was observed against E. coli and Staph. aureus (<0·1% viable cells in initial bacterial suspension). A dose-dependent fungistatic effect was observed against C. albicans. Finally, in test C discs and sterile buffer (600 ,l) were introduced in a dialysis bag and dialysed against microbial inoculum. Reduced activity was found outside the dialysis bag, demonstrating that free ammonium was able to diffuse through the dialysis membrane, displaying antiseptic properties. Conclusions:, The present protocol demonstrated that a quaternary ammonium compound remains efficient after heat polymerization with an acrylic denture base resin, both in immediate and distant microbial environments. Significance and Impact of the Study:, Such removable prosthetic devices with intrinsic antiseptic properties would contribute to improve the long-term management of denture stomatitis. [source] Diarsenic and tetraarsenic oxide inhibit cell cycle progression and bFGF- and VEGF-induced proliferation of human endothelial cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005Sang Hyeok Woo Abstract Arsenic trioxide (As2O3, diarsenic oxide) has recently been reported to induce apoptosis and inhibit the proliferation of various human cancer cells derived from solid tumors as well as hematopoietic malignancies. In this study, the in vitro effects of As2O3 and tetraasrsenic oxide (As4O6) on cell cycle regulation and basic fibroblast growth factor (bFGF)- or vascular endothelial growth factor (VEGF)-stimulated cell proliferation of human umbilical vein endothelial cells (HUVEC) were investigated. Significant dose-dependent inhibition of cell proliferation was observed when HUVEC were treated with either arsenical compound for 48 h, and flow cytometric analysis revealed that these two arsenical compounds induced cell cycle arrest at the G1 and G2/M phases,the increases in cell population at the G1 and G2/M phase were dominantly observed in As2O3 - and As4O6 -treated cells, respectively. In both arsenical compounds-treated cells, the protein levels of cyclin A and CDC25C were significantly reduced in a dose-dependent manner, concomitant to the reduced activities of CDK2- and CDC2-associated kinase. In G1 -synchronized HUVEC, the arsenical compounds prevented the cell cycle progression from G1 to S phase, which was stimulated by bFGF or VEGF, through the inhibition of growth factor-dependent signaling. These results suggest that arsenical compounds inhibit the proliferation of HUVEC via G1 and G2/M phase arrest of the cell cycle. In addition, these inhibitory effects on bFGF- or VEGF-stimulated cell proliferation suggest antiangiogenic potential of these arsenical compounds. © 2005 Wiley-Liss, Inc. [source] Gender differences in glutathione metabolism in Alzheimer's diseaseJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005Honglei Liu Abstract The mechanism underlying Alzheimer's disease (AD), an age-related neurodegenerative disease, is still an area of significant controversy. Oxidative damage of macromolecules has been suggested to play an important role in the development of AD; however, the underlying mechanism is still unclear. In this study, we showed that the concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant, was decreased in red blood cells from male AD patients compared with age- and gender-matched controls. However, there was no difference in blood GSH concentration between the female patients and female controls. The decrease in GSH content in red blood cells from male AD patients was associated with reduced activities of glutamate cysteine ligase and glutathione synthase, the two enzymes involved in de novo GSH synthesis, with no change in the amount of oxidized glutathione or the activity of glutathione reductase, suggesting that a decreased de novo GSH synthetic capacity is responsible for the decline in GSH content in AD. These results showed for the first time that GSH metabolism was regulated differently in male and female AD patients. © 2005 Wiley-Liss, Inc. [source] The maize viviparous15 locus encodes the molybdopterin synthase small subunitTHE PLANT JOURNAL, Issue 2 2006Masaharu Suzuki Summary A new Zea mays viviparous seed mutant, viviparous15 (vp15), was isolated from the UniformMu transposon-tagging population. In addition to precocious germination, vp15 has an early seedling lethal phenotype. Biochemical analysis showed reduced activities of several enzymes that require molybdenum cofactor (MoCo) in vp15 mutant seedlings. Because MoCo is required for abscisic acid (ABA) biosynthesis, the viviparous phenotype is probably caused by ABA deficiency. We cloned the vp15 mutant using a novel high-throughput strategy for analysis of high-copy Mu lines: We used MuTAIL PCR