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Sample records for renibacterium salmoninarum extracellular

  1. Inactivation of Renibacterium salmoninarum by free chlorine

    USGS Publications Warehouse

    Pascho, Ronald J.; Landolt, Marsha L.; Ongerth, Jerry E.

    1995-01-01

    Salmonid fishes contract bacterial kidney disease by vertical or horizontal transmission of the pathogenic bacterium, Renibacterium salmoninarum. Procedures to reduce vertical transmission are under evaluation, but methods are still needed to eliminate sources of waterborne R. salmoninarum. We examined the efficacy of chlorine to inactivate R. salmoninarum. The bacterium was exposed to various levels of chlorine at pH 6, 7, or 8, and at 7.5 °C or 15 °C. At pH 7 and 15 °C, 99% inactivation occurred within 18 s, even at free chlorine concentrations as low as 0.05 mg/l. Chlorine was most effective at neutral or acidic pH, and 15 °C. The inactivation curves for 7.5 °C and pH 7, or 15 °C and pH 8, deviated from first-order kinetics by exhibiting shoulders or a tailing-off effect, suggesting that chlorine and the bacterial cells were not the sole reactants. A plot of the concentration-time (Ct) products for free chlorine at pH 7 and 15 °C produced a line with a slope less than 1, indicating that the duration of exposure was more important than the concentration of free chlorine. These data indicate that R. salmoninarum is very sensitive to chlorine, and that this disinfectant may be appropriate for use in fish hatcheries rearing salmonids affected by bacterial kidney disease.

  2. Virulence of Renibacterium salmoninarum to salmonids

    USGS Publications Warehouse

    Starliper, C.E.; Smith, D.R.; Shatzer, T.

    1997-01-01

    Virulence of Renibacterium salmoninarum isolates representing five origins was evaluated in eight salmonid hosts; four origins were of Lake Michigan and the fifth was of the Pacific Northwest. The species type strain, ATCC (American Type Culture Collection) 33209, was also included. Each isolate was grown in a kidney disease medium (KDM2) supplemented with 1 % ATCC 33209 culture metabolite; serial 10-fold dilutions were prepared, and groups of fish were challenged by intraperitoneal injection with 0.1 mL of each dilution. A 70-d observation period followed, and bacterial kidney disease (BKD) was diagnosed by the fluorescent antibody technique. Virulence of isolates was quantified as a dose lethal to 50% of fish (LD50) for each host–isolate challenge. In the first set of experiments, 23 isolates were used to challenge groups of brook trout Salvelinus fontinalis. The mean LD50 was 1.087 x 106 colony-forming units per milliliter (cfu/mL; SD = 2.022 x 106), and the LD50 values ranged from 8.457 x 106 to 2.227 x 104 cfu/mL. Analysis of variance to evaluate the effect of isolate origin on virulence in brook trout revealed no significant difference (F = 1.502; P = 0.243). Susceptibilities of the other salmonid hosts were evaluated by challenge with six isolates of R. salmoninarum representing each origin and the species type strain. For many of the host–isolate challenge combinations, time to death was highly dependent on the dilution (number of bacteria) injected. In general, the isolates MCO4M, B26, and A34 (all of Lake Michigan origin) tended to be more virulent. Also, LD50 values were dispersed throughout a wider range among the more susceptible hosts. Lake trout Salvelinus namaycush, rainbow trout Oncorhynchus mykiss, and brook trout were relatively resistant to challenge with the strains, whereas coho salmon O. kisutch, domestic Atlantic salmon Saltno salar, and chinook salmon O. tshawytscha were relatively susceptible. Another challenge evaluated the effect of

  3. Shedding of Renibacterium salmoninarum by infected chinook salmon Oncorhynchus tschawytscha

    USGS Publications Warehouse

    McKibben, C.L.; Pascho, R.J.

    1999-01-01

    Laboratory studies of the transmission and pathogenesis of Renibacterium salmoninarum may describe more accurately what is occurring in the natural environment if test fish are infected by waterborne R. salmoninarum shed from infected fish. To quantify bacterial shedding by chinook salmon Oncorhynchus tschawytscha at 13??C in freshwater, groups of fish were injected intraperitoneally with R. salmoninarum at either 1.3 x 106 colony forming units (CFU) fish-1 (high-dose injection group) or 1.5 x 103 CFU fish-1 (low-dose injection group). R. salmoninarum infection levels were measured in the exposed fish by the enzyme-linked immunosorbent assay (BKD-ELISA). At regular intervals for 30 d, the numbers of R. salmoninarum shed by the injected fish were calculated on the basis of testing water samples by the membrane filtration-fluorescent antibody test (MF-FAT) and bacteriological culture. Mean BKD-ELISA optical densities (ODs) for fish in the low-dose injection group were not different from those of control fish [p > 0.05), and no R. salmoninarum were detected in water samples taken up to 30 d after injection of fish in the low-dose group. By 12 d after injection a proportion of the fish from the high-dose infection group had high (BKD-ELISA OD ??? 1.000) to severe (BKD-ELISA OD ??? 2.000) R. salmoninarum infection levels, and bacteria were detected in the water by both tests. However, measurable levels of R. salmoninarum were not consistently detected in the water until a proportion of the fish maintained high to severe infection levels for an additional 8 d. The concentrations of R salmoninarum in the water samples ranged from undetectable up to 994 cells ml-1 on the basis of the MF-FAT, and up to 1850 CFU ml-1 on the basis of bacteriological culture. The results suggest that chinook salmon infected with R. salmoninarum by injection of approximately 1 x 106 CFU fish-1 can be used as the source of infection in cohabitation challenges beginning 20 darter injection.

  4. Growth of the fish pathogen Renibacterium salmoninarum on different media.

    PubMed

    Bandín, I; Santos, Y; Barja, J I; Toranzo, A E

    1996-09-01

    In the present study, the ability of a group of Renibacterium salmoninarum strains to grow in the presence or absence of the amino acid cysteine and other mineral and organic sources of sulfur and nitrogen has been evaluated. Most of the isolates tested were able to grow on a mineral media supplemented with L-cysteine-HCl or other organic compounds, such as the vitamin thiamine and a casein hydrolysate (Bacto Casamino Acids, Difco). Bacterial growth was also recorded on commercial and specific media not supplemented with L-cysteine-HCl, or in which this amino acid was replaced by the compounds cited above.

  5. Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

    USGS Publications Warehouse

    Pascho, R.J.; Ongerth, J.E.

    2000-01-01

    The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 106 bacteria ml-1 and the bacteria exposed to chlorine at 1 mg l-1 for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 106 bacteria ml-1 and exposed to 0.8 mg l-1 free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p ??? 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 ??? 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 ??? 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated

  6. Demonstration of the salmonid humoral response to Renibacterium salmoninarum using a monoclonal antibody against salmonid immunoglobulin

    USGS Publications Warehouse

    Bartholomew, J.L.; Arkoosh , M.R.; Rohovec, J.S.

    1991-01-01

    The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytschawas compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarumpreparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarumantibody.

  7. Microevolution of Renibacterium salmoninarum: evidence for intercontinental dissemination associated with fish movements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Renibacterium salmoninarum is the causative agent of bacterial kidney disease, a major pathogen of salmonid fish species worldwide. Very low levels of intra-species genetic diversity have hampered efforts to understand the transmission dynamics and recent evolutionary history of this Gram-positive b...

  8. Recovery of Renibacterium salmoninarum from naturally infected salmonine stocks in Michigan using a modified culture protocol

    USGS Publications Warehouse

    Faisal, M.; Eissa, A.E.; Starliper, C.E.

    2010-01-01

    Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), is a fastidious and slow-growing bacterium that is extremely difficult to grow in vitro. Herein, we describe a modified primary culture protocol that encompasses a modified bacteriological culture medium and a tissue processing procedure. In order to facilitate the release of R. salmoninarum from granulomatous tissues, kidneys of infected fish were homogenized in a high speed stomacher. The kidney disease medium (KDM2), routinely used for primary culture of R. salmoninarum was modified by the addition of antibiotics and metabolites. When a relatively large inoculum of diluted kidney homogenate was streak-plate inoculated onto the modified KDM2, colonial growth of R. salmoninarum was achieved within 5-7. days, compared to the standard of two weeks or more. The modified procedure was then used to determine the prevalence of R. salmoninarum among representative captive and feral salmonid stocks in Michigan. Prevalence and clinical manifestations varied among species, strains of fish, and locations; however, R. salmoninarum isolates were biochemically homogenous. The improved primary culture procedure described in this study enabled selective and quick isolation of R. salmoninarum. Also, the isolates retrieved in this study constitute a unique biological resource for future studies of R. salmoninarum in the Laurentian Great Lakes. ?? 2009 University of Cairo.

  9. Effects of temperature on Renibacterium salmoninarum infection and transmission potential in Chinook salmon, Oncorhynchus tshawytscha (Walbaum)

    USGS Publications Warehouse

    Purcell, Maureen K.; McKibben, Constance L.; Pearman-Gillman, Schuyler; Elliott, Diane G.; Winton, James R.

    2016-01-01

    Renibacterium salmoninarum is a significant pathogen of salmonids and the causative agent of bacterial kidney disease (BKD). Water temperature affects the replication rate of pathogens and the function of the fish immune system to influence the progression of disease. In addition, rapid shifts in temperature may serve as stressors that reduce host resistance. This study evaluated the effect of shifts in water temperature on established R. salmoninarum infections. We challenged Chinook salmon with R. salmoninarum at 12°C for 2 weeks and then divided the fish into three temperature groups (8, 12 and 15°C). Fish in the 8°C group had significantly higher R. salmoninarum-specific mortality, kidney R. salmoninarum loads and bacterial shedding rates relative to the fish held at 12 or 15°C. There was a trend towards suppressed bacterial load and shedding in the 15°C group, but the results were not significant. Bacterial load was a significant predictor of shedding for the 8 and 12°C groups but not for the 15°C group. Overall, our results showed little effect of temperature stress on the progress of infection, but do support the conclusion that cooler water temperatures contribute to infection progression and increased transmission potential in Chinook salmon infected with R. salmoninarum.

  10. Prevalence of Renibacterium salmoninarum among downstream-migrating salmonids in the Columbia River

    USGS Publications Warehouse

    Sanders, J.E.; Long, J.J; Arakawa, C.K.; Bartholomew, J.L.; Rohovec, J.S.

    1992-01-01

    Bacterial kidney disease (BKD) is an important contributor to mortality of salmonids in hatcheries in the Columbia River basin. However, the impact of BKD on the survival of downstream migrants is difficult to determine because there is little information on the disease-related mortality among these fish. In this study, the impact of BKD on juvenile salmonids was examined by determining the percentage of downriver migrants infected with Renibacterium salmoninarum (the causative agent of BKD) and evaluating the effects of salt water on the progress of the disease. During the 2 years of this study, approximately 20% of the three species of migrating hatchery and wild salmonids (Oncorhynchus spp.) collected were infected with R. salmoninarum. Mortality caused by BKD increased when fish were held in salt water.

  11. Comparison of five techniques for the detection of Renibacterium salmoninarum in adult coho salmon.

    USGS Publications Warehouse

    Pascho, R.J.; Elliott, D.G.; Mallett, R.W.; Mulcahy, D.

    1987-01-01

    Samples of kidney, spleen, coelomic fluid, and blood from 56 sexually mature coho salmon Oncorhynchus kisutch were examined for infection by Renibacterium salmoninarum by five methods. The overall prevalence (all sample types combined) of R. salmoninarum in the fish was 100% by the enzyme-linked immunosorbent assay, 86% by the combined results of the direct fluorescent antibody and the direct filtration-fluorescent antibody techniques, 39% by culture, 11% by counterimmunoelectrophoresis, and 5% by agarose gel immunodiffusion. There was a significant positive correlation (P < 0.001) between the enzyme-linked immunosorbent assay absorbance levels and the counts by fluorescent antibody techniques for kidney, spleen, and coelomic fluid, and significant positive correlations (P < 0.001) in enzyme-linked immunosorbent assay absorbance levels for all four of the sample types.

  12. Evidence that coded-wire-tagging procedures can enhance transmission of Renibacterium salmoninarum in chinook salmon

    USGS Publications Warehouse

    Elliott, D.G.; Pascho, R.J.

    2001-01-01

    Binary coded wire tags (CWTs) are used extensively for identification and management of anadromous salmonid populations. A study of bacterial kidney disease (BKD) in two brood year groups of hatchery-reared spring chinook salmon Oncorhynchus tshawytscha provided strong evidence that horizontal transmission of Renibacterium salmoninarum, the causative agent of BKD, might be enhanced by CWT-marking procedures. About 4 months after CWTs were implanted in the snouts of juvenile fish, 14-16 different tissues were sampled from each of 60 fish per brood year group for histological analysis. Of the fish that were positive for R. salmoninarum by histological examination, 41% (7 of 17) of the 1988 brood year fish and 24% (10 of 42) of the 1989 brood year fish had BKD lesions confined to the head near the site of tag implantation. These lesions often resulted in the destruction of tissues of one or both olfactory organs. No focal snout infections were observed in fish that had not been marked with CWTs. Further data obtained from tissue analyses by use of an enzyme-linked immunosorbent assay and a fluorescent antibody test for detection of R. salmoninarum supported the hypothesis that infections of R. salmoninarum can be initiated in the snout tissues of CWT-marked fish and then spread to other organs. The tagging procedures might promote transmission of the pathogen among fish via contaminated tagging needles, by facilitating the entry of pathogens through the injection wound, or both. Limited evidence from this study suggested that implantation of passive integrated transponder tags in the peritoneal cavities of fish might also promote the transmission of R. salmoninarum or exacerbate existing infections. The results indicated a need for strict sanitary procedures during the tagging of fish in populations positive for R. salmoninarum to reduce the probability of enhanced horizontal transmission of the pathogen.

  13. Both msa genes in Renibacterium salmoninarum are needed for full virulence in bacterial kidney disease

    USGS Publications Warehouse

    Coady, A.M.; Murray, A.L.; Elliott, D.G.; Rhodes, L.D.

    2006-01-01

    Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile Chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression. Copyright ?? 2006, American Society for Microbiology. All Rights Reserved.

  14. Development of a specific biotinylated DNA probe for the detection of Renibacterium salmoninarum.

    PubMed Central

    Hariharan, H; Qian, B; Despres, B; Kibenge, F S; Heaney, S B; Rainnie, D J

    1995-01-01

    A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA probe, therefore, has the potential for use in the diagnosis of BKD of fish. Images Fig. 2. Fig. 3. PMID:8548693

  15. Bactericidal activity of juvenile chinook salmon macrophages against Aeromonas salmonicida after exposure to live or heat-killed Renibacterium salmoninarum or to soluble proteins produced by R. salmoninarum

    USGS Publications Warehouse

    Siegel, D.C.; Congleton, J.L.

    1997-01-01

    Macrophages isolated from the anterior kidney of juvenile chinook salmon Oncorhynchus tshawytscha in 96-well microtiter plates were exposed for 72 h to 0, 105, or 106 live or heat-killed Renibacterium salmoninarum cells per well or to 0, 0.1, 1.0, or 10 ??g/mL of R. salmoninarum soluble proteins. After treatment, the bactericidal activity of the macrophages against Aerornonas salmonicida was determined by a colorimetric assay based on the reduction of the tetrazolium dye MTT to formazan by viable bacteria. The MTT assay was modified to allow estimation of the percentage of bacteria killed by reference to a standard curve relating the number of bacteria added to microtiter wells to absorbance by formazan at 600 nm. The live and heat-killed R. salmoninarum treatments significantly (P < 0.001) increased killing of A. salmonicida by chinook salmon macrophages. In each of the five trials, significantly (P < 0.05) greater increases in killing occurred after exposure to 105 R. salmoninarum cells than to 106 R. salmoninarum cells per well. In contrast, treatment of macrophages with 10 ??g/mL R. salmoninarum soluble proteins significantly (P < 0.001) decreased killing of A. salmonicida, but treatment with lower doses did not. These results show that the bactericidal activity of chinook salmon macrophages is stimulated by exposure to R. salmoninarum cells at lower dose levels but inhibited by exposure to R. salmoninarum cells or soluble proteins at higher dose levels.

  16. Pathological and immunological responses associated with differential survival of Chinook salmon following Renibacterium salmoninarum challenge

    USGS Publications Warehouse

    Metzger, David C.; Elliott, Diane G.; Wargo, Andrew; Park, Linda K.; Purcell, Maureen K.

    2010-01-01

    Chinook salmon Oncorhynchus tshawytscha are highly susceptible to Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). Previously we demonstrated that introduced Chinook salmon from Lake Michigan, Wisconsin (WI), USA, have higher survival following R. salmoninarum challenge relative to the progenitor stock from Green River, Washington, USA. In the present study, we investigated the pathological and immunological responses that are associated with differential survival in the 2 Chinook salmon stocks following intra-peritoneal R. salmoninarum challenge of 2 different cohort years (2003 and 2005). Histological evaluation revealed delayed appearance of severe granulomatous lesions in the kidney and lower overall prevalence of membranous glomerulopathy in the higher surviving WI stock. The higher survival WI stock had a lower bacterial load at 28 d post-infection, as measured by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). However, at all other time points, bacterial load levels were similar despite higher mortality in the more susceptible Green River stock, suggesting the possibility that the stocks may differ in their tolerance to infection by the bacterium. Interferon-γ, inducible nitric oxide synthase (iNOS), Mx-1, and transferrin gene expression were up-regulated in both stocks following challenge. A trend of higher iNOS gene expression at later time points (≥28 d post-infection) was observed in the lower surviving Green River stock, suggesting the possibility that higher iNOS expression may contribute to greater pathology in that stock.

  17. Impact of stressors on transmission potential of Renibacterium salmoninarum in Chinook salmon

    USGS Publications Warehouse

    Purcell, Maureen K.; Winton, James R.

    2014-01-01

    Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD) affecting several species of Pacific salmon.  The severity of BKD can range from a chronic infection to overt disease with high mortality as in the case of large losses of adult Chinook salmon (Oncorhynchus tshawytscha) in the Great Lakes during late 1980s. The goal of this study was to empirically evaluate how environmental stressors relevant to the Great Lakes impact R. salmoninarum disease progression and bacterial shedding, the latter parameter being a proxy of horizontal transmission. In the first study (Aim 1), we focused on how endogenous host thiamine levels and dietary fatty acids impacted resistance of Chinook salmon to R. salmoninarum. Juvenile fish were fed one of four experimental diets, including a (1) thiamine replete diet formulated with fish oil, (2) thiamine deplete diet formulated with fish oil, (3) thiamine replete diet formulated with soybean oil, and (4) thiamine deplete diet formulated with soybean oil, before being challenged with buffer or R. salmoninarum. We observed significantly higher mortality in the R. salmoninarum infected groups relative to the corresponding mock controls in only the thiamine replete diet groups. We also observed a significant effect of time and diet on kidney bacterial load and bacterial shedding, with a significant trend towards higher shedding and bacterial load in the fish oil – thiamine replete diet group. However, during the course of the study, unexpected mortality occurred in all groups attributed to the myxozoan parasite Ceratomyxa shasta. Since the fish were dually-infected with C. shasta, we evaluated parasite DNA levels (parasitic load) in the kidney of sampled fish. We found that parasite load varied across time points but there was no significant effect of diet. However, parasite load did differ significantly between the mock and R. salmoninarum challenge groups with a trend towards longer persistence of C. shasta

  18. Comparison of traditional and molecular methods for detection of Renibacterium salmoninarum

    USGS Publications Warehouse

    Pascho, R.J.; Elliott, D.G.; Chase, D.M.; Cunningham, C.O.

    2002-01-01

    Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum occurs in most parts of the world where wild or cultured salmonid fishes are present. Several extensive reviews have been written on the pathogen and the disease (Fryer and Sanders, 1981; Austin and Austin, 1987; Elliott et al., 1989; Evelyn, 1993; Evenden et al., 1993; Fryer and Lannan, 1993). Bacterial kidney disease can cause serious mortality in juvenile salmonids in both fresh water and seawater, and also in prespawning adults. Although the chronic nature of the disease has hindered accurate estimates of fish losses, particularly in feral fish populations, BKD is one the most important bacterial diseases affecting cultured salmonids, with reported losses as high as 80% in stocks of Pacific salmon (Oncorhynchus spp.) and 40% in stocks of Atlantic salmon (Salmo salar) (Evenden et al., 1993).

  19. Performance of serum-free broth media for growth of Renibacterium salmoninarum

    USGS Publications Warehouse

    Starliper, C.E.; Schill, W.B.; Mathias, J.

    1998-01-01

    Growth of Renibacterium salmoninarum was compared in 14 different broth media; 13 serum-free, and 1 that contained newborn calf serum, KDM2+M. Supplementation with 1% v/v R. salmoninarum MCO4M metabolite was evaluated for 6 of the media that do not utilize it as part of their ingredients. Viable cells were enumerated on Days 10, 20, and 30 post inoculation to evaluate performance. The experiment was repeated 3 times using high, low, and medium (trials 1 to 3, respectively) cell concentrations as inoculum. In general there was no optimal medium and all performed well. The choice of which to employ depends on the ease of preparation and presence of certain ingredients that might affect subsequent assays. In trials 2 and 3, the pH was estimated using test papers at the same time as cells were counted. Maximum pH increase occurred with KDM2+M and those media containing charcoal. For most media, a simple pH determination could be used as a means to check that growth has occurred in a culture, particularly if charcoal was added directly to the media and a visual inspection could not be made to detect growth.

  20. Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis

    USGS Publications Warehouse

    Chase, D.M.; Elliott, D.G.; Pascho, R.J.

    2006-01-01

    Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

  1. Real-time PCR for quantification of viable Renibacterium salmoninarum in chum salmon Oncorhynchus keta.

    PubMed

    Suzuki, Kunio; Sakai, D K

    2007-03-13

    Quantification of msa gene mRNA of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was investigated using reverse transcription followed by real-time PCR assay on R. salmoninarum in culture, and in experimentally challenged chum salmon Oncorhynchus keta fry kidney tissues (total of 70 samples) after intraperitoneal (i.p.) injection and bath infection. Correlations of msa gene mRNA concentrations with culturable cell concentrations (as colony forming units [CFU]), determined by drop-plate culture method on selective kidney disease medium (SKDM) agar through a 12 wk incubation time, and msa gene DNA concentrations by real-time PCR assay were examined. Furthermore, ovarian fluid samples from wild chum salmon adults with no clinical signs of disease were collected from 8 rivers and from clinically infected kokanee 0. nerka and masu salmon O. masou that were reared in 1 and 2 hatcheries, respectively (total of 414 samples). All samples were examined by nested PCR assay. Then, positive samples were examined by real-time PCR assays for mRNA and DNA; mRNA was detectable at 8 log units (5.0 x 101 to 5.0 x 10(9) copies p11(-1)) with high correlation (R2 = 0.999). The mRNA concentration correlated with CFU in kidney tissue from fish infected by i.p. injection (R2 = 0.924), by bath infection (R2 = 0.502) and in culture (R2 = 0.888). R. salmoninarum was detected and quantified by real-time PCR assay for mRNA in ovarian fluid samples in both subclinically infected chum salmon adults and clinically infected kokanee and masu salmon adults; detection rates ranged from 0 to 44.4% and concentrations ranged from 9.7 x 10(2) to 5.6 x 10(5) copies pl(-1). These results indicate that real-time PCR assay for the mRNA is a rapid, sensitive and reliable method to detect and quantify the viability of R. salmoninarum in kidney and ovarian fluid samples of salmonid fishes with both clinical and subclinical infection of the pathogen.

  2. Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum

    USGS Publications Warehouse

    Weins, G.; Chien, M.S.; Winton, J.R.; Kaatari, S.L.

    1999-01-01

    Renibacterium salmoninarum, the causative agent of bacterial kidney disease, produces large quantities of a 57-58 kDa protein (p57) during growth in broth culture and during infection of salmonid fish. Biological activities of secreted p57 include agglutination of salrnonid leucocytes and rabbit erythrocytes. We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4Cl1, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a nlonomer that retains salrnonid leucocyte agglutinat~ng activity. The 3 MAbs bound a recombinant, amino-terminal fragment of p57 (211 aa) but not a carboxy-terminal fragment (315 aa) demonstrating that the neutralizing epitopes are located within the amino-terminal portion of p57. When combinations of the MAbs were used in an antigen capture ELISA. the epitopes recognized by the 3 MAbs were shown to be sterically separate. However, when the same MAb was used as both the coating and detection MAb, binding of the biotinylated detection MAb was not observed. These data indicate that the epitopes recognized by the 3 agglutination-blocking antibodies are functionally available only once per molecule and that native p57 exists as a monomer Similar ELISA results were obtained when kidney tissues from 3 naturally infected chinook salmon were assayed. Finally, a p57 monomer was purified using anion exchange and size exclusion chromatography that retained in vitro agglutinating activity. A model in which p57 is released from R. salmoninarum as a biologically active monomer during infection of salmonid fish is proposed.

  3. Evaluation by enzyme-linked immunosorbent assay (ELISA) of Renibacterium salmoninarum bacterins affected by persistence of bacterial antigens

    USGS Publications Warehouse

    Pascho, R.J.; Goodrich, T.D.; McKibben, C.L.

    1997-01-01

    Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with a bacterin containing killed Renibacterium salmoninarum cells delivered alone or in an oil-based adjuvant. We evaluated the relative abilities of the batterins to prevent the initiation or progression of infection in fish challenged by waterborne exposure to R. salmoninarum. Sixty-one days after vaccination, fish were held for 24 h in water containing either no bacteria or approximately 1.7 x 103, 1.7 x 105, or 5.3 x 106 live R. salmoninarum cells/mL. An enzyme-linked immunosorbent assay (ELISA) was used to monitor changes in the levels of R. salmoninarum antigen in live fish before and after the immersion challenges. High levels of R. salmoninarum antigens were detected by ELISA in kidney-spleen tissue homogenates from vaccinated fish immediately before the challenges. Levels of those antigens remained high in the tissues of unchallenged fish throughout the study. We found that the ELISA used in this study may be unsuitable for evaluating the efficacy of batterins because it did not distinguish antigens produced by the challenge bacteria during an infection from those of the bacterins. Groups of control and vaccinated fish also were injected with either 1.7 x 104 or 1.7 x 106 R. salmoninarum cells and served as R. salmoninarum virulence controls. Relative survival among the various subgroups in the injection challenge suggests that adverse effects might have been associated with the adjuvant used in this study. The lowest survival at both injection challenge levels was among fish vaccinated with bacteria in adjuvant.

  4. Testing of candidate non-lethal sampling methods for detection of Renibacterium salmoninarum in juvenile Chinook salmon Oncorhynchus tshawytscha

    USGS Publications Warehouse

    Elliott, Diane G.; McKibben, Constance L.; Conway, Carla M.; Purcell, Maureen K.; Chase, Dorothy M.; Applegate, Lynn M.

    2015-01-01

    Non-lethal pathogen testing can be a useful tool for fish disease research and management. Our research objectives were to determine if (1) fin clips, gill snips, surface mucus scrapings, blood draws, or kidney biopsies could be obtained non-lethally from 3 to 15 g Chinook salmon Oncorhynchus tshawytscha, (2) non-lethal samples could accurately discriminate between fish exposed to the bacterial kidney disease agent Renibacterium salmoninarum and non-exposed fish, and (3) non-lethal samples could serve as proxies for lethal kidney samples to assess infection intensity. Blood draws and kidney biopsies caused ≥5% post-sampling mortality (Objective 1) and may be appropriate only for larger fish, but the other sample types were non-lethal. Sampling was performed over 21 wk following R. salmoninarum immersion challenge of fish from 2 stocks (Objectives 2 and 3), and nested PCR (nPCR) and real-time quantitative PCR (qPCR) results from candidate non-lethal samples were compared with kidney tissue analysis by nPCR, qPCR, bacteriological culture, enzyme-linked immunosorbent assay (ELISA), fluorescent antibody test (FAT) and histopathology/immunohistochemistry. R. salmoninarum was detected by PCR in >50% of fin, gill, and mucus samples from challenged fish. Mucus qPCR was the only non-lethal assay exhibiting both diagnostic sensitivity and specificity estimates >90% for distinguishing between R. salmoninarum-exposed and non-exposed fish and was the best candidate for use as an alternative to lethal kidney sample testing. Mucus qPCR R. salmoninarum quantity estimates reflected changes in kidney bacterial load estimates, as evidenced by significant positive correlations with kidney R. salmoninaruminfection intensity scores at all sample times and in both fish stocks, and were not significantly impacted by environmentalR. salmoninarum concentrations.

  5. Renibacterium salmoninarum in spring-summer chinook salmon smolts at dams on the Columbia and Snake Rivers

    USGS Publications Warehouse

    Elliott, D.G.; Pascho, R.J.; Jackson, L.M.; Matthews, G.M.; Harmon, J.R.

    1997-01-01

    We evaluated Renibacterium salmoninarum infection in smolts of hatchery and wild spring-summer chinook salmon Oncorhynchus tshawytscha sampled during most of the out-migration at Little Goose (1988) and Lower Granite dams (1988-1991) on the Snake River and at Priest Rapids and McNary dams on the Columbia River (1988-1990). We sampled 860-2,178 fish per dam each year. Homogenates of kidney-spleen tissue from all fish were tested for the presence of R. salmoninarum antigens by the enzyme-linked immunosorbent assay (ELISA), and homogenates from 10% of the fish were examined by the fluorescent antibody technique (FAT). Although only 1-11% of fish sampled at a given dam during any 1 year exhibited lesions characteristic of bacterial kidney disease, 86-100% of the fish tested positive for R. salmoninarum antigen by ELISA, whereas 4-17% of the fish tested positive by the FAT. During most years, a majority (68-87%) of fish testing positive by the ELISA had low R. salmoninarum antigen levels, but in 1989, 53% of positive fish from Lower Granite Dam and 52% from McNary Dam showed medium-to-high antigen levels. For most years, the highest mean antigen levels were measured in fish sampled after 75% of the total out-migrants had passed a given dam. When the largest numbers of fish were being collected for bypass or downriver transportation, mean antigen levels were relatively low.

  6. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

    USGS Publications Warehouse

    Elliott, D.G.; Applegate, L.J.; Murray, A.L.; Purcell, M.K.; McKibben, C.L.

    2013-01-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

  7. Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene rosa in virulent and attenuated strains of Renibacterium salmoninarum

    USGS Publications Warehouse

    O'Farrell, C. L.; Strom, M.S.

    1999-01-01

    Virulence mechanisms utilized by the salmonid fish pathogen Renibacterium salmoninarum are poorly understood. One potential virulence factor is p57 (also designated MSA for major soluble antigen), an abundant 57 kDa soluble protein that is predominately localized on the bacterial cell surface with significant levels released into the extracellular milieu. Previous studies of an attenuated strain, MT 239, indicated that it differs from virulent strains in the amount of surface-associated p57. In this report, we show overall expression of p57 in R. salmoninarum MT 239 is considerably reduced as compared to a virulent strain, ATCC 33209. The amount of cell-associated p57 is decreased while the level of p57 in the culture supernatant is nearly equivalent between the strains. To determine if lowered amount of cell-associated p57 was due to a sequence defect in p57, a genetic comparison was performed. Two copies of the gene encoding p57 (msa1 and msa2) were found in 33209 and MT 239, as well as in several other virulent isolates. Both copies from 33209 and MT 239 were cloned and sequenced and found to be identical to each other, and identical between the 2 strains. A comparison of msa1 and msa2 within each strain showed that their sequences diverge 40 base pairs 5, to the open reading frame, while sequences 3' to the open reading frame are essentially identical for at least 225 base pairs. Northern blot analysis showed no difference in steady state levels of rosa mRNA between the 2 strains. These data suggest that while cell-surface localization of p57 may be important for R. salmoninarum virulence, the differences in localization, and total p57 expression between 33209 anti MT 239 are not due to differences in rosa sequence or differences in steady state transcript levels.

  8. Effects of Renibacterium salmoninarum on olfactory organs of Chinook salmon (Oncorhynchus tshawytscha) marked with coded wire tags

    USGS Publications Warehouse

    Elliott, Diane G.; Conway, Carla M.; Bruno, D.W.; Elliott, D.G.; Nowak, B.

    2014-01-01

    Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum can cause significant morbidity and mortality in Chinook salmon (Oncorhynchus tshawytscha), particularly in Chinook salmon of the stream (spring) life history type, which migrate to sea as yearlings rather than subyearlings. R. salmoninarum can be transmitted vertically from the female parent to the progeny in association with the egg, as well as horizontally from fish to fish. This study was conducted as part of a research project to investigate whether the prevalence and intensity of R. salmoninarum infections in adult spring Chinook salmon could affect the survival and pathogen prevalence and intensity in their progeny (Pascho et al., 1991, 1993; Elliott et al., 1995). Fish from two brood years (1988 and 1989) were reared at Dworshak National Fish Hatchery (Idaho, USA) for about 1-1/2 years, released as yearling smolts, and allowed to migrate to the Pacific Ocean for maturation. The majority of progeny fish were marked with coded wire tags (CWTs) about 4 months before they were released from the hatchery so that adult returns could be monitored. The CWTs were implanted in the snouts of the fish by an experienced team of fish markers using automated wire-tagging machines. The intended placement site was the cartilage, skeletal muscle or loose connective tissue of the snout.

  9. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

    USGS Publications Warehouse

    Pascho, Ronald J.; Chase, Dorothy M.; McKibben, Constance L.

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 × 109cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 × 104cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

  10. Swimming endurance of bull trout, lake trout, arctic char, and rainbow trout following challenge with Renibacterium salmoninarum

    USGS Publications Warehouse

    Jones, D.T.; Moffitt, C.M.

    2004-01-01

    We tested the swimming endurance of juvenile bull trout Salvelinus confluentus, lake trout S. namaycush, Arctic char S. alpinus, and rainbow trout Oncorhynchus mykiss at 9??C and 15??C to determine whether sublethal infection from a moderate challenge of Renibacterium salmoninarum administered months before testing affected the length of time fish could maintain a swimming speed of 5-6 body lengths per second in an experimental flume. Rainbow trout and Arctic char swam longer in trials than did bull trout or lake trout, regardless of challenge treatment. When we tested fish 14-23 weeks postchallenge, we found no measurable effect of R. salmoninarum on the swimming endurance of the study species except for bull trout, which showed a mixed response. We conducted additional trials with bull trout 5-8 weeks postchallenge to determine whether increasing the challenge dose would affect swimming endurance and hematocrit. In those tests, bull trout with clinical signs of disease and those exposed to the highest challenge doses had significantly reduced swimming endurance compared with unchallenged control fish. Fish hematocrit levels measured at the end of all swimming endurance tests varied among species and between test temperatures, and patterns were not always consistent between challenged and control fish.

  11. Enzyme-linked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent for salmonid bacterial kidney disease

    USGS Publications Warehouse

    Pascho, R.J.; Mulcahy, D.

    1987-01-01

    A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of a soluble fraction of Renibacterium salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. The Costar® Serocluster™ EIA microplate gave the highest absorbance and signal-to-noise ratios among seven types tested. Including Tween 80 in the wash buffer resulted in higher absorbances than Tween 20 when antigen was present. Background absorbance did not increase when Tween 80 was added to the wash buffer, but did when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37 °C and 16 h at 4 °C. Antigen attachment to antibody-coated microplate wells depended more on incubation temperature than duration; we adopted a 3-h incubation at 25 °C. Conjugate incubation for longer than 1 h at 37 °C or 3 h at 25 °C resulted in unacceptable background levels. No cross-reactions resulted from heat-extracted antigens of 10 other species of bacteria. The optimized ELISA is a 6-h test that enables detection of levels of soluble antigen as low as 2–20 ng.

  12. Mortality and kidney histopathology of Chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains

    USGS Publications Warehouse

    O'Farrell, Caroline L.; Elliott, Diane G.; Landolt, Marsha L.

    2001-01-01

    An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 x 10(3) or 1 x 10(6) bacteria fish(-1), or by a 24 h immersion in 1 x 10(5) or 1 x 10(7) bacteria ml(-1). For 22 wk fish were held in 12 degrees C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73 %). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.

  13. Mortality and kidney histopathology of chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains

    USGS Publications Warehouse

    O'Farrell, C. L.; Elliott, D.G.; Landolt, M.L.

    2000-01-01

    An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 ?? 103 or 1 ?? 106 bacteria fish-1, or by a 24 h immersion in 1 ?? 105 or 1 ?? 107 bacteria ml-1. For 22 wk fish were held in 12??C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73%). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.

  14. Vulnerability to predation and physiological stress responses in juvenile chinook salmon (Oncorhynchus tshawytscha) experimentally infected with Renibacterium salmoninarum

    USGS Publications Warehouse

    Mesa, M.G.; Poe, T.P.; Maule, A.G.; Schreck, C.B.

    1998-01-01

    We experimentally infected juvenile chinook salmon (Oncorhynchus tshawytscha) with Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease (BKD), to examine the vulnerability to predation of fish with differing levels of Rs infection and assess physiological change during progression of the disease. Immersion challenges conducted during 1992 and 1994 produced fish with either a low to moderate (1992) or high (1994) infection level of Rs during the 14-week postchallenge rearing period. When equal numbers of treatment and unchallenged control fish were subjected to predation by either northern squaw fish (Ptychocheilus oregonensis) or smallmouth bass (Micropterus dolomieui), Rs-challenged fish were eaten in significantly greater numbers than controls by nearly two to one. In 1994, we also sampled fish every 2 weeks after the challenge to determine some stressful effects of Rs infection. During disease progression in fish, plasma cortisol and lactate increased significantly whereas glucose decreased significantly. Our results indicate the role that BKD may play in predator-prey interactions, thus ascribing some ecological significance to this disease beyond that of direct pathogen-related mortality. In addition, the physiological changes observed in our fish during the chronic progression of BKD indicate that this disease is stressful, particularly during the later stages.

  15. Incidence of Renibacterium salmoninarum infections in juvenile hatchery spring chinook salmon in the Columbia and Snake Rivers

    USGS Publications Warehouse

    Maule, A.G.; Rondorf, D.W.; Beeman, J.W.; Haner, P.V.

    1996-01-01

    From 1988 through 1992, we assessed the prevalence (frequency of occurrence) and severity (degree of infection) of Renibacterium salmoninarum (RS) among fish in marked groups of Columbia River basin and Snake River basin hatchery spring chinook salmon Oncorhynchus tshawytscha before release and during their seaward migration. During the study, prevalence of RS infection decreased (from >90% to <65%) in six of the eight hatchery groups. We attributed this decrease to changes in hatchery practices that reduced vertical and horizontal transmission. Fish from Snake River hatcheries had a higher prevalence of infection when sampled at dams (mean >90%) than in the hatchery (mean <70%), but there were no differences in similar comparisons of Columbia River fish. Although prevalence and severity of RS infection were not correlated in the groups studied, it appears that fish from the Snake River were more severely infected than those from the Columbia River. Some groups of Snake River fish had higher severity of infection at dams than in the hatchery, but infection in fish from Columbia River hatcheries did not change. These differences between Snake River and Columbia River fish might have resulted from differences in river conditions and the distances from hatcheries to dams.

  16. A single Alal 39-to-Glu substitution in the Renibacterium salmoninarum virulence-associated protein p57 results in antigenic variation and is associated with enhanced p57 binding to Chinook salmon leukocytes

    USGS Publications Warehouse

    Wiens, Gregory D.; Pascho, Ron; Winton, James R.

    2002-01-01

    The gram-positive bacterium Renibacterium salmoninarum produces relatively large amounts of a 57-kDa protein (p57) implicated in the pathogenesis of salmonid bacterial kidney disease. Antigenic variation in p57 was identified by using monoclonal antibody 4C11, which exhibited severely decreased binding to R. salmoninarum strain 684 p57 and bound robustly to the p57 proteins of seven other R. salmoninarum strains. This difference in binding was not due to alterations in p57 synthesis, secretion, or bacterial cell association. The molecular basis of the 4C11 epitope loss was determined by amplifying and sequencing the two identical genes encoding p57, msa1 and msa2. The 5′ and coding sequences of the 684 msa1 and msa2 genes were identical to those of the ATCC 33209 msa1and msa2 genes except for a single C-to-A nucleotide mutation. This mutation was identified in both the msa1 and msa2 genes of strain 684 and resulted in an Ala139-to-Glu substitution in the amino-terminal region of p57. We examined whether this mutation in p57 altered salmonid leukocyte and rabbit erythrocyte binding activities. R. salmoninarum strain 684 extracellular protein exhibited a twofold increase in agglutinating activity for chinook salmon leukocytes and rabbit erythrocytes compared to the activity of the ATCC 33209 extracellular protein. A specific and quantitative p57 binding assay confirmed the increased binding activity of 684 p57. Monoclonal antibody 4C11 blocked the agglutinating activity of the ATCC 33209 extracellular protein but not the agglutinating activity of the 684 extracellular protein. These results indicate that the Ala139-to-Glu substitution altered immune recognition and was associated with enhanced biological activity of R. salmoninarum 684 p57.

  17. Comparison of two fluorescent antibody techniques (FATS) for detection and quantification of Renibacterium salmoninarum in coelomic fluid of spawning chinook salmon Oncorhynchus tshawytscha

    USGS Publications Warehouse

    Elliott, D.G.; McKibben, C.L.

    1997-01-01

    Two versions of the fluorescent antibody technique (FAT) were compared for detection and quantification of Renibacterium salmoninarum in coelomic fluid samples from naturally infected spawning chinook salmon Oncorhynchus tshawytscha. For the membrane filtration-FAT (MF-FAT), trypsin-treated samples were passed through 0.2 ??m polycarbonate filters to concentrate bacteria for direct enumeration by immunofluorescence microscopy. For the smear-FAT (S-FAT), samples were centrifuged at 8800 x g for 10 min and the pelleted material was smeared on slides for immunofluorescence staining Detected prevalences of Renibacterium salmoninarum were 1.8 to 3.4 times higher by the MF-FAT than by the S-FAT: differences were significant at p ??? 0.0002. The S-FAT consistently detected R. salmoninarum only in samples with calculated bacterial concentrations ??? 2.4 x 103 cells ml-1 by MF-FAT testing. Increasing the area examined on a filter or slide from 50 to 100 microscope fields at 1000x magnification resulted in the detection of a maximum of 4% additional positive samples by the MF-FAT and 7% additional positive samples by the S-FAT. In individual samples for which bacterial counts were obtained by both the MF-FAT and the S-FAT, the counts averaged from 47 times (??30 SD) to 175 times (??165 SD) higher by the MF-FAT. Centrifugation of samples at 10000 x g for 10 min resulted in a 4-fold increase in mean bacterial counts by the S-FAT compared with a 10-min centrifugation at 2000 x g, but the highest calculated bacterial concentration obtained by S-FAT testing was more than 6-fold lower than that obtained for the same sample by MF-FAT testing. Because of its greater sensitivity, the MF-FAT is preferable to the S-FAT for use in critical situations requiring the detection of low numbers of R. salmoninarum.

  18. Influence of infection with Renibacterium salmoninarum on susceptibility of juvenile spring chinook salmon to gas bubble trauma

    USGS Publications Warehouse

    Weiland, L.K.; Mesa, M.G.; Maule, A.G.

