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41.
Angela Chamberland Eunice Wang Aled R. Jones Lisa A. Collins-Racie Edward R. LaVallie Ying Huang Lin Liu Elisabeth A. Morris Carl R. Flannery Zhiyong Yang 《The Journal of biological chemistry》2009,284(40):27352-27359
Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV356) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV356 neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.The mammalian high-temperature requirement A (HtrA) family of serine proteases is defined by a characteristic trypsin-like serine protease domain and one or two C-terminal PDZ domains. Four mammalian HtrA proteins have been identified to date, HtrA1–4. HtrA1 (also called PRSS11) is a ubiquitously expressed extracellular serine protease which contains a signal sequence for secretion, an insulin-like growth factor (IGF)2-binding protein domain, and a Kazal-type serine protease inhibitor domain in addition to the serine protease domain and one C-terminal PDZ domain (1). HtrA1 has been implicated in the progression of several pathologies including age-related macular degeneration, cancer, Alzheimer disease, rheumatoid arthritis, and osteoarthritis (OA) (2–10). HtrA1 has also been shown to inhibit osteoblast mineralization (11).Expression of HtrA1 has been found to be elevated in articular cartilage in association with OA (5). In addition, HtrA1 levels are up-regulated in murine cartilage after experimentally induced joint damage (6). The physiological role of HtrA1 in OA disease progression as well as in other pathologies is unclear. Preliminary studies using in vitro digestion assays suggest that HtrA1 might be capable of digesting cartilage extracellular matrix (ECM) proteins such as fibromodulin, cartilage oligomeric matrix protein (COMP), fibronectin, decorin, and aggrecan (6, 12, 13). Furthermore, it was recently reported that elevated levels of HtrA1 protein (∼7-fold above normal) are present in synovial fluids obtained from OA patients and that fibronectin fragments generated by HtrA1 cleavage induced the expression of catabolic enzymes such as matrix metalloproteinases-1 (MMP-1) and MMP-3 in synovial fibroblasts (4). HtrA1 has also been shown to modulate multiple signaling pathways in vitro. It binds to transforming growth factor-β family proteins including transforming growth factor-β1 and bone morphogenetic proteins 2 and 4 and inhibits signaling mediated by these factors (14, 15). In addition, HtrA1 has been shown to cleave IGF-binding protein-5 and possibly regulate signaling mediated by IGF (16). These findings suggest that the protease HtrA1 may play a physiological role in cartilage during OA.Articular cartilage is made up of chondrocytes surrounded by the ECM comprised mainly of the proteoglycan, aggrecan, and type II collagen. During normal homeostasis there is a dynamic balance between anabolic activities such as proteoglycan synthesis as well as catabolic activities in which the ECM is destroyed. When the catabolic activities of proteases, such as MMPs and aggrecanases, offset new matrix synthesis, focal degradation and loss of articular cartilage occurs, resulting in the development of OA. In some in vitro digestion studies, we and others have shown degradation of aggrecan by recombinant HtrA1 (6, 12, 13). In the present study we set out to examine the physiological relevance of aggrecan cleavage by HtrA1 in OA disease progression. 相似文献
42.
Sarunporn Tandhavanant Aunchalee Thanwisai Direk Limmathurotsakul Sunee Korbsrisate Nicholas PJ Day Sharon J Peacock Narisara Chantratita 《BMC microbiology》2010,10(1):303
Background
Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays. 相似文献43.
Variation in heat shock proteins within tropical and desert species of poeciliid fishes 总被引:8,自引:0,他引:8
Norris CE; diIorio PJ; Schultz RJ; Hightower LE 《Molecular biology and evolution》1995,12(6):1048-1062
The 70-kilodalton heat shock protein (hsp70) family of molecular
chaperones, which contains both stress-inducible and normally abundant
constitutive members, is highly conserved across distantly related taxa.
Analysis of this protein family in individuals from an outbred population
of tropical topminnows, Poeciliopsis gracilis, showed that while
constitutive hsp70 family members showed no variation in protein isoforms,
inducibly synthesized hsp70 was polymorphic. Several species of
Poeciliopsis adapted to desert environments exhibited lower levels of
inducible hsp70 polymorphism than the tropical species, but constitutive
forms were identical to those in P. gracilis, as they were in the
confamilial species Gambusia affinis. These differences suggest that
inducible and constitutive members of this family are under different
evolutionary constraints and may indicate differences in their function
within the cell. Also, northern desert species of Poeciliopsis synthesize a
subset of the inducible hsp70 isoforms seen in tropical species. This
distribution supports the theory that ancestral tropical fish migrated
northward and colonized desert streams; the subsequent decrease in
variation of inducible hsp70 may have been due to genetic drift or a
consequence of adaptation to the desert environment. Higher levels of
variability were found when the 30- kilodalton heat shock protein (hsp30)
family was analyzed within different strains of two desert species of
Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In
both cases the distribution of hsp30 isoform diversity was similar to that
seen previously with allozyme polymorphisms.