to extract genomic sequences flanking the Mu transposons in the vp15 line. The Mu insertions specific to the vp15 line were identified by in silico subtraction using a database of MuTAIL sequences from 90 UniformMu lines. Annotation of the vp15 -specific sequences revealed a Mu insertion in a gene homologous to human MOCS2A, the small subunit of molybdopterin (MPT) synthase. Molecular analysis of two allelic mutations confirmed that Vp15 encodes a plant MPT synthase small subunit (ZmCNX7). Our results, and a related paper reporting the cloning of maize viviparous10, demonstrate robust cloning strategies based on MuTAIL-PCR. The Vp15/CNX7, together with other CNX genes, is expressed in both embryo and endosperm during seed maturation. Expression of Vp15 appears to be regulated independently of MoCo biosynthesis. Comparisons of Vp15 loci in genomes of three cereals and Arabidopsis thaliana identified a conserved sequence element in the 5, untranslated region as well as a micro-synteny among the cereals. [source] Activation loop 3 and the 170 loop interact in the active conformation of coagulation factor VIIaFEBS JOURNAL, Issue 11 2009Egon Persson The initiation of blood coagulation involves tissue factor (TF)-induced allosteric activation of factor VIIa (FVIIa), which circulates in a zymogen-like state. In addition, the (most) active conformation of FVIIa presumably relies on a number of intramolecular interactions. We have characterized the role of Gly372(223) in FVIIa, which is the sole residue in activation loop 3 that is capable of forming backbone hydrogen bonds with the unusually long 170 loop and with activation loop 2, by studying the effects of replacement with Ala [G372(223)A]. G372A-FVIIa, both in the free and TF-bound form, exhibited reduced cleavage of factor X (FX) and of peptidyl substrates, and had increased Km values compared with wild-type FVIIa. Inhibition of G372A-FVIIa·sTF by p -aminobenzamidine was characterized by a seven-fold higher Ki than obtained with FVIIa·sTF. Crystallographic and modelling data suggest that the most active conformation of FVIIa depends on the backbone hydrogen bond between Gly372(223) and Arg315(170C) in the 170 loop. Despite the reduced activity and inhibitor susceptibility, native and active site-inhibited G372A-FVIIa bound sTF with the same affinity as the corresponding forms of FVIIa, and burial of the N-terminus of the protease domain increased similarly upon sTF binding to G372A-FVIIa and FVIIa. Thus Gly372(223) in FVIIa appears to play a critical role in maturation of the S1 pocket and adjacent subsites, but does not appear to be of importance for TF binding and the ensuing allostery. [source] Species-specific responses of planktivorous fish to the introduction of a new piscivore: implications for prey fitnessFRESHWATER BIOLOGY, Issue 9 2007FRANZ HÖLKER Summary 1. Antipredator behaviour by the facultative planktivorous fish species roach (Rutilus rutilus), perch (Perca fluviatilis) and rudd (Scardinius erythrophthalmus) was studied in a multi-year whole-lake experiment to evaluate species-specific behavioural and numerical responses to the stocking of pikeperch (Sander lucioperca), a predator with different foraging behaviour than the resident predators large perch (P. fluviatilis) and pike (Esox lucius). 2. Behavioural responses to pikeperch varied greatly during the night, ranging from reduced activity (roach and small perch) and a shift in habitat (roach), to no change in the habitat use and activity of rudd. The differing responses of the different planktivorous prey species highlight the potential variation in behavioural response to predation risk from species of similar vulnerability. 3. These differences had profound effects on fitness; the density of species that exhibited an antipredator response declined only slightly (roach) or even increased (small perch), whereas the density of the species that did not exhibit an antipredator response (rudd) decreased dramatically (by more than 80%). 4. The maladaptive behaviour of rudd can be explained by a ,behavioural syndrome', i.e. the interdependence of behaviours expressed in different contexts (feeding activity, antipredator) across different situations (different densities of predators). 