    1999-01-01

    During experiments in our laboratory to assess the progression and severity of gas bubble trauma (GBT) in juvenile spring chinook salmon Oncorhynchus tshawytscha, we had the opportunity to assess the influence of Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease, on the susceptibility of salmon to GBT. We exposed fish with an established infection of Rs to 120% total dissolved gas (TDG) for 96 h and monitored severity of GBT signs in the fins and gills, Rs infection level in kidneys by using an enzyme-linked immunosorbent assay (ELISA), and mortality. Mortality occurred rapidly after exposure to 120% TDG, with a LT20 (time necessary to kill 20% of the population) of about 37 h, which is at a minimum about 16% earlier than other bioassays we have conducted using fish that had no apparent signs of disease. Fish that died early (from 31 to 36 h and from 49 to 52 h) had significantly higher infection levels (mean ?? SE ELISA absorbance = 1.532 ?? 0.108) than fish that survived for 96h (mean ?? SE ELISA absorbance = 0.828 ?? 0.137). Fish that died early also had a significantly greater number of gill filaments occluded with bubbles than those that survived 96 h. Conversely, fish that survived for 96 h had a significantly higher median fin severity ranking than those that died early. Our results indicate that fish with moderate to high levels of Rs infection are more vulnerable to the effects of dissolved gas supersaturation (DGS) and die sooner than fish with lower levels of Rs infection. However, there is a substantial amount of individual variation in susceptibility to the apparent cumulative effects of DGS and Rs infection. Collectively, our findings have important implications to programs designed to monitor the prevalence and severity of GBT in juvenile salmonids in areas like the Columbia River basin and perhaps elsewhere.

  19. Interaction of infection with Renibacterium salmoninarum and physical stress in juvenile chinook salmon: Physiological responses, disease progression, and mortality

    USGS Publications Warehouse

    Mesa, M.G.; Maule, A.G.; Schreck, C.B.

    2000-01-01

    We experimentally infected juvenile spring chinook salmon Oncorhynchus tshawytscha with Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease (BKD), in order to compare the physiological responses of Rs-infected and Rs-noninfected fish to a series of multiple, acute stressors and to determine whether exposure to these stressors worsens the infection and leads to increased mortality. After subjecting groups of fish to a waterborne challenge of Rs, we sampled them biweekly to monitor infection levels, mortality, and some stress-related physiological changes. As infections worsened, fish developed decreased hematocrits and blood glucose levels and increased levels of cortisol and lactate, indicating that BKD is stressful, particularly during the later stages. Eight weeks after the challenge, when fish had moderate to high infection levels, we subjected them, along with unchallenged control fish, to three 60-s bouts of hypoxia, struggling, and mild agitation that were separated by 48-72 h. Our results indicate that the imposition of these stressors on Rs-infected fish did not lead to higher infection levels or increased mortality when compared with diseased fish that did not receive the stressors. Furthermore, the kinetics of plasma cortisol, glucose, and lactate over a 24-h period following each application of the stressor were similar between fish with moderate to high Rs infections and those that had low or no detectable infection. Some differences in the stress responses of these two groups did exist, however. Most notably, fish with moderate to high Rs infections had higher titers of cortisol and lactate prior to each application of the stressor and also were unable to consistently elicit a significant hyperglycemia in response to the stressors. Collectively, our results should be important in understanding the impact that BKD has on the survival of juvenile salmonids, but we caution that our results represent the combined effects of one

  20. Monitoring of the in-river migration of smolts from two groups of spring chinook salmon, Oncorhynchus tshawytscha (Walbaum), with different profiles of Renibacterium salmoninarum infection

    USGS Publications Warehouse

    Pascho, R.J.; Elliott, D.G.; Achord, S.

    1993-01-01

    Broodstock segregation based on the measurement of maternal Renibacterium salmoninarum infection levels by the enzyme-linked immunosorbent assay (ELISA) and the membrane filtration-fluorescent antibody technique (MF-FAT) was previously shown to affect the prevalence and levels of bacterial kidney disease (BKD) in progeny of chinook salmon, Oncorhynchus tshawytscha (Walbaum), during hatchery rearing. Subgroups of fish from that study were marked with passive integrated transponder (PIT) tags, and monitored by PIT-tag detectors during the first 342km of their migration to the Pacific Ocean. Differences in the recovery of tagged fish were significant (P≤ 0·01) at each detection point and became more pronounced as the fish moved downstream. Cumulative recoveries of fish from the low-BKD group and the high-BKD group, respectively, were 31% and 28% after 116km, 44% and 37% after 176km, and 51% and 42% after 342km. There were no apparent differences in the migration timing of the two groups to the first detection point. The data suggested that in-river survival was higher in the progeny group from parents that had low R. salmoninarum infection levels or tested negative for R. salmoninarum (low-BKD group) than in the group female parents with high infection levels (high-BKD group).

  1. Membrane filtration – Fluorescent antibody staining procedure for detecting and quantifying Renibacterium salmoninarum in coelomic fluid of Chinook Salmon (Oncorhynchus tshawytscha)

    USGS Publications Warehouse

    Elliott, D.G.; Barila, T.Y.

    1987-01-01

    We developed a rapid method for detecting and quantifying the pathogen Renibacterium salmoninarum in coelomic fluid of spring chinook salmon (Oncorhynchus tshawytscha) by concentrating the bacteria on 0.2-μm polycarbonate filters and staining them with specific fluorescein-labeled antibody. Centrifugation of samples and resuspension of the sedimented material in phosphate-buffered saline containing Triton X-100 increased the ease of filtration. Background fluorescence was reduced by counterstaining filters with Eriochrome black T. Postfiltration staining, rinsing, and counterstaining were done in the syringe-mounted filter holders, reducing handling of the filters and possible loss of bacteria. The number of bacteria detected by the filtration – fluorescent antibody technique in a broth culture of R. salmoninarum ranged from 6.7 × 107to7.6 × 107/mL and was slightly higher than that determined by plate count (9.6 × 106/mL). Increasing the sample dilution or decreasing the number of microscope fields examined generally increased the variability of filter counts of R. salmoninarum. Using the filtration – fluorescent antibody technique, we detected the bacterium in the coelomic fluid of 85% of spawning female spring chinook salmon sampled from a hatchery population.

  2. Identifying copy number variation of the dominant virulence factors msa and p22 within genomes of the fish pathogen Renibacterium salmoninarum

    PubMed Central

    Gulla, Snorre; Feil, Edward J.; Nørstebø, Simen Foyn; Rhodes, Linda D.

    2016-01-01

    Renibacterium salmoninarum is the causative agent of bacterial kidney disease, an important disease of farmed and wild salmonid fish worldwide. Despite the wide spatiotemporal distribution of this disease and habitat pressures ranging from the natural environment to aquaculture and rivers to marine environments, little variation has been observed in the R. salmoninarum genome. Here we use the coverage depth from genomic sequencing corroborated by real-time quantitative PCR to detect copy number variation (CNV) among the genes of R. salmoninarum. CNV was primarily limited to the known dominant virulence factors msa and p22. Among 68 isolates representing the UK, Norway and North America, the msa gene ranged from two to five identical copies and the p22 gene ranged from one to five copies. CNV for these two genes co-occurred, suggesting they may be functionally linked. Isolates carrying CNV were phylogenetically restricted and originated predominantly from sites in North America, rather than the UK or Norway. Although both phylogenetic relationship and geographical origin were found to correlate with CNV status, geographical origin was a much stronger predictor than phylogeny, suggesting a role for local selection pressures in the repeated emergence and maintenance of this trait. PMID:28348850

  3. Identifying copy number variation of the dominant virulence factors msa and p22 within genomes of the fish pathogen Renibacterium salmoninarum.

    PubMed

    Brynildsrud, Ola; Gulla, Snorre; Feil, Edward J; Nørstebø, Simen Foyn; Rhodes, Linda D

    2016-04-01

    Renibacterium salmoninarum is the causative agent of bacterial kidney disease, an important disease of farmed and wild salmonid fish worldwide. Despite the wide spatiotemporal distribution of this disease and habitat pressures ranging from the natural environment to aquaculture and rivers to marine environments, little variation has been observed in the R. salmoninarum genome. Here we use the coverage depth from genomic sequencing corroborated by real-time quantitative PCR to detect copy number variation (CNV) among the genes of R. salmoninarum. CNV was primarily limited to the known dominant virulence factors msa and p22. Among 68 isolates representing the UK, Norway and North America, the msa gene ranged from two to five identical copies and the p22 gene ranged from one to five copies. CNV for these two genes co-occurred, suggesting they may be functionally linked. Isolates carrying CNV were phylogenetically restricted and originated predominantly from sites in North America, rather than the UK or Norway. Although both phylogenetic relationship and geographical origin were found to correlate with CNV status, geographical origin was a much stronger predictor than phylogeny, suggesting a role for local selection pressures in the repeated emergence and maintenance of this trait.

  4. Molecular cloning and sequence analysis of the gene coding for the 57kDa soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum

    USGS Publications Warehouse

    Chien, Maw-Sheng; Gilbert , Teresa L.; Huang, Chienjin; Landolt, Marsha L.; O'Hara, Patrick J.; Winton, James R.

    1992-01-01

    The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum, was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated Mr value of 57190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27–61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein in synthesized as a 557-amino acid precursor and processed to produce a mature protein of Mr 54505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.

  5. Temperature-mediated differences in bacterial kidney disease expression and survival in Renibacterium salmoninarum-challenged bull trout and other salmonids

    USGS Publications Warehouse

    Jones, D.T.; Moffitt, C.M.; Peters, K.K.

    2007-01-01

    Resource managers considering restoration and reconnection of watersheds to protect and enhance threatened populations of bull trout Salvelinus confluentus have little information about the consequences of bacterial kidney disease (BKD) caused by Renibacterium salmoninarum. To better understand the response of bull trout to R. salmoninarum challenge, we conducted several laboratory experiments at two water temperatures. The extent, severity, and lethality of BKD in bull trout were compared with those of similarly challenged lake trout S. namaycush, Arctic char S. alpinus, Chinook salmon Oncorhynchus tshawytscha, and rainbow trout O. mykiss. The lethal dose of bacterial cells necessary to induce 50% mortality (LD50) was 10-fold lower at the 15??C challenge than at the 9??C challenge. Of the species tested, bull trout were relatively resistant to BKD, Arctic char were the most susceptible among Salvelinus species, and Chinook salmon were the most susceptible among Oncorhynchus species tested. Mean time to death was more rapid for all fish tested at 15??C than for fish challenged at 9??C. These results suggest that infection of bull trout with BKD likely poses a low risk to successful restoration of threatened populations. ?? Copyright by the American Fisheries Society 2007.

  6. Effect of dietary vitamin E and selenium on growth, survival and the prevalence of Renibacterium salmoninarum infection in chinook salmon (Oncorhynchus tshawytscha)

    USGS Publications Warehouse

    Thorarinsson, Ragnar; Landolt, Marsha L.; Elliott, Diane G.; Pascho, Ronald J.; Hardy, Ronald W.

    1994-01-01

    Groups of juvenile spring chinook salmon naturally infected with Renibacterium salmoninarum, the causative agent of bacterial kidney disease, were fed diets containing different levels of vitamin E and selenium for 214 days in fresh water and 110 days in seawater. The fish were fed vitamin E at concentrations of either 53±3 mg (designated e) or 299±9 mg (designated E) α-tocopheryl acetate equivalence/kg dry diet in combination with sodium selenite to give selenium concentrations of either 0.038±0.008 mg (designated s) or 2.49±0.15 mg (designated S)/kg dry diet. No mortality occurred in the group fed the diet, whereas mortality was 3% in the groups fed the and diets, and 31% in the group fed the diet. At the end of the experiment, weight gain and hematocrit values were significantly greater in those fish fed the E diets compared with those fed the e diets, whereas the hepato-somatic index was significantly higher in fish fed the e diets. Glutathione peroxidase activity in blood plasma was significantly higher in fish fed the S diets compared with those fed the s diets. No definite effect of dietary vitamin E and selenium on the prevalence and severity of natural R. salmoninarum infections was demonstrated.

  7. Effect of dietary vitamin E and selenium on growth, survival and the prevalence of Renibacterium salmoninarum infection in chinook salmon (Oncorhynchus tshawytscha)

    USGS Publications Warehouse

    Thorarinsson, Ragnar; Landolt, Marsha L.; Elliott, Diane G.; Pascho, Ronald J.; Hardy, Ronald W.

    1994-01-01

    Groups of juvenile spring chinook salmon naturally infected with Renibacterium salmoninarum, the causative agent of bacterial kidney disease, were fed diets containing different levels of vitamin E and selenium for 214 days in fresh water and 110 days in seawater. The fish were fed vitamin E at concentrations of either 53±3 mg (designated e) or 299±9 mg (designated E) α-tocopheryl acetate equivalence/kg dry diet in combination with sodium selenite to give selenium concentrations of either 0.038±0.008 mg (designated s) or 2.49±0.15 mg (designated S)/kg dry diet. No mortality occurred in the group fed the SE diet, whereas mortality was 3% in the groups fed the sE and Se diets, and 31% in the group fed the se diet. At the end of the experiment, weight gain and hematocrit values were significantly greater in those fish fed the E diets compared with those fed the e diets, whereas the hepato-somatic index was significantly higher in fish fed the e diets. Glutathione peroxidase activity in blood plasma was significantly higher in fish fed the S diets compared with those fed the sdiets. No definite effect of dietary vitamin E and selenium on the prevalence and severity of natural R. salmoninarum infections was demonstrated.

  8. Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney

    USGS Publications Warehouse

    Chase, D.M.; Pascho, R.J.

    1998-01-01

    Nucleic acid-based assays have shown promise for diagnosing Renibacterium salmoninarum in tissues and body fluids of salmonids. DeVelopment of a nested polymerase chain reaction (PCR) method to detect a 320 bp DNA segment of the gene encoding the p57 protein of R. salmoninarum is described. Whereas a conventional PCR for a 383 bp segment of the p57 gene reliably detected 1000 R. salmoninarum cells per reaction in kidney tissue, the nested PCR detected as few as 10 R. salmoninarum per reaction in kidney tissue. Two DNA extraction methods for the nested PCR were compared and the correlation between replicate samples was generally higher in samples extracted by the QIAamp system compared with those extracted by the phenol/chloroform method. The specificity of the nested PCR was confirmed by testing DNA extracts of common bacterial fish pathogens and a panel of bacterial species reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for R. salmoninarum. Kidney samples from 74 naturally infected chinook Salmon were examined by the nested PCR, the ELISA, and the FAT, and the detected prevalences of R. salmoninarum were 61, 47, and 43%, respectively.

  9. Characterization of susceptibility and carrier status of burbot, Lota lota (L.), to IHNV, IPNV, Flavobacterium psychrophilum, Aeromonas salmonicida and Renibacterium salmoninarum.

    PubMed

    Polinski, M P; Fehringer, T R; Johnson, K A; Snekvik, K R; Lapatra, S E; Lafrentz, B R; Ireland, S C; Cain, K D

    2010-07-01

    In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonicida by intraperitoneal (i.p.) injection. IHNV persisted in fish for at least 28 days, whereas A. salmonicida was not re-isolated beyond 17 days post-challenge. In contrast, burbot appeared refractory to Flavobacterium psychrophilum following intramuscular (i.m.) injection and to infectious pancreatic necrosis virus (IPNV) by immersion. However, i.p injection of IPNV resulted in re-isolation of virus from fish for the duration of the 28 day challenge. Renibacterium salmoninarum appeared to induce an asymptomatic carrier state in burbot following i.p. injection, but overt manifestation of disease was not apparent. Viable bacteria persisted in fish for at least 41 days, and bacterial DNA isolated by diagnostic polymerase chain reaction was detected from burbot kidney tissue 90 days after initial exposure. This study is the first to investigate susceptibility of burbot to selected fish pathogens, and this information will aid in efforts to culture and manage this species.

  10. Brood stock segregation of spring chinook salmon Oncorhynchus tshawytscha by use of the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody technique (FAT) affects the prevalence and levels of Renibacterium salmoninarum infection in progeny

    USGS Publications Warehouse

    Pascho, Ronald J.; Elliott, Diane G.; Streufert, Jonathan M.

    1991-01-01

    A study of the effect of maternal Renibacterium salmoninarum infection levels on the prevalence and levels of bacterial kidney disease (BKD) in progeny fish was conducted at a production salmon hatchery. A total of 302 mating pairs of spring chinook salmon Oncorhynchus tshawytscha was screened in August 1988 for R. salmoninarum by an enzyme-linked immunosorbent assay (ELISA). On the basis of ELISA testing of kidney tissues from all fish and the testing of ovarian fluid samples from a subsample of the females by a direct membrane filtration fluorescent antibody technique (MF-FAT), selected egg lots were segregated into 2 groups of 30 egg lots or about 135 000 eggs each. One group contained egg lots from male and female parents that had low R. salmoninarum infection levels or tested negative for R. salmoninarum (low-BKD group), and the other group contained egg lots from female parents with relatively high R. salmoninarum infection levels and male parents with various infection levels (high-BKD group). The progeny groups were maintained in separate rearing units supplied with untreated river water, and were monitored for R. salmoninarum by the ELISA until they were released from the hatchery in April 1990. Total mortality of the juvenile fish was higher (p = 0.0001) in the high-BKD group (20%) than in the low-BKD group (10 %). Mortality in the high-BKD group was highest after the fish were moved from nursery tanks to raceways, and clinical BKD became evident in this group. During the 11 mo of raceway rearing, mortality in the high-BKD group was 17 % compared with 5 % for the low-BKD group. An ELISA analysis of smolts just before release showed an R. salmoninarum infection rate of 85 % in the high-BKD group and 62 % in the low-BKD group. Of the positive fish, 98 % in the low-BKD group and 55 % in the high-BKD group had low infection levels, whereas 36 % in the high-BKD group and only 1 % in the low-BKD group had high infection levels. The results of this research

  11. Prevalence and levels of Renibacterium salmoninarum in spring-summer Chinook salmon (Oncorhynchus tshawytscha) smolts at dams on the Columbia and Snake Rivers.

    USGS Publications Warehouse

    Elliott, D.G.; Pascho, R.J.; Jackson, L.M.; Mathews, G.M.; Harmon, J.R.

    1997-01-01

    We evaluated Renibaeterium salmoninarum infection in smolts of hatchery and wild spring-summer Chinook salmon Oncorhynchus tshawytscha sampled during most of the outmigration at Little Goose (1988) and Lower Granite dams (1988–1991) on the Snake River and at Priest Rapids and McNary dams on the Columbia River (1988–1990). We sampled 860–2,178 fish per dam each year. Homogenates of kidney–spleen tissue from all fish were tested for the presence of R. salmoninarum antigens by the enzyme-linked immunosorbent assay (ELISA), and homogenates from 10% of the fish were examined by the fluorescent antibody technique (FAT). Although only 1–11% of fish sampled at a given dam during any l year exhibited lesions characteristic of bacterial kidney disease, 86–100% of the fish tested positive for R. salmoninarum antigen by ELISA, whereas 4–17% of the fish tested positive by the FAT. During most years, a majority (68–87%) of fish testing positive by the ELISA had low R. salmoninarum antigen levels, but in 1989, 53% of positive fish from Lower Granite Dam and 52% from McNary Dam showed medium-to-high antigen levels. For most years, the highest mean antigen levels were measured in fish sampled after 75% of the total out-migrants had passed a given dam. When the largest numbers of fish were being collected for bypass or downriver transportation, mean antigen levels were relatively low.

  12. Application of isotope coded affinity tag (ICAT) analysis for the identification of differentially expressed proteins following infection of atlantic salmon (Salmo salar) with infectious hematopoietic necrosis virus (IHNV) or Renibacterium salmoninarum (BKD).

    PubMed

    Booy, A T; Haddow, J D; Ohlund, L B; Hardie, D B; Olafson, R W

    2005-01-01

    Aquaculture and commercial fisheries worldwide suffer from significant economic loss due to diseases of net-pen reared fish. In British Columbia, infectious hematopoietic necrosis (IHN) and bacterial kidney disease (BKD) epidemics occur because there are currently no commercially available drugs or fully licensed vaccines to treat these diseases. With a better understanding of the molecular mechanisms underlying these diseases, this circumstance might be significantly improved. In the present study, we have used a proteomics approach in an effort to identify and quantitate differentially expressed proteins in the liver and kidneys of diseased and healthy Atlantic salmon (Salmo salar). Isotope coded affinity tagging (ICAT), 2D gel electrophoresis, and multidimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC MS/MS) were used to identify hundreds of differentially expressed proteins. While the direct significance of changes in expression levels of many proteins remains to be elucidated, others appear to be more clearly related to the infectious process. Examples of the latter are discussed here and include, a natural killer cell enhancement factor (NKEF), procathepsin L, superoxide-producing NADPH oxidase and interferon-induced viral resistance protein Mx (IFI-Mx).

  13. Vaccination against salmonid bacterial kidney disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial kidney disease (BKD) of salmonid fishes, caused by Renibacterium salmoninarum, has presented challenges for development of effective vaccines, despite several decades of research. The only vaccine against BKD that is commercially licensed is an injectable preparation containing live cells ...

  14. Yeast extracellular proteases.

    PubMed

    Ogrydziak, D M

    1993-01-01

    Many species of yeast secrete significant amounts of protease(s). In this article, results of numerous surveys of yeast extracellular protease production have been compiled and inconsistencies in the data and limitations of the methodology have been examined. Regulation, purification, characterization, and processing of yeast extracellular proteases are reviewed. Results obtained from the sequences of cloned genes, especially the Saccharomyces cerevisiae Bar protease, the Candida albicans acid protease, and the Yarrowia lipolytica alkaline protease, have been emphasized. Biotechnological applications and the medical relevance of yeast extracellular proteases are covered. Yeast extracellular proteases have potential in beer and wine stabilization, and they probably contribute to pathogenicity of Candida spp. Yeast extracellular protease genes also provide secretion and processing signals for yeast expression systems designed for secretion of heterologous proteins. Coverage of the secretion of foreign proteases such as prochymosin, urokinase, and tissue plasminogen activator by yeast in included.

  15. Extracellular calcium sensing and extracellular calcium signaling

    NASA Technical Reports Server (NTRS)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)

    2001-01-01

    The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others

  16. Bacterial extracellular lignin peroxidase

    DOEpatents

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  17. Direct fluorescent antibody technique for the detection of bacterial kidney disease in paraffin-embedded tissues

    USGS Publications Warehouse

    Ochiai, T.; Yasutake, W.T.; Gould, R.W.

    1985-01-01

    The direct fluorescent antibody technique (FAT) was successfully used to detect the causative agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, in Bouin's solution flexed and paraffinembedded egg and tissue sections. This method is superior to gram stain and may be particularly useful in detecting the BKD organism in fish with low-grade infection.

  18. Extracellular histones inhibit efferocytosis.

    PubMed

    Friggeri, Arnaud; Banerjee, Sami; Xie, Na; Cui, Huachun; De Freitas, Andressa; Zerfaoui, Mourad; Dupont, Hervé; Abraham, Edward; Liu, Gang

    2012-07-18

    The uptake and clearance of apoptotic cells by macrophages and other phagocytic cells, a process called efferocytosis, is a major component in the resolution of inflammation. Increased concentrations of extracellular histones are found during acute inflammatory states and appear to contribute to organ system dysfunction and mortality. In these studies, we examined the potential role of histones in modulating efferocytosis. We found that phagocytosis of apoptotic neutrophils or thymocytes by macrophages was significantly diminished in the presence of histones H3 or H4, but not histone H1. Histone H3 demonstrated direct binding to macrophages, an effect that was diminished by preincubation of macrophages with the opsonins growth arrest-specific gene 6 (Gas6) and milk fat globule-epidermal growth factor (EGF) 8 (MFG-E8). Incubation of histone H3 with soluble α(v)β₅ integrin and Mer, but not with α(v)β₃, diminished its binding to macrophages. Phagocytosis of apoptotic cells by alveolar macrophages in vivo was diminished in the presence of histone H3. Incubation of histone H3 with activated protein C, a treatment that degrades histones, abrogated its inhibitory effects on efferocytosis under both in vitro and in vivo conditions. The present studies demonstrate that histones have inhibitory effects on efferocytosis, suggesting a new mechanism by which extracellular histones contribute to acute inflammatory processes and tissue injury.

  19. Extracellular matrix structure.

    PubMed

    Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

    2016-02-01

    Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented.

  20. Making recombinant extracellular matrix proteins.

    PubMed

    Ruggiero, Florence; Koch, Manuel

    2008-05-01

    A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.

  1. Extracellular RNA in aging.

    PubMed

    Dluzen, Douglas F; Noren Hooten, Nicole; Evans, Michele K

    2017-03-01

    Since the discovery of extracellular RNA (exRNA) in circulation and other bodily fluids, there has been considerable effort to catalog and assess whether exRNAs can be used as markers for health and disease. A variety of exRNA species have been identified including messenger RNA and noncoding RNA such as microRNA (miRNA), small nucleolar RNA, transfer RNA, and long noncoding RNA. Age-related changes in exRNA abundance have been observed, and it is likely that some of these transcripts play a role in aging. In this review, we summarize the current state of exRNA profiling in various body fluids and discuss age-related changes in exRNA abundance that have been identified in humans and other model organisms. miRNAs, in particular, are a major focus of current research and we will highlight and discuss the potential role that specific miRNAs might play in age-related phenotypes and disease. We will also review challenges facing this emerging field and various strategies that can be used for the validation and future use of exRNAs as markers of aging and age-related disease. WIREs RNA 2017, 8:e1385. doi: 10.1002/wrna.1385 For further resources related to this article, please visit the WIREs website.

  2. Extracellular matrix and wound healing.

    PubMed

    Maquart, F X; Monboisse, J C

    2014-04-01

    Extracellular matrix has been known for a long time as an architectural support for the tissues. Many recent data, however, have shown that extracellular matrix macromolecules (collagens, elastin, glycosaminoglycans, proteoglycans and connective tissue glycoproteins) are able to regulate many important cell functions, such as proliferation, migration, protein synthesis or degradation, apoptosis, etc., making them able to play an important role in the wound repair process. Not only the intact macromolecules but some of their specific domains, that we called "Matrikines", are also able to regulate many cell activities. In this article, we will summarize main findings showing the effects of extracellular matrix macromolecules and matrikines on connective tissue and epithelial cells, particularly in skin, and their potential implication in the wound healing process. These examples show that extracellular matrix macromolecules or some of their specific domains may play a major role in wound healing. Better knowledge of these interactions may suggest new therapeutic targets in wound healing defects.

  3. Extracellular enzyme kinetics scale with resource availability

    EPA Science Inventory

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimi...

  4. Neutrophil Extracellular Traps and Microcrystals

    PubMed Central

    2017-01-01

    Neutrophil extracellular traps represent a fascinating mechanism by which PMNs entrap extracellular microbes. The primary purpose of this innate immune mechanism is thought to localize the infection at an early stage. Interestingly, the ability of different microcrystals to induce NET formation has been recently described. Microcrystals are insoluble crystals with a size of 1–100 micrometers that have different composition and shape. Microcrystals have it in common that they irritate phagocytes including PMNs and typically trigger an inflammatory response. This review is the first to summarize observations with regard to PMN activation and NET release induced by microcrystals. Gout-causing monosodium urate crystals, pseudogout-causing calcium pyrophosphate dehydrate crystals, cholesterol crystals associated with atherosclerosis, silicosis-causing silica crystals, and adjuvant alum crystals are discussed. PMID:28373994

  5. Analysis of Yeast Extracellular Vesicles.

    PubMed

    Rodrigues, Marcio L; Oliveira, Debora L; Vargas, Gabriele; Girard-Dias, Wendell; Franzen, Anderson J; Frasés, Susana; Miranda, Kildare; Nimrichter, Leonardo

    2016-01-01

    Extracellular vesicles (EV) are important carriers of biologically active components in a number of organisms, including fungal cells. Experimental characterization of fungal EVs suggested that these membranous compartments are likely involved in the regulation of several biological events. In fungal pathogens, these events include mechanisms of disease progression and/or control, suggesting potential targets for therapeutic intervention or disease prophylaxis. In this manuscript we describe methods that have been used in the last 10 years for the characterization of EVs produced by yeast forms of several fungal species. Experimental approaches detailed in this chapter include ultracentrifugation methods for EV fractionation, chromatographic approaches for analysis of EV lipids, microscopy techniques for analysis of both intracellular and extracellular vesicular compartments, interaction of EVs with host cells, and physical chemical analysis of EVs by dynamic light scattering.

  6. Extracellular secretion of recombinant proteins

    DOEpatents

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  7. Diffusion in Brain Extracellular Space

    PubMed Central

    Syková, Eva; Nicholson, Charles

    2009-01-01

    Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many molecules in the brain. Deviations from the equation reveal loss of molecules across the blood-brain barrier, through cellular uptake, binding or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromolecules, including dextrans or proteins. Theoretical models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix and interaction of macromolecules with ECS channels. Extensive experimental studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the volume fraction of the ECS typically is about 20% and the tortuosity about 1.6 (i.e. free diffusion coefficient of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties are valuable in contexts ranging from understanding extrasynaptic volume transmission to the development of paradigms for drug delivery to the brain. PMID:18923183

  8. Mechanotransduction and extracellular matrix homeostasis

    PubMed Central

    Humphrey, Jay D.; Dufresne, Eric R.; Schwartz, Martin A.

    2015-01-01

    Preface Soft connective tissues at steady state are yet dynamic; resident cells continually read environmental cues and respond to promote homeostasis, including maintenance of the mechanical properties of the extracellular matrix that are fundamental to cellular and tissue health. The mechanosensing process involves assessment of the mechanics of the matrix by the cells through integrins and the actomyosin cytoskeleton, and is followed by a mechano-regulation process that includes the deposition, rearrangement, or removal of matrix to maintain overall form and function. Progress toward understanding the molecular, cellular, and tissue scale effects that promote mechanical homeostasis has helped identify key questions for future research. PMID:25355505

  9. The evolution of extracellular matrix.

    PubMed

    Ozbek, Suat; Balasubramanian, Prakash G; Chiquet-Ehrismann, Ruth; Tucker, Richard P; Adams, Josephine C

    2010-12-01

    We present a perspective on the molecular evolution of the extracellular matrix (ECM) in metazoa that draws on research publications and data from sequenced genomes and expressed sequence tag libraries. ECM components do not function in isolation, and the biological ECM system or "adhesome" also depends on posttranslational processing enzymes, cell surface receptors, and extracellular proteases. We focus principally on the adhesome of internal tissues and discuss its origins at the dawn of the metazoa and the expansion of complexity that occurred in the chordate lineage. The analyses demonstrate very high conservation of a core adhesome that apparently evolved in a major wave of innovation in conjunction with the origin of metazoa. Integrin, CD36, and certain domains predate the metazoa, and some ECM-related proteins are identified in choanoflagellates as predicted sequences. Modern deuterostomes and vertebrates have many novelties and elaborations of ECM as a result of domain shuffling, domain innovations and gene family expansions. Knowledge of the evolution of metazoan ECM is important for understanding how it is built as a system, its roles in normal tissues and disease processes, and has relevance for tissue engineering, the development of artificial organs, and the goals of synthetic biology.

  10. Extracellular nucleotide signaling in plants

    SciTech Connect

    Stacey, Gary

    2016-09-08

    Over the life of this funded project, our research group identified and characterized two key receptor proteins in plants; one mediating the innate immunity response to chitin and the other elucidating the key receptor for extracellular ATP. In the case of chitin recognition, we recently described the quaternary structure of this receptor, shedding light on how the receptor functions. Perhaps more importantly, we demonstrated that all plants have the ability to recognize both chitin oligomers and lipochitooligosacchardes, fundamentally changing how the community views the evolution of these systems and strategies that might be used, for example, to extend symbiotic nitrogen fixation to non-legumes. Our discovery of DORN1 opens a new chapter in plant physiology documenting conclusively that eATP is an important extracellular signal in plants, as it is in animals. At this point, we cannot predict just how far reaching this discovery may prove to be but we are convinced that eATP signaling is fundamental to plant growth and development and, hence, we believe that the future will be very exciting for the study of DORN1 and its overall function in plants.

  11. [Extracellular ribonuclease from Bacillus thuringiensis].

    PubMed

    Chepurnova, N K; Liakhov, D L; Rechinskiĭ, V O; Karpeĭskiĭ, M Ia

    1988-04-01

    The ability of the strain Bacillus thuringiensis var. subtoxicus to produce extracellular ribonuclease (ribonuclease Bt) was studied. It was found that the culture medium possesses a RNA-depolymerizing activity whose maximum is observed 4-5 hours after the beginning of the linear growth phase. A three-step chromatography of the culture extract on phosphocellulose resulted in a homogeneous enzyme with a molecular mass of 12000 Da. The enzyme showed the maximum activity towards RNA at pH 8.5, catalyzed the hydrolysis of polyribonucleotides and guanosine-2',3'-cyclophosphate. Hence, the enzyme can be related to base-nonspecific cyclizing ribonucleases showing the guanylic specificity towards nucleoside-2',3'-cyclophosphates.

  12. Extracellular adenosine triphosphate and adenosine in cancer.

    PubMed

    Stagg, J; Smyth, M J

    2010-09-30

    Adenosine triphosphate (ATP) is actively released in the extracellular environment in response to tissue damage and cellular stress. Through the activation of P2X and P2Y receptors, extracellular ATP enhances tissue repair, promotes the recruitment of immune phagocytes and dendritic cells, and acts as a co-activator of NLR family, pyrin domain-containing 3 (NLRP3) inflammasomes. The conversion of extracellular ATP to adenosine, in contrast, essentially through the enzymatic activity of the ecto-nucleotidases CD39 and CD73, acts as a negative-feedback mechanism to prevent excessive immune responses. Here we review the effects of extracellular ATP and adenosine on tumorigenesis. First, we summarize the functions of extracellular ATP and adenosine in the context of tumor immunity. Second, we present an overview of the immunosuppressive and pro-angiogenic effects of extracellular adenosine. Third, we present experimental evidence that extracellular ATP and adenosine receptors are expressed by tumor cells and enhance tumor growth. Finally, we discuss recent studies, including our own work, which suggest that therapeutic approaches that promote ATP-mediated activation of inflammasomes, or inhibit the accumulation of tumor-derived extracellular adenosine, may constitute effective new means to induce anticancer activity.

  13. Neutrophil Extracellular Traps Go Viral

    PubMed Central

    Schönrich, Günther; Raftery, Martin J.

    2016-01-01

    Neutrophils are the most numerous immune cells. Their importance as the first line of defense against bacterial and fungal pathogens is well described. In contrast, the role of neutrophils in controlling viral infections is less clear. Bacterial and fungal pathogens can stimulate neutrophils extracellular traps (NETs) in a process called NETosis. Although NETosis has previously been described as a special form of programmed cell death, there are forms of NET production that do not end with the demise of neutrophils. As an end result of NETosis, genomic DNA complexed with microbicidal proteins is expelled from neutrophils. These structures can kill pathogens or at least prevent their local spread within host tissue. On the other hand, disproportionate NET formation can cause local or systemic damage. Only recently, it was recognized that viruses can also induce NETosis. In this review, we discuss the mechanisms by which NETs are produced in the context of viral infection and how this may contribute to both antiviral immunity and immunopathology. Finally, we shed light on viral immune evasion mechanisms targeting NETs. PMID:27698656

  14. Extracellular Matrix and Liver Disease

    PubMed Central

    Arriazu, Elena; Ruiz de Galarreta, Marina; Cubero, Francisco Javier; Varela-Rey, Marta; Pérez de Obanos, María Pilar; Leung, Tung Ming; Lopategi, Aritz; Benedicto, Aitor; Abraham-Enachescu, Ioana

    2014-01-01

    Abstract Significance: The extracellular matrix (ECM) is a dynamic microenvironment that undergoes continuous remodeling, particularly during injury and wound healing. Chronic liver injury of many different etiologies such as viral hepatitis, alcohol abuse, drug-induced liver injury, obesity and insulin resistance, metabolic disorders, and autoimmune disease is characterized by excessive deposition of ECM proteins in response to persistent liver damage. Critical Issues: This review describes the main collagenous and noncollagenous components from the ECM that play a significant role in pathological matrix deposition during liver disease. We define how increased myofibroblasts (MF) from different origins are at the forefront of liver fibrosis and how liver cell-specific regulation of the complex scarring process occurs. Recent Advances: Particular attention is paid to the role of cytokines, growth factors, reactive oxygen species, and newly identified matricellular proteins in the regulation of fibrillar type I collagen, a field to which our laboratory has significantly contributed over the years. We compile data from recent literature on the potential mechanisms driving fibrosis resolution such as MF’ apoptosis, senescence, and reversal to quiescence. Future Directions: We conclude with a brief description of how epigenetics, an evolving field, can regulate the behavior of MF and of how new “omics” tools may advance our understanding of the mechanisms by which the fibrogenic response to liver injury occurs. Antioxid. Redox Signal. 21, 1078–1097. PMID:24219114

  15. Extracellular Control of Limb Regeneration

    NASA Astrophysics Data System (ADS)

    Calve, S.; Simon, H.-G.

    Adult newts possess the ability to completely regenerate organs and appendages. Immediately after limb loss, the extracellular matrix (ECM) undergoes dramatic changes that may provide mechanical and biochemical cues to guide the formation of the blastema, which is comprised of uncommitted stem-like cells that proliferate to replace the lost structure. Skeletal muscle is a known reservoir for blastema cells but the mechanism by which it contributes progenitor cells is still unclear. To create physiologically relevant culture conditions for the testing of primary newt muscle cells in vitro, the spatio-temporal distribution of ECM components and the mechanical properties of newt muscle were analyzed. Tenascin-C and hyaluronic acid (HA) were found to be dramatically upregulated in the amputated limb and were co-expressed around regenerating skeletal muscle. The transverse stiffness of muscle measured in situ was used as a guide to generate silicone-based substrates of physiological stiffness. Culturing newt muscle cells under different conditions revealed that the cells are sensitive to both matrix coating and substrate stiffness: Myoblasts on HA-coated soft substrates display a rounded morphology and become more elongated as the stiffness of the substrate increases. Coating of soft substrates with matrigel or fibronectin enhanced cell spreading and eventual cell fusion.

  16. Extracellular vesicles in parasitic diseases

    PubMed Central

    Marcilla, Antonio; Martin-Jaular, Lorena; Trelis, Maria; de Menezes-Neto, Armando; Osuna, Antonio; Bernal, Dolores; Fernandez-Becerra, Carmen; Almeida, Igor C.; del Portillo, Hernando A.

    2014-01-01

    Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs) in parasitic diseases, acting both in parasite–parasite inter-communication as well as in parasite–host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens. PMID:25536932

  17. Regulation of innate immunity by extracellular nucleotides

    PubMed Central

    Gorini, Stefania; Gatta, Lucia; Pontecorvo, Laura; Vitiello, Laura; la Sala, Andrea

    2013-01-01

    Extracellular ATP (eATP) is the most abundant among extracellular nucleotides and is commonly considered as a classical danger signal, which stimulates immune responses in the presence of tissue injury. In fact, increased nucleotide concentration in the extracellular space is generally closely associated with tissue stress or damage. However non-lytic nucleotide release may also occur in many cell types under a variety of conditions. Extracellular nucleotides are sensed by a class of plasma membrane receptors called P2 purinergic receptors (P2Rs). P2 receptors are expressed by all immunological cells and their activation elicits different responses. Extracellular ATP can act as an initiator or terminator of immune responses being able to induce different effects on immune cells depending on the pattern of P2 receptors engaged, the duration of the stimulus and its concentration in the extracellular milieu. Millimolar (high) concentrations of extracellular ATP, induce predominantly proinflammatory effects, while micromolar (low) doses exert mainly tolerogenic/immunosuppressive action. Moreover small, but significant differences in the pattern of P2 receptor expression in mice and humans confer diverse capacities of ATP in regulating the immune response. PMID:23358447

  18. Extracellular vesicles in coronary artery disease.

    PubMed

    Boulanger, Chantal M; Loyer, Xavier; Rautou, Pierre-Emmanuel; Amabile, Nicolas

    2017-02-02

    Membrane vesicles released in the extracellular space are composed of a lipid bilayer enclosing soluble cytosolic material and nuclear components. Extracellular vesicles include apoptotic bodies, exosomes, and microvesicles (also known previously as microparticles). Originating from different subcellular compartments, the role of extracellular vesicles as regulators of transfer of biological information, acting locally and remotely, is now acknowledged. Circulating vesicles released from platelets, erythrocytes, leukocytes, and endothelial cells contain potential valuable biological information for biomarker discovery in primary and secondary prevention of coronary artery disease. Extracellular vesicles also accumulate in human atherosclerotic plaques, where they affect major biological pathways, including inflammation, proliferation, thrombosis, calcification, and vasoactive responses. Extracellular vesicles also recapitulate the beneficial effect of stem cells to treat cardiac consequences of acute myocardial infarction, and now emerge as an attractive alternative to cell therapy, opening new avenues to vectorize biological information to target tissues. Although interest in microvesicles in the cardiovascular field emerged about 2 decades ago, that for extracellular vesicles, in particular exosomes, started to unfold a decade ago, opening new research and therapeutic avenues. This Review summarizes current knowledge on the role of extracellular vesicles in coronary artery disease, and their emerging potential as biomarkers and therapeutic agents.

  19. Proteomics of extracellular vesicles: Exosomes and ectosomes.

    PubMed

    Choi, Dong-Sic; Kim, Dae-Kyum; Kim, Yoon-Keun; Gho, Yong Song

    2015-01-01

    Almost all bacteria, archaea, and eukaryotic cells shed extracellular vesicles either constitutively or in a regulated manner. These nanosized membrane vesicles are spherical, bilayered proteolipids that harbor specific subsets of proteins, DNAs, RNAs, and lipids. Recent research has facilitated conceptual advancements in this emerging field that indicate that extracellular vesicles act as intercellular communicasomes by transferring signals to their target cell via surface ligands and delivering receptors and functional molecules. Recent progress in mass spectrometry-based proteomic analyses of mammalian extracellular vesicles derived from diverse cell types and body fluids has resulted in the identification of several thousand vesicular proteins that provide us with essential clues to the molecular mechanisms involved in vesicle cargo sorting and biogenesis. Furthermore, cell-type- or disease-specific vesicular proteins help us to understand the pathophysiological functions of extracellular vesicles and contribute to the discovery of diagnostic and therapeutic target proteins. This review focuses on the high-throughput mass spectrometry-based proteomic analyses of mammalian extracellular vesicles (i.e., exosomes and ectosomes), EVpedia (a free web-based integrated database of high-throughput data for systematic analyses of extracellular vesicles; http://evpedia.info), and the intravesicular protein-protein interaction network analyses of mammalian extracellular vesicles. The goal of this article is to encourage further studies to construct a comprehensive proteome database for extracellular vesicles that will help us to not only decode the biogenesis and cargo-sorting mechanisms during vesicle formation but also elucidate the pathophysiological roles of these complex extracellular organelles.

  20. Mitogenic properties of major extracellular proteins.

    PubMed

    Lévesque, J P; Hatzfeld, A; Hatzfeld, J

    1991-08-01

    The major plasma and extracellular matrix proteins are multifunctional molecules. Some, such as fibrinogen or C3, have one domain that binds adhesion receptors and another that specifically binds and activates a separate, mitogenic receptor. In this review, Jean-Pierre Lévesque, Antoinette Hatzfeld and Jacques Hatzfeld describe adhesion and mitogenic receptors that bind to distinct domains of the same extracellular matrix protein and discuss the possibility of common ancestral genes for cell adhesion molecules, extracellular matrix proteins, integrins, immunoglobulins, growth factors and their receptors.