相似文献
44.
PJ?MumbyEmail author JD?Hedley JRM?Chisholm CD?Clark H?Ripley J?Jaubert 《Coral reefs (Online)》2004,23(2):171-183
Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m2 pixels and 6 spectral bands with 0.25-m2 pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead
Porites (total colony mortality within 6 months), old dead (>6 months) Porites,
Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites
colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m2 plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m2 grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m2 quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of
Porites spp.). At the scale of the reef (all ten 25-m2 quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living
Porites, recently dead Porites
and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m2 of reef, which represents 3,700 plots of 25 m2 each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m2 and 0.25-m2 image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m2 pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey). 相似文献
45.
White Nicholas J Ashley Elizabeth A Recht Judith Delves Michael J Ruecker Andrea Smithuis Frank M Eziefula Alice C Bousema Teun Drakeley Chris Chotivanich Kesinee Imwong Mallika Pukrittayakamee Sasithon Prachumsri Jetsumon Chu Cindy Andolina Chiara Bancone Germana Hien Tran T Mayxay Mayfong Taylor Walter RJ von Seidlein Lorenz Price Ric N Barnes Karen I Djimdé Abdoulaye ter Kuile Feiko Gosling Roly Chen Ingrid Dhorda Mehul J Stepniewska Kasia Guérin Philippe Woodrow Charles J Dondorp Arjen M Day Nicholas PJ Nosten Francois H 《Malaria journal》2014,13(1):1-14
Background
Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness.Methods
Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits.Results
Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions.Conclusions
These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic ‘supermales’ could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings. 相似文献46.
C. S. Herrington A. K. Graham D. M. J. Flannery J. Burns J. O''D. McGee 《The Histochemical journal》1990,22(10):545-554
Summary It is generally assumed that nucleic acid association duringin situ hybridization reactions is similar to that of nucleic acid association in solution. This assumption has been investigated by detecting closely homologous human papillomavirus types 6 and 11 byin situ hybridization as a model for the evaluation of stringency conditions in clinical biopsies.By examining matched and mismatched, labelled and target sequences under various stringency conditions, empirical DNA-DNA stability curves and their derivative equations for tissue melting temperatures (Tmt) were derived. The corresponding values for Tmt are 10–20°C higher than their solution equivalents. These data, supported by polymerase chain reaction experiments, demonstrate that closely homologous viral DNAs cross linked in tissue by formaldehyde fixation do not interact with the corresponding labelled probes as predicted from solution kinetic equations. This not only has theoretical implications but is also relevant to the accuracy of clinical diagnostic testing. 相似文献
47.
48.
Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopic in situ hybridization. 总被引:8,自引:2,他引:6 下载免费PDF全文
A rapid method is described for non isotopic in situ mapping of single copy genes directly on G-banded chromosomes by "one-step" regular light microscopy. It is based on hybridizing biotinylated probes to metaphase chromosomes. Biotin residues are detected by rabbit antibiotin antibody and anti-rabbit Ig labelled with peroxidase or colloidal gold. The peroxidase reaction product or colloidal gold signals are amplified by silver precipitation. The final product is a black silver dot at the gene locus on a purple G-banded chromosome. N-ras and alpha-1-antitrypsin genes have been mapped using plasmids with inserts of 1.5 and 1.3kb to 1p13.1 and the junction of 14q31/32 respectively. The signal to noise ratio in these experiments ranged from 32:1-46:1. This technology is at least as sensitive as radioisotopic in situ hybridization and gives results within 1 day of hybridization and has much better resolution. Additionally, genes are visualized by regular light microscopy without specialized techniques such as reflection contrast, fluorescence or phase microscopy. This methodology should facilitate more precise chromosomal gene localization. 相似文献
49.
50.
Alessandro Pecego Martins Romano Zouraide Guerra Antunes Costa Daniel Garkauskas Ramos Maria Auxiliadora Andrade Valéria de Sá Jayme Marco Ant?nio Barreto de Almeida Kátia Campomar Vettorello Melissa Mascheretti Brendan Flannery 《PLoS neglected tropical diseases》2014,8(3)
Due to the risk of severe vaccine-associated adverse events, yellow fever vaccination in Brazil is only recommended in areas considered at risk for disease. From September 2008 through June 2009, two outbreaks of yellow fever in previously unvaccinated populations resulted in 21 confirmed cases with 9 deaths (case-fatality, 43%) in the southern state of Rio Grande do Sul and 28 cases with 11 deaths (39%) in Sao Paulo state. Epizootic deaths of non-human primates were reported before and during the outbreak. Over 5.5 million doses of yellow fever vaccine were administered in the two most affected states. Vaccine-associated adverse events were associated with six deaths due to acute viscerotropic disease (0.8 deaths per million doses administered) and 45 cases of acute neurotropic disease (5.6 per million doses administered). Yellow fever vaccine recommendations were revised to include areas in Brazil previously not considered at risk for yellow fever. 相似文献