5. Our study extends previous studies, that have typically been limited to more controlled situations, by illustrating the variability in intensity of phenotypic responses to predators, and the consequences for population density, in a large whole-lake setting. [source] Influence of factor VIII:C and factor IX activity in plasmas of haemophilic dogs on the activated partial thromboplastin time measured with two commercial reagentsHAEMOPHILIA, Issue 3 2000R. Mischke The present study is based on 145 plasma samples with a reduced activity of factor VIII:C (range: 0.009,0.62 IU mL,1) and 28 samples with a reduced factor IX activity (range: 0.035,0.55 IU mL,1). The samples were collected from dogs with haemophilia A (n=22) or haemophilia B (n=3), some of these during substitution therapy. For all samples the activated partial thromboplastin time (APTT) was measured with two commercial reagents containing kaolin as a contact activator. In each case, the deficiency of factor VIII:C or IX was reflected in abnormal results of the APTT. This was true for both reagents. A significant correlation (P < 0.001) was found between factor VIII:C activity and APTT (reagent 1, Pathromtin®; Spearman's rank correlation coefficient, rS=,0.731, reagent 2, PTT-Reagenz; rS=,0.875) as well as between factor IX activity and APTT (reagent 1, rS=,0.819; reagent 2, rS=,0.955]. In each case, the relationship between coagulation factor activity and APTT could be proven most precisely by geometric regression. The results of this study illustrate the applicability of commercial APTT test kits as a sensitive screening test of factor VIII:C and IX deficiencies in canine plasma. [source] The neural response to changing semantic and perceptual complexity during language processingHUMAN BRAIN MAPPING, Issue 3 2010David J. Sharp Abstract Speech comprehension involves processing at different levels of analysis, such as acoustic, phonetic, and lexical. We investigated neural responses to manipulating the difficulty of processing at two of these levels. Twelve subjects underwent positron emission tomographic scanning while making decisions based upon the semantic relatedness between heard nouns. We manipulated perceptual difficulty by presenting either clear or acoustically degraded speech, and semantic difficulty by varying the degree of semantic relatedness between words. Increasing perceptual difficulty was associated with greater activation of the left superior temporal gyrus, an auditory-perceptual region involved in speech processing. Increasing semantic difficulty was associated with reduced activity in both superior temporal gyri and increased activity within the left angular gyrus, a heteromodal region involved in accessing word meaning. Comparing across all the conditions, we also observed increased activation within the left inferior prefrontal cortex as the complexity of language processing increased. These results demonstrate a flexible system for language processing, where activity within distinct parts of the network is modulated as processing demands change. Hum Brain Mapp, 2010. © 2009 Wiley-Liss, Inc. [source] Population-based controlled study of social support, self-perceived stress, activity and work issues, and access to health care in inflammatory bowel diseaseINFLAMMATORY BOWEL DISEASES, Issue 4 2008Linda Rogala RN Abstract Background: The Manitoba IBD Cohort Study is a longitudinal, population-based study of multiple determinants of health outcomes in persons with inflammatory bowel disease (IBD) diagnosed within 7 years at enrollment. In this cross-sectional substudy we compared IBD participants' levels of social support, self-perceived stress, disability, and access to healthcare with those of a matched community sample. Methods: IBD participants (n = 388) were interviewed using the Canadian Community Health Surveys (CCHS) 1.1 and 1.2 to assess psychosocial variables. The national CCHS data were accessed to extract a community comparison group, matched on age, sex, and geographic residence. Results: Compared to the community sample, IBD participants received more tangible, affective, or emotional support in the past year and were more likely to have experienced a positive social interaction. Those with IBD were as likely to be employed as those in the community sample, although they reported greater rates of reduced activity and days missed. Work was not identified as a significant source of stress, but physical health was more likely to be identified as a main stressor by those with active IBD compared to the non-IBD sample. Individuals with IBD were twice as likely to report unmet healthcare needs than the community sample; however, there was agreement across both groups regarding common barriers, including long waits and availability. Conclusions: While the disease may contribute to greater interference with work quality and daily activities, IBD patients have similar levels of stress and appear to have enhanced social supports relative to those in the community without IBD. (Inflamm Bowel Dis 2008) [source] Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistanceJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2000J.P. Grill Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N,-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (Z,pH) was