  1. Extracellular matrix proteins of dentine.

    PubMed

    Butler, W T; Ritchie, H H; Bronckers, A L

    1997-01-01

    Bone and dentine extracellular matrix proteins are similar, consisting primarily of type I collagen, acidic proteins and proteoglycans. Although collagen forms the lattice for deposition of calcium and phosphate for formation of carbonate apatite, the non-collagenous proteins are believed to control initiation and growth of the crystals. Despite this similarity, dentine contains three unique proteins apparently absent from bone and other tissue: dentine phosphophoryn (DPP), dentine matrix protein 1 (DMP1) and dentine sialoprotein (DSP). DPP and DMP1 are acidic phosphoproteins probably involved in the control of mineralization processes. DPP may localize in gap regions of collagen and initiate apatite crystal formation by binding large quantities of calcium in a conformation that promotes this process. Extensive studies have been conducted in our laboratory on the nature, biosynthesis, localization and gene structure of DSP. Immunolocalization studies showed that rat DSP, a 53 kDa sialic acid-rich glycoprotein, was synthesized by young and mature odontoblasts, and by dental pulp cells and pre-ameloblasts, but not by ameloblasts, osteoblasts, chondrocytes or other cell types. The cDNA sequence indicated that DSP was a 366-residue protein with several potential N-glycosylation sites, as well as phosphorylation sites, but that the amino acid sequence was dissimilar to that of other known proteins. Northern blot analysis detected several mRNA species near 4.6 and 1.5 kb, indicative of alternative splicing events. Evidence for two DSP genes was obtained, further complicating this picture. Recent in situ hybridization studies utilizing rat and mouse molars and incisors indicated that DSP mRNA was expressed by young odontoblasts and odontoblasts in animals of all ages. Transcripts were also observed in pre-ameloblasts. The expression of DSP mRNA ceased when these cells matured to become secretory ameloblasts. DSP transcripts were not detected in osteoblasts or other cell

  2. The extracellular RNA complement of Escherichia coli

    PubMed Central

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-01

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. PMID:25611733

  3. Vertebrate extracellular preovulatory and postovulatory egg coats.

    PubMed

    Menkhorst, Ellen; Selwood, Lynne

    2008-11-01

    Extracellular egg coats deposited by maternal or embryonic tissues surround all vertebrate conceptuses during early development. In oviparous species, the time of hatching from extracellular coats can be considered equivalent to the time of birth in viviparous species. Extracellular coats must be lost during gestation for implantation and placentation to occur in some viviparous species. In the most recent classification of vertebrate extracellular coats, Boyd and Hamilton (Cleavage, early development and implantation of the egg. In: Parkes AS (ed.), Marshall's Physiology of Reproduction, vol. 2, 3rd ed. London: Longmans, Green & Co; 1961:1-126) defined the coat synthesized by the oocyte during oogenesis as primary and the coat deposited by follicle cells surrounding the oocyte as secondary. Tertiary egg coats are those synthesized and deposited around the primary or secondary coat by the maternal reproductive tract. This classification is difficult to reconcile with recent data collected using modern molecular biological techniques that can accurately establish the site of coat precursor synthesis and secretion. We propose that a modification to the classification by Boyd and Hamilton is required. Vertebrate egg coats should be classed as belonging to the following two broad groups: the preovulatory coat, which is deposited during oogenesis by the oocyte or follicle cells, and the postovulatory coats, which are deposited after fertilization by the reproductive tract or conceptus. This review discusses the origin and classification of vertebrate extracellular preovulatory and postovulatory coats and illustrates what is known about coat homology between the vertebrate groups.

  4. Extracellular purines, purinergic receptors and tumor growth

    PubMed Central

    Di Virgilio, F; Adinolfi, E

    2017-01-01

    Virtually, all tumor cells as well as all immune cells express plasma membrane receptors for extracellular nucleosides (adenosine) and nucleotides (ATP, ADP, UTP, UDP and sugar UDP). The tumor microenvironment is characterized by an unusually high concentration of ATP and adenosine. Adenosine is a major determinant of the immunosuppressive tumor milieu. Sequential hydrolysis of extracellular ATP catalyzed by CD39 and CD73 is the main pathway for the generation of adenosine in the tumor interstitium. Extracellular ATP and adenosine mold both host and tumor responses. Depending on the specific receptor activated, extracellular purines mediate immunosuppression or immunostimulation on the host side, and growth stimulation or cytotoxicity on the tumor side. Recent progress in this field is providing the key to decode this complex scenario and to lay the basis to harness the potential benefits for therapy. Preclinical data show that targeting the adenosine-generating pathway (that is, CD73) or adenosinergic receptors (that is, A2A) relieves immunosuppresion and potently inhibits tumor growth. On the other hand, growth of experimental tumors is strongly inhibited by targeting the P2X7 ATP-selective receptor of cancer and immune cells. This review summarizes the recent data on the role played by extracellular purines (purinergic signaling) in host–tumor interaction and highlights novel therapeutic options stemming from recent advances in this field. PMID:27321181

  5. Extracellular histones in tissue injury and inflammation.

    PubMed

    Allam, Ramanjaneyulu; Kumar, Santhosh V R; Darisipudi, Murthy N; Anders, Hans-Joachim

    2014-05-01

    Neutrophil NETosis is an important element of host defense as it catapults chromatin out of the cell to trap bacteria, which then are killed, e.g., by the chromatin's histone component. Also, during sterile inflammation TNF-alpha and other mediators trigger NETosis, which elicits cytotoxic effects on host cells. The same mechanism should apply to other forms of regulated necrosis including pyroptosis, necroptosis, ferroptosis, and cyclophilin D-mediated regulated necrosis. Beyond these toxic effects, extracellular histones also trigger thrombus formation and innate immunity by activating Toll-like receptors and the NLRP3 inflammasome. Thereby, extracellular histones contribute to the microvascular complications of sepsis, major trauma, small vessel vasculitis as well as acute liver, kidney, brain, and lung injury. Finally, histones prevent the degradation of extracellular DNA, which promotes autoimmunization, anti-nuclear antibody formation, and autoimmunity in susceptible individuals. Here, we review the current evidence on the pathogenic role of extracellular histones in disease and discuss how to target extracellular histones to improve disease outcomes.

  6. The complex extracellular biology of Streptomyces.

    PubMed

    Chater, Keith F; Biró, Sandor; Lee, Kye Joon; Palmer, Tracy; Schrempf, Hildgund

    2010-03-01

    Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.

  7. Extracellular enzyme kinetics scale with resource availability

    USGS Publications Warehouse

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

    2014-01-01

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

  8. Genetic effects of ELISA-based segregation for control of bacterial kidney disease in Chinook salmon (Oncorhynchus tshawytscha)

    USGS Publications Warehouse

    Hard, J.J.; Elliott, D.G.; Pascho, R.J.; Chase, D.M.; Park, L.K.; Winton, J.R.; Campton, D.E.

    2006-01-01

    We evaluated genetic variation in ability of Chinook salmon (Oncorhynchus tshawytscha) to resist two bacterial pathogens: Renibacterium salmoninarum, the agent of bacterial kidney disease (BKD), and Listonella anguillarum, an agent of vibriosis. After measuring R. salmoninarum antigen in 499 adults by enzyme-linked immunosorbent assay (ELISA), we mated each of 12 males with high or low antigen levels to two females with low to moderate levels and exposed subsets of their progeny to each pathogen separately. We found no correlation between R. salmoninarum antigen level in parents and survival of their progeny following pathogen exposure. We estimated high heritability for resistance to R. salmoninarum (survival h2 = 0.890 ?? 0.256 (mean ?? standard error)) independent of parental antigen level, but low heritability for resistance to L. anguillarum (h2 = 0.128 ?? 0.078). The genetic correlation between these survivals (rA = -0.204 ?? 0.309) was near zero. The genetic and phenotypic correlations between survival and antigen levels among surviving progeny exposed to R. salmoninarum were both negative (rA = -0.716 ?? 0.140; rP = -0.378 ?? 0.041), indicating that variation in antigen level is linked to survival. These results suggest that selective culling of female broodstock with high antigen titers, which is effective in controlling BKD in salmon hatcheries, will not affect resistance of their progeny. ?? 2006 NRC.

  9. Extracellular vesicles: Exosomes, microvesicles, and friends

    PubMed Central

    Stoorvogel, Willem

    2013-01-01

    Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function. PMID:23420871

  10. Extracellular recombinant protein production from Escherichia coli.

    PubMed

    Ni, Ye; Chen, Rachel

    2009-11-01

    Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.

  11. Oxidative and other posttranslational modifications in extracellular vesicle biology.

    PubMed

    Szabó-Taylor, Katalin; Ryan, Brent; Osteikoetxea, Xabier; Szabó, Tamás G; Sódar, Barbara; Holub, Marcsilla; Németh, Andrea; Pálóczi, Krisztina; Pállinger, Éva; Winyard, Paul; Buzás, Edit I

    2015-04-01

    Extracellular vesicles including exosomes, microvesicles and apoptotic vesicles, are phospholipid bilayer surrounded structures secreted by cells universally, in an evolutionarily conserved fashion. Posttranslational modifications such as oxidation, citrullination, phosphorylation and glycosylation play diverse roles in extracellular vesicle biology. Posttranslational modifications orchestrate the biogenesis of extracellular vesicles. The signals extracellular vesicles transmit between cells also often function via modulating posttranslational modifications of target molecules, given that extracellular vesicles are carriers of several active enzymes catalysing posttranslational modifications. Posttranslational modifications of extracellular vesicles can also contribute to disease pathology by e.g. amplifying inflammation, generating neoepitopes or carrying neoepitopes themselves.

  12. Involvement of extracellular matrix constituents in breast cancer

    SciTech Connect

    Lochter, Andre; Bissell, Mina J

    1995-06-01

    It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

  13. Biotechnological Aspects of Microbial Extracellular Electron Transfer

    PubMed Central

    Kato, Souichiro

    2015-01-01

    Extracellular electron transfer (EET) is a type of microbial respiration that enables electron transfer between microbial cells and extracellular solid materials, including naturally-occurring metal compounds and artificial electrodes. Microorganisms harboring EET abilities have received considerable attention for their various biotechnological applications, in addition to their contribution to global energy and material cycles. In this review, current knowledge on microbial EET and its application to diverse biotechnologies, including the bioremediation of toxic metals, recovery of useful metals, biocorrosion, and microbial electrochemical systems (microbial fuel cells and microbial electrosynthesis), were introduced. Two potential biotechnologies based on microbial EET, namely the electrochemical control of microbial metabolism and electrochemical stimulation of microbial symbiotic reactions (electric syntrophy), were also discussed. PMID:26004795

  14. Nanomechanics of the Cartilage Extracellular Matrix

    NASA Astrophysics Data System (ADS)

    Han, Lin; Grodzinsky, Alan J.; Ortiz, Christine

    2011-08-01

    Cartilage is a hydrated biomacromolecular fiber composite located at the ends of long bones that enables proper joint lubrication, articulation, loading, and energy dissipation. Degradation of extracellular matrix molecular components and changes in their nanoscale structure greatly influence the macroscale behavior of the tissue and result in dysfunction with age, injury, and diseases such as osteoarthritis. Here, the application of the field of nanomechanics to cartilage is reviewed. Nanomechanics involves the measurement and prediction of nanoscale forces and displacements, intra- and intermolecular interactions, spatially varying mechanical properties, and other mechanical phenomena existing at small length scales. Experimental nanomechanics and theoretical nanomechanics have been applied to cartilage at varying levels of material complexity, e.g., nanoscale properties of intact tissue, the matrix associated with single cells, biomimetic molecular assemblies, and individual extracellular matrix biomolecules (such as aggrecan, collagen, and hyaluronan). These studies have contributed to establishing a fundamental mechanism-based understanding of native and engineered cartilage tissue function, quality, and pathology.

  15. Circulating Extracellular RNA Markers of Liver Regeneration

    PubMed Central

    Yan, Irene K.; Wang, Xue; Asmann, Yan W.; Haga, Hiroaki; Patel, Tushar

    2016-01-01

    Background and Aims Although a key determinant of hepatic recovery after injury is active liver regeneration, the ability to detect ongoing regeneration is lacking. The restoration of liver mass after hepatectomy involves systemic changes with coordinated changes in gene expression guiding regenerative responses, activation of progenitor cells, and proliferation of quiescent hepatocytes. We postulated that these responses involve intercellular communication involving extracellular RNA and that these could represent biomarkers of active regenerative responses. Methods RNA sequencing was performed to identify temporal changes in serum extracellular non-coding RNA after partial hepatectomy in C57BL/6 male mice. Tissue expression of selected RNA was performed by microarray analysis and validated using qRT-PCR. Digital PCR was used to detect and quantify serum expression of selected RNA. Results A peak increase in extracellular RNA content occurred six hours after hepatectomy. RNA sequencing identified alterations in several small non-coding RNA including known and novel microRNAs, snoRNAs, tRNA, antisense and repeat elements after partial hepatectomy. Combinatorial effects and network analyses identified signal regulation, protein complex assembly, and signal transduction as the most common biological processes targeted by miRNA that altered. miR-1A and miR-181 were most significantly altered microRNA in both serum and in hepatic tissues, and their presence in serum was quantitated using digital PCR. Conclusions Extracellular RNA selectively enriched during acute regeneration can be detected within serum and represent biomarkers of ongoing liver regeneration in mice. The ability to detect ongoing active regeneration would improve the assessment of hepatic recovery from liver injury. PMID:27415797

  16. Extracellular cyclophilins in health and disease

    PubMed Central

    Bukrinsky, Michael

    2014-01-01

    Background Extracellular cyclophilins (eCyPs) are pro-inflammatory factors implicated in pathogenesis of a number of inflammatory diseases. Most pathogenic activities of eCyPs are related to their chemotactic activity towards leukocytes, which is mediated by eCyP receptor on target cells, CD147, and involves peptydil-prolyl cis-trans isomerase activity of cyclophilins. This activity is inhibited by cyclosporine A (CsA) and non-immunosuppressive derivatives of this drug. Accumulating evidence for the role of eCyPs in disease pathogenesis stimulated research on the mechanisms of eCyP-initiated events, resulting in identification of multiple signaling pathways, characterization of a variety of effector molecules released from eCyP-treated cells, and synthesis of CsA derivatives specifically blocking eCyPs. However, a number of important questions related to the mode of action of eCyPs remain unanswered. Scope of review In this article, we integrate available information on release and function of extracellular cyclophilins into a unified model, focusing on outstanding issues that need to be clarified. Major conclusions Extracellular cyclophilins are critical players in pathogenesis of a number of inflammatory diseases. Their mechanism of action involves interaction with the receptor, CD147, and initiation of a poorly characterized signal transduction process culminating in chemotaxis and production of pro-inflammatory factors. General significance Extracellular cyclophilins present an attractive target for therapeutic interventions that can be used to alleviate symptoms and consequences of acute and chronic inflammation. PMID:25445705

  17. Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms

    PubMed Central

    Gorgojo, Juan; Scharrig, Emilia; Gómez, Ricardo M.; Harvill, Eric T.; Rodríguez, Maria Eugenia

    2017-01-01

    B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts. PMID:28095485

  18. Extracellular Aldonolactonase from Myceliophthora thermophila▿ †

    PubMed Central

    Beeson, William T.; Iavarone, Anthony T.; Hausmann, Corinne D.; Cate, Jamie H. D.; Marletta, Michael A.

    2011-01-01

    Fungi secrete many different enzymes to deconstruct lignocellulosic biomass, including several families of hydrolases, oxidative enzymes, and many uncharacterized proteins. Here we describe the isolation, characterization, and primary sequence analysis of an extracellular aldonolactonase from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile). The lactonase is a 48-kDa glycoprotein with a broad pH optimum. The enzyme catalyzes the hydrolysis of glucono-δ-lactone and cellobiono-δ-lactone with an apparent second-order rate constant, kcat/Km, of ∼1 × 106 M−1 s−1 at pH 5.0 and 25°C but is unable to hydrolyze xylono-γ-lactone or arabino-γ-lactone. Sequence analyses of the lactonase show that it has distant homology to cis-carboxy-muconate lactonizing enzymes (CMLE) as well as 6-phosphogluconolactonases present in some bacteria. The M. thermophila genome contains two predicted extracellular lactonase genes, and expression of both genes is induced by the presence of pure cellulose. Homologues of the M. thermophila lactonase, which are also predicted to be extracellular, are present in nearly all known cellulolytic ascomycetes. PMID:21075873

  19. Effective extracellular trehalose production by Cellulosimicrobium cellulans.

    PubMed

    Seto, A; Yoshijima, H; Toyomasu, K; Ogawa, H-O; Kakuta, H; Hosono, K; Ueda, K; Beppu, T

    2004-06-01

    A bacterium isolated from a petal of Casa Blanca Lily (ST26 strain) produced a marked amount of extracellular trehalose (alpha- d-glucopyranosyl-[1,1]-alpha- d-glucopyranose) in culture medium containing glucose. 16S rDNA-based phylogeny showed that ST26 belongs to, or is related to, Cellulosimicrobium cellulans, a close relative of Cellulomonas spp. Various Cellulomonas strains obtained from culture collections also showed extracellular trehalose productivity, suggesting that trehalose production is a common property of this bacterial genus. ST26 accumulated trehalose in medium supplied with glucose but not with sucrose, glycerol or maltose. Effective extracellular trehalose production by ST26 was achieved by supplying 0.5-1% ammonium sulfate and 0.5-1% CaCO(3). The addition of CaCO(3) adjusted the pH of the culture to around 5.0. The optimized culture conditions yielded trehalose from glucose at a conversion rate of 61%. The addition of ammonium sulfate greatly reduced the dry cell weight of ST26 and intracellular content of trehalose, which suggests that the addition of ammonium sulfate makes ST26 cells leak trehalose into the medium. ST26 effectively propagated in minimal medium containing trehalose as a sole carbon source, which suggests that trehalose serves as a carbohydrate reserve of this organism.

  20. [Pharmacology of the extracellular calcium ion receptor].

    PubMed

    Ruat, Martial

    2003-01-01

    The calcium sensing receptor (CaSR) belongs to family 3 of G-protein coupled receptors. The CaSR, expressed at the surface of the parathyroid cells, controls parathyroid hormone (PTH) secretion and is the main regulator of calcium homeostasis. Its activity is regulated by small changes in the physiological concentrations of calcium and magnesium ions present in the serum and extracellular fluids, leading to the stimulation of the phospholipases C and A2. Molecules that potentiate the effect of extracellular calcium are called calcimimetics. They reduce the PTH level in vivo and have been proposed to be of therapeutic benefit for the treatment of both primary and secondary hyperparathyroidism. The blocking of CaSR by a calcilytic molecule results in the increase in serum PTH and might be of interest in the treatment of osteoporosis. The CaSR is also expressed in the thyroid, kidney, bone and in neuronal and glial cell populations, where it should be involved in the complex responses associated with calcium and magnesium ions present in the extracellular fluids.

  1. Extracellular DNA: A Bridge to Cancer.

    PubMed

    Hawes, Martha C; Wen, Fushi; Elquza, Emad

    2015-10-15

    DNase I is a secreted enzyme whose function has been presumed to control "waste management" in the human system, by degrading DNA that leaks from dead and dying cells. Emerging studies have instead yielded evidence that DNase I plays a central role in newly defined dynamics of immune and autoimmune diseases, as well as cancer and vascular disorders, including thrombosis. Cancer cells have been reported to be associated with distinctive extracellular structures that facilitate aggregation and implantation. The fact that DNA is a component of such structures and that it plays a role in cancer development is illustrated by direct evidence: DNase I added to tumor cells eliminates the structures and inhibits tumorigenicity of some cancer cell lines. DNase I injected into experimental animals, moreover, results in significant inhibition of metastasis. Despite independent observations of such phenomena in diverse cancers for over 50 years, the potential for using DNase I as a clinical tool to prevent or treat cancer remains unexplored. The discovery of neutrophil extracellular traps has yielded a conceptual framework for interpreting how extracellular DNA may function in cancer development and why it may prove to be an important clinical target in stopping cancer outside the cell.

  2. Differential detergent sensitivity of extracellular vesicle subpopulations.

    PubMed

    Osteikoetxea, Xabier; Sódar, Barbara; Németh, Andrea; Szabó-Taylor, Katalin; Pálóczi, Krisztina; Vukman, Krisztina V; Tamási, Viola; Balogh, Andrea; Kittel, Ágnes; Pállinger, Éva; Buzás, Edit Irén

    2015-10-14

    Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers. In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy. Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20. Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.

  3. Listeria monocytogenes induces mast cell extracellular traps.

    PubMed

    Campillo-Navarro, Marcia; Leyva-Paredes, Kahiry; Donis-Maturano, Luis; González-Jiménez, Marco; Paredes-Vivas, Yuriria; Cerbulo-Vázquez, Arturo; Serafín-López, Jeanet; García-Pérez, Blanca; Ullrich, Stephen E; Flores-Romo, Leopoldo; Pérez-Tapia, Sonia M; Estrada-Parra, Sergio; Estrada-García, Iris; Chacón-Salinas, Rommel

    2017-02-01

    Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when β-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.

  4. Airway and Extracellular Matrix Mechanics in COPD

    PubMed Central

    Bidan, Cécile M.; Veldsink, Annemiek C.; Meurs, Herman; Gosens, Reinoud

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases worldwide, and is characterized by airflow obstruction that is not fully reversible with treatment. Even though airflow obstruction is caused by airway smooth muscle contraction, the extent of airway narrowing depends on a range of other structural and functional determinants that impact on active and passive tissue mechanics. Cells and extracellular matrix in the airway and parenchymal compartments respond both passively and actively to the mechanical stimulation induced by smooth muscle contraction. In this review, we summarize the factors that regulate airway narrowing and provide insight into the relative contributions of different constituents of the extracellular matrix and their biomechanical impact on airway obstruction. We then review the changes in extracellular matrix composition in the airway and parenchymal compartments at different stages of COPD, and finally discuss how these changes impact airway narrowing and the development of airway hyperresponsiveness. Finally, we position these data in the context of therapeutic research focused on defective tissue repair. As a conclusion, we propose that future works should primarily target mild or early COPD, prior to the widespread structural changes in the alveolar compartment that are more characteristic of severe COPD. PMID:26696894

  5. Extracellular killing of inhaled pneumococci in rats

    SciTech Connect

    Coonrod, J.D.; Marple, S.; Holmes, G.P.; Rehm, S.R.

    1987-12-01

    Early clearance of inhaled Staphylococcus aureus is believed to be caused by phagocytosis by alveolar macrophages. In murine models inhaled pneumococci are cleared even more rapidly than S. aureus. Conventional opsonins appear to play no role in this clearance, and recently it has been shown that murine alveolar lining material contains free fatty acids and other soluble factors that are directly bactericidal for pneumococci. To determine whether non-phagocytic factors are involved in pneumococcal clearance, we compared the site of killing of inhaled pneumococci and S. aureus in rats using histologic methods and bronchoalveolar lavage. Spontaneous lysis of pneumococci was prevented by use of autolysin-defective pneumococci or by substitution of ethanolamine for choline in the cell wall. Histologic studies showed that the percent of inhaled staphylococci associated with alveolar macrophages always exceeded the percent of staphylococci cleared, whereas there was little association of pneumococci with macrophages during clearance. Analysis of the intracellular or extracellular location of iron 59 in bronchoalveolar lavage fluid of rats that had inhaled aerosols of /sup 59/Fe-labeled bacteria suggested that staphylococci were killed predominantly in macrophages and pneumococci in the extracellular space. When /sup 59/Fe-labeled pneumococci or staphylococci were ingested and killed by macrophages in vitro, the /sup 59/Fe remained with the macrophages, suggesting that the extracellular location of /sup 59/Fe during pneumococcal killing in vivo was not caused by rapid turnover of /sup 59/Fe in macrophages. Studies of the site of killing of inhaled type 25 pneumococci labeled exclusively in the cell wall with carbon 14-ethanolamine confirmed the results obtained with /sup 59/Fe-labeled pneumococci. Thus, early killing of inhaled pneumococci, unlike staphylococci, appears to take place outside of macrophages.

  6. Microbial extracellular polysaccharides and plagioclase dissolution

    SciTech Connect

    Welch, S.A.; Barker, W.W.; Banfield, J.F.

    1999-05-01

    Bytownite feldspar was dissolved in batch reactors in solutions of starch (glucose polymer), gum xanthan (glucose, mannose, glucuronic acid), pectin (poly-galacturonic acid), and four alginates (mannuronic and guluronic acid) with a range of molecular weights (low, medium, high and uncharacterized) to evaluate the effect of extracellular microbial polymers on mineral dissolution rates. Solutions were analyzed for dissolved Si and Al as an indicator of feldspar dissolution. At neutral pH, feldspar dissolution was inhibited by five of the acid polysaccharides, gum xanthan, pectin, alginate low, alginate medium, alginate high, compared to an organic-free control. An uncharacterized alginate substantially enhanced both Si and Al release from the feldspar. Starch, a neutral polysaccharide, had no apparent effect. Under mildly acidic conditions, initial pH {approx} 4, all of the polymers enhanced feldspar dissolution compared to the inorganic controls. Si release from feldspar in starch solution exceeded the control by a factor of three. Pectin and gum xanthan increased feldspar dissolution by a factor of 10, and the alginates enhanced feldspar dissolution by a factor of 50 to 100. Si and Al concentrations increased with time, even though solutions were supersaturated with respect to several possible secondary phases. Under acidic conditions, initial pH {approx} 3, below the pK{sub a} of the carboxylic acid groups, dissolution rates increased, but the relative increase due to the polysaccharides is lower, approximately a factor of two to ten. Microbial extracellular polymers play a complex role in mineral weathering. Polymers appear to inhibit dissolution under some conditions, possibly by irreversibly binding to the mineral surfaces. The extracellular polysaccharides can also enhance dissolution by providing protons and complexing with ions in solution.

  7. Mechanism of cryoprotection by extracellular polymeric solutes.

    PubMed Central

    Takahashi, T; Hirsh, A; Erbe, E; Williams, R J

    1988-01-01

    To elucidate the means by which polymer solutions protect cells from freezing injury, we cooled human monocytes to -80 degrees C or below in the presence of various polymers. Differential scanning calorimetric studies showed that those polymers which protect cells best have a limiting glass transition temperature (T'g) of approximately -20 degrees C; those with a T'g significantly higher or lower did not protect. Freeze-etch electron micrographs indicated that intracellular ice crystals had formed during this freezing procedure, but remained smaller than approximately 300 nm in the same proportion of cells as survived rapid thawing. We propose that cryoprotection of slowly frozen monocytes by polymers is a consequence of a T'g of -20 degrees C in the extracellular solution. In our hypothesis, the initial concentration and viscosity of protective polymer solutions reduce the extent and rate of cell water loss to extracellular ice and limit the injurious osmotic stress, which cells face during freezing at moderate rates to -20 degrees C. Below -20 degrees C, glass formation prevents further osmotic stress by isolating cells from extracellular ice crystals, virtually eliminating cell water loss at lower temperatures. On the other hand, the protective polymer solutions will allow some diffusion of water away from cells at temperatures above T'g. If conditions are correct, cells will concentrate the cytoplasm sufficiently during the initial cooling to T'g to avoid lethal intracellular freezing between T'g and the intracellular Tg, which has been depressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits. Images FIGURE 9 FIGURE 9 PMID:2462928

  8. Anomalous extracellular diffusion in rat cerebellum.

    PubMed

    Xiao, Fanrong; Hrabe, Jan; Hrabetova, Sabina

    2015-05-05

    Extracellular space (ECS) is a major channel transporting biologically active molecules and drugs in the brain. Diffusion-mediated transport of these substances is hindered by the ECS structure but the microscopic basis of this hindrance is not fully understood. One hypothesis proposes that the hindrance originates in large part from the presence of dead-space (DS) microdomains that can transiently retain diffusing molecules. Because previous theoretical and modeling work reported an initial period of anomalous diffusion in similar environments, we expected that brain regions densely populated by DS microdomains would exhibit anomalous extracellular diffusion. Specifically, we targeted granular layers (GL) of rat and turtle cerebella that are populated with large and geometrically complex glomeruli. The integrative optical imaging (IOI) method was employed to evaluate diffusion of fluorophore-labeled dextran (MW 3000) in GL, and the IOI data analysis was adapted to quantify the anomalous diffusion exponent dw from the IOI records. Diffusion was significantly anomalous in rat GL, where dw reached 4.8. In the geometrically simpler turtle GL, dw was elevated but not robustly anomalous (dw = 2.6). The experimental work was complemented by numerical Monte Carlo simulations of anomalous ECS diffusion in several three-dimensional tissue models containing glomeruli-like structures. It demonstrated that both the duration of transiently anomalous diffusion and the anomalous exponent depend on the size of model glomeruli and the degree of their wrapping. In conclusion, we have found anomalous extracellular diffusion in the GL of rat cerebellum. This finding lends support to the DS microdomain hypothesis. Transiently anomalous diffusion also has a profound effect on the spatiotemporal distribution of molecules released into the ECS, especially at diffusion distances on the order of a few cell diameters, speeding up short-range diffusion-mediated signals in less permeable

  9. Anomalous Extracellular Diffusion in Rat Cerebellum

    PubMed Central

    Xiao, Fanrong; Hrabe, Jan; Hrabetova, Sabina

    2015-01-01

    Extracellular space (ECS) is a major channel transporting biologically active molecules and drugs in the brain. Diffusion-mediated transport of these substances is hindered by the ECS structure but the microscopic basis of this hindrance is not fully understood. One hypothesis proposes that the hindrance originates in large part from the presence of dead-space (DS) microdomains that can transiently retain diffusing molecules. Because previous theoretical and modeling work reported an initial period of anomalous diffusion in similar environments, we expected that brain regions densely populated by DS microdomains would exhibit anomalous extracellular diffusion. Specifically, we targeted granular layers (GL) of rat and turtle cerebella that are populated with large and geometrically complex glomeruli. The integrative optical imaging (IOI) method was employed to evaluate diffusion of fluorophore-labeled dextran (MW 3000) in GL, and the IOI data analysis was adapted to quantify the anomalous diffusion exponent dw from the IOI records. Diffusion was significantly anomalous in rat GL, where dw reached 4.8. In the geometrically simpler turtle GL, dw was elevated but not robustly anomalous (dw = 2.6). The experimental work was complemented by numerical Monte Carlo simulations of anomalous ECS diffusion in several three-dimensional tissue models containing glomeruli-like structures. It demonstrated that both the duration of transiently anomalous diffusion and the anomalous exponent depend on the size of model glomeruli and the degree of their wrapping. In conclusion, we have found anomalous extracellular diffusion in the GL of rat cerebellum. This finding lends support to the DS microdomain hypothesis. Transiently anomalous diffusion also has a profound effect on the spatiotemporal distribution of molecules released into the ECS, especially at diffusion distances on the order of a few cell diameters, speeding up short-range diffusion-mediated signals in less permeable

  10. EXTRACELLULAR POLYSACCHARIDES OF AZOTOBACTER VINELANDII1

    PubMed Central

    Cohen, Gary H.; Johnstone, Donald B.

    1964-01-01

    Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone. Extracellular polysaccharides of Azotobacter vinelandii. J. Bacteriol. 88:329–338. 1964.—Extracellular polysaccharides synthetized by Azotobacter vinelandii strains 155, 102, and 3A were shown to be carboxylic acid heteropolysaccharides of apparent high molecular weight. Cells were grown in a nitrogen-free, mineral broth medium with 2% sucrose. Extracellular slime was recovered by centrifugation and purified by repeated alcohol precipitation and Sevag deproteinization. Capsular polysaccharide was recovered from washed cells by mild alkaline digestion. Methods of isolation and purification appeared to provide polysaccharide showing no evidence of heterogeneity when examined by chemical and physical methods. Infrared analysis of purified slime from the three strains suggested fundamental structural similarities. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed slime polysaccharide indicated that the polymers contained in common galacturonic acid, [α] d-glucose, and rhamnose at a ratio of approximately 43:2:1, as well as a hexuronic acid lactone, probably mannurono-lactone. However, as shown by chemical and infrared analysis, minor differences did exist; namely, slime from strain 155 and 102 contained o-acetyl groups, whereas slime from strain 3A contained none. A sialic acid-like component (1.5% of dry weight of the polysaccharide, calculated as N-acetyl neuraminic acid), was found only in the slime of strain 155. Capsular polysaccharide composition closely resembled that for slime. It is of interest that the major slime components were identical whether the energy source provided for the cells was sucrose, glucose, fructose, or ethanol. PMID:14203348

  11. Extracellular Signatures as Indicators of Processing Methods

    SciTech Connect

    Wahl, Karen L.

    2012-01-09

    As described in other chapters within this volume, many aspects of microbial cells vary with culture conditions and therefore can potentially be analyzed as forensic signatures of growth conditions. In addition to changes or variations in components of the microbes themselves, extracellular materials indicative of production processes may remain associated with the final bacterial product. It is well recognized that even with considerable effort to make pure products such as fine chemicals or pharmaceuticals, trace impurities from components or synthesis steps associated with production processes can be detected in the final product. These impurities can be used as indicators of production source or methods, such as to help connect drugs of abuse to supply chains. Extracellular residue associated with microbial cells could similarly help to characterize production processes. For successful growth of microorganisms on culture media there must be an available source of carbon, nitrogen, inorganic phosphate and sulfur, trace metals, water and vitamins. The pH, temperature, and a supply of oxygen or other gases must also be appropriate for a given organism for successful culture. The sources of these components and the range in temperature, pH and other variables has adapted over the years with currently a wide range of possible combinations of media components, recipes and parameters to choose from for a given organism. Because of this wide variability in components, mixtures of components, and other parameters, there is the potential for differentiation of cultured organisms based on changes in culture conditions. The challenge remains how to narrow the field of potential combinations and be able to attribute variations in the final bacterial product and extracellular signatures associated with the final product to information about the culture conditions or recipe used in the production of that product.

  12. Extracellular matrix signaling in morphogenesis and repair.

    PubMed

    Clause, Kelly C; Barker, Thomas H

    2013-10-01

    The extracellular matrix (ECM) is critically important for many cellular processes including growth, differentiation, survival, and morphogenesis. Cells remodel and reshape the ECM by degrading and reassembling it, playing an active role in sculpting their surrounding environment and directing their own phenotypes. Both mechanical and biochemical molecules influence ECM dynamics in multiple ways; by releasing small bioactive signaling molecules, releasing growth factors stored within the ECM, eliciting structural changes to matrix proteins which expose cryptic sites and by degrading matrix proteins directly. The dynamic reciprocal communication between cells and the ECM plays a fundamental roll in tissue development, homeostasis, and wound healing.

  13. Wingful, an extracellular feedback inhibitor of Wingless

    PubMed Central

    Gerlitz, Offer; Basler, Konrad

    2002-01-01

    Secreted peptide signals control many fundamental processes during animal development. Proper responses to these signals require cognate inducible feedback antagonists. Here we report the identification of a novel Drosophila Wingless (Wg) target gene, wingful (wf), and show that it encodes a potent extracellular feedback inhibitor of Wg. In contrast to the cytoplasmic protein Naked cuticle (Nkd), the only known Wg feedback antagonist, Wf functions during larval stages, when Nkd function is dispensable. We propose that Wf may provide feedback control for the long-range morphogen activities of Wg. PMID:12000788

  14. The evolution of metazoan extracellular matrix

    PubMed Central

    2012-01-01

    The modular domain structure of extracellular matrix (ECM) proteins and their genes has allowed extensive exon/domain shuffling during evolution to generate hundreds of ECM proteins. Many of these arose early during metazoan evolution and have been highly conserved ever since. Others have undergone duplication and divergence during evolution, and novel combinations of domains have evolved to generate new ECM proteins, particularly in the vertebrate lineage. The recent sequencing of several genomes has revealed many details of this conservation and evolution of ECM proteins to serve diverse functions in metazoa. PMID:22431747

  15. Defining the extracellular matrix using proteomics

    PubMed Central

    Byron, Adam; Humphries, Jonathan D; Humphries, Martin J

    2013-01-01

    The cell microenvironment has a profound influence on the behaviour, growth and survival of cells. The extracellular matrix (ECM) provides not only mechanical and structural support to cells and tissues but also binds soluble ligands and transmembrane receptors to provide spatial coordination of signalling processes. The ability of cells to sense the chemical, mechanical and topographical features of the ECM enables them to integrate complex, multiparametric information into a coherent response to the surrounding microenvironment. Consequently, dysregulation or mutation of ECM components results in a broad range of pathological conditions. Characterization of the composition of ECM derived from various cells has begun to reveal insights into ECM structure and function, and mechanisms of disease. Proteomic methodologies permit the global analysis of subcellular systems, but extracellular and transmembrane proteins present analytical difficulties to proteomic strategies owing to the particular biochemical properties of these molecules. Here, we review advances in proteomic approaches that have been applied to furthering our understanding of the ECM microenvironment. We survey recent studies that have addressed challenges in the analysis of ECM and discuss major outcomes in the context of health and disease. In addition, we summarize efforts to progress towards a systems-level understanding of ECM biology. PMID:23419153

  16. Solute partitioning and filtration by extracellular matrices

    PubMed Central

    Hofmann, Christina L.; Ferrell, Nicholas; Schnell, Lisa; Dubnisheva, Anna; Zydney, Andrew L.; Yurchenco, Peter D.; Roy, Shuvo

    2009-01-01

    The physiology of glomerular filtration remains mechanistically obscure despite its importance in disease. The correspondence between proteinuria and foot process effacement suggests podocytes as the locus of the filtration barrier. If so, retained macromolecules ought to accumulate at the filtration barrier, an effect called concentration polarization. Literature data indicate macromolecule concentrations decrease from subendothelial to subepithelial glomerular basement membrane (GBM), as would be expected if the GBM were itself the filter. The objective of this study was to obtain insights into the possible role of the GBM in protein retention by performing fundamental experimental and theoretical studies on the properties of three model gels. Solute partitioning and filtration through thin gels of a commercially available laminin-rich extracellular matrix, Matrigel, were measured using a polydisperse polysaccharide tracer molecule, Ficoll 70. Solute partitioning into laminin gels and lens basement membrane (LBM) were measured using Ficoll 70. A novel model of a laminin gel was numerically simulated, as well as a mixed structure-random-fiber model for LBM. Experimental partitioning was predicted by numerical simulations. Sieving coefficients through thin gels of Matrigel were size dependent and strongly flux dependent. The observed flux dependence arose from compression of the gel in response to the applied pressure. Gel compression may alter solute partitioning into extracellular matrix at physiologic pressures present in the glomerular capillary. This suggests a physical mechanism coupling podocyte structure to permeability characteristics of the GBM. PMID:19587146

  17. Extracellular polymers of ozonized waste activated sludge.

    PubMed

    Liu, J C; Lee, C H; Lai, J Y; Wang, K C; Hsu, Y C; Chang, B V

    2001-01-01

    Effect of ozonation on characteristics of waste activated sludge was investigated in the current study. Concentrations of cell-bound extracellular polymers (washed ECPs) did not change much upon ozonation, whereas the sum of cell-bound and soluble extracellular polymers (unwashed ECPs) increased with increasing ozone dose. Washed ECPs in original sludge as divided by molecular weight distribution was 39% < 1,000 Da (low MW), 30% from 1,000 to 10,000 Da (medium MW), and 31% > 10,000 Da (high MW). It was observed that the low-MW fraction decreased, and the high-MW fraction increased in ozonized sludge. The unwashed ECPs were characterized as 44% in low MW, 30% in medium MW, and 26% in high MW. Both low-MW and medium-MW fractions of unwashed ECPs decreased while high-MW fraction increased in ozonized sludge. The dewaterability of ozonized sludge, assessed by capillary suction time (CST) and specific resistance to filtration (SRF), deteriorated with ozone dose. The optimal dose of cationic polyelectrolyte increased with increasing ozone dose. The production rate and the accumulated amount of methane gas of ozonized sludge were also higher.

  18. Targeting extracellular ROS signaling of tumor cells.

    PubMed

    Bauer, Georg

    2014-04-01

    Expression of membrane-associated NADPH oxidase (NOX1) represents a characteristic feature of malignant cells. NOX1-derived extracellular superoxide anions are the basis for autocrine stimulation of proliferation, but also drive the HOCl and the NO/peroxynitrite signaling pathways. This may cause the elimination of transformed cells. Tumor cells express membrane-associated catalase that efficiently protects the cells against apoptosis-inducing reactive oxygen species (ROS) signaling. Membrane-associated superoxide dismutase (SOD) plays a co-modulatory protective role that is functionally interrelated with the protective effect mediated by catalase. Due to the co-localization of NOX1, catalase and SOD on the outer membrane of tumor cells, specific inhibition of membrane-associated SOD causes superoxide anion-dependent inhibition of catalase. This establishes a strong apoptotic signaling through the NO/peroxynitrite pathway. In parallel, it causes a drastic decrease in the concentration of proliferation-stimulating H2O2. Knowledge of the biochemical network on the surface of tumor cells should, therefore, allow development of specific novel strategies for tumor therapy, based on the specific features of tumor cell-specific extracellular ROS interactions.

  19. [Glutamic acid as a universal extracellular signal].

    PubMed

    Yoneda, Yukio

    2015-08-01

    The prevailing view is that both glutamic (Glu) and gamma-aminobutyric (GABA) acids play a role as an amino acid neurotransmitter released from neurons. However, little attention has been paid to the possible expression and functionality of signaling machineries required for amino acidergic neurotransmission in cells other than central neurons. In line with our first demonstration of the presence of Glu receptors outside the brain, in this review I will outline our recent findings accumulated since then on the physiological and pathological significance of neuronal amino acids as an extracellular signal essential for homeostasis in a variety of phenotypic cells. In undifferentiated neural progenitor cells, for instance, functional expression is seen with different signaling machineries used for glutamatergic and GABAergic neurotransmission in neurons. Moreover, Glu plays a role in mechanisms underlying suppression of proliferation for self-replication in undifferentiated mesenchymal stem cells. There is more accumulating evidence for neuronal amino acids playing a role as an extracellular autocrine or paracrine signal commonly used in different phenotypic cells. Evaluation of drugs currently used could be thus beneficial for the efficient prophylaxis and/or the therapy of a variety of diseases relevant to disturbance of amino acid signaling in diverse organs.

  20. Upper threshold of extracellular neural stimulation

    PubMed Central

    Pangratz-Fuehrer, Susanne; Suh, Bongsoo; Mathieson, Keith; Naik, Natasha; Palanker, Daniel

    2012-01-01

    It is well known that spiking neurons can produce action potentials in response to extracellular stimulation above certain threshold. It is widely assumed that there is no upper limit to somatic stimulation, except for cellular or electrode damage. Here we demonstrate that there is an upper stimulation threshold, above which no action potential can be elicited, and it is below the threshold of cellular damage. Existence of this upper stimulation threshold was confirmed in retinal ganglion cells (RGCs) at pulse durations ranging from 5 to 500 μs. The ratio of the upper to lower stimulation thresholds varied typically from 1.7 to 7.6, depending on pulse duration. Computational modeling of extracellular RGC stimulation explained the upper limit by sodium current reversal on the depolarized side of the cell membrane. This was further confirmed by experiments in the medium with a low concentration of sodium. The limited width of the stimulation window may have important implications in design of the electro-neural interfaces, including neural prosthetics. PMID:22993266

  1. Upper threshold of extracellular neural stimulation.

    PubMed

    Boinagrov, David; Pangratz-Fuehrer, Susanne; Suh, Bongsoo; Mathieson, Keith; Naik, Natasha; Palanker, Daniel

    2012-12-01

    It is well known that spiking neurons can produce action potentials in response to extracellular stimulation above certain threshold. It is widely assumed that there is no upper limit to somatic stimulation, except for cellular or electrode damage. Here we demonstrate that there is an upper stimulation threshold, above which no action potential can be elicited, and it is below the threshold of cellular damage. Existence of this upper stimulation threshold was confirmed in retinal ganglion cells (RGCs) at pulse durations ranging from 5 to 500 μs. The ratio of the upper to lower stimulation thresholds varied typically from 1.7 to 7.6, depending on pulse duration. Computational modeling of extracellular RGC stimulation explained the upper limit by sodium current reversal on the depolarized side of the cell membrane. This was further confirmed by experiments in the medium with a low concentration of sodium. The limited width of the stimulation window may have important implications in design of the electro-neural interfaces, including neural prosthetics.