involved in Lactobacillus bile salt resistance. [source] The pivotal role of the alternative NF-,B pathway in maintenance of basal bone homeostasis and osteoclastogenesis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2010Niroshani S Soysa Abstract The alternative NF-,B pathway consists predominantly of NF-,B-inducing kinase (NIK), I,B kinase , (IKK,), p100/p52, and RelB. The hallmark of the alternative NF-,B signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. The physiologic relevance of alternative NF-,B activation in bone biology, however, is not well understood. To elucidate the role of the alternative pathway in bone homeostasis, we first analyzed alymphoplasic (aly/aly) mice, which have a defective NIK and are unable to process p100, resulting in the absence of p52. We observed increased bone mineral density (BMD) and bone volume, indicating an osteopetrotic phenotype. These mice also have a significant defect in RANKL-induced osteoclastogenesis in vitro and in vivo. NF-,B DNA-binding assays revealed reduced activity of RelA, RelB, and p50 and no binding activity of p52 in aly/aly osteoclast nuclear extracts after RANKL stimulation. To determine the role of p100 itself without the influence of a concomitant lack of p52, we used p100,/, mice, which specifically lack the p100 inhibitor but still express p52. p100,/, mice have an osteopenic phenotype owing to the increased osteoclast and decreased osteoblast numbers that was rescued by the deletion of one allele of the relB gene. Deletion of both allele of relB resulted in a significantly increased bone mass owing to decreased osteoclast activity and increased osteoblast numbers compared with wild-type (WT) controls, revealing a hitherto unknown role for RelB in bone formation. Our data suggest a pivotal role of the alternative NF-,B pathway, especially of the inhibitory role of p100, in both basal and stimulated osteoclastogenesis and the importance of RelB in both bone formation and resorption. © 2010 American Society for Bone and Mineral Research [source] ,-Amyloid inhibits integrated mitochondrial respiration and key enzyme activitiesJOURNAL OF NEUROCHEMISTRY, Issue 1 2002C. S. Casley Abstract Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), ,-ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post-mortem Alzheimer's disease brain. ,-Amyloid is strongly implicated in Alzheimer's pathology and can be formed intracellularly in neurones. We have investigated the possibility that ,-amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the ,-amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimer's brain. Mitochondrial respiration, electron transport chain complex activities, ,-ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. ,-Amyloid caused a significant reduction in state 3 and state 4 mitochondrial respiration that was further diminished by the addition of nitric oxide. Cytochrome oxidase, ,-ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by ,-amyloid. The Km of cytochrome oxidase for reduced cytochrome c was raised by ,-amyloid. We conclude that ,-amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimer's disease. [source] Development of a potent and selective GPR7 (NPBW1) agonist: a systematic structure,activity study of neuropeptide BJOURNAL OF PEPTIDE SCIENCE, Issue 6 2007Maki Kanesaka Abstract Neuropeptide B (NPB) has been recently identified as an endogenous ligand for GPR7 (NPBW1) and GPR8 (NPBW2) and has been shown to possess a relatively high selectivity for GPR7. In order to identify useful experimental tools to address physiological roles of GPR7, we synthesized a series of NPB analogs based on modification of an unbrominated form of 23 amino acids with amidated C -terminal, Br(,)NPB-23-NH2. We confirmed that truncation of the N -terminal Trp residue resulted in almost complete loss of the binding affinity of NPB for GPR7 and GPR8, supporting the special importance of this residue for binding. Br(,)NPB-23-NH2 analogs in which each amino acid in positions 4, 5, 7, 8, 9, 10, 12 and 21 was replaced with alanine or glycine exhibited potent binding affinity comparable to the parent peptide. In contrast, replacement of Tyr11 with alanine reduced the binding affinity for both GPR7 and GPR8 four fold. Of particular interest, several NPB analogs in which the consecutive amino acids from Pro4 to Val13 were replaced with several units of 5-aminovaleric acid (Ava) linkers retained their potent affinity for GPR7. Furthermore, these Ava-substituted NPB analogs exhibited potent agonistic activities for GPR7 expressed in HEK293 cells. Among the Ava-substituted NPB analogs, analog 15 (Ava-5) and 17 (Ava-3) exhibited potency comparable to the parent peptide for GPR7 with significantly reduced activity for GPR8, resulting in high selectivity for GPR7. These highly potent and selective NPB analogs may be useful pharmacological tools to investigate the physiological and pharmacological roles of GPR7. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Measurement of in situ Monomer Sorption in Poly(propylene)MACROMOLECULAR RAPID COMMUNICATIONS, Issue 18 2004Jochem T. M. Pater Abstract Summary: An experimental method has been developed to compare the amount of monomer absorbed in freshly produced poly(propylene) with the amount of monomer absorbed in the same material after degassing. It has been found that propylene sorption in freshly produced poly(propylene) is significantly higher than the sorption in the same but degassed polymer. The difference depends on the degree of drying and is time-dependent. This fact can be an explanation for reduced activity often observed in the transition from liquid- to gas-phase polymerization. Pressure profile during the pressure-swing part of the experiment. [source] The impact of predation risk from small mustelids on prey populationsMAMMAL REVIEW, Issue 3-4 2000Kai Norrdahl ABSTRACT Small mustelids are ,snake-like' mammals adapted to hunt small rodents, which are their principal prey, in tunnels leaving practically no refuge for the prey. Prey rodents have adaptive behaviours to situations where the predation risk from mustelids is high, including reduced activity and escape by climbing. Small mustelids may affect prey population dynamics directly through killing (increased mortality) and/or indirectly through behavioural changes in prey as a response to the presence of mustelids (predation risk). The Predator-Induced Breeding Suppression hypothesis (PIBS) states that a trade-off between survival and reproduction should lead to delayed breeding under temporarily high predation risk, so that the mere presence of predators may reduce reproductive output. Current results suggest that small mustelids mainly affect prey population growth rate directly through killing. In many cyclic rodent populations, small mustelid predation is a major mortality factor, and experimental evidence supports the hypothesis that these predators drive prolonged summer declines in prey. In contrast, the evidence for PIBS is controversial. Experimental evidence shows that the indirect effects of small mustelids on prey populations are negligible during the best breeding season. However, in other seasons, the presence of predators may indirectly affect prey populations, although this has not been studied experimentally. Prey rodents may decrease mobility as a response to high predation risk by small mustelids, and this reduction in mobility decreases feeding. Reduced feeding affects the energy reserves of voles, and may delay maturation or lower the size of the first litter. [source] Ancestral roles of eukaryotic frataxin: mitochondrial frataxin function and heterologous expression of hydrogenosomal Trichomonas homologues in trypanosomesMOLECULAR MICROBIOLOGY, Issue 1 2008Shaojun Long Summary Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe,S cluster-containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin-depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe,S cluster biogenesis and protection from reactive oxygen species. [source] Positioning and the specific sequence of each 13-mer motif are critical for activity of the plasmid RK2 replication originMOLECULAR MICROBIOLOGY, Issue 5 2005Lukasz Kowalczyk Summary The minimal replication origin of the broad-host-range plasmid RK2, oriV, contains five iterons which are binding sites for the plasmid-encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT-rich region containing four 13-mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13-mer motifs have been constructed and analysed for replication activity in vivo and in vitro. The data show that the replacement of oriV 13-mers by similar but not identical 13-mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13-mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of single-nucleotide substitutions demonstrates very restricted sequence requirements depending on the 13-mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli. Our experiments demonstrate considerable complexity in the plasmid AT-rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence-specific protein interactions within the 13-mers during assembly of replication complexes at the plasmid origin. [source] Neurological aspects of osteopetrosisNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 2 2003C. G. Steward The osteopetroses are caused by reduced activity of osteoclasts