  2. Probing extracellular Sonic hedgehog in neurons.

    PubMed

    Eitan, Erez; Petralia, Ronald S; Wang, Ya-Xian; Indig, Fred E; Mattson, Mark P; Yao, Pamela J

    2016-08-15

    The bioactivity of Sonic hedgehog (Shh) depends on specific lipid modifications; a palmitate at its N-terminus and a cholesterol at its C-terminus. This dual-lipid modification makes Shh molecules lipophilic, which prevents them from diffusing freely in extracellular space. Multiple lines of evidence indicate that Shh proteins are carried by various forms of extracellular vesicles (EVs). It also has been shown, for instance, that in some tissues Shh proteins are transported to neighboring cells directly via filopodia. We have previously reported that Shh proteins are expressed in hippocampal neurons. In this study we show that, in the hippocampus and cerebellum of postnatal day (P)2 rats, Shh is mostly found near or on the membrane surface of small neurites or filopodia. We also examined cultured hippocampal neurons where we observed noticeable and widespread Shh-immunolabeled vesicles located outside neurons. Through immunoelectron microscopy and biochemical analysis, we find Shh-containing EVs with a wide range of sizes. Unlike robust Shh activity in EVs isolated from cells overexpressing an N-terminal Shh fragment construct, we did not detect measurable Shh activity in EVs purified from the medium of cultured hippocampal neurons. These results suggest the complexity of the transcellular Shh signaling mechanisms in neurons.

  3. Extracellular superoxide dismutase of boar seminal plasma.

    PubMed

    Kowalowka, M; Wysocki, P; Fraser, L; Strzezek, J

    2008-08-01

    Superoxide dismutase (SOD) is an enzymatic component of the antioxidant defense system that protects spermatozoa by catalysing the dismutation of superoxide anions to hydrogen peroxide and oxygen. Age and season effects on SOD activity in the seminal plasma were measured in boars at the onset of 8 months through a 35-month period. It was found that age-related changes in SOD activity in the seminal plasma were markedly higher in boars less than 2 years of age. However, it appeared that SOD activity was established at the early sexual maturity age (8-12 months). There were variations in SOD activity throughout the season, being significantly higher in spring and autumn than in summer. A secretory extracellular form of SOD (EC-SOD) was purified to homogeneity (350-fold) from boar seminal plasma, using a three-step purification protocol (affinity chromatography followed by ion exchange and ceramic hydroxyapatite chromatography). The molecular properties and specificity of SOD (molecular mass, isoelectric point, optimum pH, thermostability and susceptibility to inhibitors) confirmed that the purified enzyme is an extracellular form of Cu/Zn-superoxide dismutase occurring in boar seminal plasma. The results of this study indicate that EC-SOD is an important antioxidant enzyme of boar seminal plasma, which plays an important physiological role in counteracting oxidative stress in spermatozoa.

  4. How Neutrophil Extracellular Traps Become Visible

    PubMed Central

    2016-01-01

    Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as released nuclear DNA associated with histones and granule proteins, which form an extracellular web-like structure that is able to entrap and occasionally kill certain microbes. Furthermore, NETs have been shown to contribute to several noninfectious disease conditions when released by activated neutrophils during inflammation. The identification of NETs has mainly been succeeded by various microscopy techniques, for example, immunofluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Since the last years the development and improvement of new immunofluorescence-based techniques enabled optimized visualization and quantification of NETs. On the one hand in vitro live-cell imaging led to profound new ideas about the mechanisms involved in the formation and functionality of NETs. On the other hand different intravital, in vivo, and in situ microscopy techniques led to deeper insights into the role of NET formation during health and disease. This paper presents an overview of the main used microscopy techniques to visualize NETs and describes their advantages as well as disadvantages. PMID:27294157

  5. The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps

    PubMed Central

    Cabezas-Olcoz, Jonathan; Wang, Steven X.; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E.

    2016-01-01

    Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix. PMID:27622514

  6. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

    PubMed Central

    Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

    2016-01-01

    The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge. PMID:27989272

  7. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles.

    PubMed

    Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

    2016-01-01

    The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, "Basics of Extracellular Vesicles," uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform "Coursera" and is free of charge.

  8. Vaccination against bacterial kidney disease: Chapter 22

    USGS Publications Warehouse

    Elliott, Diane G.; Wiens, Gregory D.; Hammell, K. Larry; Rhodes, Linda D.; Edited by Gudding, Roar; Lillehaug, Atle; Evensen, Øystein

    2014-01-01

    Bacterial kidney disease (BKD) of salmonid fishes, caused by Renibacterium salmoninarum, has been recognized as a serious disease in salmonid fishes since the 1930s. This chapter discusses the occurrence and significance, etiology, and pathogenesis of BKD. It then describes the different vaccination procedures and the effects and side-effects of vaccination. Despite years of research, however, only a single vaccine has been licensed for prevention of BKD, and has demonstrated variable efficacy. Therefore, in addition to a presentation of the current status of BKD vaccination, a discussion of potential future directions for BKD vaccine development is included in the chapter. This discussion is focused on the unique characteristics of R. salmoninarum and its biology, as well as aspects of the salmonid immune system that might be explored specifically to develop more effective vaccines for BKD prevention.

  9. Extracellular RNAs: development as biomarkers of human disease

    PubMed Central

    Quinn, Joseph F.; Patel, Tushar; Wong, David; Das, Saumya; Freedman, Jane E.; Laurent, Louise C.; Carter, Bob S.; Hochberg, Fred; Keuren-Jensen, Kendall Van; Huentelman, Matt; Spetzler, Robert; Kalani, M. Yashar S.; Arango, Jorge; Adelson, P. David; Weiner, Howard L.; Gandhi, Roopali; Goilav, Beatrice; Putterman, Chaim; Saugstad, Julie A.

    2015-01-01

    Ten ongoing studies designed to test the possibility that extracellular RNAs may serve as biomarkers in human disease are described. These studies, funded by the NIH Common Fund Extracellular RNA Communication Program, examine diverse extracellular body fluids, including plasma, serum, urine and cerebrospinal fluid. The disorders studied include hepatic and gastric cancer, cardiovascular disease, chronic kidney disease, neurodegenerative disease, brain tumours, intracranial haemorrhage, multiple sclerosis and placental disorders. Progress to date and the plans for future studies are outlined. PMID:26320940

  10. A cohabitation challenge to compare the efficacies of vaccines for bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha

    USGS Publications Warehouse

    Alcorn, S.; Murray, A.L.; Pascho, R.J.; Varney, J.

    2005-01-01

    The relative efficacies of 1 commercial and 5 experimental vaccines for bacterial kidney disease (BKD) were compared through a cohabitation waterborne challenge. Groups of juvenile chinook salmon Oncorhynchus tshawytscha were vaccinated with one of the following: (1) killed Renibacterium salmoninarum ATCC 33209 (Rs 33209) cells; (2) killed Rs 33209 cells which had been heated to 37??C for 48 h, a process that destroys the p57 protein; (3) killed R. salmoninarum MT239 (Rs MT239) cells; (4) heated Rs MT239 cells; (5) a recombinant version of the p57 protein (r-p57) emulsified in Freund's incomplete adjuvant (FIA); (6) the commercial BKD vaccine Renogen; (7) phosphate-buffered saline (PBS) emulsified with an equal volume of FIA; or (8) PBS alone. Following injection, each fish was marked with a subcutaneous fluorescent latex tag denoting its treatment group and the vaccinated fish were combined into sham and disease challenge tanks. Two weeks after these fish were vaccinated, separate groups of fish were injected with either PBS or live R. salmoninarum GL64 and were placed inside coated-wire mesh cylinders (liveboxes) in the sham and disease challenge tanks, respectively. Mortalities in both tanks were recorded for 285 d. Any mortalities among the livebox fish were replaced with an appropriate cohort (infected with R. salmoninarum or healthy) fish. None of the bacterins evaluated in this study induced protective immunity against the R. salmoninarum shed from the infected livebox fish. The percentage survival within the test groups in the R. salmoninarum challenge tank ranged from 59% (heated Rs MT239 bacterin) to 81 % (PBS emulsified with FIA). There were no differences in the percentage survival among the PBS-, PBS/FIA-, r-p57-and Renogen-injected groups. There also were no differences in survival among the bacterin groups, regardless of whether the bacterial cells had been heated or left untreated prior to injection. ?? Inter-Research 2005.

  11. A cohabitation challenge to compare the efficacies of vaccines for bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha.

    PubMed

    Alcorn, Stewart; Murray, Anthony L; Pascho, Ronald J; Varney, Jed

    2005-02-28

    The relative efficacies of 1 commercial and 5 experimental vaccines for bacterial kidney disease (BKD) were compared through a cohabitation waterborne challenge. Groups of juvenile chinook salmon Oncorhynchus tshawytscha were vaccinated with one of the following: (1) killed Renibacterium salmoninarum ATCC 33209 (Rs 33209) cells; (2) killed Rs 33209 cells which had been heated to 37 degrees C for 48 h, a process that destroys the p57 protein; (3) killed R. salmoninarum MT239 (Rs MT239) cells; (4) heated Rs MT239 cells; (5) a recombinant version of the p57 protein (r-p57) emulsified in Freund's incomplete adjuvant (FIA); (6) the commercial BKD vaccine Renogen; (7) phosphate-buffered saline (PBS) emulsified with an equal volume of FIA; or (8) PBS alone. Following injection, each fish was marked with a subcutaneous fluorescent latex tag denoting its treatment group and the vaccinated fish were combined into sham and disease challenge tanks. Two weeks after these fish were vaccinated, separate groups of fish were injected with either PBS or live R. salmoninarum GL64 and were placed inside coated-wire mesh cylinders (liveboxes) in the sham and disease challenge tanks, respectively. Mortalities in both tanks were recorded for 285 d. Any mortalities among the livebox fish were replaced with an appropriate cohort (infected with R. salmoninarum or healthy) fish. None of the bacterins evaluated in this study induced protective immunity against the R. salmoninarum shed from the infected livebox fish. The percentage survival within the test groups in the R. salmoninarum challenge tank ranged from 59% (heated Rs MT239 bacterin) to 81% (PBS emulsified with FIA). There were no differences in the percentage survival among the PBS-, PBS/FIA-, r-p57- and Renogen-injected groups. There also were no differences in survival among the bacterin groups, regardless of whether the bacterial cells had been heated or left untreated prior to injection.

  12. Identification of Phosphorylation Sites on Extracellular Corneal Epithelial Cell Maspin

    PubMed Central

    Narayan, Malathi; Mirza, Shama P.; Twining, Sally S.

    2011-01-01

    Maspin, a 42-kDa non classical serine protease inhibitor (serpin) is expressed by epithelial cells of various tissues including the cornea. The protein localizes to the nucleus and cytosol, and is present in the extracellular space. While extracellular maspin regulates corneal stromal fibroblast adhesion and inhibits angiogenesis during wound healing in the cornea, the molecular mechanism of its extracellular functions is unclear. We hypothesized that identifying post-translational modifications of maspin, such as phosphorylation, may help decipher its mode of action. The focus of this study was on the identification of phosphorylation sites on extracellular maspin, since the extracellular form of the molecule is implicated in several functions. Multi-stage fragmentation mass spectrometry was used to identify sites of phosphorylation on extracellular corneal epithelial cell maspin. A total of eight serine and threonine phosphorylation sites (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310, Ser316) were identified on the extracellular forms of the molecule. Phosphorylation of tyrosine residues on extracellular maspin was not detected on extracellular maspin from corneal epithelial cell, in contrast to breast epithelial cells. This study provides the basis for further investigation into the functional role of phosphorylation of corneal epithelial maspin. PMID:21365746

  13. The NIH Extracellular RNA Communication Consortium.

    PubMed

    Ainsztein, Alexandra M; Brooks, Philip J; Dugan, Vivien G; Ganguly, Aniruddha; Guo, Max; Howcroft, T Kevin; Kelley, Christine A; Kuo, Lillian S; Labosky, Patricia A; Lenzi, Rebecca; McKie, George A; Mohla, Suresh; Procaccini, Dena; Reilly, Matthew; Satterlee, John S; Srinivas, Pothur R; Church, Elizabeth Stansell; Sutherland, Margaret; Tagle, Danilo A; Tucker, Jessica M; Venkatachalam, Sundar

    2015-01-01

    The Extracellular RNA (exRNA) Communication Consortium, funded as an initiative of the NIH Common Fund, represents a consortium of investigators assembled to address the critical issues in the exRNA research arena. The overarching goal is to generate a multi-component community resource for sharing fundamental scientific discoveries, protocols, and innovative tools and technologies. The key initiatives include (a) generating a reference catalogue of exRNAs present in body fluids of normal healthy individuals that would facilitate disease diagnosis and therapies, (b) defining the fundamental principles of exRNA biogenesis, distribution, uptake, and function, as well as development of molecular tools, technologies, and imaging modalities to enable these studies,

  14. Why regenerative medicine needs an extracellular matrix.

    PubMed

    Prestwich, Glenn D; Healy, Kevin E

    2015-01-01

    Regenerative medicine is now coming of age. Many attempts at cell therapy have failed to show significant efficacy, and the umbrella term 'stem cell therapy' is perceived in some quarters as hype or just expensive and unnecessary medical tourism. Here we present a short editorial in three parts. First, we examine the importance of using a semisynthetic extracellular matrix (ECM) mimetic, or sECM, to deliver and retain therapeutic cells at the site of administration. Second, we describe one approach in which biophysical and biochemical properties are tailored to each tissue type, which we call "design for optimal functionality." Third, we describe an alternative approach to sECM design and implementation, called "design for simplicity," in which a deconstructed, minimalist sECM is employed and biology is allowed to perform the customization in situ. We opine that an sECM, whether minimal or instructive, is an essential contributor to improve the outcomes of cell-based therapies.

  15. Extracellular Vesicles in Chronic Obstructive Pulmonary Disease

    PubMed Central

    Kadota, Tsukasa; Fujita, Yu; Yoshioka, Yusuke; Araya, Jun; Kuwano, Kazuyoshi; Ochiya, Takahiro

    2016-01-01

    Chronic obstructive pulmonary disease (COPD) is characterized by the progression of irreversible airflow limitation and is a leading cause of morbidity and mortality worldwide. Although several crucial mechanisms of COPD pathogenesis have been studied, the precise mechanism remains unknown. Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are released from almost all cell types and are recognized as novel cell–cell communication tools. They have been shown to carry and transfer a wide variety of molecules, such as microRNAs, messenger RNAs, and proteins, which are involved in physiological functions and the pathology of various diseases. Recently, EVs have attracted considerable attention in pulmonary research. In this review, we summarize the recent findings of EV-mediated COPD pathogenesis. We also discuss the potential clinical usefulness of EVs as biomarkers and therapeutic agents for the treatment of COPD. PMID:27801806

  16. Pneumococcal MSCRAMM targeting of the extracellular matrix

    PubMed Central

    Paterson, Gavin K.; Orihuela, Carlos J.

    2010-01-01

    The attachment of bacteria to host cells and tissues and their subsequent invasion and dissemination are key processes during disease pathogenesis. In this issue of Molecular Microbiology, Jensch and co-workers provide further molecular insight into these events during infection with the Gram-positive bacterium Streptococcus pneumoniae. Their characterization of PavB, a bacterial surface protein with orthologues in other streptococci, shows it to bind the extracellar matrix components fibronection and plasminogen by virtue of repetitive sequences designated Streptococcal Surface Repeats (SSURE). In mice, a pavB mutant showed reduced nasopharyngeal colonisation and was attenuated in a lung infection model. As discussed here in the context of the pneumococcus, the study of PavB highlights the central role during microbal pathogenesis of targetting the extracellular matrix by so-called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). PMID:20444102

  17. The (dys)functional extracellular matrix☆

    PubMed Central

    Freedman, Benjamin R.; Bade, Nathan D.; Riggin, Corinne N.; Zhang, Sijia; Haines, Philip G.; Ong, Katy L.; Janmey, Paul A.

    2016-01-01

    The extracellular matrix (ECM) is a major component of the biomechanical environment with which cells interact, and it plays important roles in both normal development and disease progression. Mechanical and biochemical factors alter the biomechanical properties of tissues by driving cellular remodeling of the ECM. This review provides an overview of the structural, compositional, and mechanical properties of the ECM that instruct cell behaviors. Case studies are reviewed that highlight mechanotransduction in the context of two distinct tissues: tendons and the heart. Although these two tissues demonstrate differences in relative cell–ECM composition and mechanical environment, they share similar mechanisms underlying ECM dysfunction and cell mechanotransduction. Together, these topics provide a framework for a fundamental understanding of the ECM and how it may vary across normal and diseased tissues in response to mechanical and biochemical cues. This article is part of a Special Issue entitled: Mechanobiology. PMID:25930943

  18. Extracellular Matrices (ECM) for Tissue Repair.

    PubMed

    Polanco, Thais O; Xylas, Joanna; Lantis, John C

    2016-04-01

    Persistence of skin wounds due to underlying disease, bacterial contamination, and/or repeated trauma, causes a chronic condition where a functional extracellular matrix (ECM) cannot be established and the normal wound-healing cascade is unable to progress. These open chronic wounds leave the body susceptible to infection and present a major healthcare problem. To this end, a broad range of biologic ECM scaffolds have been developed that can provide other therapeutic options aside from traditional wound care approaches. These tissue engineered ECM scaffolds aim to facilitate the restoration of functional skin-like tissue by altering the chronic wound environment and facilitating cellular attachment, proliferation, and differentiation. This discussion will center on reviewing current ECM scaffolds and highlighting their properties and mechanism of action with respect to the clinical application in chronic, non-healing wounds.

  19. Achondrogenesis type II, abnormalities of extracellular matrix.

    PubMed

    Horton, W A; Machado, M A; Chou, J W; Campbell, D

    1987-09-01

    Immune and lectin histochemical and microchemical methods were employed to study growth cartilage from seven cases of achondrogenesis type II (Langer-Saldino). The normal architecture of the epiphyseal and growth plate cartilage was replaced by a morphologically heterogeneous tissue. Some areas were comprised of vascular canals surrounded by extensive fibrous tissue and enlarged cells that had the appearance and histochemical characteristics of hypertrophic chondrocytes. Other areas contained a mixture of cells ranging from small to the enlarged chondrocytes. The extracellular matrix in the latter areas was more abundant and had characteristics of both precartilage mesenchymal matrix and typical cartilage matrix; it contained types I and II collagen, cartilage proteoglycan, fibronectin, and peanut agglutinin binding glycoconjugate(s). Peptide mapping of cyanogen bromide cartilage collagen peptides revealed the presence of types I and II collagen. These observations could be explained by a defect in the biosynthesis of type II collagen or in chondrocyte differentiation.

  20. Neutrophil extracellular traps in tissue pathology.

    PubMed

    Nakazawa, Daigo; Kumar, Santosh; Desai, Jyaysi; Anders, Hans-Joachim

    2017-03-01

    Neutrophil extracellular traps (NETs) are innate immune systems against invading pathogens. NETs are characterized as released DNA mixed with cytoplasmic antimicrobial proteins such as myeloperoxidase, proteinase3 and neutrophil elastase. While NETs are thought to have an important role in host defense, recent work has suggested that NETs contribute to tissue injury in non-infectious disease states. Uncontrolled NET formation in autoimmune diseases, metabolic disorders, cancers and thrombotic diseases can exacerbate a disease or even be a major initiator of tissue injury. But spotting NETs in tissues is not easy. Here we review the available histopathological evidence on the presence of NETs in a variety of diseases. We discuss technical difficulties and potential sources of misinterpretation while trying to detect NETs in tissue samples.

  1. Extracellular Matrix Revisited: Roles in Tissue Engineering

    PubMed Central

    2016-01-01

    The extracellular matrix (ECM) is a heterogeneous, connective network composed of fibrous glycoproteins that coordinate in vivo to provide the physical scaffolding, mechanical stability, and biochemical cues necessary for tissue morphogenesis and homeostasis. This review highlights some of the recently raised aspects of the roles of the ECM as related to the fields of biophysics and biomedical engineering. Fundamental aspects of focus include the role of the ECM as a basic cellular structure, for novel spontaneous network formation, as an ideal scaffold in tissue engineering, and its essential contribution to cell sheet technology. As these technologies move from the laboratory to clinical practice, they are bound to shape the vast field of tissue engineering for medical transplantations. PMID:27230457

  2. Extracellular nicotinamide phosphoribosyltransferase, a new cancer metabokine

    PubMed Central

    Grolla, Ambra A; Travelli, Cristina

    2016-01-01

    Abstract In this review, we focus on the secreted form of nicotinamide phosphoribosyltransferase (NAMPT); extracellular NAMPT (eNAMPT), also known as pre‐B cell colony‐enhancing factor or visfatin. Although intracellular NAMPT is a key enzyme in controlling NAD metabolism, eNAMPT has been reported to function as a cytokine, with many roles in physiology and pathology. Circulating eNAMPT has been associated with several metabolic and inflammatory disorders, including cancer. Because cytokines produced in the tumour micro‐environment play an important role in cancer pathogenesis, in part by reprogramming cellular metabolism, future improvements in cancer immunotherapy will require a better understanding of the crosstalk between cytokine action and tumour biology. In this review, the knowledge of eNAMPT in cancer will be discussed, focusing on its immunometabolic function as a metabokine, its secretion, its mechanism of action and possible roles in the cancer micro‐environment. PMID:27128025

  3. Neutrophil extracellular traps in cancer progression.

    PubMed

    Cools-Lartigue, Jonathan; Spicer, Jonathan; Najmeh, Sara; Ferri, Lorenzo

    2014-11-01

    Neutrophils are being increasingly recognized as an important element in tumor progression. They have been shown to exert important effects at nearly every stage of tumor progression with a number of studies demonstrating that their presence is critical to tumor development. Novel aspects of neutrophil biology have recently been elucidated and its contribution to tumorigenesis is only beginning to be appreciated. Neutrophil extracellular traps (NETs) are neutrophil-derived structures composed of DNA decorated with antimicrobial peptides. They have been shown to trap and kill microorganisms, playing a critical role in host defense. However, their contribution to tumor development and metastasis has recently been demonstrated in a number of studies highlighting NETs as a potentially important therapeutic target. Here, studies implicating NETs as facilitators of tumor progression and metastasis are reviewed. In addition, potential mechanisms by which NETs may exert these effects are explored. Finally, the ability to target NETs therapeutically in human neoplastic disease is highlighted.

  4. Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps

    PubMed Central

    Behnen, Martina; Möller, Sonja; Brozek, Antonia; Klinger, Matthias; Laskay, Tamás

    2017-01-01

    The inflammatory microenvironment is commonly characterized by extracellular acidosis (pH < 7.35). Sensitivity to pH, CO2 or bicarbonate concentrations allows neutrophils to react to changes in their environment and to detect inflamed areas in the tissue. One important antimicrobial effector mechanism is the production of neutrophil extracellular traps (NETs), which are released during a programmed reactive oxygen species (ROS)-dependent cell death, the so-called NETosis. Although several functions of neutrophils have been analyzed under acidic conditions, the effect of extracellular acidosis on NETosis remains mainly unexplored and the available experimental results are contradictory. We performed a comprehensive study with the aim to elucidate the effect of extracellular acidosis on ROS-dependent NETosis of primary human neutrophils and to identify the underlying mechanisms. The study was performed in parallel in a CO2–bicabonate-buffered culture medium, which mimics in vivo conditions, and under HEPES-buffered conditions to verify the effect of pH independent of CO2 or bicarbonate. We could clearly show that extracellular acidosis (pH 6.5, 6.0, and 5.5) and intracellular acidification inhibit the release of ROS-dependent NETs upon stimulation of neutrophils with phorbol myristate acetate and immobilized immune complexes. Moreover, our findings suggest that the diminished NET release is a consequence of reduced ROS production and diminished glycolysis of neutrophils under acidic conditions. It was suggested previously that neutrophils can sense the border of inflamed tissue by the pH gradient and that a drop in pH serves as an indicator for the progress of inflammation. Following this hypothesis, our data indicate that an acidic inflammatory environment results in inhibition of extracellular operating effector mechanisms of neutrophils such as release of ROS and NETs. This way the release of toxic components and tissue damage can be avoided. However, we

  5. Ciliary Extracellular Vesicles: Txt Msg Organelles.

    PubMed

    Wang, Juan; Barr, Maureen M

    2016-04-01

    Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV-cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and Caenorhabditis elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. Caenorhabditis elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport-dependent manner. Caenorhabditis elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia-EV interactions. Until the 21st century, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies.

  6. Identification of a Receptor for Extracellular Renalase

    PubMed Central

    Wang, Ling; Velazquez, Heino; Chang, John; Safirstein, Robert; Desir, Gary V.

    2015-01-01

    Background An increased risk for developing essential hypertension, stroke and diabetes is associated with single nucleotide gene polymorphisms in renalase, a newly described secreted flavoprotein with oxidoreductase activity. Gene deletion causes hypertension, and aggravates acute ischemic kidney (AKI) and cardiac injury. Independent of its intrinsic enzymatic activities, extracellular renalase activates MAPK signaling and prevents acute kidney injury (AKI) in wild type (WT) mice. Therefore, we sought to identity the receptor for extracellular renalase. Methods and Results RP-220 is a previously identified, 20 amino acids long renalase peptide that is devoid of any intrinsic enzymatic activity, but it is equally effective as full-length recombinant renalase at protecting against toxic and ischemic injury. Using biotin transfer studies with RP-220 in the human proximal tubular cell line HK-2 and protein identification by mass spectrometry, we identified PMCA4b as a renalase binding protein. This previously characterized plasma membrane ATPase is involved in cell signaling and cardiac hypertrophy. Co-immunoprecipitation and co-immunolocalization confirmed protein-protein interaction between endogenous renalase and PMCA4b. Down-regulation of endogenous PMCA4b expression by siRNA transfection, or inhibition of its enzymatic activity by the specific peptide inhibitor caloxin1b each abrogated RP-220 dependent MAPK signaling and cytoprotection. In control studies, these maneuvers had no effect on epidermal growth factor mediated signaling, confirming specificity of the interaction between PMCA4b and renalase. Conclusions PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling. PMID:25906147

  7. Ciliary extracellular vesicles: Txt msg orgnlls

    PubMed Central

    Wang, Juan; Barr, Maureen M.

    2016-01-01

    Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV-cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and C. elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. C. elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport (IFT)-dependent manner. C. elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia-EV interactions, suggest the cilium may be an important organelle as an EV donor or as an EV target. Until the past few decades, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies. PMID:26983828

  8. Role of extracellular superoxide dismutase in hypertension.

    PubMed

    Gongora, Maria Carolina; Qin, Zhenyu; Laude, Karine; Kim, Ha Won; McCann, Louise; Folz, J Rodney; Dikalov, Sergey; Fukai, Tohru; Harrison, David G

    2006-09-01

    We previously found that angiotensin II-induced hypertension increases vascular extracellular superoxide dismutase (ecSOD), and proposed that this is a compensatory mechanism that blunts the hypertensive response and preserves endothelium-dependent vasodilatation. To test this hypothesis, we studied ecSOD-deficient mice. ecSOD(-/-) and C57Blk/6 mice had similar blood pressure at baseline; however, the hypertension caused by angiotensin II was greater in ecSOD(-/-) compared with wild-type mice (168 versus 147 mm Hg, respectively; P<0.01). In keeping with this, angiotensin II increased superoxide and reduced endothelium-dependent vasodilatation in small mesenteric arterioles to a greater extent in ecSOD(-/-) than in wild-type mice. In contrast to these findings in resistance vessels, angiotensin II paradoxically improved endothelium-dependent vasodilatation, reduced intracellular and extracellular superoxide, and increased NO production in aortas of ecSOD(-/-) mice. Whereas aortic expression of endothelial NO synthase, Cu/ZnSOD, and MnSOD were not altered in ecSOD(-/-) mice, the activity of Cu/ZnSOD was increased by 80% after angiotensin II infusion. This was associated with a concomitant increase in expression of the copper chaperone for Cu/ZnSOD in the aorta but not in the mesenteric arteries. Moreover, the angiotensin II-induced increase in aortic reduced nicotinamide-adenine dinucleotide phosphate oxidase activity was diminished in ecSOD(-/-) mice as compared with controls. Thus, during angiotensin II infusion, ecSOD reduces hypertension, minimizes vascular superoxide production, and preserves endothelial function in resistance arterioles. We also identified novel compensatory mechanisms involving upregulation of copper chaperone for Cu/ZnSOD, increased Cu/ZnSOD activity, and decreased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity in larger vessels. These compensatory mechanisms preserve large vessel function when ecSOD is absent in

  9. Bioengineering Human Myocardium on Native Extracellular Matrix

    PubMed Central

    Guyette, Jacques P.; Charest, Jonathan M; Mills, Robert W; Jank, Bernhard J.; Moser, Philipp T.; Gilpin, Sarah E.; Gershlak, Joshua R.; Okamoto, Tatsuya; Gonzalez, Gabriel; Milan, David J.; Gaudette, Glenn R.; Ott, Harald C.

    2015-01-01

    Rationale More than 25 million individuals suffer from heart failure worldwide, with nearly 4,000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only about 2,500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. Objective The objective of this study is to translate previous work to human scale and clinically relevant cells, for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human iPS-derived cardiac myocytes. Methods and Results To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiac myocytes derived from non-transgenic human induced pluripotent stem cells (iPSCs) and generated tissues of increasing three-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole heart scaffolds with human iPSC-derived cardiac myocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue, showed electrical conductivity, left ventricular pressure development, and metabolic function. Conclusions Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human iPS-derived cardiac myocytes, and enable

  10. Filter based phase distortions in extracellular spikes

    PubMed Central

    Yael, Dorin

    2017-01-01

    Extracellular recordings are the primary tool for extracting neuronal spike trains in-vivo. One of the crucial pre-processing stages of this signal is the high-pass filtration used to isolate neuronal spiking activity. Filters are characterized by changes in the magnitude and phase of different frequencies. While filters are typically chosen for their effect on magnitudes, little attention has been paid to the impact of these filters on the phase of each frequency. In this study we show that in the case of nonlinear phase shifts generated by most online and offline filters, the signal is severely distorted, resulting in an alteration of the spike waveform. This distortion leads to a shape that deviates from the original waveform as a function of its constituent frequencies, and a dramatic reduction in the SNR of the waveform that disrupts spike detectability. Currently, the vast majority of articles utilizing extracellular data are subject to these distortions since most commercial and academic hardware and software utilize nonlinear phase filters. We show that this severe problem can be avoided by recording wide-band signals followed by zero phase filtering, or alternatively corrected by reversed filtering of a narrow-band filtered, and in some cases even segmented signals. Implementation of either zero phase filtering or phase correction of the nonlinear phase filtering reproduces the original spike waveforms and increases the spike detection rates while reducing the number of false negative and positive errors. This process, in turn, helps eliminate subsequent errors in downstream analyses and misinterpretations of the results. PMID:28358895

  11. Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1987 Annual Report.

    SciTech Connect

    Kaattari, Stephen

    1988-06-01

    Bacterial kidney disease (BKD) has been and remains a chronic contributory problem limiting the productivity of salmon in the Columbia River Basin. Control of this disease will not come easily, but it would lead to a tremendous increase in the health and numbers of salmon populations. Vaccination of salmon to Renibacterium salmoninarum (KDB) is a potentially successful method of controlling this disease. To date, however, no successful vaccine has been developed for general use. A possible solution to this problem, and thus the goal of this research, is to isolate the antigenic components of KDB and enhance their ability to activate the host defenses. This will be accomplished by the chemical modification of these antigens with potent immunomodulatory substances. These modified antigens will then be tested for their effectiveness in inducing immunity to BKD and thereby preventing the disease. The goal of the project's fourth year was to test the immunogenicity and prophylactic value in coho salmon (Oncorhynchus kisutch) of various--chemical conjugates of Renibacterium salmoninarum cell and major antigens. This was accomplished by assessing the serum antibody response, the cellular immune response (chemiluminescence), and the kinetics of mortality after lethal injections of the bacteria. The studies completed this year have: (1) identified immunization procedures which enhance the induction of high levels of antibody; (2) identified functionally distinct serum antibodies which may possess different abilities to protect salmon against BKD; (3) begun the isolation and characterization of anti-R. salmoninarum antibodies which may correlate with varying degrees of protection; (4) identified chemiluminescence as a potential method for assessing cellular immunity to bacterial kidney disease; and (5) characterized two monoclonal antibodies to R. salmoninarum which will be of benefit in the diagnosis of this disease.

  12. Effects of Extracellular Calcium on Cell Membrane Resealing during Sonoporation

    NASA Astrophysics Data System (ADS)

    Zhou, Yun; Cui, Jianmin; Deng, Cheri X.

    2006-05-01

    Sonoporation has been exploited as a novel strategy for intracellular drug and gene delivery. In sonoporation, ultrasound application generates transient pores or openings in the cell membrane that allow entry of extracellular agents normally not permeable to the cell membrane. In order to improve the sonoporation outcome, we seek to obtain improved understanding of the sonoporation mechanism and investigate the factors affecting sonoporation process. We established a voltage clamp technique for real time measurement of sonoporation at single cell level using Xenopus oocytes as a model system. As both cell survival and intracellular delivery efficiency of drug or genes depend on the sonoporation dynamic process, and Calcium plays important roles in cellular processes, we focus on studying of the effect of extracellular Calcium concentration on the formation, extension, and resealing of membrane pores in sonoporation. We obtained experimental results demonstrating that the cell membrane reseals in the order of seconds in the presence of physiological level of extracellular [Ca]. We measured the resealing as function of extracellular [Ca] (0-1.8mM) and observed that the resealing rate decreases as extracellular [Ca] decreases from normal physiological level. No resealing was demonstrated when 1mM EGTA was added in the extracellular medium to chelate the [Ca] extracellularly. Our experimental findings suggest that extracellular Calcium plays an important role in controlling membrane resealing in sonoporation and thus the sonoporation outcome such as cell survival and delivery efficiency.

  13. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers?

    PubMed Central

    Le, Thu H.

    2016-01-01

    Extracellular vesicles from the urine and circulation have gained significant interest as potential diagnostic biomarkers in renal diseases. Urinary extracellular vesicles contain proteins from all sections of the nephron, whereas most studied circulating extracellular vesicles are derived from platelets, immune cells, and the endothelium. In addition to their diagnostic role as markers of kidney and vascular damage, extracellular vesicles may have functional significance in renal health and disease by facilitating communication between cells and protecting against kidney injury and bacterial infection in the urinary tract. However, the current understanding of extracellular vesicles has derived mostly from studies with very small numbers of patients or in vitro data. Moreover, accurate assessment of these vesicles remains a challenge, in part because of a lack of consensus in the methodologies to measure extracellular vesicles and the inability of most techniques to capture the entire size range of these vesicles. However, newer techniques and standardized protocols to improve the detection of extracellular vesicles are in development. A clearer understanding of the composition and biology of extracellular vesicles will provide insights into their pathophysiologic, diagnostic, and therapeutic roles. PMID:26251351

  14. Release of extracellular ATP by bacteria during growth

    PubMed Central

    2013-01-01

    Background Adenosine triphosphate (ATP) is used as an intracellular energy source by all living organisms. It plays a central role in the respiration and metabolism, and is the most important energy supplier in many enzymatic reactions. Its critical role as the energy storage molecule makes it extremely valuable to all cells. Results We report here the detection of extracellular ATP in the cultures of a variety of bacterial species. The levels of the extracellular ATP in bacterial cultures peaked around the end of the log phase and decreased in the stationary phase of growth. Extracellular ATP levels were dependent on the cellular respiration as bacterial mutants lacking cytochrome bo oxidase displayed lower extracellular ATP levels. We have also shown that Escherichia coli (E. coli) and Salmonella actively depleted extracellular ATP and an ATP supplement in culture media enhanced the stationary survival of E. coli and Salmonella. In addition to E. coli and Salmonella the presence of the extracellular ATP was observed in a variety of bacterial species that contain human pathogens such as Acinetobacter, Pseudomonas, Klebsiella and Staphylococcus. Conclusion Our results indicate that extracellular ATP is produced by many bacterial species during growth and extracellular ATP may serve a role in the bacterial physiology. PMID:24364860

  15. Extracellular taurine in the substantia nigra: taurine-glutamate interaction.

    PubMed

    García Dopico, José; Perdomo Díaz, Juan; Alonso, Teofilo Jorge; González Hernández, Tomás; Castro Fuentes, Rafael; Rodríguez Díaz, Manuel

    2004-05-15

    Taurine has been proposed as an inhibitory transmitter in the substantia nigra (SN), but the mechanisms involved in its release and uptake remain practically unexplored. We studied the extracellular pool of taurine in the rat's SN by using microdialysis methods, paying particular attention to the taurine-glutamate (GLU) interaction. Extracellular taurine increased after cell depolarization with high-K(+) in a Ca(2+)-dependent manner, being modified by the local perfusion of GLU, GLU receptor agonists, and zinc. Nigral administration of taurine increased the extracellular concentration of gamma-aminobutyric acid (GABA) and GLU, the transmitters of the two main inputs of the SN. The modification of the glial metabolism with fluocitrate and L-methionine sulfoximine also changed the extracellular concentration of taurine. The complex regulation of the extracellular pool of taurine, its interaction with GABA and GLU, and the involvement of glial cells in its regulation suggest a volume transmission role for taurine in the SN.

  16. Gram-negative and Gram-positive bacterial extracellular vesicles.

    PubMed

    Kim, Ji Hyun; Lee, Jaewook; Park, Jaesung; Gho, Yong Song

    2015-04-01

    Like mammalian cells, Gram-negative and Gram-positive bacteria release nano-sized membrane vesicles into the extracellular environment either in a constitutive manner or in a regulated manner. These bacterial extracellular vesicles are spherical bilayered proteolipids enriched with bioactive proteins, lipids, nucleic acids, and virulence factors. Recent progress in this field supports the critical pathophysiological functions of these vesicles in both bacteria-bacteria and bacteria-host interactions. This review provides an overview of the current understanding on Gram-negative and Gram-positive bacterial extracellular vesicles, especially regarding the biogenesis, components, and functions in poly-species communities. We hope that this review will stimulate additional research in this emerging field of bacterial extracellular vesicles and contribute to the development of extracellular vesicle-based diagnostic tools and effective vaccines against pathogenic Gram-negative and Gram-positive bacteria.

  17. Extracellular ice phase transitions in insects.

    PubMed

    Hawes, T C

    2014-01-01

    At temperatures below their temperature of crystallization (Tc), the extracellular body fluids of insects undergo a phase transition from liquid to solid. Insects that survive the transition to equilibrium (complete freezing of the body fluids) are designated as freeze tolerant. Although this phenomenon has been reported and described in many Insecta, current nomenclature and theory does not clearly delineate between the process of transition (freezing) and the final solid phase itself (the frozen state). Thus freeze tolerant insects are currently, by convention, described in terms of the temperature at which the crystallization of their body fluids is initiated, Tc. In fact, the correct descriptor for insects that tolerate freezing is the temperature of equilibrium freezing, Tef. The process of freezing is itself a separate physical event with unique physiological stresses that are associated with ice growth. Correspondingly there are a number of insects whose physiological cryo-limits are very specifically delineated by this transitional envelope. The distinction also has considerable significance for our understanding of insect cryobiology: firstly, because the ability to manage endogenous ice growth is a fundamental segregator of cryotype; and secondly, because our understanding of internal ice management is still largely nascent.

  18. Extracellular enzymatic activity of Microsporum canis isolates.

    PubMed

    Papini, R; Mancianti, F

    The enzymatic activity of 70 feline and canine Microsporum canis isolates was determined by the Api-Zym test. The liquid phase of cultures, inoculated into Tryptic Soy Broth, was used to examine 19 enzymes. Considerable differences were observed among the extracellular enzymatic patterns. All the isolates produced alkaline phosphatase and beta-glucosidase, while lipase (C14), trypsin, chymotrypsin, beta-glucuronidase, and alpha-fucosidase activity was never revealed. Esterase (C4) activity was present in 57 samples (81%), esterase lipase (C8) in 31 (44%), leucine arylamidase in 35 (50%), valine arylamidase and cystine arylamidase in 7 (10%), acid phosphatase in 64 (91%), naphthol-AS-BI-phosphohydrolase in 60 (86%), alpha-galactosidase in 5 (7%), beta-galactosidase in 6 (8%), alpha-glucosidase in 25 (36%), N-acetyl-beta-glucosaminidase in 41 (58%), and alpha-mannosidase in 51 (73%). The beta-galactosidase activity of M. canis has not been reported previously. Remarkable variations of intensity for each enzymatic activity were also detected. It is believed that these results could provide basic data for further investigations on the pathogenic role of enzymes secreted by M. canis.

  19. Extracellular Matrix, a Hard Player in Angiogenesis

    PubMed Central

    Mongiat, Maurizio; Andreuzzi, Eva; Tarticchio, Giulia; Paulitti, Alice

    2016-01-01

    The extracellular matrix (ECM) is a complex network of proteins, glycoproteins, proteoglycans, and polysaccharides. Through multiple interactions with each other and the cell surface receptors, not only the ECM determines the physical and mechanical properties of the tissues, but also profoundly influences cell behavior and many physiological and pathological processes. One of the functions that have been extensively explored is its impingement on angiogenesis. The strong impact of the ECM in this context is both direct and indirect by virtue of its ability to interact and/or store several growth factors and cytokines. The aim of this review is to provide some examples of the complex molecular mechanisms that are elicited by these molecules in promoting or weakening the angiogenic processes. The scenario is intricate, since matrix remodeling often generates fragments displaying opposite effects compared to those exerted by the whole molecules. Thus, the balance will tilt towards angiogenesis or angiostasis depending on the relative expression of pro- or anti-angiogenetic molecules/fragments composing the matrix of a given tissue. One of the vital aspects of this field of research is that, for its endogenous nature, the ECM can be viewed as a reservoir to draw from for the development of new more efficacious therapies to treat angiogenesis-dependent pathologies. PMID:27809279

  20. Extracellular proteases from eight psychrotolerant Antarctic strains.

    PubMed

    Vazquez, Susana C; Coria, Silvia H; MacCormack, Walter P

    2004-01-01

    Extracellular proteases from 8 Antarctic psychrotolerant Pseudomonas sp. strains were purified and characterised. All of them are neutral metalloproteases, have an apparent molecular mass of 45kDa, optimal activity at 40 degrees C and pH 7-9, retaining significant activity at pH 5-11. With the exception of P96-18, which is less stable, all retain more than 50% activity after 3 h of incubation at pH 5-9 and show low thermal stability (their half-life times range from 20 to 60 min at 40 degrees C and less than 5 min at 50 degrees C). These proteases can be used in commercial processes carried out at neutral pH and moderate temperatures, and are of special interest for their application in mixtures of enzymes where final thermal selective inactivation is needed. Results also highlight the relevance of Antarctic biotopes for the isolation of protease-producing enzymes active at low temperatures.