which results in defective remodelling of bone and increased bone density. They range from a devastating neurometabolic disease, through severe malignant infantile osteopetrosis (OP) to two more benign conditions principally affecting adults [autosomal dominant OP (ADO I and II)]. In many patients the disease is caused by defects in either the proton pump [the a3 subunit of vacuolar-type H(+)-ATPase, encoded by the gene variously termed ATP6i or TCIRG1] or the ClC-7 chloride channel (ClCN7 gene). These pumps are responsible for acidifying the bone surface beneath the osteoclast. Although generally thought of as bone diseases, the most serious consequences of the osteopetroses are seen in the nervous system. Cranial nerves, blood vessels and the spinal cord are compressed by either gradual occlusion or lack of growth of skull foramina. Most patients with OP have some degree of optic atrophy and many children with severe forms of autosomal recessive OP are rendered blind; optic decompression is frequently attempted to prevent the latter. Auditory, facial and trigeminal nerves may also be affected, and hydrocephalus can develop. Stenosis of both arterial supply (internal carotid and vertebral arteries) and venous drainage may occur. The least understood form of the disease is neuronopathic OP [OP and infantile neuroaxonal dystrophy, MIM (Mendelian inheritance in man) 600329] which causes rapid neurodegeneration and death within the first year. Although characterized by the finding of widespread axonal spheroids and accumulation of ceroid lipofuscin, the biochemical basis of this disease remains unknown. The neurological complications of this disease and other variants are presented in the context of the latest classification of the disease. [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] Both antisense and sense expression of biotin carboxyl carrier protein isoform 2 inactivates the plastid acetyl-coenzyme A carboxylase in Arabidopsis thalianaTHE PLANT JOURNAL, Issue 4 2002Jay J. Thelen Summary To further characterize the role of biotin carboxyl carrier protein isoform 2 (BCCP2) in acetyl-coenzyme A carboxylase (ACCase) function and fatty acid biosynthesis, plants with reduced or increased expression of this protein were characterized. Analysis of 38 independent Arabidopsis lines expressing antisense BCCP2 transcript behind a constitutive promoter showed no significant phenotype, though antisense transcript was highly expressed. In developing seed, BCCP2 protein was reduced by an average of 38% resulting in a 9% average decrease in fatty acid content in mature seed. Over-expression of BCCP2 behind a seed-specific napin promoter increased the amount of holo-BCCP2 in developing seed by an average of twofold, as determined with anti-biotin antibodies. Surprisingly, the average fatty acid content of T2 seed from over-expression lines was 23% lower than wild-type seed. These plants also exhibited reduced seed setting in 18 of 20 T1 lines which was coincident with increased individual seed mass. Quantification of total BCCP2 in developing siliques using anti-BCCP2 antibodies indicated that as much as 60% of total plastidial BCCP2 was in the non-biotinylated form (apo-BCCP2). Although apo-BCCP2 was highly over-expressed in developing seed, accumulation of other ACCase subunits was unaffected. The specific activity of ACCase was up to 65% lower in developing seed of over-expression lines versus wild type. This was attributed to the assembly of biologically inactive (non-biotinylated) ACCase complexes. Consistent with ACCase exerting control over de novo fatty acid synthesis, reduced activity in developing seed resulted in lower oil content, altered fatty acid composition and reduced seed setting. [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] The Protective Effect of Bee Venom against Ethanol-Induced Hepatic Injury via Regulation of the Mitochondria-Related Apoptotic PathwayBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2010Kyung-Hyun Kim Death of hepatocytes is a characteristic feature of chronic liver disease for various causes. Bee venom (Apis mellifera) has been traditionally used for the treatment of various chronic diseases, such as chronic inflammatory arthritis and chronic liver disease. However, the precise mechanism for bee venom in chronic liver disease is not still cleared. To assess the effects of bee venom in chronic liver disease, we investigated the potential role of the bee venom in the ethanol-induced hepatocyte apoptosis. Bee venom treatment inhibited the apoptotic cell morphology and increased the cell viability in ethanol-induced hepatocyte apoptosis. With ethanol treatment, bee venom-treated hepatocytes increased activity of Bcl-2 and Bcl-xL, reduced activity of Bax, Caspase and PARP. In conclusion, bee venom treatment in ethanol-induced hepatocyte apoptosis occurred through the regulation of Bcl family with subsequent inactivation of the Caspase and PARP. These results suggest that bee venom could be an effective agent to reduce ethanol-induced hepatocyte apoptosis. [source] Neural correlates of deficient response inhibition in mentally disordered violent individualsBEHAVIORAL SCIENCES & THE LAW, Issue 1 2008Ian Barkataki Ph.D. In this study, response inhibition and associated neural activation during a motor inhibition paradigm were investigated in (i) men with antisocial personality disorder (APD) with a history of violence (n,=,14), (ii) men with schizophrenia with a history of violence (n,=,12), (iii) men with schizophrenia without a history of violence (n,=,12), and (iv) healthy control subjects (n,=,14) using functional magnetic resonance imaging (fMRI). At the behavioural level, individuals with schizophrenia showed impaired performance across all conditions, whereas an increased error rate was seen in the APD group only during the conditions requiring inhibition. At the neural level, both violent groups showed reduced thalamic activity, compared with controls, in association with modulation of inhibition by task demands. In addition, the violent schizophrenia group, compared with controls, showed reduced activity in the caudate nucleus during the condition requiring inhibition. It is concluded that violence may not be specifically associated with impaired voluntary inhibition in schizophrenia but this is likely in APD. Reduced thalamic function, perhaps due to its known association with sensorimotor disturbances, is implicated in violent behaviour across both disorders. In addition, caudate dysfunction may contribute, given its role in timing and temporal processing as well as suppression of motor actions, to deficient inhibition and violent behaviour in schizophrenia. Copyright © 2008 John Wiley & Sons, Ltd. [source] The Role of the Conserved Threonine in P450BM3 Oxygen Activation: Substrate-Determined Hydroxylation Activity of the Thr268Ala MutantCHEMBIOCHEM, Issue 2 2008Max J. Cryle Dr. Abstract The hydroxylation activity of the Thr268Ala mutant of P450BM3 has been shown to occur to varying degrees with small alterations in the structure of a fatty-acid substrate. Ten substrates were investigated, including straight chain, branched chain and cis -cyclopropyl substituted fatty acids with a straight-chain length that varied between 12 and 16 carbon atoms. The efficacy of the hydroxylation activity appeared to be governed by the chain length of the substrate. Substrates possessing 14 to 15 carbons afforded the highest levels of activity, which were comparable with the wild-type enzyme. Outside of this window, straight-chain fatty acids showed reduced activity over the other substrate types. These results provide a cautionary tale concerning the loss of ferryl activity in such cytochrome P450 threonine to alanine mutants, as the nature of the substrate can determine the extent to which hydroxylation chemistry is abolished. [source] Hypogonadism-related symptoms: development and evaluation of an empirically derived self-rating instrument (HRS ,Hypogonadism Related Symptom Scale')ANDROLOGIA, Issue 5 2009J. Wiltink Summary While self-report screening instruments are highly sensitive to hypogonadism in the ageing male, they have lacked specificity as evidenced by low or absent correlations with testosterone. The purpose of this paper was to develop an economical and specific screening instrument for identifying hypogonadal ageing men. Based on a comprehensive study of physical, somatoform and affective complaints, sexual behaviour and function and hormonal parameters of 263 outpatients aged 40 years and above (M = 56.2; 40,84 years) recruited from six andrological outpatient departments in Germany, we identified those items correlating significantly with testosterone. By factor analyses, five factors were identified: ,reduced activity', ,dissatisfaction with sexual function', ,negative self-concept of physical fitness', ,reduced sexual desire' and ,hot flushes'. The corresponding scales were reliable and only moderately inter-correlated. Consistent correlations were found with the level of testosterone, ageing male scales (Androgen Deficiency in the Aging Male, Aging Male Survey), specific affective, somatoform and sexual functioning scales and potential determinants of low testosterone (body mass index, physical inactivity, etc.). While further validation is needed, the new Hypogonadism Related Symptoms Scale appears to be a promising hypogonadism screening tool. [source] | |||||||||||||||||||||||||||||||