  1. Extracellular proton release by stimulated neutrophils

    SciTech Connect

    van Zwieten, R.; Wever, R.; Hamers, M.N.; Weening, R.S.; Roos, D.

    1981-07-01

    We have tried to elucidate the mechanism of phagosome acidification in human neutrophils. Assuming that phenomena occurring at the plasma membrane reflect reactions in the phagocytic vacuoles, we have stimulated human neutrophils with agents that induce a ''respiratory burst,'' and we have measured the release of protons into the extracellular medium. Phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine and serum-opsonized zymosan particles each caused a rapid release of protons, concomitant with the increase in oxygen consumption. The stimulated release of protons was strictly coupled to the increase respiration of the cells, because inhibition of the respiration of either anaerobiosis, chlorpromazine, or glycolytic inhibitors also inhibited the release of protons. Also, in the presence of the above-mentioned stimulating agents, neutrophils from three patients with chronic granulomatous disease enhanced neither respiration not proton release. In normal cells, the ratio of deltaH+/-deltaO2 was 1.04 +/- 0.19 (mean +/ SD, n . 13). The mechanism of this proton release is not clear. The amount of lactic and carbonic acid produced by stimulated neutrophils was inadequate to explain the amount of protons released. Perhydroxyl radicals were also ruled out as the source of the protons. Because the cells did not release measurable amounts of phosphate ions, a phosphate-hydroxyl-ion antiport was also excluded. Finally, the lack of any effect of uncouplers renders it unlikely that a respiration-driven proton gradient is built up across the plasma membrane.

  2. Mechanics of composite cytoskeletal and extracellular networks

    NASA Astrophysics Data System (ADS)

    Das, Moumita

    2014-03-01

    Living cells sense and respond to mechanical forces in their surroundings. This mechanical response is mainly due to the cell cytoskeleton, and its interaction with the extracellular matrix (ECM). The cell cytoskeleton is a composite polymeric scaffold made of many different types of protein filaments and crosslinking proteins. Two major filament systems in the cytoskeleton are actin filaments (F-actin) and microtubules (MTs). Actin filaments are semiflexible, while the much stiffer MTs behave as rigid rods. I shall discuss theories that help understand how the direct coupling to the surrounding F-actin matrix allows intracellular MTs to bear large compressive forces and controls the range of force transmission along the MTs, and how the MTs not only enhance the stiffness of the cell cytoskeleton, but can also dramatically endow an initially nearly incompressible F-actin matrix with enhanced compressibility relative to its shear compliance. A second source of compositeness in the cytoskeleton is the presences of different types of crosslinkers that can interact cooperatively leading to enhanced mechanical rigidity and tunable response. Like the cytoskeleton, the ECM is also a polymeric composite. It is primarily composed of a mesh of fibrous proteins, mainly stiff collagen filaments, and a comparatively flexible gel of proteoglycans and hyaluronan. I shall discuss a model that shows how the interplay between the collagen network and the background elastic gel leads to a mechanically robust ECM.

  3. Tetraspanins in extracellular vesicle formation and function.

    PubMed

    Andreu, Zoraida; Yáñez-Mó, María

    2014-01-01

    Extracellular vesicles (EVs) represent a novel mechanism of intercellular communication as vehicles for intercellular transfer of functional membrane and cytosolic proteins, lipids, and RNAs. Microvesicles, ectosomes, shedding vesicles, microparticles, and exosomes are the most common terms to refer to the different kinds of EVs based on their origin, composition, size, and density. Exosomes have an endosomal origin and are released by many different cell types, participating in different physiological and/or pathological processes. Depending on their origin, they can alter the fate of recipient cells according to the information transferred. In the last two decades, EVs have become the focus of many studies because of their putative use as non-invasive biomarkers and their potential in bioengineering and clinical applications. In order to exploit this ability of EVs many aspects of their biology should be deciphered. Here, we review the mechanisms involved in EV biogenesis, assembly, recruitment of selected proteins, and genetic material as well as the uptake mechanisms by target cells in an effort to understand EV functions and their utility in clinical applications. In these contexts, the role of proteins from the tetraspanin superfamily, which are among the most abundant membrane proteins of EVs, will be highlighted.

  4. Getting to know the extracellular vesicle glycome.

    PubMed

    Gerlach, Jared Q; Griffin, Matthew D

    2016-04-01

    Extracellular vesicles (EVs) are a diverse population of complex biological particles with diameters ranging from approximately 20 to 1000 nm. Tremendous interest in EVs has been generated following a number of recent, high-profile reports describing their potential utility in diagnostic, prognostic, drug delivery, and therapeutic roles. Subpopulations, such as exosomes, are now known to directly participate in cell-cell communication and direct material transfer. Glycomics, the 'omic' portion of the glycobiology field, has only begun to catalog the surface oligosaccharide and polysaccharide structures and also the carbohydrate-binding proteins found on and inside EVs. The EV glycome undoubtedly contains vital clues essential to better understanding the function, biogenesis, release and transfer of vesicles, however getting at this information is technically challenging and made even more so because of the small physical size of the vesicles and the typically minute yield from physiological-scale biological samples. Vesicle micro-heterogeneity which may be related to specific vesicle origins and functions presents a further challenge. A number of primary studies carried out over the past decade have turned up specific and valuable clues regarding the composition and roles of glycan structures and also glycan binding proteins involved EV biogenesis and transfer. This review explores some of the major EV glycobiological research carried out to date and discusses the potential implications of these findings across the life sciences.

  5. The NIH Extracellular RNA Communication Consortium

    PubMed Central

    Ainsztein, Alexandra M.; Brooks, Philip J.; Dugan, Vivien G.; Ganguly, Aniruddha; Guo, Max; Howcroft, T. Kevin; Kelley, Christine A.; Kuo, Lillian S.; Labosky, Patricia A.; Lenzi, Rebecca; McKie, George A.; Mohla, Suresh; Procaccini, Dena; Reilly, Matthew; Satterlee, John S.; Srinivas, Pothur R.; Church, Elizabeth Stansell; Sutherland, Margaret; Tagle, Danilo A.; Tucker, Jessica M.; Venkatachalam, Sundar

    2015-01-01

    The Extracellular RNA (exRNA) Communication Consortium, funded as an initiative of the NIH Common Fund, represents a consortium of investigators assembled to address the critical issues in the exRNA research arena. The overarching goal is to generate a multi-component community resource for sharing fundamental scientific discoveries, protocols, and innovative tools and technologies. The key initiatives include (a) generating a reference catalogue of exRNAs present in body fluids of normal healthy individuals that would facilitate disease diagnosis and therapies, (b) defining the fundamental principles of exRNA biogenesis, distribution, uptake, and function, as well as development of molecular tools, technologies, and imaging modalities to enable these studies, (c) identifying exRNA biomarkers of disease, (d) demonstrating clinical utility of exRNAs as therapeutic agents and developing scalable technologies required for these studies, and (e) developing a community resource, the exRNA Atlas, to provide the scientific community access to exRNA data, standardized exRNA protocols, and other useful tools and technologies generated by funded investigators. PMID:26320938

  6. Extracellular matrix components in peripheral nerve regeneration.

    PubMed

    Gonzalez-Perez, Francisco; Udina, Esther; Navarro, Xavier

    2013-01-01

    Injured axons of the peripheral nerve are able to regenerate and, eventually, reinnervate target organs. However, functional recovery is usually poor after severe nerve injuries. The switch of Schwann cells to a proliferative state, secretion of trophic factors, and the presence of extracellular matrix (ECM) molecules (such as collagen, laminin, or fibronectin) in the distal stump are key elements to create a permissive environment for axons to grow. In this review, we focus attention on the ECM components and their tropic role in axonal regeneration. These components can also be used as molecular cues to guide the axons through artificial nerve guides in attempts to better mimic the natural environment found in a degenerating nerve. Most used scaffolds tested are based on natural molecules that form the ECM, but use of synthetic polymers and functionalization of hydrogels are bringing new options. Progress in tissue engineering will eventually lead to the design of composite artificial nerve grafts that may replace the use of autologous nerve grafts to sustain regeneration over long gaps.

  7. Vascular Extracellular Matrix and Arterial Mechanics

    PubMed Central

    WAGENSEIL, JESSICA E.; MECHAM, ROBERT P.

    2009-01-01

    An important factor in the transition from an open to a closed circulatory system was a change in vessel wall structure and composition that enabled the large arteries to store and release energy during the cardiac cycle. The component of the arterial wall in vertebrates that accounts for these properties is the elastic fiber network organized by medial smooth muscle. Beginning with the onset of pulsatile blood flow in the developing aorta, smooth muscle cells in the vessel wall produce a complex extracellular matrix (ECM) that will ultimately define the mechanical properties that are critical for proper function of the adult vascular system. This review discusses the structural ECM proteins in the vertebrate aortic wall and will explore how the choice of ECM components has changed through evolution as the cardiovascular system became more advanced and pulse pressure increased. By correlating vessel mechanics with physiological blood pressure across animal species and in mice with altered vessel compliance, we show that cardiac and vascular development are physiologically coupled, and we provide evidence for a universal elastic modulus that controls the parameters of ECM deposition in vessel wall development. We also discuss mechanical models that can be used to design better tissue-engineered vessels and to test the efficacy of clinical treatments. PMID:19584318

  8. Extracellular matrix, supramolecular organisation and shape.

    PubMed Central

    Scott, J E

    1995-01-01

    Connective tissue function is defined as the formation and maintenance of shape, without which centralised physiologies (circulatory, digestive or nervous) could not have evolved. Two elements, inextensible (collagenous) fibrils and compression-resistant interfibrillar soluble polymers (proteoglycans), cope with all usual stresses. Relationships between the two are highly specific, as demonstrated by electron histochemistry based on Cupromeronic blue and critical electrolyte concentration (CEC) methodologies. Recent ideas on (1) the protofibrillar or modular structure of collagen fibrils, (2) the nature of specific binding sites for proteoglycans on fibrils, and (3) fundamental similarities in secondary and tertiary structures of the glycosaminoglycans (hyaluronan, chondroitin, keratan and dermatan sulphates) are described. They have greatly illuminated the study of extracellular matrix structure and function in normal, pathological (osteogenesis imperfecta) and ageing tissues. The small proteoglycans are proposed to be tissue organisers, orienting and ordering the collagen fibrils--thus shaping the tissue at a molecular and ultimately macro level. These interfibrillar structures are based on their bifunctional character, the protein parts binding to collagen fibrils at specific sites and the glycosaminoglycans duplexing and aggregating to hold the proteins and hence the collagen fibrils at defined distances from each other, rather like yardsticks. Examples of the way these functions work in specific tissues are drawn from the cornea and vitreous humour of the eye and developing tendon. Images Fig. 3 (cont.) Fig. 3 PMID:7591990

  9. Extracellular alkaline proteinase of Colletotrichum gloeosporioides.

    PubMed

    Dunaevsky, Ya E; Matveeva, A R; Beliakova, G A; Domash, V I; Belozersky, M A

    2007-03-01

    The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C, with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase--via hydrolysis of cell wall--provides for penetration of the fungus into the tissues of the host plant.

  10. Protein Dynamics in the Plant Extracellular Space

    PubMed Central

    Guerra-Guimarães, Leonor; Pinheiro, Carla; Chaves, Inês; Barros, Danielle R.; Ricardo, Cândido P.

    2016-01-01

    The extracellular space (ECS or apoplast) is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF). The present review is focused on APF proteomics papers and intends to draw information on the metabolic processes occurring in the ECS under abiotic and biotic stresses, as well as under non-challenged conditions. The large majority of the proteins detected are involved in “cell wall organization and biogenesis”, “response to stimulus” and “protein metabolism”. It becomes apparent that some proteins are always detected, irrespective of the experimental conditions, although with different relative contribution. This fact suggests that non-challenged plants have intrinsic constitutive metabolic processes of stress/defense in the ECS. In addition to the multiple functions ascribed to the ECS proteins, should be considered the interactions established between themselves and with the plasma membrane and its components. These interactions are crucial in connecting exterior and interior of the cell, and even simple protein actions in the ECS can have profound effects on plant performance. The proteins of the ECS are permanently contributing to the high dynamic nature of this plant compartment, which seems fundamental to plant development and adaptation to the environmental conditions. PMID:28248232

  11. Characterization of Alternaria infectoria extracellular vesicles

    PubMed Central

    Silva, Branca M.A.; Prados-Rosales, Rafael; Espadas-Moreno, Javier; Wolf, Julie M.; Luque-Garcia, Jose L.; Gonçalves, Teresa; Casadevall, Arturo

    2015-01-01

    Many fungi use membrane vesicles to transport complex molecules across their cell walls. Like mammalian exosomes, fungal vesicles contain lipids, proteins, and polysaccharides, many of which are associated with virulence. Here we identify and characterize extracellular vesicles (EVs) in Alternaria infectoria, a ubiquitous, environmental filamentous fungus that is also an opportunistic human pathogen. Examination of the A. infectoria EVs revealed a morphology similar to that of vesicles described in other fungal species. Of note, proteomic analysis detected a reduced number of vesicle-associated proteins. There were two prevalent categories among the 20 identified proteins, including the polysaccharide metabolism group, probably related to plant host invasion or biosynthesis/degradation of cell wall components, and the nuclear proteins, especially DNA repair enzymes. We also found enzymes related to pigment synthesis, adhesion to the host cell, and trafficking of vesicles/organelles/molecules. This is the first time EV secretions have been identified in a filamentous fungus. We believe that these vesicles might have a role in virulence. PMID:24576997

  12. Extracellular Vesicles and Autophagy in Osteoarthritis

    PubMed Central

    Guo, Weimin; Chen, Mingxue; Huang, Jingxiang; Yuan, Zhiguo; Zhang, Yu; Wang, Mingjie; Li, Penghao; Wang, Aiyuan; Wang, Yu; Sui, Xiang; Zhang, Li; Xu, Wenjing; Lu, Shibi

    2016-01-01

    Osteoarthritis (OA) is a type of chronic joint disease that is characterized by the degeneration and loss of articular cartilage and hyperplasia of the synovium and subchondral bone. There is reasonable knowledge about articular cartilage physiology, biochemistry, and chondrocyte metabolism. However, the etiology and pathogenesis of OA remain unclear and need urgent clarification to guide the early diagnosis and treatment of OA. Extracellular vesicles (EVs) are small membrane-linking particles that are released from cells. In recent decades, several special biological properties have been found in EV, especially in terms of cartilage. Autophagy plays a critical role in the regulation of cellular homeostasis. Likewise, more and more research has gradually focused on the effect of autophagy on chondrocyte proliferation and function in OA. The synthesis and release of EV are closely associated with autophagy. At the same time, both EV and autophagy play a role in OA development. Based on the mechanism of EV and autophagy in OA development, EV may be beneficial in the early diagnosis of OA; on the other hand, the combination of EV and autophagy-related regulatory drugs may provide insight into possible OA therapeutic strategies. PMID:28078284

  13. Relevance of extracellular DNA in rhizosphere

    NASA Astrophysics Data System (ADS)

    Pietramellara, Giacomo; Ascher, Judith; Baraniya, Divyashri; Arfaioli, Paola; Ceccherini, Maria Teresa; Hawes, Martha

    2013-04-01

    One of the most promising areas for future development is the manipulation of the rhizosphere to produce sustainable and efficient agriculture production systems. Using Omics approaches, to define the distinctive features of eDNA systems and structures, will facilitate progress in rhizo-enforcement and biocontrol studies. The relevance of these studies results clear when we consider the plethora of ecological functions in which eDNA is involved. This fraction can be actively extruded by living cells or discharged during cellular lysis and may exert a key role in the stability and variability of the soil bacterial genome, resulting also a source of nitrogen and phosphorus for plants due to the root's capacity to directly uptake short DNA fragments. The adhesive properties of the DNA molecule confer to eDNA the capacity to inhibit or kill pathogenic bacteria by cation limitation induction, and to facilitate formation of biofilm and extracellular traps (ETs), that may protect microorganisms inhabiting biofilm and plant roots against pathogens and allelopathic substances. The ETs are actively extruded by root border cells when they are dispersed in the rhizosphere, conferring to plants the capacity to extend an endogenous pathogen defence system outside the organism. Moreover, eDNA could be involved in rhizoremediation in heavy metal polluted soil acting as a bioflotation reagent.

  14. Protein Dynamics in the Plant Extracellular Space.

    PubMed

    Guerra-Guimarães, Leonor; Pinheiro, Carla; Chaves, Inês; Barros, Danielle R; Ricardo, Cândido P

    2016-07-13

    The extracellular space (ECS or apoplast) is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF). The present review is focused on APF proteomics papers and intends to draw information on the metabolic processes occurring in the ECS under abiotic and biotic stresses, as well as under non-challenged conditions. The large majority of the proteins detected are involved in "cell wall organization and biogenesis", "response to stimulus" and "protein metabolism". It becomes apparent that some proteins are always detected, irrespective of the experimental conditions, although with different relative contribution. This fact suggests that non-challenged plants have intrinsic constitutive metabolic processes of stress/defense in the ECS. In addition to the multiple functions ascribed to the ECS proteins, should be considered the interactions established between themselves and with the plasma membrane and its components. These interactions are crucial in connecting exterior and interior of the cell, and even simple protein actions in the ECS can have profound effects on plant performance. The proteins of the ECS are permanently contributing to the high dynamic nature of this plant compartment, which seems fundamental to plant development and adaptation to the environmental conditions.

  15. Micro- and macrorheology of jellyfish extracellular matrix.

    PubMed

    Gambini, Camille; Abou, Bérengère; Ponton, Alain; Cornelissen, Annemiek J M

    2012-01-04

    Mechanical properties of the extracellular matrix (ECM) play a key role in tissue organization and morphogenesis. Rheological properties of jellyfish ECM (mesoglea) were measured in vivo at the cellular scale by passive microrheology techniques: microbeads were injected in jellyfish ECM and their Brownian motion was recorded to determine the mechanical properties of the surrounding medium. Microrheology results were compared with macrorheological measurements performed with a shear rheometer on slices of jellyfish mesoglea. We found that the ECM behaved as a viscoelastic gel at the macroscopic scale and as a much softer and heterogeneous viscoelastic structure at the microscopic scale. The fibrous architecture of the mesoglea, as observed by differential interference contrast and scanning electron microscopy, was in accord with these scale-dependent mechanical properties. Furthermore, the evolution of the mechanical properties of the ECM during aging was investigated by measuring microrheological properties at different jellyfish sizes. We measured that the ECM in adult jellyfish was locally stiffer than in juvenile ones. We argue that this stiffening is a consequence of local aggregations of fibers occurring gradually during aging of the jellyfish mesoglea and is enhanced by repetitive muscular contractions of the jellyfish.

  16. Crosstalk between glia, extracellular matrix and neurons.

    PubMed

    Song, Inseon; Dityatev, Alexander

    2017-03-08

    Extracellular matrix (ECM) molecules in the central nervous system form highly organized ECM structures around cell somata, axon initial segments, and synapses and play prominent roles in early development by guiding cell migration, neurite outgrowth and synaptogenesis, and by regulating closure of the critical period of development, synaptic plasticity and stability, cognitive flexibility, and axonal regeneration in adults. Major components of neural ECM, including chondroitin sulfate proteoglycans (CSPGs), tenascin-R and hyaluronic acid, are synthesized by both neurons and glial cells. The expression of these molecules is dynamically regulated during brain development in physiological conditions, shaping both neuronal and glial functions through multitude of molecular mechanisms. Upregulation of particular CSPGs and other ECM molecules, in particular by reactive astrocytes, after CNS injuries, during aging, neuroinflammation, and neurodegeneration on the one hand results in formation of growth-impermissive environment and impaired synaptic plasticity. On the other hand, ECM appeared to have a neuroprotective effect, at least in the form of perineuronal nets. CSPGs-degrading matrix metalloproteinases (MMPs) and several members of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family of proteases are secreted by neurons and glia and may drive neural ECM remodeling in physiological conditions as well as after brain injury and other brain disorders. Thus, targeting expression of specific ECM molecules, associated glycans and degrading enzymes may lead to development of new therapeutic strategies promoting regeneration and synaptic plasticity.

  17. Extracellular matrix mechanics in lung parenchymal diseases.

    PubMed

    Suki, Béla; Bates, Jason H T

    2008-11-30

    In this review, we examine how the extracellular matrix (ECM) of the lung contributes to the overall mechanical properties of the parenchyma, and how these properties change in disease. The connective tissues of the lung are composed of cells and ECM, which includes a variety of biological macromolecules and water. The macromolecules that are most important in determining the mechanical properties of the ECM are collagen, elastin, and proteoglycans. We first discuss the various components of the ECM and how their architectural organization gives rise to the mechanical properties of the parenchyma. Next, we examine how mechanical forces can affect the physiological functioning of the lung parenchyma. Collagen plays an especially important role in determining the homeostasis and cellular responses to injury because it is the most important load-bearing component of the parenchyma. We then demonstrate how the concept of percolation can be used to link microscopic pathologic alterations in the parenchyma to clinically measurable lung function during the progression of emphysema and fibrosis. Finally, we speculate about the possibility of using targeted tissue engineering to optimize treatment of these two major lung diseases.

  18. Extracellular recordings of field potentials from single cardiomyocytes.

    PubMed

    Klauke, Norbert; Smith, Godfrey L; Cooper, Jon

    2006-10-01

    Open microfluidic channels were used to separate the extracellular space around a cardiomyocyte into three compartments: the cell ends and a central partition (insulating gap). The microchannels were filled with buffer solution and overlaid with paraffin oil, thus forming the cavities for the cell ends. The central part of the cardiomyocyte rested on the partition between two adjacent microchannels and was entirely surrounded by the paraffin oil. This arrangement increased the extracellular electrical resistance to > 20 MOmega and facilitated the recording of the time course of the change in extracellular voltage and current during subthreshold and suprathreshold stimuli. The waveform of the extracellular current and voltage in response to an extracellular depolarizing stimulus comprised an initial monophasic signal followed by a biphasic signal with a delay of 2-15 ms. The latter was associated with a transient contraction and therefore caused by an action potential. The biphasic signal became monophasic after the depolarization of one cell end by raised extracellular [K+]. This form of differential recording revealed the repolarization phase of the action potential. At rest, the sarcomere length within the gap was 12% +/- 4.8% longer than outside the gap, but intracellular Ca2+ transients occurred to the same extent as that observed in the outer pools. This data demonstrate the feasibility of the use of a microfluidic bath design to limit the extracellular resistance between two ends of an isolated cardiomyocyte.

  19. The dark side of extracellular ATP in kidney diseases.

    PubMed

    Solini, Anna; Usuelli, Vera; Fiorina, Paolo

    2015-05-01

    Intracellular ATP is the most vital source of cellular energy for biologic systems, whereas extracellular ATP is a multifaceted mediator of several cell functions via its interaction, in an autocrine or paracrine manner, with P2 purinergic receptors expressed on the cell surface. These ionotropic and metabotropic P2 purinergic receptors modulate a variety of physiologic events upon the maintenance of a highly sensitive "set point," the derangement of which may lead to the development of key pathogenic mechanisms during acute and chronic diseases. Growing evidence suggests that extracellular ATP signaling via P2 purinergic receptors may be involved in different renal pathologic conditions. For these reasons, investigators and pharmaceutical companies are actively exploring novel strategies to antagonize or block these receptors with the goal of reducing extracellular ATP production or accelerating extracellular ATP clearance. Targeting extracellular ATP signaling, particularly through the P2X7 receptor, has considerable translational potential, given that novel P2X7-receptor inhibitors are already available for clinical use (e.g., CE224,535, AZD9056, and GSK1482160). This review summarizes the current evidence regarding the involvement of extracellular ATP and its P2 purinergic receptor-mediated signaling in physiologic and pathologic processes in the kidney; potential therapeutic options targeting extracellular ATP purinergic receptors are analyzed as well.

  20. Extracellular Recordings of Field Potentials from Single Cardiomyocytes

    PubMed Central

    Klauke, Norbert; Smith, Godfrey L.; Cooper, Jon

    2006-01-01

    Open microfluidic channels were used to separate the extracellular space around a cardiomyocyte into three compartments: the cell ends and a central partition (insulating gap). The microchannels were filled with buffer solution and overlaid with paraffin oil, thus forming the cavities for the cell ends. The central part of the cardiomyocyte rested on the partition between two adjacent microchannels and was entirely surrounded by the paraffin oil. This arrangement increased the extracellular electrical resistance to >20 MΩ and facilitated the recording of the time course of the change in extracellular voltage and current during subthreshold and suprathreshold stimuli. The waveform of the extracellular current and voltage in response to an extracellular depolarizing stimulus comprised an initial monophasic signal followed by a biphasic signal with a delay of 2–15 ms. The latter was associated with a transient contraction and therefore caused by an action potential. The biphasic signal became monophasic after the depolarization of one cell end by raised extracellular [K+]. This form of differential recording revealed the repolarization phase of the action potential. At rest, the sarcomere length within the gap was 12% ± 4.8% longer than outside the gap, but intracellular Ca2+ transients occurred to the same extent as that observed in the outer pools. This data demonstrate the feasibility of the use of a microfluidic bath design to limit the extracellular resistance between two ends of an isolated cardiomyocyte. PMID:16844752

  1. Focus on Extracellular Vesicles: Development of Extracellular Vesicle-Based Therapeutic Systems

    PubMed Central

    Ohno, Shin-ichiro; Drummen, Gregor P. C.; Kuroda, Masahiko

    2016-01-01

    Many types of cells release phospholipid membrane vesicles thought to play key roles in cell-cell communication, antigen presentation, and the spread of infectious agents. Extracellular vesicles (EVs) carry various proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs), like a “message in a bottle” to cells in remote locations. The encapsulated molecules are protected from multiple types of degradative enzymes in body fluids, making EVs ideal for delivering drugs. This review presents an overview of the potential roles of EVs as natural drugs and novel drug-delivery systems. PMID:26861303

  2. Lung extracellular matrix and redox regulation

    PubMed Central

    Watson, Walter H.; Ritzenthaler, Jeffrey D.; Roman, Jesse

    2016-01-01

    Pulmonary fibrosis affects millions worldwide and, even though there has been a significant investment in understanding the processes involved in wound healing and maladaptive repair, a complete understanding of the mechanisms responsible for lung fibrogenesis eludes us, and interventions capable of reversing or halting disease progression are not available. Pulmonary fibrosis is characterized by the excessive expression and uncontrolled deposition of extracellular matrix (ECM) proteins resulting in erosion of the tissue structure. Initially considered an ‘end-stage’ process elicited after injury, these events are now considered pathogenic and are believed to contribute to the course of the disease. By interacting with integrins capable of signal transduction and by influencing tissue mechanics, ECM proteins modulate processes ranging from cell adhesion and migration to differentiation and growth factor expression. In doing so, ECM proteins help orchestrate complex developmental processes and maintain tissue homeostasis. However, poorly controlled deposition of ECM proteins promotes inflammation, fibroproliferation, and aberrant differentiation of cells, and has been implicated in the pathogenesis of pulmonary fibrosis, atherosclerosis and cancer. Considering their vital functions, ECM proteins are the target of investigation, and oxidation–reduction (redox) reactions have emerged as important regulators of the ECM. Oxidative stress invariably accompanies lung disease and promotes ECM expression directly or through the overproduction of pro-fibrotic growth factors, while affecting integrin binding and activation. In vitro and in vivo investigations point to redox reactions as targets for intervention in pulmonary fibrosis and related disorders, but studies in humans have been disappointing probably due to the narrow impact of the interventions tested, and our poor understanding of the factors that regulate these complex reactions. This review is not meant to

  3. Glioblastoma extracellular vesicles: reservoirs of potential biomarkers

    PubMed Central

    Redzic, Jasmina S; Ung, Timothy H; Graner, Michael W

    2014-01-01

    Glioblastoma multiforme (GBM) is the most frequent and most devastating of the primary central nervous system tumors, with few patients living beyond 2 years postdiagnosis. The damage caused by the disease and our treatments for the patients often leave them physically and cognitively debilitated. Generally, GBMs appear after very short clinical histories and are discovered by imaging (using magnetic resonance imaging [MRI]), and the diagnosis is validated by pathology, following surgical resection. The treatment response and diagnosis of tumor recurrence are also tracked by MRI, but there are numerous problems encountered with these monitoring modalities, such as ambiguous interpretation and forms of pseudoprogression. Diagnostic, prognostic, and predictive biomarkers would be an immense boon in following treatment schemes and in determining recurrence, which often requires an invasive intracranial biopsy to verify imaging data. Extracellular vesicles (EVs) are stable, membrane-enclosed, virus-sized particles released from either the cell surface or from endosomal pathways that lead to the systemic release of EVs into accessible biofluids, such as serum/plasma, urine, cerebrospinal fluid, and saliva. EVs carry a wide variety of proteins, nucleic acids, lipids, and other metabolites, with many common features but with enough individuality to be able to identify the cell of origin of the vesicles. These components, if properly interrogated, could allow for the identification of tumor-derived EVs in biofluids, indicating tumor progression, relapse, or treatment failure. That knowledge would allow clinicians to continue with treatment regimens that were actually effective or to change course if the therapies were failing. Here, we review the features of GBM EVs, in terms of EV content and activities that may lead to the use of EVs as serially accessible biomarkers for diagnosis and treatment response in neuro-oncology. PMID:24634586

  4. Extracellular Matrix Assembly in Diatoms (Bacillariophyceae)1

    PubMed Central

    Wustman, Brandon A.; Lind, Jan; Wetherbee, Richard; Gretz, Michael R.

    1998-01-01

    Achnanthes longipes is a marine, biofouling diatom that adheres to surfaces via adhesive polymers extruded during motility or organized into structures called stalks that contain three distinct regions: the pad, shaft, and collar. Four monoclonal antibodies (AL.C1–AL.C4) and antibodies from two uncloned hybridomas (AL.E1 and AL.E2) were raised against the extracellular adhesives of A. longipes. Antibodies were screened against a hot-water-insoluble/hot-bicarbonate-soluble-fraction. The hot-water-insoluble/hot-bicarbonate-soluble fraction was fractionated to yield polymers in three size ranges: F1, ≥ 20,000,000 Mr; F2, ≅100,000 Mr; and F3, <10,000 Mr relative to dextran standards. The ≅100,000-Mr fraction consisted of highly sulfated (approximately 11%) fucoglucuronogalactans (FGGs) and low-sulfate (approximately 2%) FGGs, whereas F1 was composed of O-linked FGG (F2)-polypeptide (F3) complexes. AL.C1, AL.C2, AL.C4, AL.E1, and AL.E2 recognized carbohydrate complementary regions on FGGs, with antigenicity dependent on fucosyl-containing side chains. AL.C3 was unique in that it had a lower affinity for FGGs and did not label any portion of the shaft. Enzyme-linked immunosorbent assay and immunocytochemistry indicated that low-sulfate FGGs are expelled from pores surrounding the raphe terminus, creating the cylindrical outer layers of the shaft, and that highly sulfated FGGs are extruded from the raphe, forming the central core. Antibody-labeling patterns and other evidence indicated that the shaft central-core region is related to material exuded from the raphe during cell motility. PMID:9536061

  5. Biological reference materials for extracellular vesicle studies.

    PubMed

    Valkonen, S; van der Pol, E; Böing, A; Yuana, Y; Yliperttula, M; Nieuwland, R; Laitinen, S; Siljander, P R M

    2017-02-15

    Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated. The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively). Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories.

  6. Metabolic requirements for neutrophil extracellular traps formation

    PubMed Central

    Rodríguez-Espinosa, Oscar; Rojas-Espinosa, Oscar; Moreno-Altamirano, María Maximina Bertha; López-Villegas, Edgar Oliver; Sánchez-García, Francisco Javier

    2015-01-01

    As part of the innate immune response, neutrophils are at the forefront of defence against infection, resolution of inflammation and wound healing. They are the most abundant leucocytes in the peripheral blood, have a short lifespan and an estimated turnover of 1010 to 1011 cells per day. Neutrophils efficiently clear microbial infections by phagocytosis and by oxygen-dependent and oxygen-independent mechanisms. In 2004, a new neutrophil anti-microbial mechanism was described, the release of neutrophil extracellular traps (NETs) composed of DNA, histones and anti-microbial peptides. Several microorganisms, bacterial products, as well as pharmacological stimuli such as PMA, were shown to induce NETs. Neutrophils contain relatively few mitochondria, and derive most of their energy from glycolysis. In this scenario we aimed to analyse some of the metabolic requirements for NET formation. Here it is shown that NETs formation is strictly dependent on glucose and to a lesser extent on glutamine, that Glut-1, glucose uptake, and glycolysis rate increase upon PMA stimulation, and that NET formation is inhibited by the glycolysis inhibitor, 2-deoxy-glucose, and to a lesser extent by the ATP synthase inhibitor oligomycin. Moreover, when neutrophils were exposed to PMA in glucose-free medium for 3 hr, they lost their characteristic polymorphic nuclei but did not release NETs. However, if glucose (but not pyruvate) was added at this time, NET release took place within minutes, suggesting that NET formation could be metabolically divided into two phases; the first, independent from exogenous glucose (chromatin decondensation) and, the second (NET release), strictly dependent on exogenous glucose and glycolysis. PMID:25545227

  7. Lung extracellular matrix and redox regulation.

    PubMed

    Watson, Walter H; Ritzenthaler, Jeffrey D; Roman, Jesse

    2016-08-01

    Pulmonary fibrosis affects millions worldwide and, even though there has been a significant investment in understanding the processes involved in wound healing and maladaptive repair, a complete understanding of the mechanisms responsible for lung fibrogenesis eludes us, and interventions capable of reversing or halting disease progression are not available. Pulmonary fibrosis is characterized by the excessive expression and uncontrolled deposition of extracellular matrix (ECM) proteins resulting in erosion of the tissue structure. Initially considered an 'end-stage' process elicited after injury, these events are now considered pathogenic and are believed to contribute to the course of the disease. By interacting with integrins capable of signal transduction and by influencing tissue mechanics, ECM proteins modulate processes ranging from cell adhesion and migration to differentiation and growth factor expression. In doing so, ECM proteins help orchestrate complex developmental processes and maintain tissue homeostasis. However, poorly controlled deposition of ECM proteins promotes inflammation, fibroproliferation, and aberrant differentiation of cells, and has been implicated in the pathogenesis of pulmonary fibrosis, atherosclerosis and cancer. Considering their vital functions, ECM proteins are the target of investigation, and oxidation-reduction (redox) reactions have emerged as important regulators of the ECM. Oxidative stress invariably accompanies lung disease and promotes ECM expression directly or through the overproduction of pro-fibrotic growth factors, while affecting integrin binding and activation. In vitro and in vivo investigations point to redox reactions as targets for intervention in pulmonary fibrosis and related disorders, but studies in humans have been disappointing probably due to the narrow impact of the interventions tested, and our poor understanding of the factors that regulate these complex reactions. This review is not meant to

  8. Extracellular electron transfer mechanisms between microorganisms and minerals

    SciTech Connect

    Shi, Liang; Dong, Hailiang; Reguera, Gemma; Beyenal, Haluk; Lu, Anhuai; Liu, Juan; Yu, Han-Qing; Fredrickson, James K.

    2016-08-30

    Electrons can be transferred from microorganisms to multivalent metal ions that are associated with minerals and vice versa. As the microbial cell envelope is neither physically permeable to minerals nor electrically conductive, microorganisms have evolved strategies to exchange electrons with extracellular minerals. In this Review, we discuss the molecular mechanisms that underlie the ability of microorganisms to exchange electrons, such as c-type cytochromes and microbial nanowires, with extracellular minerals and with microorganisms of the same or different species. Microorganisms that have extracellular electron transfer capability can be used for biotechnological applications, including bioremediation, biomining and the production of biofuels and nanomaterials.

  9. Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture.

    PubMed

    Kii, Isao; Nishiyama, Takashi; Li, Minqi; Matsumoto, Ken-Ichi; Saito, Mitsuru; Amizuka, Norio; Kudo, Akira

    2010-01-15

    Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.

  10. Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN.

    PubMed

    Kumar, Santhosh V R; Kulkarni, Onkar P; Mulay, Shrikant R; Darisipudi, Murthy N; Romoli, Simone; Thomasova, Dana; Scherbaum, Christina R; Hohenstein, Bernd; Hugo, Christian; Müller, Susanna; Liapis, Helen; Anders, Hans-Joachim

    2015-10-01

    Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN.

  11. Improved Methods of Producing and Administering Extracellular Vesicles | Poster

    Cancer.gov

    An efficient method of producing purified extracellular vesicles (EVs), in conjunction with a method that blocks liver macrophages from clearing EVs from the body, has produced promising results for the use of EVs in cancer therapy.

  12. Placental Extracellular Vesicles and Feto-Maternal Communication

    PubMed Central

    Tong, M.; Chamley, L.W.

    2015-01-01

    The human placenta is an anatomically unique structure that extrudes a variety of extracellular vesicles into the maternal blood (including syncytial nuclear aggregates, microvesicles, and nanovesicles). Large quantities of extracellular vesicles are produced by the placenta in both healthy and diseased pregnancies. Since their first description more than 120 years ago, placental extracellular vesicles are only now being recognized as important carriers for proteins, lipids, and nucleic acids, which may play a crucial role in feto-maternal communication. Here, we summarize the current literature on the cargos of placental extracellular vesicles and the known effects of such vesicles on maternal cells/systems, especially those of the maternal immune and vascular systems. PMID:25635060

  13. Effects of ionizing radiation on extracellular matrix

    NASA Astrophysics Data System (ADS)

    Mohamed, F.; Bradley, D. A.; Winlove, C. P.

    2007-09-01

    The extracellular matrix is a ubiquitous and important component of tissues. We investigated the effects of ionizing radiation on the physical properties of its principal macromolecular components, pericardial collagen, ligament elastin and hyaluronan, a representative glycosaminoglycan. Samples were exposed to X-rays from an electron linear accelerator in the range of 10-100 Gy to cover the range of irradiation exposure during radiotherapy. A uniaxial mechanical testing protocol was used to characterize the fibrous proteins. For pericardial tissue the major change was an increase in the elastic modulus in the toe region of the curve (⩽20% strain), from 23±18 kPa for controls to 57±22 kPa at a dose of 10 Gy ( p=0.01, α=0.05). At larger strain (⩾20% strain), the elastic modulus in the linear region decreased from 1.92±0.70 MPa for control pericardium tissue to 1.31±0.56 MPa ( p=0.01, α=0.05) for 10 Gy X-irradiated sample. Similar observations have been made previously on tendon collagen at larger strains. For elastin, the stress-strain relationship was linear up to 30% strain, but the elastic modulus decreased significantly with irradiation (controls 626±65 kPa, irradiated 474±121 kPa ( p=0.02, α=0.05), at 10 Gy X-irradiation). The results suggest that for collagen the primary effect of irradiation is generation of additional cross-links, while for elastin chain scissions are important. The viscosity of HA (at 1.25% w/v and 0.125% w/v) was measured by both cone and plate and capillary viscometry, the former providing measurement at uniform shear rate and the latter providing a more sensitive indication of changes at low viscosity. Both techniques revealed a dose-dependent reduction in viscosity (from 3400±194 cP for controls to 1500±88 cP at a shear rate of 2 s -1 and dose of 75 Gy), again suggesting depolymerization.

  14. The extracellular matrix of plants: Molecular, cellular and developmental biology

    SciTech Connect

    1996-12-31

    A symposium entitled ``The Extracellular Matrix of Plants: Molecular, Cellular and Developmental Biology was held in Tamarron, Colorado, March 15--21, 1996. The following topics were explored in addresses by 43 speakers: structure and biochemistry of cell walls; biochemistry, molecular biology and biosynthesis of lignin; secretory pathway and synthesis of glycoproteins; biosynthesis of matrix polysaccharides, callose and cellulose; role of the extracellular matrix in plant growth and development; plant cell walls in symbiosis and pathogenesis.

  15. Extracellular potassium homeostasis: insights from hypokalemic periodic paralysis.

    PubMed

    Cheng, Chih-Jen; Kuo, Elizabeth; Huang, Chou-Long

    2013-05-01

    Extracellular potassium makes up only about 2% of the total body's potassium store. The majority of the body potassium is distributed in the intracellular space, of which about 80% is in skeletal muscle. Movement of potassium in and out of skeletal muscle thus plays a pivotal role in extracellular potassium homeostasis. The exchange of potassium between the extracellular space and skeletal muscle is mediated by specific membrane transporters. These include potassium uptake by Na(+), K(+)-adenosine triphosphatase and release by inward-rectifier K(+) channels. These processes are regulated by circulating hormones, peptides, ions, and by physical activity of muscle as well as dietary potassium intake. Pharmaceutical agents, poisons, and disease conditions also affect the exchange and alter extracellular potassium concentration. Here, we review extracellular potassium homeostasis, focusing on factors and conditions that influence the balance of potassium movement in skeletal muscle. Recent findings that mutations of a skeletal muscle-specific inward-rectifier K(+) channel cause hypokalemic periodic paralysis provide interesting insights into the role of skeletal muscle in extracellular potassium homeostasis. These recent findings are reviewed.

  16. Economical evolution: microbes reduce the synthetic cost of extracellular proteins.

    PubMed

    Smith, Daniel R; Chapman, Matthew R

    2010-08-24

    Protein evolution is not simply a race toward improved function. Because organisms compete for limited resources, fitness is also affected by the relative economy of an organism's proteome. Indeed, many abundant proteins contain relatively high percentages of amino acids that are metabolically less taxing for the cell to make, thus reducing cellular cost. However, not all abundant proteins are economical, and many economical proteins are not particularly abundant. Here we examined protein composition and found that the relative synthetic cost of amino acids constrains the composition of microbial extracellular proteins. In Escherichia coli, extracellular proteins contain, on average, fewer energetically expensive amino acids independent of their abundance, length, function, or structure. Economic pressures have strategically shaped the amino acid composition of multicomponent surface appendages, such as flagella, curli, and type I pili, and extracellular enzymes, including type III effector proteins and secreted serine proteases. Furthermore, in silico analysis of Pseudomonas syringae, Mycobacterium tuberculosis, Saccharomyces cerevisiae, and over 25 other microbes spanning a wide range of GC content revealed a broad bias toward more economical amino acids in extracellular proteins. The synthesis of any protein, especially those rich in expensive aromatic amino acids, represents a significant investment. Because extracellular proteins are lost to the environment and not recycled like other cellular proteins, they present a greater burden on the cell, as their amino acids cannot be reutilized during translation. We hypothesize that evolution has optimized extracellular proteins to reduce their synthetic burden on the cell.

  17. Cyanobacterial reuse of extracellular organic carbon in microbial mats

    PubMed Central

    Stuart, Rhona K; Mayali, Xavier; Lee, Jackson Z; Craig Everroad, R; Hwang, Mona; Bebout, Brad M; Weber, Peter K; Pett-Ridge, Jennifer; Thelen, Michael P

    2016-01-01

    Cyanobacterial organic matter excretion is crucial to carbon cycling in many microbial communities, but the nature and bioavailability of this C depend on unknown physiological functions. Cyanobacteria-dominated hypersaline laminated mats are a useful model ecosystem for the study of C flow in complex communities, as they use photosynthesis to sustain a more or less closed system. Although such mats have a large C reservoir in the extracellular polymeric substances (EPSs), the production and degradation of organic carbon is not well defined. To identify extracellular processes in cyanobacterial mats, we examined mats collected from Elkhorn Slough (ES) at Monterey Bay, California, for glycosyl and protein composition of the EPS. We found a prevalence of simple glucose polysaccharides containing either α or β (1,4) linkages, indicating distinct sources of glucose with differing enzymatic accessibility. Using proteomics, we identified cyanobacterial extracellular enzymes, and also detected activities that indicate a capacity for EPS degradation. In a less complex system, we characterized the EPS of a cyanobacterial isolate from ES, ESFC-1, and found the extracellular composition of biofilms produced by this unicyanobacterial culture were similar to that of natural mats. By tracing isotopically labeled EPS into single cells of ESFC-1, we demonstrated rapid incorporation of extracellular-derived carbon. Taken together, these results indicate cyanobacteria reuse excess organic carbon, constituting a dynamic pool of extracellular resources in these mats. PMID:26495994

  18. Extracellular Matrix Scaffolds for Tissue Engineering and Regenerative Medicine.

    PubMed

    Yi, Sheng; Ding, Fei; Gong, Leiiei; Gu, Xiaosong

    2017-01-01

    The extracellular matrix is produced by the resident cells in tissues and organs, and secreted into the surrounding medium to provide biophysical and biochemical support to the surrounding cells due to its content of diverse bioactive molecules. Recently, the extracellular matrix has been used as a promising approach for tissue engineering. Emerging studies demonstrate that extracellular matrix scaffolds are able to create a favorable regenerative microenvironment, promote tissue-specific remodeling, and act as an inductive template for the repair and functional reconstruction of skin, bone, nerve, heart, lung, liver, kidney, small intestine, and other organs. In the current review, we will provide a critical overview of the structure and function of various types of extracellular matrix, the construction of three-dimensional extracellular matrix scaffolds, and their tissue engineering applications, with a focus on translation of these novel tissue engineered products to the clinic. We will also present an outlook on future perspectives of the extracellular matrix in tissue engineering and regenerative medicine.

  19. Brood stock segregation for the control of bacterial kidney disease can affect mortality of progeny chinook salmon (Oncorhynchus tshawytscha) in seawater

    USGS Publications Warehouse

    Elliott, Diane G.; Pascho, Ronald J.; Palmisano, Aldo N.

    1995-01-01

    Segregation of spring chinook salmon (Oncorhynchus tshawytscha) brood stock based on the measurement of maternal Renibacterium salmoninarum infection levels by the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody technique (FAT) was previously shown to affect the prevalence and levels of bacterial kidney disease (BKD) in progeny fish during hatchery rearing. Smolts from that study were subjected to standardized fish health and condition evaluation procedures 2 weeks before the conclusion of hatchery rearing and release of the fish for migration to the Pacific Ocean. The results suggested that the general health of the smolts in the progeny group from parents that had low R. salmoninarum infection levels or tested negative for R. salmoninarum (low-BKD group) was better than that of the smolts in the progeny group from female parents with high R. salmoninarum infection levels (high-BKD group). Testing by the ELISA showed that the overall severity of R. salmoninarum infection also was lower in the smolts from the low-BKD group. Subgroups of smolts from the study were acclimated to tanks of seawater for extended holding. After a 22-day acclimation period and 98 days in full-strength (29 ppt salinity) seawater, total mortality was 12% in the low-BKD group and 44% in the high-BKD group. All of the mortality in the low-BKD group and 85% of the mortality in the high-BKD group occurred after the fish were transferred to full-strength seawater. Testing of kidney tissues from all dead fish by the FAT revealed that 85% of the fish that died in the high-BKD group had high R. salmoninarum numbers, indicating that BKD was the cause of death. In contrast, none of the fish that died in the low-BKD group had detectable numbers of R. salmoninarum. We concluded that brood stock segregation by use of the ELISA and the FAT can affect mortality and the R. salmoninarum status of progeny chinook salmon for as long as 21 months after hatching, even after the fish have

  20. Content and persistence of extracellular DNA in native soils

    NASA Astrophysics Data System (ADS)

    Blagodatskaya, Evgenia; Blagodatsky, Sergey; Anderson, Traute-Heidi; Kuzyakov, Yakov

    2014-05-01

    The long-term persistence of soil extracellular DNA is questionable because of high potential activity of nucleases produced by soil microorganisms. By the other hand, the relative persistence of DNA-like biopolymers could be due to their adsorption on clay minerals and humus substances in soil. High-specific and ultra sensitive reagent PicoGreenTM (Molecular Probes) permits the quantitative assessment of microbial dsDNA in diluted soil extracts giving a good tool for tracing the DNA fate in soil. Our goal was to determine intracellular and extracellular DNA content in cambisol (loamy sand) and in chernozem (silty loam) soils and to investigate the possible adsorption and degradation of extracellular DNA in soil. Optimized procedure of mechanical and enzymatic destruction of cell walls was used for direct extraction of microbial DNA with Tris-EDTA buffer (Blagodatskaya et al., 2003). Extracellular dsDNA was determined in distilled water and in Tris-EDTA extracts without enzymatic or mechanical treatments. DNA content was determined after addition of PicoGreen to diluted soil extracts. Degradation of extracellular DNA was traced during 24 h incubation of 2 µg lambda-phage DNA in soil. Possible DNA adsorption to soil matrix was determined by recovery of lambda -phage DNA added to autoclaved soil. Extracellular dsDNA was absent in water extracts of both soils. The content of extracellular dsDNA extracted by Tris-EDTA buffer was 0.46 µg/g in chernozem and 1.59 µg/g in cambisol amounting 0.43 and 2.8% of total dsDNA content in these soils, respectively. 100% and 64.8% of added extracellular lambda -phage dsDNA was found in cambisol and chernozem soils, respectively, in 5 h after application. 39% and 73.5% of added DNA disappeared in cambisol and in chernozem, respectively, during 24 h incubation. Degradation rate of extracellular DNA depended on microbial biomass content, which was 2.5 times higher in chernozem as compared to cambisol. Maximum adsorption of DNA by

  1. [Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

    PubMed

    Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

    2013-01-01

    After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

  2. Enzymatic Production of Extracellular Reactive Oxygen Species by Marine Microorganisms

    NASA Astrophysics Data System (ADS)

    Diaz, J. M.; Andeer, P. F.; Hansel, C. M.

    2014-12-01

    Reactive oxygen species (ROS) serve as intermediates in a myriad of biogeochemically important processes, including cell signaling pathways, cellular oxidative stress responses, and the transformation of both nutrient and toxic metals such as iron and mercury. Abiotic reactions involving the photo-oxidation of organic matter were once considered the only important sources of ROS in the environment. However, the recent discovery of substantial biological ROS production in marine systems has fundamentally shifted this paradigm. Within the last few decades, marine phytoplankton, including diatoms of the genus Thalassiosira, were discovered to produce ample extracellular quantities of the ROS superoxide. Even more recently, we discovered widespread production of extracellular superoxide by phylogenetically and ecologically diverse heterotrophic bacteria at environmentally significant levels (up to 20 amol cell-1 hr-1), which has introduced the revolutionary potential for substantial "dark" cycling of ROS. Despite the profound biogeochemical importance of extracellular biogenic ROS, the cellular mechanisms underlying the production of this ROS have remained elusive. Through the development of a gel-based assay to identify extracellular ROS-producing proteins, we have recently found that enzymes typically involved in antioxidant activity also produce superoxide when molecular oxygen is the only available electron acceptor. For example, large (~3600 amino acids) heme peroxidases are involved in extracellular superoxide production by a bacterium within the widespread Roseobacter clade. In Thalassiosira spp., extracellular superoxide is produced by flavoproteins such as glutathione reductase and ferredoxin NADP+ reductase. Thus, extracellular ROS production may occur via secreted and/or cell surface enzymes that modulate between producing and degrading ROS depending on prevailing geochemical and/or ecological conditions.

  3. Management of bacterial kidney disease in Chinook Salmon hatcheries based on broodstock testing by enzyme-linked immunosorbent assay: A multiyear study

    USGS Publications Warehouse

    Munson, A. Douglas; Elliott, Diane G.; Johnson, Keith

    2010-01-01

    From the mid-1980s through the early 1990s, outbreaks of bacterial kidney disease (BKD) caused by Renibacterium salmoninarum continued in Chinook salmon Oncorhynchus tshawytscha in Idaho Department of Fish and Game (IDFG) hatcheries despite the use of three control methods: (1) injection of returning adult fish with erythromycin to reduce prespawning BKD mortality and limit vertical transmission of R. salmoninarum, (2) topical disinfection of green eggs with iodophor, and (3) prophylactic treatments of juvenile fish with erythromycin-medicated feed. In addition, programs to manage BKD through measurement of R. salmoninarum antigen levels in kidney tissues from spawning female Chinook salmon by an enzyme-linked immunosorbent assay (ELISA) were tested over 13–15 brood years at three IDFG hatcheries. The ELISA results were used for either (1) segregated rearing of progeny from females with high ELISA optical density (OD) values (usually ≥0.25), which are indicative of high R. salmoninarum antigen levels, or (2) culling of eggs from females with high ELISA OD values. The ELISA-based culling program had the most profound positive effects on the study populations. Mortality of juvenile fish during rearing was significantly lower at each hatchery for brood years derived from culling compared with brood years for which culling was not practiced. The prevalence of R. salmoninarum in juvenile fish, as evidenced by detection of the bacterium in kidney smears by the direct fluorescent antibody test, also decreased significantly at each hatchery. In addition, the proportions of returning adult females with kidney ELISA OD values of 0.25 or more decreased 56–85% for fish reared in brood years during which culling was practiced, whereas the proportions of ELISA-negative adults increased 55–58%. This management strategy may allow IDFG Chinook salmon hatcheries to reduce or eliminate prophylactic erythromycin-medicated feed treatments. We recommend using ELISA

  4. The extracellular matrix modulates the hallmarks of cancer.

    PubMed

    Pickup, Michael W; Mouw, Janna K; Weaver, Valerie M

    2014-12-01

    The extracellular matrix regulates tissue development and homeostasis, and its dysregulation contributes to neoplastic progression. The extracellular matrix serves not only as the scaffold upon which tissues are organized but provides critical biochemical and biomechanical cues that direct cell growth, survival, migration and differentiation and modulate vascular development and immune function. Thus, while genetic modifications in tumor cells undoubtedly initiate and drive malignancy, cancer progresses within a dynamically evolving extracellular matrix that modulates virtually every behavioral facet of the tumor cells and cancer-associated stromal cells. Hanahan and Weinberg defined the hallmarks of cancer to encompass key biological capabilities that are acquired and essential for the development, growth and dissemination of all human cancers. These capabilities include sustained proliferation, evasion of growth suppression, death resistance, replicative immortality, induced angiogenesis, initiation of invasion, dysregulation of cellular energetics, avoidance of immune destruction and chronic inflammation. Here, we argue that biophysical and biochemical cues from the tumor-associated extracellular matrix influence each of these cancer hallmarks and are therefore critical for malignancy. We suggest that the success of cancer prevention and therapy programs requires an intimate understanding of the reciprocal feedback between the evolving extracellular matrix, the tumor cells and its cancer-associated cellular stroma.

  5. Extracellular Metabolites from Industrial Microalgae and Their Biotechnological Potential.

    PubMed

    Liu, Lu; Pohnert, Georg; Wei, Dong

    2016-10-20

    Industrial microalgae, as a big family of promising producers of renewable biomass feedstock, have been commercially exploited for functional food, living feed and feed additives, high-value chemicals in nutraceuticals, cosmeceuticals, and chemical reagents. Recently, microalgae have also been considered as a group that might play an important role in biofuel development and environmental protection. Almost all current products of industrial microalgae are derived from their biomass; however, large amounts of spent cell-free media are available from mass cultivation that is mostly unexploited. In this contribution we discuss that these media, which may contain a remarkable diversity of bioactive substances are worthy to be recovered for further use. Obviously, the extracellular metabolites from industrial microalgae have long been neglected in the development of production methods for valuable metabolites. With the advances in the last ten years, more and more structures and properties from extracellular metabolites have been identified, and the potential utilization over wide fields is attracting attention. Some of these extracellular metabolites can be potentially used as drugs, antioxidants, growth regulators or metal chelators. The purpose of this review is to provide an overview of the known extracellular metabolites from industrial microalgae which might be of commercial interest. The attention mainly focuses on the reports of extracellular bioactive metabolites and their potential application in biotechnology.

  6. Propagation of thrombosis by neutrophils and extracellular nucleosome networks

    PubMed Central

    Pfeiler, Susanne; Stark, Konstantin; Massberg, Steffen; Engelmann, Bernd

    2017-01-01

    Neutrophils, early mediators of the innate immune defense, are recruited to developing thrombi in different types of thrombosis. They amplify intravascular coagulation by stimulating the tissue factor-dependent extrinsic pathway via inactivation of endogenous anticoagulants, enhancing factor XII activation or decreasing plasmin generation. Neutrophil-dependent prothrombotic mechanisms are supported by the externalization of decondensed nucleosomes and granule proteins that together form neutrophil extracellular traps. These traps, either in intact or fragmented form, are causally involved in various forms of experimental thrombosis as first indicated by their role in the enhancement of both microvascular thrombosis during bacterial infection and carotid artery thrombosis. Neutrophil extracellular traps can be induced by interactions of neutrophils with activated platelets; vice versa, these traps enhance adhesion of platelets via von Willebrand factor. Neutrophil-induced microvascular thrombus formation can restrict the dissemination and survival of blood-borne bacteria and thereby sustain intravascular immunity. Dysregulation of this innate immune pathway may support sepsis-associated coagulopathies. Notably, neutrophils and extracellular nucleosomes, together with platelets, critically promote fibrin formation during flow restriction-induced deep vein thrombosis. Neutrophil extracellular traps/extracellular nucleosomes are increased in thrombi and in the blood of patients with different vaso-occlusive pathologies and could be therapeutically targeted for the prevention of thrombosis. Thus, during infections and in response to blood vessel damage, neutrophils and externalized nucleosomes are major promoters of intravascular blood coagulation and thrombosis. PMID:27927771

  7. Multistep hopping and extracellular charge transfer in microbial redox chains.

    PubMed

    Pirbadian, Sahand; El-Naggar, Mohamed Y

    2012-10-28

    Dissimilatory metal-reducing bacteria are microorganisms that gain energy by transferring respiratory electrons to extracellular solid-phase electron acceptors. In addition to its importance for physiology and natural environmental processes, this form of metabolism is being investigated for energy conversion and fuel production in bioelectrochemical systems, where microbes are used as biocatalysts at electrodes. One proposed strategy to accomplish this extracellular charge transfer involves forming a conductive pathway to electrodes by incorporating redox components on outer cell membranes and along extracellular appendages known as microbial nanowires within biofilms. To describe extracellular charge transfer in microbial redox chains, we employed a model based on incoherent hopping between sites in the chain and an interfacial treatment of electrochemical interactions with the surrounding electrodes. Based on this model, we calculated the current-voltage (I-V) characteristics and found the results to be in good agreement with I-V measurements across and along individual microbial nanowires produced by the bacterium Shewanella oneidensis MR-1. Based on our analysis, we propose that multistep hopping in redox chains constitutes a viable strategy for extracellular charge transfer in microbial biofilms.

  8. Optogenetic approaches addressing extracellular modulation of neural excitability

    PubMed Central

    Ferenczi, Emily A.; Vierock, Johannes; Atsuta-Tsunoda, Kyoko; Tsunoda, Satoshi P.; Ramakrishnan, Charu; Gorini, Christopher; Thompson, Kimberly; Lee, Soo Yeun; Berndt, Andre; Perry, Chelsey; Minniberger, Sonja; Vogt, Arend; Mattis, Joanna; Prakash, Rohit; Delp, Scott; Deisseroth, Karl; Hegemann, Peter

    2016-01-01

    The extracellular ionic environment in neural tissue has the capacity to influence, and be influenced by, natural bouts of neural activity. We employed optogenetic approaches to control and investigate these interactions within and between cells, and across spatial scales. We began by developing a temporally precise means to study microdomain-scale interactions between extracellular protons and acid-sensing ion channels (ASICs). By coupling single-component proton-transporting optogenetic tools to ASICs to create two-component optogenetic constructs (TCOs), we found that acidification of the local extracellular membrane surface by a light-activated proton pump recruited a slow inward ASIC current, which required molecular proximity of the two components on the membrane. To elicit more global effects of activity modulation on ‘bystander’ neurons not under direct control, we used densely-expressed depolarizing (ChR2) or hyperpolarizing (eArch3.0, eNpHR3.0) tools to create a slow non-synaptic membrane current in bystander neurons, which matched the current direction seen in the directly modulated neurons. Extracellular protons played contributory role but were insufficient to explain the entire bystander effect, suggesting the recruitment of other mechanisms. Together, these findings present a new approach to the engineering of multicomponent optogenetic tools to manipulate ionic microdomains, and probe the complex neuronal-extracellular space interactions that regulate neural excitability. PMID:27045897

  9. Extracellular Metabolites from Industrial Microalgae and Their Biotechnological Potential

    PubMed Central

    Liu, Lu; Pohnert, Georg; Wei, Dong

    2016-01-01

    Industrial microalgae, as a big family of promising producers of renewable biomass feedstock, have been commercially exploited for functional food, living feed and feed additives, high-value chemicals in nutraceuticals, cosmeceuticals, and chemical reagents. Recently, microalgae have also been considered as a group that might play an important role in biofuel development and environmental protection. Almost all current products of industrial microalgae are derived from their biomass; however, large amounts of spent cell-free media are available from mass cultivation that is mostly unexploited. In this contribution we discuss that these media, which may contain a remarkable diversity of bioactive substances are worthy to be recovered for further use. Obviously, the extracellular metabolites from industrial microalgae have long been neglected in the development of production methods for valuable metabolites. With the advances in the last ten years, more and more structures and properties from extracellular metabolites have been identified, and the potential utilization over wide fields is attracting attention. Some of these extracellular metabolites can be potentially used as drugs, antioxidants, growth regulators or metal chelators. The purpose of this review is to provide an overview of the known extracellular metabolites from industrial microalgae which might be of commercial interest. The attention mainly focuses on the reports of extracellular bioactive metabolites and their potential application in biotechnology. PMID:27775594

  10. Salt stress represses production of extracellular proteases in Bacillus pumilus.

    PubMed

    Liu, R F; Huang, C L; Feng, H

    2015-05-11

    Bacillus pumilus is able to secrete subtilisin-like prote-ases, one of which has been purified and characterized biochemically, demonstrating great potential for use in industrial applications. In the current study, the biosynthesis and transcription of extracellular pro-teases in B. pumilus (BA06) under salt stress were investigated using various methods, including a proteolytic assay, zymogram analysis, and real-time PCR. Our results showed that total extracellular proteolytic activity, both in fermentation broth and on milk-containing agar plates, was considerably repressed by salt in a dosage-dependent manner. As Bacillus species usually secret multiple extracellular proteases, a vari-ety of individual extracellular protease encoding genes were selected for real-time PCR analysis. It was shown that proteases encoded by the aprE and aprX genes were the major proteases in the fermentation broth in terms of their transcripts in B. pumilus. Further, transcription of aprE, aprX, and epr genes was indeed repressed by salt stress. In con-trast, transcription of other genes (e.g., vpr and wprA) was not repressed or significantly affected by the salt. Conclusively, salt stress represses total extracellular proteolytic activity in B. pumilus, which can largely be ascribed to suppression of the major protease-encoding genes (aprE, aprX) at the transcriptional level. In contrast, transcription of other pro-tease-encoding genes (e.g., vpr, wprA) was not repressed by salt stress.

  11. Vitamin A Deficiency and Alterations in the Extracellular Matrix

    PubMed Central

    Barber, Teresa; Esteban-Pretel, Guillermo; Marín, María Pilar; Timoneda, Joaquín

    2014-01-01

    Vitamin A or retinol which is the natural precursor of several biologically active metabolites can be considered the most multifunctional vitamin in mammals. Its deficiency is currently, along with protein malnutrition, the most serious and common nutritional disorder worldwide. It is necessary for normal embryonic development and postnatal tissue homeostasis, and exerts important effects on cell proliferation, differentiation and apoptosis. These actions are produced mainly by regulating the expression of a variety of proteins through transcriptional and non-transcriptional mechanisms. Extracellular matrix proteins are among those whose synthesis is known to be modulated by vitamin A. Retinoic acid, the main biologically active form of vitamin A, influences the expression of collagens, laminins, entactin, fibronectin, elastin and proteoglycans, which are the major components of the extracellular matrix. Consequently, the structure and macromolecular composition of this extracellular compartment is profoundly altered as a result of vitamin A deficiency. As cell behavior, differentiation and apoptosis, and tissue mechanics are influenced by the extracellular matrix, its modifications potentially compromise organ function and may lead to disease. This review focuses on the effects of lack of vitamin A in the extracellular matrix of several organs and discusses possible molecular mechanisms and pathologic implications. PMID:25389900

  12. In vitro Determination of Extracellular Proteins from Xylella fastidiosa

    PubMed Central

    Mendes, Juliano S.; Santiago, André S.; Toledo, Marcelo A. S.; Horta, Maria A. C.; de Souza, Alessandra A.; Tasic, Ljubica; de Souza, Anete P.

    2016-01-01

    The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3–30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components. PMID:28082960

  13. Monitoring of Extracellular Matrix Formation using Nanosecond Pulsed Laser

    NASA Astrophysics Data System (ADS)

    Ishihara, Miya; Sato, Masato; Mitani, Genya; Nagai, Toshihiro; Kutsuna, Toshiharu; Mochida, Joji; Kikuchi, Makoto

    There is a new demand in the field of tissue engineering for evaluation technology of extracellular matrix because the extracellular matrix plays an important role in the function of skeletal tissue such as articular cartilage. We previously proposed a noninvasive method of viscoelastic characterization of tissue phantom, based on the photoacoustic measurement. The purpose of this study was to verify the applicability of the photoacoustic measurement method for monitoring of the development of extracellular matrix using tissue engineering technology. The decay times measured by the photoacoustic method were varied with culture periods when tissue-engineered articular cartilages with various culture periods (-12 weeks) were used as samples. Tissue-engineered cartilage cultured for a long period showed shorter decay times, indicating that the samples approached an elastic solid from a rheological viewpoint. By comparison between biochemical analyses and biomechanical studies, we proved that the photoacoustic signal was a good indicator for evaluating extracellular matrix formation because the change of the photoacoustic decay times would reflect the production of an extracellular matrix.

  14. A Look inside the Listeria monocytogenes Biofilms Extracellular Matrix

    PubMed Central

    Colagiorgi, Angelo; Di Ciccio, Pierluigi; Zanardi, Emanuela; Ghidini, Sergio; Ianieri, Adriana

    2016-01-01

    Listeria monocytogenes is a foodborne pathogen able to persist in food industry and is responsible for a severe illness called listeriosis. The ability of L. monocytogenes to persist in environments is due to its capacity to form biofilms that are a sessile community of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS’s). In this review, we summarized recent efforts performed in order to better characterize the polymeric substances that compose the extracellular matrix (ECM) of L. monocytogenes biofilms. EPS extraction and analysis led to the identification of polysaccharides, proteins, extracellular DNA, and other molecules within the listerial ECM. All this knowledge will be useful for increasing food protection, suggesting effective strategies for the minimization of persistence of L. monocytogenes in food industry environments. PMID:27681916

  15. Functional Advantages Conferred by Extracellular Prokaryotic Membrane Vesicles

    PubMed Central

    Manning, Andrew J.; Kuehn, Meta J.

    2015-01-01

    The absence of subcellular organelles is a characteristic typically used to distinguish prokaryotic from eukaryotic cells. But recent discoveries do not support this dogma. Over the past 50 years, researchers have begun to appreciate and characterize Gram-negative bacterial outer membrane derived vesicles and Gram-positive and archaeal membrane vesicles. These extracellular, membrane-bound organelles can perform a variety of functions, including binding and delivery of DNA, transport of virulence factors, protection of the cell from outer membrane targeting antimicrobials, and ridding the cell of toxic envelope proteins. Here we review the contributions of these extracellular organelles to prokaryotic physiology and compare these with the contributions of the bacterial interior membrane bound organelles responsible for harvesting light energy and for generating magnetic crystals of heavy metals. Understanding the roles of these multifunctional extracellular vesicle organelles as microbial tools will help us to better realize the diverse interactions that occur in our polymicrobial world. PMID:23615201

  16. Extracellular matrix as a driver for lung regeneration.

    PubMed

    Balestrini, Jenna L; Niklason, Laura E

    2015-03-01

    Extracellular matrix has manifold roles in tissue mechanics, guidance of cellular behavior, developmental biology, and regenerative medicine. Over the past several decades, various pre-clinical and clinical studies have shown that many connective tissues may be replaced and/or regenerated using suitable extracellular matrix scaffolds. More recently, decellularization of lung tissue has shown that gentle removal of cells can leave behind a "footprint" within the matrix that may guide cellular adhesion, differentiation and homing following cellular repopulation. Fundamental issues like understanding matrix composition and micro-mechanics remain difficult to tackle, largely because of a lack of available assays and tools for systematically characterizing intact matrix from tissues and organs. This review will critically examine the role of engineered and native extracellular matrix in tissue and lung regeneration, and provide insights into directions for future research and translation.

  17. Functional advantages conferred by extracellular prokaryotic membrane vesicles.

    PubMed

    Manning, Andrew J; Kuehn, Meta J

    2013-01-01

    The absence of subcellular organelles is a characteristic typically used to distinguish prokaryotic from eukaryotic cells. But recent discoveries do not support this dogma. Over the past 50 years, researchers have begun to appreciate and characterize Gram-negative bacterial outer membrane-derived vesicles and Gram-positive and archaeal membrane vesicles. These extracellular, membrane-bound organelles can perform a variety of functions, including binding and delivery of DNA, transport of virulence factors, protection of the cell from outer membrane targeting antimicrobials and ridding the cell of toxic envelope proteins. Here, we review the contributions of these extracellular organelles to prokaryotic physiology and compare these with the contributions of the bacterial interior membrane-bound organelles responsible for harvesting light energy and for generating magnetic crystals of heavy metals. Understanding the roles of these multifunctional extracellular vesicle organelles as microbial tools will help us to better realize the diverse interactions that occur in our polymicrobial world.

  18. Modeling extracellular electrical stimulation: II. Computational validation and numerical results.

    PubMed

    Tahayori, Bahman; Meffin, Hamish; Dokos, Socrates; Burkitt, Anthony N; Grayden, David B

    2012-12-01

    The validity of approximate equations describing the membrane potential under extracellular electrical stimulation (Meffin et al 2012 J. Neural Eng. 9 065005) is investigated through finite element analysis in this paper. To this end, the finite element method is used to simulate a cylindrical neurite under extracellular stimulation. Laplace's equations with appropriate boundary conditions are solved numerically in three dimensions and the results are compared to the approximate analytic solutions. Simulation results are in agreement with the approximate analytic expressions for longitudinal and transverse modes of stimulation. The range of validity of the equations describing the membrane potential for different values of stimulation and neurite parameters are presented as well. The results indicate that the analytic approach can be used to model extracellular electrical stimulation for realistic physiological parameters with a high level of accuracy.

  19. Unidirectional cell crawling model guided by extracellular cues.

    PubMed

    Wang, Zhanjiang; Geng, Yuxu

    2015-03-01

    Cell migration is a highly regulated and complex cellular process to maintain proper homeostasis for various biological processes. Extracellular environment was identified as the main affecting factors determining the direction of cell crawling. It was observed experimentally that the cell prefers migrating to the area with denser or stiffer array of microposts. In this article, an integrated unidirectional cell crawling model was developed to investigate the spatiotemporal dynamics of unidirectional cell migration, which incorporates the dominating intracellular biochemical processes, biomechanical processes and the properties of extracellular micropost arrays. The interpost spacing and the stiffness of microposts are taken into account, respectively, to study the mechanism of unidirectional cell locomotion and the guidance of extracellular influence cues on the direction of unidirectional cell crawling. The model can explain adequately the unidirectional crawling phenomena observed in experiments such as "spatiotaxis" and "durotaxis," which allows us to obtain further insights into cell migration.

  20. Only scratching the cell surface: extracellular signals in cerebrum development.

    PubMed

    Hébert, Jean M

    2013-08-01

    Numerous roles have been identified for extracellular signals such as Fibroblast Growth Factors (FGFs), Transforming Growth Factor-βs (TGFβs), Wingless-Int proteins (WNTs), and Sonic Hedgehog (SHH) in assigning fates to cells during development of the cerebrum. However, several fundamental questions remain largely unexplored. First, how does the same extracellular signal instruct precursor cells in different locations or at different stages to adopt distinct fates? And second, how does a precursor cell integrate multiple signals to adopt a specific fate? Answers to these questions require knowing the mechanisms that underlie each cell type's competence to respond to certain extracellular signals. This brief review provides illustrative examples of potential mechanisms that begin to bridge the gap between cell surface and cell fate during cerebrum development.

  1. Imaging hydrated microbial extracellular polymers: Comparative analysis by electron microscopy

    SciTech Connect

    Dohnalkova, A.C.; Marshall, M. J.; Arey, B. W.; Williams, K. H.; Buck, E. C.; Fredrickson, J. K.

    2011-01-01

    Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigating microscale associations. Electron microscopy has been used extensively for geomicrobial investigations and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions using conventional electron microscopy approaches of imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding nature of interactions between microbial extracellular polymers and their environment.

  2. Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate.

    PubMed

    Li, Man-Song; Holstead, Ryan G; Wang, Wuyang; Linsdell, Paul

    2011-01-01

    The CFTR contributes to Cl⁻ and HCO₃⁻ transport across epithelial cell apical membranes. The extracellular face of CFTR is exposed to varying concentrations of Cl⁻ and HCO₃⁻ in epithelial tissues, and there is evidence that CFTR is sensitive to changes in extracellular anion concentrations. Here we present functional evidence that extracellular Cl⁻ and HCO₃⁻ regulate anion conduction in open CFTR channels. Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO₃⁻ than when it contains Cl⁻. This difference appears to reflect differences in the ability of extracellular HCO₃⁻ and Cl⁻ to interact with and repel intracellular blocking anions from the pore. Strong block by endogenous cytosolic anions leading to reduced CFTR channel currents in intact cells occurs at physiologically relevant HCO₃⁻ concentrations and membrane potentials and can result in up to ∼50% inhibition of current amplitude. We propose that channel block by cytosolic anions is a previously unrecognized, physiologically relevant mechanism of channel regulation that confers on CFTR channels sensitivity to different anions in the extracellular fluid. We further suggest that this anion sensitivity represents a feedback mechanism by which CFTR-dependent anion secretion could be regulated by the composition of the secretions themselves. Implications for the mechanism and regulation of CFTR-dependent secretion in epithelial tissues are discussed.

  3. Production of extracellular fatty acid using engineered Escherichia coli

    PubMed Central

    2012-01-01

    Background As an alternative for economic biodiesel production, the microbial production of extracellular fatty acid from renewable resources is receiving more concerns recently, since the separation of fatty acid from microorganism cells is normally involved in a series of energy-intensive steps. Many attempts have been made to construct fatty acid producing strains by targeting genes in the fatty acid biosynthetic pathway, while few studies focused on the cultivation process and the mass transfer kinetics. Results In this study, both strain improvements and cultivation process strategies were applied to increase extracellular fatty acid production by engineered Escherichia coli. Our results showed overexpressing ‘TesA and the deletion of fadL in E. coli BL21 (DE3) improved extracellular fatty acid production, while deletion of fadD didn’t strengthen the extracellular fatty acid production for an undetermined mechanism. Moreover, the cultivation process controls contributed greatly to extracellular fatty acid production with respect to titer, cell growth and productivity by adjusting the temperature, adding ampicillin and employing on-line extraction. Under optimal conditions, the E. coli strain (pACY-‘tesA-ΔfadL) produced 4.8 g L−1 extracellular fatty acid, with the specific productivity of 0.02 g h−1 g−1dry cell mass, and the yield of 4.4% on glucose, while the ratios of cell-associated fatty acid versus extracellular fatty acid were kept below 0.5 after 15 h of cultivation. The fatty acids included C12:1, C12:0, C14:1, C14:0, C16:1, C16:0, C18:1, C18:0. The composition was dominated by C14 and C16 saturated and unsaturated fatty acids. Using the strain pACY-‘tesA, similar results appeared under the same culture conditions and the titer was also much higher than that ever reported previously, which suggested that the supposedly superior strain did not necessarily perform best for the efficient production of desired product. The strain p

  4. Response of zonal chondrocytes to extracellular matrix-hydrogels.

    PubMed

    Hwang, Nathaniel S; Varghese, Shyni; Lee, H Janice; Theprungsirikul, Parnduangjai; Canver, Adam; Sharma, Blanka; Elisseeff, Jennifer

    2007-09-04

    We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses.

  5. RESPONSE OF ZONAL CHONDROCYTES TO EXTRACELLULAR MATRIX-HYDROGELS

    PubMed Central

    Hwang, Nathaniel S.; Varghese, Shyni; Lee, H. Janice; Theprungsirikul, Parnduangjai; Canver, Adam; Sharma, Blanka; Elisseeff, Jennifer

    2009-01-01

    We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses. PMID:17692846

  6. Catabolism of host-derived compounds during extracellular bacterial infections

    PubMed Central

    Meadows, Jamie A.; Wargo, Matthew J.

    2014-01-01

    Efficient catabolism of host-derived compounds is essential for bacterial survival and virulence. While these links in intracellular bacteria are well-studied, such studies in extracellular bacteria lag behind, mostly for technical reasons. The field has identified important metabolic pathways, but the mechanisms by which they impact infection and in particular, establishing the importance of a compound’s catabolism versus alternate metabolic roles has been difficult. In this review we will examine evidence for catabolism during extracellular bacterial infections in animals and known or potential roles in virulence. In the process, we point out key gaps in the field that will require new or newly adapted techniques. PMID:24038340

  7. Extracellular vesicles round off communication in the nervous system

    PubMed Central

    Budnik, Vivian; Ruiz-Cañada, Catalina; Wendler, Franz

    2016-01-01

    Functional neural competence and integrity require interactive exchanges among sensory and motor neurons, interneurons and glial cells. Recent studies have attributed some of the tasks needed for these exchanges to extracellular vesicles (such as exosomes and microvesicles), which are most prominently involved in shuttling reciprocal signals between myelinating glia and neurons, thus promoting neuronal survival, the immune response mediated by microglia, and synapse assembly and plasticity. Such vesicles have also been identified as important factors in the spread of neurodegenerative disorders and brain cancer. These extracellular vesicle functions add a previously unrecognized level of complexity to transcellular interactions within the nervous system. PMID:26891626

  8. Extracellular proteins limit the dispersal of biogenic nanoparticles

    USGS Publications Warehouse

    Moreau, J.W.; Weber, P.K.; Martin, M.C.; Gilbert, B.; Hutcheon, I.D.; Banfield, J.F.

    2007-01-01

    High-spatial-resolution secondary ion microprobe spectrometry, synchrotron radiation-based Fourier-transform infrared spectroscopy, and polyacrylamide gel analysis demonstrated the intimate association of proteins with spheroidal aggregates of biogenic zinc sulfide nanocrystals, an example of extracellular biomineralization. Experiments involving synthetic zinc sulfide nanoparticles and representative amino acids indicated a driving role for cysteine in rapid nanoparticle aggregation. These findings suggest that microbially derived extracellular proteins can limit the dispersal of nanoparticulate metal-bearing phases, such as the mineral products of bioremediation, that may otherwise be transported away from their source by subsurface fluid flow.

  9. Extracellular Vesicles as New Players in Cellular Senescence

    PubMed Central

    Urbanelli, Lorena; Buratta, Sandra; Sagini, Krizia; Tancini, Brunella; Emiliani, Carla

    2016-01-01

    Cell senescence is associated with the secretion of many factors, the so-called “senescence-associated secretory phenotype”, which may alter tissue microenvironment, stimulating the organism to clean up senescent cells and replace them with newly divided ones. Therefore, although no longer dividing, these cells are still metabolically active and influence the surrounding tissue. Much attention has been recently focused not only on soluble factors released by senescent cells, but also on extracellular vesicles as conveyors of senescence signals outside the cell. Here, we give an overview of the role of extracellular vesicles in biological processes and signaling pathways related to senescence and aging. PMID:27571072

  10. Modulation of Human Cardiac TRPM7 Current by Extracellular Acidic pH Depends upon Extracellular Concentrations of Divalent Cations

    PubMed Central

    Mačianskienė, Regina; Almanaitytė, Mantė; Jekabsone, Aistė; Mubagwa, Kanigula

    2017-01-01

    TRPM7 channels participate in a variety of physiological/pathological processes. TRPM7 currents are modulated by protons but opposing effects of external pH (pHo) (potentiation vs inhibition) have been reported. TRPM7 has been less studied in human cardiomyocytes than in heart-derived non-cardiomyocyte cells. We used the whole-cell patch-clamp technique on isolated human atrial cardiomyocytes to investigate the impact of an acidic pHo on the TRPM7 current. With voltage-dependent and other ion channels inhibited, cardiomyocytes were challenged with external acidification in either the presence or the absence of extracellular divalent cations. TRPM7 outward and inward currents were increased by acidic pHo in extracellular medium containing Ca2+ and Mg2+, but suppressed by acidic pHo in the absence of extracellular Ca2+ and Mg2+. The potentiating effect in the presence of extracellular divalents occurred at pHo below 6 and was voltage-dependent. The inhibitory effect in the absence of extracellular divalents was already marked at pHo of 6 and was practically voltage-independent. TRPM7 current density was higher in cardiomyocytes from patients with history of coronary vascular disease and the difference compared to cardiomyocytes from patients without history of myocardial ischemia increased with acidic pHo. We demonstrate that proton-induced modification of TRPM7 currents depends on the presence of extracellular Ca2+ and Mg2+. Variability of the TRPM7 current density in human cardiomyocytes is related to the clinical history, being higher in atrial fibrillation and in ischemic cardiomyopathy. PMID:28129376

  11. Focus on Extracellular Vesicles: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles.

    PubMed

    Zhang, Bin; Yeo, Ronne Wee Yeh; Tan, Kok Hian; Lim, Sai Kiang

    2016-02-06

    The intense research focus on stem and progenitor cells could be attributed to their differentiation potential to generate new cells to replace diseased or lost cells in many highly intractable degenerative diseases, such as Alzheimer disease, multiple sclerosis, and heart diseases. However, experimental and clinical studies have increasingly attributed the therapeutic efficacy of these cells to their secretion. While stem and progenitor cells secreted many therapeutic molecules, none of these molecules singly or in combination could recapitulate the functional effects of stem cell transplantations. Recently, it was reported that extracellular vesicles (EVs) could recapitulate the therapeutic effects of stem cell transplantation. Based on the observations reported thus far, the prevailing hypothesis is that stem cell EVs exert their therapeutic effects by transferring biologically active molecules such as proteins, lipids, mRNA, and microRNA from the stem cells to injured or diseased cells. In this respect, stem cell EVs are similar to EVs from other cell types. They are both primarily vehicles for intercellular communication. Therefore, the differentiating factor is likely due to the composition of their cargo. The cargo of EVs from different cell types are known to include a common set of proteins and also proteins that reflect the cell source of the EVs and the physiological or pathological state of the cell source. Hence, elucidation of the stem cell EV cargo would provide an insight into the multiple physiological or biochemical changes necessary to affect the many reported stem cell-based therapeutic outcomes in a variety of experimental models and clinical trials.

  12. Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1986 Annual Report.

    SciTech Connect

    Kaattari, Stephen L.

    1987-06-01

    Bacterial kidney disease (BRD) has been and remains a chronic contributory problem limiting the productivity of salmon of the Columbia River Basin. Control of this disease will not come easily, but it would lead to a tremendous increase in the health and numbers of salmon populations. Vaccination of salmon of Renibacterium salmoninarum (KDB) is a potentially successful method of controlling this disease. To date, however, no successful vaccine has been developed for general use. A possible solution to this problem,and thus the goal of this research, is to isolate the antigenic components of KDB and enhance their ability to activate the host defenses. This will be accomplished by the chemical modification of these antigens with potent immunomodulatory substances. These modified antigens will then be tested for their effectiveness in inducing immunity to BKD and thereby preventing the disease. The goal of the project's third year was to test the immunogenicity and prophylactic value in coho salmon (Oncorhynchus kisutch) of various chemical conjugates of Renibacterium salmoninarum cells and major antigens. This was accomplished by assessing the serum antibody response, the cellular immune response (cellular proliferation), and the kinetics of mortality after Lethal injections of the bacterium. An important facet of this research is the identification and isolation of virulence factors. These studies are not only important to the dissection of the mechanism of pathogenesis of bacterial kidney disease, but the purification of such a factor(s) will insure the production of a more potent vaccine. The studies completed this year have: (1) identified antigenic material which protect; (2) identified antigenic material which can exacerbate the disease; (3) identified a possibly major mechanism of pathogenesis via the interference with antibody; (4) the general ability to produce delineated a western blot technique for identification of infected fish; (5) described the use of

  13. Pathogens associated with native and exotic trout populations in Shenandoah National Park and the relationships to fish stocking practices

    USGS Publications Warehouse

    Panek, Frank M.; Atkinson, James; Coll, John

    2008-01-01

    Restrictive fish stocking policies in National Parks were developed as early as 1936 in order to preserve native fish assemblages and historic genetic diversity. Despite recent efforts to understand the effects of non-native or exotic fish introductions, park managers have limited information regarding the effects of these introductions on native fish communities. Shenandoah National Park was established in 1936 and brook trout (Salvelinus fontinalis) restoration within selected streams in the park began in 1937 in collaboration with the Virginia Department of Game and Inland Fisheries (VDGIF). An analysis of tissue samples from brook, brown (Salmo trutta), and rainbow trout (Oncorhynchus mykiss) from 29 streams within the park from 1998–2002 revealed the presence of Renibacterium salmoninarum, Yersinia ruckeri, and infectious pancreatic necrosis virus (IPNv). In order to investigate the relationships of the occurrence of fish pathogens with stocking histories we classified the streams into three categories: 1) streams with no record of stocking, 2) streams that are known to have been stocked historically, and 3) streams that were historically stocked within the park and continue to be stocked downstream of the park boundary. The occurrences of pathogens were summarized relative to this stocking history. Renibacterium salmoninarum, the causative agent of bacterial kidney disease, was the most prevalent pathogen found, occurring in all three species and stream stocking categories, and appears to be endemic to the park. Two other pathogens, Yersinia ruckeri and infectious pancreatic necrosis virus were also described from brook trout populations within the park. IPNv was only found in brook trout populations in streams with prior stocking histories. Yersinia ruckeri was only found in brook trout in steams that have never been stocked and like R. salmoninarum, is likely endemic.

  14. Extracellular Xylella fastidiosa genomic DNA enhances biofilm formation in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa (Xf) is a Gram negative, xylem-limited bacterium that causes Pierce’s Disease (PD) of grapevine, as well as other diseases of economically important crops and landscape plants. Many bacteria produce large amounts of extracellular DNA, which may function as a matrix component in b...

  15. The macrophage chemotactic activity of Edwardsiella tarda extracellular products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The chemoattractant capabilities of Edwardsiella tarda extracellular products (ECP) were investigated from two isolates, the virulent FL6-60 parent and less virulent RET-04 mutant. Chemotaxis and chemokinesis were assayed in vitro using blind well chambers with peritoneal macrophages obtained from ...

  16. EXTRACELLULAR HEAT SHOCK PROTEINS: A NEW LOCATION, A NEW FUNCTION

    PubMed Central

    De Maio, Antonio; Vazquez, Daniel

    2015-01-01

    The expression of heat shock proteins (hsp) is a basic and well conserved cellular response to an array of stresses. These proteins are involved in the repair of cellular damage induced by the stress, which is necessary for the salutary resolution from the insult. Moreover, they confer protection from subsequent insults, which has been coined stress tolerance. Since these proteins are expressed in subcellular compartments, it was thought that their function during stress conditions was circumscribed to the intracellular environment. However, it is now well established that hsp can also be present outside cells where they appear to display a function different than the well understood chaperone role. Extracellular hsp act as alert stress signals priming other cells, particularly of the immune system, to avoid the propagation of the insult and favor resolution. Since the majority of hsp do not possess a secretory peptide signal, they are likely be exported by a non-classical secretory pathway. Different mechanisms have been proposed to explain the export of hsp, including translocation across the plasma membrane and release associated with lipid vesicles, as well as the passive release after cell death by necrosis. Extracellular hsp appear in various flavors, including membrane-bound and membrane-free forms. All of these variants of extracellular hsp suggest that their interactions with cells may be quite diverse, both in target cell types and the activation signaling pathways. This review addresses some of our current knowledge about the release and relevance of extracellular hsp. PMID:23807250

  17. Biology and proteomics of extracellular vesicles: harnessing their clinical potential.

    PubMed

    D'Souza-Schorey, Crislyn; Di Vizio, Dolores

    2014-06-01

    Extracellular membrane vesicles have recently emerged as versatile mediators of intercellular communication, pathogenesis, drug and gene delivery and as potentially rich reservoirs of clinical biomarkers. Channeling their properties toward patient care is dependent on technological progress in approaches used for their analysis and molecular profiling.

  18. Extracellular NAD(+): a danger signal hindering regulatory T cells.

    PubMed

    Adriouch, Sahil; Haag, Friedrich; Boyer, Olivier; Seman, Michel; Koch-Nolte, Friedrich

    2012-11-01

    Endogenous danger signals released during cell damage contribute to alert the immune system. Typically, their release results in the activation and maturation of innate immune cells, and the production of pro-inflammatory cytokines. In addition, extracellular NAD(+) stimulates immune responses by hindering regulatory T cells (Tregs), and could, therefore, represent the prototype of a new category of danger signals.

  19. Changes in Acetylcholine Extracellular Levels during Cognitive Processes

    ERIC Educational Resources Information Center

    Pepeu, Giancarlo; Giovannini, Maria Grazia

    2004-01-01

    Measuring the changes in neurotransmitter extracellular levels in discrete brain areas is considered a tool for identifying the neuronal systems involved in specific behavioral responses or cognitive processes. Acetylcholine (ACh) is the first neurotransmitter whose diffusion from the central nervous system was investigated and whose extracellular…

  20. Differential induction of redox sensitive extracellular phenolic amides in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study focuses on the differential induction of extracellular phenolic amides that accumulate in potato cell suspensions during the first few hours of the interaction between these plant cells and bacterial pathogens or pathogen-related elicitors. Using suspension cells of Solanum tuberosum we ...

  1. Model-based source localization of extracellular action potentials.

    PubMed

    Somogyvári, Zoltán; Zalányi, László; Ulbert, István; Erdi, Péter

    2005-09-30

    A new model-based analysis method was set up for revealing information encrypted in extracellular spatial potential patterns of neocortical action potentials. Spikes were measured by extracellular linear multiple microelectrode in vivo cat's primary auditory cortex and were analyzed based on current source density (CSD) distribution models. Validity of the monopole and other point source approximations were tested on the measured potential patterns by numerical fitting. We have found, that point source models could not provide accurate description of the measured patterns. We introduced a new model of the CSD distribution on a spiking cell, called counter-current model (CCM). This new model was shown to provide better description of the spatial current distribution of the cell during the initial negative deflection of the extracellular action potential, from the onset of the spike to the negative peak. The new model was tested on simulated extracellular potentials. We proved numerically, that all the parameters of the model could be determined accurately based on measurements. Thus, fitting of the CCM allowed extraction of these parameters from the measurements. Due to model fitting, CSD could be calculated with much higher accuracy as done with the traditional method because distance dependence of the spatial potential patterns was explicitly taken into consideration in our method. Average CSD distribution of the neocortical action potentials was calculated and spatial decay constant of the dendritic trees was determined by applying our new method.

  2. Extracellular matrix components direct porcine muscle stem cell behavior

    SciTech Connect

    Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J.

    2010-02-01

    In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.

  3. Sortilin mediates vascular calcification via its recruitment into extracellular vesicles

    PubMed Central

    Goettsch, Claudia; Hutcheson, Joshua D.; Aikawa, Masanori; Iwata, Hiroshi; Pham, Tan; Nykjaer, Anders; Kjolby, Mads; Rogers, Maximillian; Michel, Thomas; Shibasaki, Manabu; Hagita, Sumihiko; Kramann, Rafael; Singh, Sasha A.

    2016-01-01

    Vascular calcification is a common feature of major cardiovascular diseases. Extracellular vesicles participate in the formation of microcalcifications that are implicated in atherosclerotic plaque rupture; however, the mechanisms that regulate formation of calcifying extracellular vesicles remain obscure. Here, we have demonstrated that sortilin is a key regulator of smooth muscle cell (SMC) calcification via its recruitment to extracellular vesicles. Sortilin localized to calcifying vessels in human and mouse atheromata and participated in formation of microcalcifications in SMC culture. Sortilin regulated the loading of the calcification protein tissue nonspecific alkaline phosphatase (TNAP) into extracellular vesicles, thereby conferring its calcification potential. Furthermore, SMC calcification required Rab11-dependent trafficking and FAM20C/casein kinase 2–dependent C-terminal phosphorylation of sortilin. In a murine model, Sort1-deficiency reduced arterial calcification but did not affect bone mineralization. Additionally, transfer of sortilin-deficient BM cells to irradiated atherosclerotic mice did not affect vascular calcification, indicating a primary role of SMC-derived sortilin. Together, the results of this study identify sortilin phosphorylation as a potential therapeutic target for ectopic calcification/microcalcification and may clarify the mechanism that underlies the genetic association between the SORT1 gene locus and coronary artery calcification. PMID:26950419

  4. Extracellular Recognition of Oomycetes during Biotrophic Infection of Plants.

    PubMed

    Raaymakers, Tom M; Van den Ackerveken, Guido

    2016-01-01

    Extracellular recognition of pathogens by plants constitutes an important early detection system in plant immunity. Microbe-derived molecules, also named patterns, can be recognized by pattern recognition receptors (PRRs) on the host cell membrane that trigger plant immune responses. Most knowledge on extracellular pathogen detection by plants comes from research on bacterial and fungal pathogens. For oomycetes, that comprise some of the most destructive plant pathogens, mechanisms of extracellular pattern recognition have only emerged recently. These include newly recognized patterns, e.g., cellulose-binding elicitor lectin, necrosis and ethylene-inducing peptide 1-like proteins (NLPs), and glycoside hydrolase 12, as well as their receptors, e.g., the putative elicitin PRR elicitin response and the NLP PRR receptor-like protein 23. Immunity can also be triggered by the release of endogenous host-derived patterns, as a result of oomycete enzymes or damage. In this review we will describe the types of patterns, both pathogen-derived exogenous and plant-derived endogenous ones, and what is known about their extracellular detection during (hemi-)biotrophic oomycete infection of plants.

  5. Increased intra- and extracellular granzyme expression in patients with tuberculosis.

    PubMed

    Garcia-Laorden, M Isabel; Blok, Dana C; Kager, Liesbeth M; Hoogendijk, Arie J; van Mierlo, Gerard J; Lede, Ivar O; Rahman, Wahid; Afroz, Rumana; Ghose, Aniruddha; Visser, Caroline E; Md Zahed, Abu Shahed; Husain, Md Anwar; Alam, Khan Mashrequl; Chandra Barua, Pravat; Hassan, Mahtabuddin; Hossain, Ahmed; Tayab, Md Abu; Day, Nick; Dondorp, Arjen M; de Vos, Alex F; van der Poll, Tom

    2015-09-01

    Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Granzymes (gzms) are proteases mainly found in cytotoxic lymphocytes, but also extracellularly. While the role of gzms in target cell death has been widely characterized, considerable evidence points towards broader roles related to infectious and inflammatory responses. To investigate the expression of the gzms in TB, intracellular gzms A, B and K were measured by flow cytometry in lymphocyte populations from peripheral blood mononuclear cells from 18 TB patients and 12 healthy donors from Bangladesh, and extracellular levels of gzmA and B were measured in serum from 58 TB patients and 31 healthy controls. TB patients showed increased expression of gzmA in CD8(+) T, CD4(+) T and CD56(+) T, but not NK, cells, and of gzmB in CD8(+) T cells, when compared to controls. GzmK expression was not altered in TB patients in any lymphocyte subset. The extracellular levels of gzmA and, to a lesser extent, of gzmB, were increased in TB patients, but did not correlate with intracellular gzm expression in lymphocyte subsets. Our results reveal enhanced intra- and extracellular expression of gzmA and B in patients with pulmonary TB, suggesting that gzms are part of the host response to tuberculosis.

  6. Filtration recovery of extracellular DNA from environmental water samples

    EPA Science Inventory

    qPCR methods are able to analyze DNA from microbes within hours of collecting water samples, providing the promptest notification and public awareness possible when unsafe pathogenic levels are reached. Health risk, however, may be overestimated by the presence of extracellular ...

  7. Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

    PubMed

    Doke, M; Fukamachi, H; Morisaki, H; Arimoto, T; Kataoka, H; Kuwata, H

    2016-08-01

    Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg(2+) and Ca(2+) for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs.

  8. Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration.

    PubMed

    Richter, Katrin; Schicklberger, Marcus; Gescher, Johannes

    2012-02-01

    An extension of the respiratory chain to the cell surface is necessary to reduce extracellular electron acceptors like ferric iron or manganese oxides. In the past few years, more and more compounds were revealed to be reduced at the surface of the outer membrane of Gram-negative bacteria, and the list does not seem to have an end so far. Shewanella as well as Geobacter strains are model organisms to discover the biochemistry that enables the dissimilatory reduction of extracellular electron acceptors. In both cases, c-type cytochromes are essential electron-transferring proteins. They make the journey of respiratory electrons from the cytoplasmic membrane through periplasm and over the outer membrane possible. Outer membrane cytochromes have the ability to catalyze the last step of the respiratory chains. Still, recent discoveries provided evidence that they are accompanied by further factors that allow or at least facilitate extracellular reduction. This review gives a condensed overview of our current knowledge of extracellular respiration, highlights recent discoveries, and discusses critically the influence of different strategies for terminal electron transfer reactions.

  9. Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

    NASA Technical Reports Server (NTRS)

    Globus. Ruth K.; Almeida, Eduardo A. C.; Searby, Nancy D.; Bowley, Susan M. (Technical Monitor)

    2000-01-01

    Spaceflight adversely affects the skeleton, posing a substantial risk to astronaut's health during long duration missions. The reduced bone mass observed in growing animals following spaceflight is due at least in part to inadequate bone formation by osteoblasts. Thus, it is of central importance to identify basic cellular mechanisms underlying normal bone formation. The fundamental ideas underlying our research are that interactions between extracellular matrix proteins, integrin adhesion receptors, cytoplasmic signaling and cytoskeletal proteins are key ingredients for the proper functioning of osteoblasts, and that gravity impacts these interactions. As an in vitro model system we used primary fetal rat calvarial cells which faithfully recapitulate osteoblast differentiation characteristically observed in vivo. We showed that specific integrin receptors ((alpha)3(beta)1), ((alpha)5(beta)1), ((alpha)8(betal)1) and extracellular matrix proteins (fibronectin, laminin) were needed for the differentiation of immature osteoblasts. In the course of maturation, cultured osteoblasts switched from depending on fibronectin and laminin for differentiation to depending on these proteins for their very survival. Furthermore, we found that manipulating the gravity vector using ground-based models resulted in activation of key intracellular survival signals generated by integrin/extracellular matrix interactions. We are currently testing the in vivo relevance of some of these observations using targeted transgenic technology. In conclusion, mechanical factors including gravity may participate in regulating survival via cellular interactions with the extracellular matrix. This leads us to speculate that microgravity adversely affects the survival of osteoblasts and contributes to spaceflight-induced osteoporosis.

  10. Another dimension to calcium signaling: a look at extracellular calcium.

    PubMed

    Hofer, Aldebaran M

    2005-03-01

    Cell biologists know the calcium ion best as a vital intracellular second messenger that governs countless cellular functions. However, the recent identification of cell-surface detectors for extracellular Ca(2+) has prompted consideration of whether Ca(2+) also functions as a signaling molecule in the extracellular milieu. The cast of Ca(2+) sensors includes the well-characterized extracellular-Ca(2+)-sensing receptor, a G-protein-coupled receptor originally isolated from the parathyroid gland. In addition, other receptors, channels and membrane proteins, such as gap junction hemichannels, metabotropic glutamate receptors, HERG K(+) channels and the receptor Notch, are all sensitive to external [Ca(2+)] fluctuations. A recently cloned Ca(2+) sensor (CAS) in Arabidopsis extends this concept to the plant kingdom. Emerging evidence indicates that [Ca(2+)] in the local microenvironment outside the cell undergoes alterations potentially sufficient to exert biological actions through these sensor proteins. The extracellular space might therefore constitute a much more dynamic Ca(2+) signaling compartment than previously appreciated.

  11. Extracellular Recognition of Oomycetes during Biotrophic Infection of Plants

    PubMed Central

    Raaymakers, Tom M.; Van den Ackerveken, Guido

    2016-01-01

    Extracellular recognition of pathogens by plants constitutes an important early detection system in plant immunity. Microbe-derived molecules, also named patterns, can be recognized by pattern recognition receptors (PRRs) on the host cell membrane that trigger plant immune responses. Most knowledge on extracellular pathogen detection by plants comes from research on bacterial and fungal pathogens. For oomycetes, that comprise some of the most destructive plant pathogens, mechanisms of extracellular pattern recognition have only emerged recently. These include newly recognized patterns, e.g., cellulose-binding elicitor lectin, necrosis and ethylene-inducing peptide 1-like proteins (NLPs), and glycoside hydrolase 12, as well as their receptors, e.g., the putative elicitin PRR elicitin response and the NLP PRR receptor-like protein 23. Immunity can also be triggered by the release of endogenous host-derived patterns, as a result of oomycete enzymes or damage. In this review we will describe the types of patterns, both pathogen-derived exogenous and plant-derived endogenous ones, and what is known about their extracellular detection during (hemi-)biotrophic oomycete infection of plants. PMID:27446136

  12. Flavin Electron Shuttles Dominate Extracellular Electron Transfer by Shewanella oneidensis

    PubMed Central

    Kotloski, Nicholas J.; Gralnick, Jeffrey A.

    2013-01-01

    ABSTRACT Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δbfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. PMID:23322638

  13. Nanofiltration concentration of extracellular glutathione produced by engineered Saccharomyces cerevisiae.

    PubMed

    Sasaki, Kengo; Hara, Kiyotaka Y; Kawaguchi, Hideo; Sazuka, Takashi; Ogino, Chiaki; Kondo, Akihiko

    2016-01-01

    This study aimed to optimize extracellular glutathione production by a Saccharomyces cerevisiae engineered strain and to concentrate the extracellular glutathione by membrane separation processes, including ultrafiltration (UF) and nanofiltration (NF). Synthetic defined (SD) medium containing 20 g L(-1) glucose was fermented for 48 h; the fermentation liquid was passed through an UF membrane to remove macromolecules. Glutathione in this permeate was concentrated for 48 h to 545.1 ± 33.6 mg L(-1) using the NF membrane; this was a significantly higher concentration than that obtained with yeast extract peptone dextrose (YPD) medium following 96 h NF concentration (217.9 ± 57.4 mg L(-1)). This higher glutathione concentration results from lower cellular growth in SD medium (final OD600 = 6.9 ± 0.1) than in YPD medium (final OD600 = 11.0 ± 0.6) and thus higher production of extracellular glutathione (16.0 ± 1.3 compared to 9.2 ± 2.1 mg L(-1) in YPD medium, respectively). Similar fermentation and membrane processing of sweet sorghum juice containing 20 g L(-1) total sugars provided 240.3 ± 60.6 mg L(-1) glutathione. Increased extracellular production of glutathione by this engineered strain in SD medium and subsequent UF permeation and NF concentration in shortend time may help realize industrial recovery of extracellular glutathione.

  14. ELISA-Based Segregation of Adult Spring Chinook Salmon for Control of Bacterial Kidney Disease, Annual Report FY 1989.

    SciTech Connect

    Kaattari, Stephen L.; Winton, James R.

    1989-12-01

    Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a serious disease of salmonid fish worldwide. The disease has a major impact on spring chinook salmon populations in the Columbia River system. There is strong evidence that R. safmoninarum can be transmitted from parent to progeny, and therefore culling of gametes from infected parents should obviate this mode of transmission. This report presents the results from the first year of our four year study to investigate segregation of broodstock as a tool for controlling BKD. The segregations will use Enzyme-Linked Immunosorbent Assays (ELISAs) as detection systems to identify, in tissues of infected fish, proteins produced by R. salmoninarum. A first step in the development of the described detection systems was the optimization of the production of important antigenic proteins from R. salmoninarum. Different culture media were qualitatively and quantitatively evaluated for their ability to support production of cellular and soluble proteins. The major factor affecting antigen quality was the presence and absence of calf serum. Media components and R. salmoninarum growth products could not be separated during harvest of proteins from the cultures containing serum. This caused problems with the quantitation of actual bacterial proteins in the preparation. Thus media without serum is currently employed. Two independent ELISA techniques for the identification of infected parents were examined. One technique is based on polyclonal antisera produced in rabbits and the second is based on mouse monoclonal antibodies (Mabs). To develop the latter system, several Mabs against a major R. salmoninarum antigenic protein were produced. These Mabs were used for the detection of R. salmoninarum antigens in infected fish and also to characterize proteins produced by the bacterium. Both ELISAs were deemed suitable for the segregation of parents into the high and low BKD groups required for this study. An

  15. A common theme in extracellular fluids of beetles: extracellular superoxide dismutases crucial for balancing ROS in response to microbial challenge.

    PubMed

    Gretscher, René R; Streicher, Priska E; Strauß, Anja S; Wielsch, Natalie; Stock, Magdalena; Wang, Ding; Boland, Wilhelm; Burse, Antje

    2016-04-12

    Extracellular Cu/Zn superoxide dismutases (SODs) are critical for balancing the level of reactive oxygen species in the extracellular matrix of eukaryotes. In the present study we have detected constitutive SOD activity in the haemolymph and defensive secretions of different leaf beetle species. Exemplarily, we have chosen the mustard leaf beetle, Phaedon cochleariae, as representative model organism to investigate the role of extracellular SODs in antimicrobial defence. Qualitative and quantitative proteome analyses resulted in the identification of two extracellular Cu/Zn SODs in the haemolymph and one in the defensive secretions of juvenile P. cochleariae. Furthermore, quantitative expression studies indicated fat body tissue and defensive glands as the main synthesis sites of these SODs. Silencing of the two SODs revealed one of them, PcSOD3.1, as the only relevant enzyme facilitating SOD activity in haemolymph and defensive secretions in vivo. Upon challenge with the entomopathogenic fungus, Metarhizium anisopliae, PcSOD3.1-deficient larvae exhibited a significantly higher mortality compared to other SOD-silenced groups. Hence, our results serve as a basis for further research on SOD regulated host-pathogen interactions. In defensive secretions PcSOD3.1-silencing affected neither deterrent production nor activity against fungal growth. Instead, we propose another antifungal mechanism based on MRJP/yellow proteins in the defensive exudates.

  16. A common theme in extracellular fluids of beetles: extracellular superoxide dismutases crucial for balancing ROS in response to microbial challenge

    PubMed Central

    Gretscher, René R.; Streicher, Priska E.; Strauß, Anja S.; Wielsch, Natalie; Stock, Magdalena; Wang, Ding; Boland, Wilhelm; Burse, Antje

    2016-01-01

    Extracellular Cu/Zn superoxide dismutases (SODs) are critical for balancing the level of reactive oxygen species in the extracellular matrix of eukaryotes. In the present study we have detected constitutive SOD activity in the haemolymph and defensive secretions of different leaf beetle species. Exemplarily, we have chosen the mustard leaf beetle, Phaedon cochleariae, as representative model organism to investigate the role of extracellular SODs in antimicrobial defence. Qualitative and quantitative proteome analyses resulted in the identification of two extracellular Cu/Zn SODs in the haemolymph and one in the defensive secretions of juvenile P. cochleariae. Furthermore, quantitative expression studies indicated fat body tissue and defensive glands as the main synthesis sites of these SODs. Silencing of the two SODs revealed one of them, PcSOD3.1, as the only relevant enzyme facilitating SOD activity in haemolymph and defensive secretions in vivo. Upon challenge with the entomopathogenic fungus, Metarhizium anisopliae, PcSOD3.1-deficient larvae exhibited a significantly higher mortality compared to other SOD-silenced groups. Hence, our results serve as a basis for further research on SOD regulated host-pathogen interactions. In defensive secretions PcSOD3.1-silencing affected neither deterrent production nor activity against fungal growth. Instead, we propose another antifungal mechanism based on MRJP/yellow proteins in the defensive exudates. PMID:27068683

  17. Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium.

    PubMed

    Laurent, Louise C; Abdel-Mageed, Asim B; Adelson, P David; Arango, Jorge; Balaj, Leonora; Breakefield, Xandra; Carlson, Elizabeth; Carter, Bob S; Majem, Blanca; Chen, Clark C; Cocucci, Emanuele; Danielson, Kirsty; Courtright, Amanda; Das, Saumya; Abd Elmageed, Zakaria Y; Enderle, Daniel; Ezrin, Alan; Ferrer, Marc; Freedman, Jane; Galas, David; Gandhi, Roopali; Huentelman, Matthew J; Van Keuren-Jensen, Kendall; Kalani, Yashar; Kim, Yong; Krichevsky, Anna M; Lai, Charles; Lal-Nag, Madhu; Laurent, Clara D; Leonardo, Trevor; Li, Feng; Malenica, Ivana; Mondal, Debasis; Nejad, Parham; Patel, Tushar; Raffai, Robert L; Rubio, Renee; Skog, Johan; Spetzler, Robert; Sun, Jie; Tanriverdi, Kahraman; Vickers, Kasey; Wang, Liang; Wang, Yaoyu; Wei, Zhiyun; Weiner, Howard L; Wong, David; Yan, Irene K; Yeri, Ashish; Gould, Stephen

    2015-01-01

    Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.

  18. Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium

    PubMed Central

    Laurent, Louise C.; Abdel-Mageed, Asim B.; Adelson, P. David; Arango, Jorge; Balaj, Leonora; Breakefield, Xandra; Carlson, Elizabeth; Carter, Bob S.; Majem, Blanca; Chen, Clark C.; Cocucci, Emanuele; Danielson, Kirsty; Courtright, Amanda; Das, Saumya; Elmageed, Zakaria Y. Abd; Enderle, Daniel; Ezrin, Alan; Ferrer, Marc; Freedman, Jane; Galas, David; Gandhi, Roopali; Huentelman, Matthew J.; Van Keuren-Jensen, Kendall; Kalani, Yashar; Kim, Yong; Krichevsky, Anna M.; Lai, Charles; Lal-Nag, Madhu; Laurent, Clara D.; Leonardo, Trevor; Li, Feng; Malenica, Ivana; Mondal, Debasis; Nejad, Parham; Patel, Tushar; Raffai, Robert L.; Rubio, Renee; Skog, Johan; Spetzler, Robert; Sun, Jie; Tanriverdi, Kahraman; Vickers, Kasey; Wang, Liang; Wang, Yaoyu; Wei, Zhiyun; Weiner, Howard L.; Wong, David; Yan, Irene K.; Yeri, Ashish; Gould, Stephen

    2015-01-01

    Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field. PMID:26320937

  19. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

    PubMed Central

    Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

    2016-01-01

    Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease. PMID:27336156

  20. Electrophoretic and antigenic characterisation of Dermatophilus congolensis extracellular products.

    PubMed

    Ambrose, N C; el Jack, M A; McOrist, S; Boid, R

    1997-12-01

    Dermatophilus congolensis is the causative agent of bovine dermatophilosis and lumpy wool in sheep. Two field isolates of D. congolensis, one each from a cow in Ghana and a sheep in Scotland, were cultured for 24-72 h in a synthetic medium based on RPMI-1640. Culture filtrates were examined by SDS-PAGE and considered to contain extracellular products released by growing hyphae and filaments. Electrophoretic profiles of culture filtrates of the two isolates contained common bands and bands that were unique to each isolate. The composition of extracellular products altered with increasing culture periods indicating that specific products were released at different stages of growth. Culture filtrate prepared in the presence of serine protease and metalloprotease inhibitors contained more and better defined bands than that prepared without protease inhibitors indicating the presence of proteases in culture filtrates. Western blot analysis of extracellular products using a panel of sera showed that the two isolates from different host species and distant geographical locations contained cross-reactive antigens. Natural and experimental infections stimulated antibody responses to antigens in culture filtrates, sera from animals that were disease free but in-contact with dermatophilosis-infected animals also contained antibodies to extracellular antigens. The antigens recognised by most sera had molecular weights of 200 kDa in the bovine isolate, 170 kDa in the ovine isolate and 67, 27 and 52-55 kDa in both isolates. The number of antigenic bands of both isolates was positively correlated with the intensity of challenge and the severity of infection: antibodies in sera from disease-free cattle in Ghana recognised more antigens than sera from disease-free sheep in Scotland and more antigens were recognised by sera from chronically-infected Ghanaian cattle than by sera from experimentally-infected calves and sheep. The latter developed antibodies to antigens of 27 and 24 k

  1. EXTRACELLULAR POLYANIONS IN DIGESTED SLUDGE: MEASUREMENT AND RELATIONSHIP TO SLUDGE DEWATERABILITY. (R823486)

    EPA Science Inventory

    The polyanionic fraction of digested sludge extracellular material was quantified using an in situ dye adsorption method, and the relationships between measured extracellular polyanion (ECPA)
    concentrations and sludge dewaterability were investigated. Measured ECPA concentrat...

  2. Properties of extracellular DNA from the cerebrospinal fluid and blood plasma during Parkinson's disease.

    PubMed

    Glebova, K V; Konorova, I L; Poleshchuk, V V; Baidakova, G V; Veiko, N N

    2014-04-01

    The cerebrospinal fluid of patients with Parkinson's disease was shown to contain extracellular DNA. Extracellular DNA concentration in the cerebrospinal fluid was 3.3-fold lower than in blood plasma from these patients. HPLC-mass spectrometry analysis showed that the pool of extracellular DNA from the liquor is characterized by a lower content of deoxythymidine, but greater amounts of deoxycytidine and deoxyguanosine than the pool of extracellular DNA from the plasma. The level of deoxyguanosine was 2 times lower than that of deoxycytidine (as differentiated from plasma extracellular DNA with similar content of these substances). Our findings indicate that extracellular DNA from the cerebrospinal fluid contains considerable amounts of modified deoxyguanosine. These data attest to significant differences in the quantitative and qualitative characteristics of extracellular DNA from the blood and cerebrospinal fluid of patients. Specific features of extracellular DNA from the cerebrospinal fluid of patients suggest its involvement in the pathogenesis of Parkinson's disease.

  3. Challenges posed by extracellular vesicles from eukaryotic microbes

    PubMed Central

    Wolf, Julie M.; Casadevall, Arturo

    2014-01-01

    Extracellular vesicles (EV) produced by eukaryotic microbes play an important role during infection. EV release is thought to benefit microbial invasion by delivering a high concentration of virulence factors to distal host cells or to the cytoplasm of a host cell. EV can significantly impact the outcome of host-pathogen interaction in a cargo-dependent manner. Release of EV from eukaryotic microbes poses unique challenges when compared to their bacterial or archaeal counterparts. Firstly, the membrane-bound organelles within eukaryotes facilitate multiple mechanisms of vesicle generation. Secondly, the fungal cell wall poses a unique barrier between the vesicle release site at the plasma membrane and its destined extracellular environment. This review focuses on these eukaryotic-specific aspects of vesicle synthesis and release. PMID:25460799

  4. Graphene Multielectrode Arrays as a Versatile Tool for Extracellular Measurements.

    PubMed

    Kireev, Dmitry; Seyock, Silke; Lewen, Johannes; Maybeck, Vanessa; Wolfrum, Bernhard; Offenhäusser, Andreas

    2017-04-03

    Graphene multielectrode arrays (GMEAs) presented in this work are used for cardio and neuronal extracellular recordings. The advantages of the graphene as a part of the multielectrode arrays are numerous: from a general flexibility and biocompatibility to the unique electronic properties of graphene. The devices used for extensive in vitro studies of a cardiac-like cell line and cortical neuronal networks show excellent ability to extracellularly detect action potentials with signal to noise ratios in the range of 45 ± 22 for HL-1 cells and 48 ± 26 for spontaneous bursting/spiking neuronal activity. Complex neuronal bursting activity patterns as well as a variety of characteristic shapes of HL-1 action potentials are recorded with the GMEAs. This paper illustrates that the potential applications of the GMEAs in biological and medical research are still numerous and diverse.

  5. Extracellular potassium accumulation in voltage-clamped frog ventricular muscle.

    PubMed Central

    Cleemann, L; Morad, M

    1979-01-01

    1. Application of voltage clamp pulses (1--10 sec) to frog ventricular strips causes temporary changes in the extracellular K concentration. 2. The changes in the extracellular K concentration can be estimated from (a) slowly decaying post-clamp after-potentials, (b) changes in the action potential duration, and (c) measurements with a K-selective micro-electrode. 3. The depolarization of the resting potential and the shortening of the action potential are present in approximately the same proportions during voltage-clamp induced extracellular K accumulation and during perfusion with a K-ricn Ringer solution but small consistent differences are noticed. 4. The measurements of the after-potential, the action potential shortening, and the K-electrode response were analysed as indicators of extracellular K+ activity and it was concluded that the after-potential provides the most convenient and reliable estimate of the absolute magnitude of the voltage-clamp induced extracellular K accumulation. 5. The depolarizing after-potentials decay more slowly than the hyperpolarizing after-potentials but it is found that this reflects the selectivity of the membrane to K+ concentrations as predicted by the Nernst or the Goldman equations. 6. Analysis of the redistribution of accumulated K+ from the decay of the after-potential suggests that the major part of the redistribution process can be described by a single time constant (2--4 sec). A much longer time constant is required for a smaller component of the 'tail' in order to bring [K]o to the normal resting state. 7. N-shaped relations similar to the 'steady state' current-voltage relation are obtained when the post-clamp after-potential, the action potential shortening, and the K-electrode response are plotted versus the clamped membrane potential. The maxima of these curves are located around -40 mV and the minima around -20 mV. 8. In spite of a significant outward membrane current (1--1.5 microamperemeter) in the minimum

  6. Telocytes and Their Extracellular Vesicles—Evidence and Hypotheses

    PubMed Central

    Cretoiu, Dragos; Xu, Jiahong; Xiao, Junjie; Cretoiu, Sanda M.

    2016-01-01

    Entering the new millennium, nobody believed that there was the possibility of discovering a new cellular type. Nevertheless, telocytes (TCs) were described as a novel kind of interstitial cell. Ubiquitously distributed in the extracellular matrix of any tissue, TCs are regarded as cells with telopodes involved in intercellular communication by direct homo- and heterocellular junctions or by extracellular vesicle (EVs) release. Their discovery has aroused the interest of many research groups worldwide, and many researchers regard them as potentially regenerative cells. Given the experience of our laboratory, where these cells were first described, we review the evidence supporting the fact that TCs release EVs, and discuss alternative hypotheses about their future implications. PMID:27529228

  7. Neutrophil extracellular traps - the dark side of neutrophils.

    PubMed

    Sørensen, Ole E; Borregaard, Niels

    2016-05-02

    Neutrophil extracellular traps (NETs) were discovered as extracellular strands of decondensed DNA in complex with histones and granule proteins, which were expelled from dying neutrophils to ensnare and kill microbes. NETs are formed during infection in vivo by mechanisms different from those originally described in vitro. Citrullination of histones by peptidyl arginine deiminase 4 (PAD4) is central for NET formation in vivo. NETs may spur formation of autoantibodies and may also serve as scaffolds for thrombosis, thereby providing a link among infection, autoimmunity, and thrombosis. In this review, we present the mechanisms by which NETs are formed and discuss the physiological and pathophysiological consequences of NET formation. We conclude that NETs may be of more importance in autoimmunity and thrombosis than in innate immune defense.

  8. Functional properties of extracellular domains of transducer receptor gp130.

    PubMed

    Kostjukova, M N; Tupitsyn, N N

    2011-04-01

    Cytokine receptor molecules have been shown to have extracellular domains of complex structure and a multi-step activation system. Glycoprotein gp130 is a typical transducer of cytokine signal; it functions by forming multicomponent receptor complexes and transferring signals of tens of cytokines from the IL-6 family. Structural organization and basic functioning principles of gp130 are well known, as well as related signal pathways, which function during normal differentiation and are involved in pathogenesis of many tumors. The role of gp130 in IL-6-dependent tumors is best studied. In this review, based on extensive accumulated data, we examine the functional significance of certain parts of gp130 extracellular domains. Potentials of a recently developed method for estimation of receptor activation at the level of epitope structure are discussed.

  9. Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi

    NASA Astrophysics Data System (ADS)

    van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

    Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

  10. Microbial extracellular enzymes and the marine carbon cycle.

    PubMed

    Arnosti, Carol

    2011-01-01

    Extracellular enzymes initiate microbial remineralization of organic matter by hydrolyzing substrates to sizes sufficiently small to be transported across cell membranes. As much of marine primary productivity is processed by heterotrophic microbes, the substrate specificities of extracellular enzymes, the rates at which they function in seawater and sediments, and factors controlling their production, distribution, and active lifetimes, are central to carbon cycling in marine systems. In this review, these topics are considered from biochemical, microbial/molecular biological, and geochemical perspectives. Our understanding of the capabilities and limitations of heterotrophic microbial communities has been greatly advanced in recent years, in part through genetic and genomic approaches. New methods to measure enzyme activities in the field are needed to keep pace with these advances and to pursue intriguing evidence that patterns of enzyme activities in different environments are linked to differences in microbial community composition that may profoundly affect the marine carbon cycle.

  11. Neutrophil extracellular trap (NET) impact on deep vein thrombosis.

    PubMed

    Fuchs, Tobias A; Brill, Alexander; Wagner, Denisa D

    2012-08-01

    Deep vein thrombosis (DVT) is a major health problem that requires improved prophylaxis and treatment. Inflammatory conditions such as infection, cancer, and autoimmune diseases are risk factors for DVT. We and others have recently shown that extracellular DNA fibers produced in inflammation and known as neutrophil extracellular traps (NETs) contribute to experimental DVT. NETs stimulate thrombus formation and coagulation and are abundant in thrombi in animal models of DVT. It appears that, in addition to fibrin and von Willebrand factor, NETs represent a third thrombus scaffold. Here, we review how NETs stimulate thrombosis and discuss known and potential interactions of NETs with endothelium, platelets, red blood cells, and coagulation factors and how NETs could influence thrombolysis. We propose that drugs that inhibit NET formation or facilitate NET degradation may prevent or treat DVT.

  12. Capturing relevant extracellular matrices for investigating cell migration

    PubMed Central

    Keely, Patricia; Nain, Amrinder

    2015-01-01

    Much progress in understanding cell migration has been determined by using classic two-dimensional (2D) tissue culture platforms. However, increasingly, it is appreciated that certain properties of cell migration in vivo are not represented by strictly 2D assays. There is much interest in creating relevant three-dimensional (3D) culture environments and engineered platforms to better represent features of the extracellular matrix and stromal microenvironment that are not captured in 2D platforms. Important to this goal is a solid understanding of the features of the extracellular matrix—composition, stiffness, topography, and alignment—in different tissues and disease states and the development of means to capture these features PMID:26918156

  13. Extracellular hyperosmolality and body temperature during physical exercise in dogs

    NASA Technical Reports Server (NTRS)

    Kozlowski, S.; Greenleaf, J. E.; Turlejska, E.; Nazar, K.

    1980-01-01

    The purpose of this study was to test the hypothesis that thermoregulation during exercise can be affected by extracellular fluid hyperosmolality without changing the plasma Na(+) concentration. The effects of preexercise venous infusions of hypertonic mannitol and NaCl solutions on rectal temperature responses were compared in dogs running at moderate intensity for 60 min on a treadmill. Plasma Na(+) concentration was increased by 12 meq after NaCl infusion, and decreased by 9 meq after mannitol infusion. Both infusions increased plasma by 15 mosmol/kg. After both infusions, rectal temperature was essentially constant during 60 min rest. However, compared with the noninfusion exercise increase in osmolality of 1.3 C, rectal temperature increased by 1.9 C after both postinfusion exercise experiments. It was concluded that inducing extracellular hyperosmolality, without elevating plasma, can induce excessive increases in rectal temperature during exericse but not at rest.

  14. Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study

    PubMed Central

    Zaborowski, MikoŁaj P.; Balaj, Leonora; Breakefield, Xandra O.; Lai, Charles P.

    2015-01-01

    The release of extracellular vesicles (EVs), including exosomes and microvesicles, is a phenomenon shared by many cell types as a means of communicating with other cells and also potentially removing cell contents. The cargo of EVs includes the proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate. EVs released into the extracellular space can enter body fluids and potentially reach distant tissues. Once taken up by neighboring and/or distal cells, EVs can transfer functional cargo that may alter the status of recipient cells, thereby contributing to both physiological and pathological processes. In this article, we will focus on EV composition, mechanisms of uptake, and their biological effects on recipient cells. We will also discuss established and recently developed methods used to study EVs, including isolation, quantification, labeling and imaging protocols, as well as RNA analysis. PMID:26955082

  15. Neuroprotective Effects of Glutamate Antagonists and Extracellular Acidity

    NASA Astrophysics Data System (ADS)

    Kaku, David A.; Giffard, Rona G.; Choi, Dennis W.

    1993-06-01

    Glutamate antagonists protect neurons from hypoxic injury both in vivo and in vitro, but in vitro studies have not been done under the acidic conditions typical of hypoxia-ischemia in vivo. Consistent with glutamate receptor antagonism, extracellular acidity reduced neuronal death in murine cortical cultures that were deprived of oxygen and glucose. Under these acid conditions, N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isox-azolepropionate-kainate antagonists further reduced neuronal death, such that some neurons tolerated prolonged oxygen and glucose deprivation almost as well as did astrocytes. Neuroprotection induced by this combination exceeded that induced by glutamate antagonists alone, suggesting that extracellular acidity has beneficial effects beyond the attenuation of ionotropic glutamate receptor activation.

  16. Specialisation of extracellular matrix for function in tendons and ligaments.

    PubMed

    Birch, Helen L; Thorpe, Chavaunne T; Rumian, Adam P

    2013-01-01

    Tendons and ligaments are similar structures in terms of their composition, organisation and mechanical properties. The distinction between them stems from their anatomical location; tendons form a link between muscle and bone while ligaments link bones to bones. A range of overlapping functions can be assigned to tendon and ligaments and each structure has specific mechanical properties which appear to be suited for particular in vivo function. The extracellular matrix in tendon and ligament varies in accordance with function, providing appropriate mechanical properties. The most useful framework in which to consider extracellular matrix differences therefore is that of function rather than anatomical location. In this review we discuss what is known about the relationship between functional requirements, structural properties from molecular to gross level, cellular gene expression and matrix turnover. The relevance of this information is considered by reviewing clinical aspects of tendon and ligament repair and reconstructive procedures.

  17. Lipid-targeting peptide probes for extracellular vesicles

    PubMed Central

    Flynn, Aaron D.; Yin, Hang

    2017-01-01

    Extracellular vesicles released from cells are under intense investigation for their roles in cell-cell communication and cancer progression. However, individual vesicles have been difficult to probe as their small size renders them invisible by conventional light microscopy. However, as a consequence of their small size these vesicles possess highly curved lipid membranes that offer an unconventional target for curvature-sensing probes. In this article, we present a strategy for using peptide-based biosensors to detect highly curved membranes and the negatively charged membrane lipid phosphatidylserine, we delineate several assays used to validate curvature- and lipid-targeting mechanisms, and we explore potential applications in probing extracellular vesicles released from sources such as apoptotic cells, cancer cells, or activated platelets. PMID:26909741

  18. Extracellular DNA and histones: double-edged swords in immunothrombosis.

    PubMed

    Gould, T J; Lysov, Z; Liaw, P C

    2015-06-01

    The existence of extracellular DNA in human plasma, also known as cell-free DNA (cfDNA), was first described in the 1940s. In recent years, there has been a resurgence of interest in the functional significance of cfDNA, particularly in the context of neutrophil extracellular traps (NETs). cfDNA and histones are key components of NETs that aid in the host response to infection and inflammation. However, cfDNA and histones may also exert harmful effects by triggering coagulation, inflammation, and cell death and by impairing fibrinolysis. In this article, we will review the pathologic nature of cfDNA and histones in macrovascular and microvascular thrombosis, including venous thromboembolism, cancer, sepsis, and trauma. We will also discuss the prognostic value of cfDNA and histones in these disease states. Understanding the molecular and cellular pathways regulated by cfDNA and histones may provide novel insights to prevent pathological thrombus formation and vascular occlusion.

  19. Extracellular matrix mediates epithelial effects on chondrogenesis in vitro.

    PubMed

    Solursh, M; Jensen, K L; Zanetti, N C; Linsenmayer, T F; Reiter, R S

    1984-10-01

    It has been previously observed that single chick embryonic limb mesenchymal cells can differentiate into chondrocytes without cell-cell interactions when cultured in collagen or agarose gels. In the present study, limb ectoderm, but not dermis, inhibits chondrogenesis when placed on such collagen gel cultures. The inhibitory influence can be transmitted extensive distances in the gel, even when the ectoderm is placed on a porous filter. Collagen gels, preconditioned with limb ectoderms, are also inhibitory to chondrogenesis. On the other hand, chondrogenesis is less inhibited by ectoderm when the mesenchymal cells are placed in agarose. These results suggest that the antichondrogenic effect of limb ectoderm is mediated through alterations of the collagenous extracellular matrix and support the idea that the extracellular matrix must be considered as an organized, functional unit capable of regulating cell differentiation.

  20. Sea Ice Microorganisms: Environmental Constraints and Extracellular Responses

    PubMed Central

    Ewert, Marcela; Deming, Jody W.

    2013-01-01

    Inherent to sea ice, like other high latitude environments, is the strong seasonality driven by changes in insolation throughout the year. Sea-ice organisms are exposed to shifting, sometimes limiting, conditions of temperature and salinity. An array of adaptations to survive these and other challenges has been acquired by those organisms that inhabit the ice. One key adaptive response is the production of extracellular polymeric substances (EPS), which play multiple roles in the entrapment, retention and survival of microorganisms in sea ice. In this concept paper we consider two main areas of sea-ice microbiology: the physico-chemical properties that define sea ice as a microbial habitat, imparting particular advantages and limits; and extracellular responses elicited in microbial inhabitants as they exploit or survive these conditions. Emphasis is placed on protective strategies used in the face of fluctuating and extreme environmental conditions in sea ice. Gaps in knowledge and testable hypotheses are identified for future research. PMID:24832800

  1. Optimizing analog-to-digital converters for sampling extracellular potentials.

    PubMed

    Artan, N Sertac; Xu, Xiaoxiang; Shi, Wei; Chao, H Jonathan

    2012-01-01

    In neural implants, an analog-to-digital converter (ADC) provides the delicate interface between the analog signals generated by neurological processes and the digital signal processor that is tasked to interpret these signals for instance for epileptic seizure detection or limb control. In this paper, we propose a low-power ADC architecture for neural implants that process extracellular potentials. The proposed architecture uses the spike detector that is readily available on most of these implants in a closed-loop with an ADC. The spike detector determines whether the current input signal is part of a spike or it is part of noise to adaptively determine the instantaneous sampling rate of the ADC. The proposed architecture can reduce the power consumption of a traditional ADC by 62% when sampling extracellular potentials without any significant impact on spike detection accuracy.

  2. Analysis of cellular and extracellular DNA in fingerprints

    SciTech Connect

    Button, Julie M.

    2014-09-09

    It has been previously shown that DNA can be recovered from latent fingerprints left on various surfaces [R. A. H. van Oorschot and M. K. Jones, Nature 387, 767 (1997)]. However, the source of the DNA, extracellular versus cellular origin, is difficult to determine. If the DNA is cellular, it is believed to belong to skin cells while extracellular DNA is believed to originate from body fluids such as sweat [D. J. Daly et. al, Forensic Sci. Int. Genet. 6, 41-46 (2012); V. V. Vlassov et. al, BioEssays 29, 654-667 (2007)]. The origin of the DNA in fingerprints has implications for processing and interpretation of forensic evidence. The determination of the origin of DNA in fingerprints is further complicated by the fact that the DNA in fingerprints tends to be at a very low quantity [R. A. H. van Oorschot and M. K. Jones, Nature 387, 767 (1997)]. This study examined fingerprints from five volunteers left on sterilized glass slides and plastic pens. Three fingerprints were left on each glass slide (thumb, index, and middle fingers) while the pens were held as if one was writing with them. The DNA was collected from the objects using the wet swabbing technique (TE buffer). Following collection, the cellular and extracellular components of each sample were separated using centrifugation and an acoustofluidics system. Centrifugation is still the primary separation technique utilized in forensics laboratories, while acoustic focusing uses sound waves to focus large particles (cells) into low pressure nodes, separating them from the rest of the sample matrix. After separation, all samples were quantified using real-time quantitative PCR (qPCR). The overall trend is that there is more DNA in the extracellular fractions than cellular fractions for both centrifugation and acoustofluidic processing. Additionally, more DNA was generally collected from the pen samples than the samples left on glass slides.

  3. Ratiometric Imaging of Extracellular pH in Dental Biofilms.

    PubMed

    Schlafer, Sebastian; Dige, Irene

    2016-03-09

    The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.

  4. Extracellular Proteinases of Yeasts and Yeastlike Fungi1

    PubMed Central

    Ahearn, D. G.; Meyers, S. P.; Nichols, R. A.

    1968-01-01

    Approximately 800 yeasts and other fungi, representing over 70 species, were tested for extracellular caseinolysis. Isolates of a variety of genera, including Aureobasidium, Cephalosporium, Endomycopsis, Kluyveromyces, and numerous sporobolomycetes, demonstrated significant proteolytic activity. Caseinolysis was not necessarily correlated with gelatin liquefaction or with albuminolysis. Numerous fungi showed significant proteolysis at 5 C. The most active organisms were isolates of Candida lipolytica, Aureobasidium pullulans, Candida punicea, and species of Cephalosporium. Taxonomic and ecological implications of proteolytic activity are discussed. Images Fig. 1 PMID:5692110

  5. Microfluidic filtration system to isolate extracellular vesicles from blood.

    PubMed

    Davies, Ryan T; Kim, Junho; Jang, Su Chul; Choi, Eun-Jeong; Gho, Yong Song; Park, Jaesung

    2012-12-21

    Extracellular vesicles are released by various cell types, particularly tumor cells, and may be potential targets for blood-based cancer diagnosis. However, studies performed on blood-borne vesicles to date have been limited by lack of effective, standardized purification strategies. Using in situ prepared nanoporous membranes, we present a simple strategy employing a microfluidic filtration system to isolate vesicles from whole blood samples. This method can be applied to purify nano-sized particles from blood allowing isolation of intact extracellular vesicles, avoiding the need for laborious and potentially damaging centrifugation steps or overly specific antibody-based affinity purification. Porous polymer monoliths were integrated as membranes into poly(methyl methacrylate) microfluidic chips by benchtop UV photopolymerization through a mask, allowing precise positioning of membrane elements while preserving simplicity of device preparation. Pore size could be manipulated by changing the ratio of porogenic solvent to prepolymer solution, and was tuned to a size proper for extraction of vesicles. Using the membrane as a size exclusion filter, we separated vesicles from cells and large debris by injecting whole blood under pressure through the microfluidic device. To enhance isolation purity, DC electrophoresis was employed as an alternative driving force to propel particles across the filter and increase the separation efficiency of vesicles from proteins. From the whole blood of melanoma-grown mice, we isolated extracellular vesicles and performed RT-PCR to verify their contents of RNA. Melan A mRNA derived from melanoma tumor cells were found enriched in filtered samples, confirming the recovery of vesicles via their cargo. This filtration system can be incorporated into other on-chip processes enabling integrated sample preparation for the downstream analysis of blood-based extracellular vesicles.

  6. Recent advances in understanding the extracellular calcium-sensing receptor

    PubMed Central

    Colella, Matilde; Gerbino, Andrea; Hofer, Aldebaran M.; Curci, Silvana

    2016-01-01

    The extracellular calcium-sensing receptor (CaR), a ubiquitous class C G-protein-coupled receptor (GPCR), is responsible for the control of calcium homeostasis in body fluids. It integrates information about external Ca 2+ and a surfeit of other endogenous ligands into multiple intracellular signals, but how is this achieved? This review will focus on some of the exciting concepts in CaR signaling and pharmacology that have emerged in the last few years. PMID:27803801

  7. Extracellular Alkalinization as a Defense Response in Potato Cells

    PubMed Central

    Moroz, Natalia; Fritch, Karen R.; Marcec, Matthew J.; Tripathi, Diwaker; Smertenko, Andrei; Tanaka, Kiwamu

    2017-01-01

    A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we demonstrate extracellular alkalinization as a defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-derived elicitors in a dose- and time-dependent manner. We also assessed the defense response against a variety of potato pathogens, such as protists (Phytophthora infestans and Spongospora subterranea) and fungi (Verticillium dahliae and Colletotrichum coccodes). Our results show that extracellular pH increases within 30 min in proportion to the number of pathogen spores added. Consistently with the alkalinization effect, the higher transcription level of several defense-related genes and production of reactive oxygen species was observed. Our results demonstrate that the alkalinization response is an effective marker to study early stages of defense response in potatoes. PMID:28174578

  8. Extracellular Transglucosylase and α-Amylase of Streptococcus equinus1

    PubMed Central

    Boyer, Ernest W.; Hartman, Paul A.

    1971-01-01

    Culture filtrates of Streptococcus equinus 1091 contained α-amylase and transglucosylase. The effects of calcium carbonate, age of inoculum, concentration of maltose, and duration of the fermentation on α-amylase and transglucosylase production were determined. The extracellular α-amylase was purified 48-fold and was free of transglucosylase activity. The α-amylase (amylose substrate) required Cl− for maximum activity; ethylenediaminetetraacetic acid (EDTA) partially inhibited activity, but CaCl2 prevented EDTA inhibition. The temperature optimum was 38 C at pH 7.0, and the pH optimum was 7.0 at 37 C in the presence of CaCl2. Predominant final products of amylose hydrolysis, in order of decreasing prevalence, were maltose, maltotriose, maltotetraose, and glucose. The α-amylase showed no evidence of multiple attack. The extracellular transglucosylase was purified 27-fold, but a small amount of α-amylase remained. Transglucosylase activity (amylose substrate) was not increased in the presence of CaCl2. The temperature optimum was 37 C at pH 6.5, and the pH optimum was 6.0 at 37 C. Carbohydrates that served as acceptors for the transglucosylase to degrade amylose were, in order of decreasing acceptor efficiency: d-glucose, d-mannose, l-sorbose, maltose, sucrose, and trehalose. The extracellular transglucosylase of S. equinus 1091 synthesized higher maltodextrins in the medium when the cells were grown in the presence of maltose. Images PMID:4995651

  9. The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis.

    PubMed

    Al Shammari, Basim; Shiomi, Takayuki; Tezera, Liku; Bielecka, Magdalena K; Workman, Victoria; Sathyamoorthy, Tarangini; Mauri, Francesco; Jayasinghe, Suwan N; Robertson, Brian D; D'Armiento, Jeanine; Friedland, Jon S; Elkington, Paul T

    2015-08-01

    A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis-infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction.

  10. Extracellular Antibody Drug Conjugates Exploiting the Proximity of Two Proteins

    PubMed Central

    Marshall, David J; Harried, Scott S; Murphy, John L; Hall, Chad A; Shekhani, Mohammed S; Pain, Christophe; Lyons, Conner A; Chillemi, Antonella; Malavasi, Fabio; Pearce, Homer L; Thorson, Jon S; Prudent, James R

    2016-01-01

    The human Na+/K+-ATPase (NKA) is a plasma membrane ion pump that uses ATP to help maintain the resting potential of all human cells. Inhibition of the NKA leads to cell swelling and death. The results of this investigation show that on cancer cells, the NKA either comes in close proximity to, associate with or complexes to important cancer-related proteins, and thus can be targeted with a new type of precision therapy called the extracellular drug conjugate or EDC. The EDCs reported here exhibit EC50 values in the low to mid-picomolar range, and signal to noise ratios > 1,000:1, both of which are dependent on the cell surface expression of the NKA and corresponding cancer-related target. We demonstrate that a potent small molecule inhibitor of the NKA can be covalently attached to antibodies targeting CD20, CD38, CD56, CD147, or dysadherin, to create a series of selective and powerful EDCs that kill cancer cells extracellularly by a mechanism resembling necrosis. This is therefore a framework for the development of a new type of precision therapy wherein exquisite selectivity is achieved for targeting extracellular disease-related proteins. PMID:27434591

  11. Mycobacterium massiliense Induces Macrophage Extracellular Traps with Facilitating Bacterial Growth

    PubMed Central

    Yoon, Yina; Na, Yirang; Kim, Bum-Joon; Seok, Seung Hyeok

    2016-01-01

    Human neutrophils have been known to release neutrophil extracellular traps (NETs), antimicrobial DNA structures capable of capturing and killing microbes. Recently, a similar phenomenon has been reported in macrophages infected with various pathogens. However, a role for macrophages extracellular traps (METs) in host defense responses against Mycobacterium massiliense (M. mass) has yet to be described. In this study, we show that M. mass, a rapid growing mycobacterium (RGM), also induces the release of METs from PMA-differentiated THP-1 cells. Intriguingly, this process is not dependent on NADPH oxidase activity, which regulates NET formation. Instead, M. mass-induced MET formation partially depends on calcium influx and requires phagocytosis of high bacterial load. The METs consist of a DNA backbone embedded with microbicidal proteins such as histone, MPO and elastase. Released METs entrap M. mass and prevent their dissemination, but do not have bactericidal activity. Instead, they result in enhanced bacterial growth. In this regard, METs were considered to provide interaction of M. mass with cells and an environment for bacterial aggregation, which may facilitate mycobacterial survival and growth. In conclusion, our results demonstrate METs as an innate defense response against M. mass infection, and suggest that extracellular traps play a multifaceted role in the interplay between host and bacteria. PMID:27191593

  12. Structural basis of Smoothened regulation by its extracellular domains

    NASA Astrophysics Data System (ADS)

    Byrne, Eamon F. X.; Sircar, Ria; Miller, Paul S.; Hedger, George; Luchetti, Giovanni; Nachtergaele, Sigrid; Tully, Mark D.; Mydock-McGrane, Laurel; Covey, Douglas F.; Rambo, Robert P.; Sansom, Mark S. P.; Newstead, Simon; Rohatgi, Rajat; Siebold, Christian

    2016-07-01

    Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

  13. Extracellular Alkalinization as a Defense Response in Potato Cells.

    PubMed

    Moroz, Natalia; Fritch, Karen R; Marcec, Matthew J; Tripathi, Diwaker; Smertenko, Andrei; Tanaka, Kiwamu

    2017-01-01

    A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we demonstrate extracellular alkalinization as a defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-derived elicitors in a dose- and time-dependent manner. We also assessed the defense response against a variety of potato pathogens, such as protists (Phytophthora infestans and Spongospora subterranea) and fungi (Verticillium dahliae and Colletotrichum coccodes). Our results show that extracellular pH increases within 30 min in proportion to the number of pathogen spores added. Consistently with the alkalinization effect, the higher transcription level of several defense-related genes and production of reactive oxygen species was observed. Our results demonstrate that the alkalinization response is an effective marker to study early stages of defense response in potatoes.

  14. Extracellular rigidity sensing by talin isoform–specific mechanical linkages

    PubMed Central

    Austen, Katharina; Ringer, Pia; Mehlich, Alexander; Chrostek-Grashoff, Anna; Kluger, Carleen; Klingner, Christoph; Sabass, Benedikt; Zent, Roy; Rief, Matthias; Grashoff, Carsten

    2015-01-01

    The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule–calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages upon cell adhesion, which are indispensable for cells to probe tissue stiffness. Talin linkages are exposed to a range of piconewton (pN) forces and bear, on average, 7–10 pN during cell adhesion depending on their association with f-actin and vinculin. Disruption of talin’s mechanical engagement does not impair integrin activation and initial cell adhesion but prevents focal adhesion reinforcement and thus extracellular rigidity sensing. Intriguingly, talin mechanics are isoform-specific so that expression of either talin-1 or talin-2 modulates extracellular rigidity sensing. PMID:26523364

  15. The Extracellular Matrix In Development and Morphogenesis: A Dynamic View

    PubMed Central

    Rozario, Tania; DeSimone, Douglas W.

    2009-01-01

    The extracellular matrix (ECM) is synthesized and secreted by embryonic cells beginning at the earliest stages of development. Our understanding of ECM composition, structure and function has grown considerably in the last several decades and this knowledge has revealed that the extracellular microenvironment is critically important for cell growth, survival, differentiation and morphogenesis. ECM and the cellular receptors that interact with it mediate both physical linkages with the cytoskeleton and the bidirectional flow of information between the extracellular and intracellular compartments. This review considers the range of cell and tissue functions attributed to ECM molecules and summarizes recent findings specific to key developmental processes. The importance of ECM as a dynamic repository for growth factors is highlighted along with more recent studies implicating the 3-dimensional organization and physical properties of the ECM as it relates to cell signaling and the regulation of morphogenetic cell behaviors. Embryonic cell and tissue generated forces and mechanical signals arising from ECM adhesion represent emerging areas of interest in this field. PMID:19854168

  16. Problems with extracellular recording of electrical activity in gastrointestinal muscle.

    PubMed

    Sanders, Kenton M; Ward, Sean M; Hennig, Grant W

    2016-12-01

    Motility patterns of the gastrointestinal tract are important for efficient processing of nutrients and waste. Peristalsis and segmentation are based on rhythmic electrical slow waves that generate the phasic contractions fundamental to gastrointestinal motility. Slow waves are generated and propagated actively by interstitial cells of Cajal (ICC), and these events conduct to smooth muscle cells to elicit excitation-contraction coupling. Extracellular electrical recording has been utilized to characterize slow-wave generation and propagation and abnormalities that might be responsible for gastrointestinal motility disorders. Electrode array recording and digital processing are being used to generate data for models of electrical propagation in normal and pathophysiological conditions. Here, we discuss techniques of extracellular recording as applied to gastrointestinal organs and how mechanical artefacts might contaminate these recordings and confound their interpretation. Without rigorous controls for movement, current interpretations of extracellular recordings might ascribe inaccurate behaviours and electrical anomalies to ICC networks and gastrointestinal muscles, bringing into question the findings and validity of models of gastrointestinal electrophysiology developed from these recordings.

  17. Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes.

    PubMed Central

    Ganz, T

    1987-01-01

    Human polymorphonuclear leukocytes (PMN) contain three antimicrobial and cytotoxic peptides which belong to a family of mammalian granulocyte peptides named defensins. To determine their potential availability for extracellular microbicidal or cytotoxic events, we quantified the extracellular release of defensins after stimulation of human PMN with phorbol myristate acetate and opsonized zymosan. As determined by enzyme immunoassay and confirmed by polyacrylamide gel electrophoresis and densitometry, 10(6) human PMN contained 4 to 5 micrograms of defensins. After stimulation with a high concentration of phorbol myristate acetate (1 microgram/ml), about 8% of PMN defensins were found in the media. Release of defensins correlated best with the release of azurophil granule marker beta-glucuronidase or elastase and poorly with the release of either the specific granule marker lactoferrin or cytoplasmic lactate dehydrogenase. Phagocytosis of opsonized zymosan resulted in the extracellular release of less than 3% of PMN defensins. The factors responsible for less release of defensins into media relative to the release of other azurophil granule proteins may include heterogeneity of azurophil granules and the affinity of defensins for cellular surfaces and opsonized particles. In vivo, defensins are most likely to reach effective microbicidal or cytotoxic concentrations in PMN-rich exudates (pus), in confined environments of the phagolysosomes, or in intercellular clefts between PMN and their targets. PMID:3643886

  18. Extracellular proteins of Cryptococcus neoformans and host antibody response.

    PubMed Central

    Chen, L C; Pirofski, L A; Casadevall, A

    1997-01-01

    Proteins secreted by the fungal pathogen Cryptococcus neoformans may be involved in invasion and could be useful in vaccine design. Despite the medical importance of this fungus, little is known about its extracellular proteins or the immune response to these antigens. To study C. neoformans extracellular proteins, 12 strains were metabolically radiolabeled and protein supernatants were analyzed. Both strain- and growth condition-dependent differences were observed. Enzymatic assays of filtered culture supernatants revealed butyrate esterase and caprylate esterase lipase activity for 11 of 12 strains, as well as acid phosphatase, naphthol-AS-BI-phosphohydrolase, and beta-glucosidase activities in some strains. Serum from infected rodents immunoprecipitated several secreted proteins, consistent with in vivo expression and development of an antibody response. For strain 24067, two immunodominant species, of approximately 75 and 30 kDa, were recognized. The relative intensity of the autoradiographic bands depended on the route of infection for both rats and mice. In summary, our results indicate that (i) there are multiple proteins in C. neoformans culture supernatants, (ii) there are strain differences in supernatant protein profiles, (iii) there are differences in supernatant protein profile depending on the growth conditions, (iv) there are several new extracellular and/or cell-associated enzymatic activities, and (v) antibodies to several supernatant proteins are made in the course of infection. PMID:9199426

  19. Extracellularly activatable nanocarriers for drug delivery to tumors

    PubMed Central

    Yeo, Yoon

    2014-01-01

    Introduction Nanoparticles for drug delivery to tumors need to satisfy two seemingly conflicting requirements: they should maintain physical and chemical stability during circulation and be able to interact with target cells and release drug at desired locations with no substantial delay. Unique microenvironment of tumors and externally-applied stimuli provide a useful means to maintain a balance between the two requirements. Areas covered We discuss nanoparticulate drug carriers that maintain stable structures in normal conditions but respond to stimuli for spatiotemporal control of drug delivery. We first define the desired effects of extracellular activation of nanoparticles and frequently used stimuli and review examples of extracellularly activated nanoparticles. Expert opinion Several challenges remain in developing extracellularly activatable nanoparticles. First, some of the stimuli-responsive NPs undergo incremental changes in response to stimuli, losing circulation stability. Second, the applicability of stimuli in clinical settings is limited due to the occasional occurrence of the activating conditions in normal tissues. Third, the construction of stimuli-responsive nanoparticles involves increasing complexity in nanoparticle structure and production methods. Future efforts are needed to identify new targeting conditions and increase the contrast between activated and non-activated NPs, while keeping the production methods simple and scalable. PMID:24950343

  20. Spike library based simulator for extracellular single unit neuronal signals.

    PubMed

    Thorbergsson, P T; Jorntell, H; Bengtsson, F; Garwicz, M; Schouenborg, J; Johansson, A

    2009-01-01

    A well defined set of design criteria is of great importance in the process of designing brain machine interfaces (BMI) based on extracellular recordings with chronically implanted micro-electrode arrays in the central nervous system (CNS). In order to compare algorithms and evaluate their performance under various circumstances, ground truth about their input needs to be present. Obtaining ground truth from real data would require optimal algorithms to be used, given that those exist. This is not possible since it relies on the very algorithms that are to be evaluated. Using realistic models of the recording situation facilitates the simulation of extracellular recordings. The simulation gives access to a priori known signal characteristics such as spike times and identities. In this paper, we describe a simulator based on a library of spikes obtained from recordings in the cat cerebellum and observed statistics of neuronal behavior during spontaneous activity. The simulator has proved to be useful in the task of generating extracellular recordings with realistic background noise and known ground truth to use in the evaluation of algorithms for spike detection and sorting.

  1. Influence of extracellular zinc on M1 microglial activation

    PubMed Central

    Higashi, Youichirou; Aratake, Takaaki; Shimizu, Shogo; Shimizu, Takahiro; Nakamura, Kumiko; Tsuda, Masayuki; Yawata, Toshio; Ueba, Tetuya; Saito, Motoaki

    2017-01-01

    Extracellular zinc, which is released from hippocampal neurons in response to brain ischaemia, triggers morphological changes in microglia. Under ischaemic conditions, microglia exhibit two opposite activation states (M1 and M2 activation), which may be further regulated by the microenvironment. We examined the role of extracellular zinc on M1 activation of microglia. Pre-treatment of microglia with 30–60 μM ZnCl2 resulted in dose-dependent increases in interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNFα) secretion when M1 activation was induced by lipopolysaccharide administration. In contrast, the cell-permeable zinc chelator TPEN, the radical scavenger Trolox, and the P2X7 receptor antagonist A438079 suppressed the effects of zinc pre-treatment on microglia. Furthermore, endogenous zinc release was induced by cerebral ischaemia–reperfusion, resulting in increased expression of IL-1β, IL-6, TNFα, and the microglial M1 surface marker CD16/32, without hippocampal neuronal cell loss, in addition to impairments in object recognition memory. However, these effects were suppressed by the zinc chelator CaEDTA. These findings suggest that extracellular zinc may prime microglia to enhance production of pro-inflammatory cytokines via P2X7 receptor activation followed by reactive oxygen species generation in response to stimuli that trigger M1 activation, and that these inflammatory processes may result in deficits in object recognition memory. PMID:28240322

  2. Diffusion in the extracellular space in brain and tumors

    NASA Astrophysics Data System (ADS)

    Verkman, A. S.

    2013-08-01

    Diffusion of solutes and macromolecules in the extracellular space (ECS) in brain is important for non-synaptic intercellular communication, extracellular ionic buffering, and delivery of drugs and metabolites. Diffusion in tumor ECS is important for delivery of anti-tumor drugs. The ECS in brain comprises ˜20% of brain parenchymal volume and contains cell-cell gaps down to ˜50 nm. We have developed fluorescence methods to quantify solute diffusion in the ECS, allowing measurements deep in solid tissues using microfiberoptics with micron tip size. Diffusion through the tortuous ECS in brain is generally slowed by ˜3-5-fold compared with that in water, with approximately half of the slowing due to tortuous ECS geometry and half due to the mildly viscous extracellular matrix (ECM). Mathematical modeling of slowed diffusion in an ECS with reasonable anatomical accuracy is in good agreement with experiment. In tumor tissue, diffusion of small macromolecules is only mildly slowed (<3-fold slower than in water) in superficial tumor, but is greatly slowed (>10-fold) at a depth of few millimeters as the tumor tissue becomes more compact. Slowing by ECM components such as collagen contribute to the slowed diffusion. Therefore, as found within cells, cellular crowding and highly tortuous transport can produce only minor slowing of diffusion in the ECS.

  3. Pseudomonas deceptionensis DC5-mediated synthesis of extracellular silver nanoparticles.

    PubMed

    Jo, Jae H; Singh, Priyanka; Kim, Yeon J; Wang, Chao; Mathiyalagan, Ramya; Jin, Chi-Gyu; Yang, Deok C

    2016-09-01

    The biological synthesis of metal nanoparticles is of great interest in the field of nanotechnology. The present work highlights the extracellular biological synthesis of silver nanoparticles using Pseudomonas deceptionensis DC5. The particles were synthesized in the culture supernatant within 48 h of incubation. Extracellular synthesis of silver nanoparticles in the culture supernatant was confirmed by ultraviolet-visible spectroscopy, which showed the absorption peak at 428 nm, and also under field emission transmission electron microscopy which displayed the spherical shape. In addition, the particles were characterized by X-ray diffraction spectroscopy, which corresponds to the crystalline nature of nanoparticles, and energy-dispersive X-ray analysis which exhibited the intense peak at 3 keV, resembling the silver nanoparticles. Further, the synthesized nanoparticles were examined by elemental mapping which displayed the dominance of the silver element in the synthesized product, and dynamic light scattering which showed the distribution of silver nanoparticles with respect to intensity, volume, and number of particles. Moreover, the silver nanoparticles have been found to be quite active in antimicrobial activity and biofilm inhibition activity against pathogenic microorganisms. Thus, the present work emphasized the prospect of using the P. deceptionensis DC5 to achieve the extracellular synthesis of silver nanoparticles in a facile and environmental manner.

  4. Applying Proteomics to Investigate Extracellular Matrix in Health and Disease.

    PubMed

    Randles, Michael; Lennon, Rachel

    2015-01-01

    The molecular composition of basement membranes (BMs) has traditionally been investigated by candidate-based approaches leading to the identification of key structural components as described in previous chapters. Laminins, collagen IV, nidogens, perlecan, and type XV/XVIII collagen are integral to BMs with isoforms showing tissue specificity. More recently the application of mass spectrometry (MS)-based proteomics has led to the discovery of many more structural and regulatory components of BMs and more broadly, extracellular matrix (ECM). These investigations have revealed tissue-specific signatures of between 100 and 150 ECM components, demonstrating the complexity of the extracellular niche. In addition to providing a structural scaffold for cells, ECM is a dynamic extracellular environment capable of regulating the physical properties of tissues. Global investigations of ECM with proteomics in turn enable systems level analyses and when applied to health and disease states these investigations provide insights into pathways regulating matrix dysregulation. This chapter focuses on the methods used to extract ECM and on the analysis of its composition using MS-based proteomics, and it provides examples of how these approaches have been used to investigate health and disease states.

  5. Structural basis for Smoothened regulation by its extracellular domains

    PubMed Central

    Miller, Paul S.; Hedger, George; Luchetti, Giovanni; Nachtergaele, Sigrid; Tully, Mark D.; Mydock-McGrane, Laurel; Covey, Douglas F.; Rambo, Robert P.; Sansom, Mark S. P.; Rohatgi, Rajat; Siebold, Christian

    2016-01-01

    Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How such large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened (SMO), which contains two distinct ligand-binding sites in its TMD and CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain (LD). Structure-guided mutations show that the interface between the CRD, LD and TMD stabilises the inactive state of SMO. Unexpectedly, we find a cholesterol molecule bound to SMO in the CRD-binding site. Mutations predicted to prevent cholesterol binding impair the ability of SMO to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-LD-TMD interface. Our work elucidates the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains. PMID:27437577

  6. Horizontal Transmission of Cytosolic Sup35 Prions by Extracellular Vesicles

    PubMed Central

    Liu, Shu; Hossinger, André; Hofmann, Julia P.; Denner, Philip

    2016-01-01

    ABSTRACT Prions are infectious protein particles that replicate by templating their aggregated state onto soluble protein of the same type. Originally identified as the causative agent of transmissible spongiform encephalopathies, prions in yeast (Saccharomyces cerevisiae) are epigenetic elements of inheritance that induce phenotypic changes of their host cells. The prototype yeast prion is the translation termination factor Sup35. Prions composed of Sup35 or its modular prion domain NM are heritable and are transmitted vertically to progeny or horizontally during mating. Interestingly, in mammalian cells, protein aggregates derived from yeast Sup35 NM behave as true infectious entities that employ dissemination strategies similar to those of mammalian prions. While transmission is most efficient when cells are in direct contact, we demonstrate here that cytosolic Sup35 NM prions are also released into the extracellular space in association with nanometer-sized membrane vesicles. Importantly, extracellular vesicles are biologically active and are taken up by recipient cells, where they induce self-sustained Sup35 NM protein aggregation. Thus, in mammalian cells, extracellular vesicles can serve as dissemination vehicles for protein-based epigenetic information transfer. PMID:27406566

  7. Extracellular enzyme activities and nutrient availability during artificial groundwater recharge.

    PubMed

    Kolehmainen, Reija E; Korpela, Jaana P; Münster, Uwe; Puhakka, Jaakko A; Tuovinen, Olli H

    2009-02-01

    Natural organic matter (NOM) removal is the main objective of artificial groundwater recharge (AGR) for drinking water production and biodegradation plays a substantial role in this process. This study focused on the biodegradation of NOM and nutrient availability for microorganisms in AGR by the determination of extracellular enzyme activities (EEAs) and nutrient concentrations along a flow path in an AGR aquifer (Tuusula Water Works, Finland). Natural groundwater in the same area but outside the influence of recharge was used as a reference. Determination of the specific alpha-d-glucosidase (alpha-Glu), beta-d-glucosidase (beta-Glu), phosphomonoesterase (PME), leucine aminopeptidase (LAP) and acetate esterase (AEST) activities by fluorogenic model substrates revealed major increases in the enzymatic hydrolysis rates in the aquifer within a 10m distance from the basin. The changes in the EEAs along the flow path occurred simultaneously with decreases in nutrient concentrations. The results support the assumption that the synthesis of extracellular enzymes in aquatic environments is up and down regulated by nutrient availability. The EEAs in the basin sediment and pore water samples (down to 10cm) were in the same order of magnitude as in the basin water, suggesting similar nutritional conditions. Phosphorus was likely to be the limiting nutrient at this particular AGR site. Furthermore, the extracellular enzymes functioned in a synergistic and cooperative way.

  8. Extracellular Genomic DNA Mediates Enhancement of Xylella fastidiosa Biofilm Formation in Vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa (Xf) produces extracellular DNA in PD3 liquid medium. This extracellular DNA could enhance biofilm formation, a factor in successful establishment of Xf in planta. The relative amounts of extracellular DNA were positively correlated with planktonic growth and biofilm formation in ...

  9. Commercial Cow Milk Contains Physically Stable Extracellular Vesicles Expressing Immunoregulatory TGF-β

    PubMed Central

    Bennink, Miranda B.; Broeren, Mathijs G. A.; van Caam, Arjan P. M.; Koenders, Marije I.; van Lent, Peter L. E. M.; van den Berg, Wim B.; de Vries, Marieke; van der Kraan, Peter M.; van de Loo, Fons A. J.

    2015-01-01

    Scope Extracellular vesicles, including exosomes, have been identified in all biological fluids and rediscovered as an important part of the intercellular communication. Breast milk also contains extracellular vesicles and the proposed biological function is to enhance the antimicrobial defense in newborns. It is, however, unknown whether extracellular vesicles are still present in commercial milk and, more importantly, whether they retained their bioactivity. Here, we characterize the extracellular vesicles present in semi-skimmed cow milk available for consumers and study their effect on T cells. Methods and Results Extracellular vesicles from commercial milk were isolated and characterized. Milk-derived extracellular vesicles contained several immunomodulating miRNAs and membrane protein CD63, characteristics of exosomes. In contrast to RAW 267.4 derived extracellular vesicles the milk-derived extracellular vesicles were extremely stable under degrading conditions, including low pH, boiling and freezing. Milk-derived extracellular vesicles were easily taken up by murine macrophages in vitro. Furthermore, we found that they can facilitate T cell differentiation towards the pathogenic Th17 lineage. Using a (CAGA)12-luc reporter assay we showed that these extracellular vesicles carried bioactive TGF-β, and that anti-TGF-β antibodies blocked Th17 differentiation. Conclusion Our findings show that commercial milk contains stable extracellular vesicles, including exosomes, and carry immunoregulatory cargo. These data suggest that the extracellular vesicles present in commercial cow milk remains intact in the gastrointestinal tract and exert an immunoregulatory effect. PMID:25822997

  10. Potential functional applications of extracellular vesicles: a report by the NIH Common Fund Extracellular RNA Communication Consortium.

    PubMed

    Quesenberry, Peter J; Aliotta, Jason; Camussi, Giovanni; Abdel-Mageed, Asim B; Wen, Sicheng; Goldberg, Laura; Zhang, Huang-Ge; Tetta, Ciro; Franklin, Jeffrey; Coffey, Robert J; Danielson, Kirsty; Subramanya, Vinita; Ghiran, Ionita; Das, Saumya; Chen, Clark C; Pusic, Kae M; Pusic, Aya D; Chatterjee, Devasis; Kraig, Richard P; Balaj, Leonora; Dooner, Mark

    2015-01-01

    The NIH Extracellular RNA Communication Program's initiative on clinical utility of extracellular RNAs and therapeutic agents and developing scalable technologies is reviewed here. Background information and details of the projects are presented. The work has focused on modulation of target cell fate by extracellular vesicles (EVs) and RNA. Work on plant-derived vesicles is of intense interest, and non-mammalian sources of vesicles may represent a very promising source for different therapeutic approaches. Retro-viral-like particles are intriguing. Clearly, EVs share pathways with the assembly machinery of several other viruses, including human endogenous retrovirals (HERVs), and this convergence may explain the observation of viral-like particles containing viral proteins and nucleic acid in EVs. Dramatic effect on regeneration of damaged bone marrow, renal, pulmonary and cardiovascular tissue is demonstrated and discussed. These studies show restoration of injured cell function and the importance of heterogeneity of different vesicle populations. The potential for neural regeneration is explored, and the capacity to promote and reverse neoplasia by EV exposure is described. The tremendous clinical potential of EVs underlies many of these projects, and the importance of regulatory issues and the necessity of general manufacturing production (GMP) studies for eventual clinical trials are emphasized. Clinical trials are already being pursued and should expand dramatically in the near future.

  11. Modulation of extracellular neurotransmitter levels in the nucleus accumbens by a taurine uptake inhibitor.

    PubMed

    Olive, M F; Mehmert, K K; Hodge, C W

    2000-12-15

    Using in vivo microdialysis, we examined the effect of local perfusion of the taurine uptake inhibitor guanidinoethyl sulfonate on extracellular levels of various neurotransmitters in the rat nucleus accumbens. Guanidinoethyl sulfonate (500 microM-50 mM) produced a concentration-dependent increase in extracellular taurine levels. While 500 microM and 5 mM concentrations of guanidinoethyl sulfonate were largely without effect, 50 mM guanidinoethyl sulfonate produced a significant decrease in extracellular levels of aspartate, glutamate and glycine, with no effect on extracellular dopamine levels. These results indicate that guanidinoethyl sulfonate can modulate extracellular amino acid levels in the nucleus accumbens.

  12. Enzymatic degradation of monocrotophos by extracellular fungal OP hydrolases.

    PubMed

    Jain, Rachna; Garg, Veena

    2013-11-01

    The present study explores the potential of extracellular fungal organophosphate (OP) hydrolase for the degradation of monocrotophos. Extracellular OP hydrolases were isolated and purified from five different fungal isolates viz. Aspergillus niger (M1), Aspergillus flavus (M2), Penicillium aculeatum (M3), Fusarium pallidoroseum (M4), and Macrophomina sp. (M5) by AmSO4 precipitation, dialysis, and G-100 chromatography. M3 showed highest percentage yield of 68.81 followed by 55.41 % for M1. Each of the purified enzyme fraction constituted of two different subunits of 33- and 67-kDa molecular weight. Optimum enzyme fraction (150 μg ml(-1)) rapidly degraded monocrotophos within 120 h in phosphorus-free liquid culture medium (CZM) with K deg of 0.0368, 0.0138, 0.048, 0.016, 0.0138, and 0.048 day(-1) and half-life of 0.79, 2.11, 0.6, 1.8, and 2.11 days for M1, M2, M3, M4, and M5, respectively. The results were further confirmed by high performance thin layer chromatography and Fourier transform infrared which indicate the disappearance of monocrotophos by hydrolytic cleavage of vinyl phosphate bond. The overall order of enzymatic degradation was found to be P. aculeatum > A. niger > F. pallidoroseum > A. flavus = Macrophomina sp. Hence, the study concludes that extracellular OP hydrolases efficiently degraded monocrotophos and could be used as a potential candidate for the detoxification of this neurotoxin pesticide.

  13. Extracellular Proteins: Novel Key Components of Metal Resistance in Cyanobacteria?

    PubMed Central

    Giner-Lamia, Joaquín; Pereira, Sara B.; Bovea-Marco, Miquel; Futschik, Matthias E.; Tamagnini, Paula; Oliveira, Paulo

    2016-01-01

    Metals are essential for all living organisms and required for fundamental biochemical processes. However, when in excess, metals can turn into highly-toxic agents able to disrupt cell membranes, alter enzymatic activities, and damage DNA. Metal concentrations are therefore tightly controlled inside cells, particularly in cyanobacteria. Cyanobacteria are ecologically relevant prokaryotes that perform oxygenic photosynthesis and can be found in many different marine and freshwater ecosystems, including environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been widely studied in cyanobacteria. So far, most studies have focused on how cells are capable of controlling their internal metal pools, with a strong bias toward the analysis of intracellular processes. Ultrastructure, modulation of physiology, dynamic changes in transcription and protein levels have been studied, but what takes place in the extracellular environment when cells are exposed to an unbalanced metal availability remains largely unknown. The interest in studying the subset of proteins present in the extracellular space has only recently begun and the identification and functional analysis of the cyanobacterial exoproteomes are just emerging. Remarkably, metal-related proteins such as the copper-chaperone CopM or the iron-binding protein FutA2 have already been identified outside the cell. With this perspective, we aim to raise the awareness that metal-resistance mechanisms are not yet fully known and hope to motivate future studies assessing the role of extracellular proteins on bacterial metal homeostasis, with a special focus on cyanobacteria. PMID:27375598

  14. Extracellular DNA in Single- and Multiple-Species Unsaturated Biofilms

    PubMed Central

    Steinberger, R. E.; Holden, P. A.

    2005-01-01

    The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA. PMID:16151131

  15. Fibrillin assemblies: extracellular determinants of tissue formation and fibrosis.

    PubMed

    Olivieri, Jacopo; Smaldone, Silvia; Ramirez, Francesco

    2010-12-02

    The extracellular matrix (ECM) plays a key role in tissue formation, homeostasis and repair, mutations in ECM components have catastrophic consequences for organ function and therefore, for the fitness and survival of the organism. Collagen, fibrillin and elastin polymers represent the architectural scaffolds that impart specific mechanic properties to tissues and organs. Fibrillin assemblies (microfibrils) have the additional function of distributing, concentrating and modulating local transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) signals that regulate a plethora of cellular activities, including ECM formation and remodeling. Fibrillins also contain binding sites for integrin receptors, which induce adaptive responses to changes in the extracellular microenvironment by reorganizing the cytoskeleton, controlling gene expression, and releasing and activating matrix-bound latent TGF-β complexes. Genetic evidence has indicated that fibrillin-1 and fibrillin-2 contribute differently to the organization and structural properties of non-collagenous architectural scaffolds, which in turn translate into discrete regulatory outcomes of locally released TGF-β and BMP signals. Additionally, the study of congenital dysfunctions of fibrillin-1 has yielded insights into the pathogenesis of acquired connective tissue disorders of the connective tissue, such as scleroderma. On the one hand, mutations that affect the structure or expression of fibrillin-1 perturb microfibril biogenesis, stimulate improper latent TGF-β activation, and give rise to the pleiotropic manifestations in Marfan syndrome (MFS). On the other hand, mutations located around the integrin-binding site of fibrillin-1 perturb cell matrix interactions, architectural matrix assembly and extracellular distribution of latent TGF-β complexes, and lead to the highly restricted fibrotic phenotype of Stiff Skin syndrome. Understanding the molecular similarities and differences between

  16. Plasma extracellular RNA profiles in healthy and cancer patients

    PubMed Central

    Yuan, Tiezheng; Huang, Xiaoyi; Woodcock, Mark; Du, Meijun; Dittmar, Rachel; Wang, Yuan; Tsai, Susan; Kohli, Manish; Boardman, Lisa; Patel, Tushar; Wang, Liang

    2016-01-01

    Extracellular vesicles are selectively enriched in RNA that has potential as disease biomarkers. To systemically characterize circulating extracellular RNA (exRNA) profiles, we performed RNA sequencing analysis on plasma extracellular vesicles derived from 50 healthy individuals and 142 cancer patients. Of ~12.6 million raw reads for each individual, the number of mappable reads aligned to RNA references was ~5.4 million including miRNAs (~40.4%), piwiRNAs (~40.0%), pseudo-genes (~3.7%), lncRNAs (~2.4%), tRNAs (~2.1%), and mRNAs (~2.1%). By expression stability testing, we identified a set of miRNAs showing relatively consistent expression, which may serve as reference control for exRNA quantification. By performing multivariate analysis of covariance, we identified significant associations of these exRNAs with age, sex and different types of cancers. In particular, down-regulation of miR-125a-5p and miR-1343-3p showed an association with all cancer types tested (false discovery rate <0.05). We developed multivariate statistical models to predict cancer status with an area under the curve from 0.68 to 0.92 depending cancer type and staging. This is the largest RNA-seq study to date for profiling exRNA species, which has not only provided a baseline reference profile for circulating exRNA, but also revealed a set of RNA candidates for reference controls and disease biomarkers. PMID:26786760

  17. Extracellular NO signalling from a mechanically stimulated osteocyte.

    PubMed

    Vatsa, Aviral; Smit, Theo H; Klein-Nulend, Jenneke

    2007-01-01

    Bone remodelling is a dynamic process that requires the coordinated interaction of osteocytes, osteoblasts, and osteoclasts, collaborating in basic multicellular units (BMUs). Communication between these cells can be by extracellular soluble molecules as well as directly propagating intercellular signalling molecules. Key to the understanding of bone remodelling is osteocyte mechanosensing and chemical signalling to the surrounding cells, since osteocytes are believed to be the mechanosensors of bone, responding to mechanical stresses. Nitric oxide (NO) is an important parameter to study osteocyte activation following mechanical loading. It is a small short-lived molecule, which makes its real-time, quantitative monitoring difficult. However, recently we demonstrated that DAR-4M AM chromophore can be used for real-time quantitative monitoring of intracellular NO production in individual cells following mechanical loading. Here we studied if a single mechanically stimulated osteocyte communicates with, and thus activates its surrounding cells via extracellular soluble factors. We monitored quantitatively intracellular NO production in the stimulated osteocyte and in its surrounding osteocytes, which were not interconnected. Mechanical stimulation by microneedle of a single-MLO-Y4 osteocyte-like cell upregulated the average intracellular NO production by 94% in the stimulated cell, and by 31-150% in the surrounding osteocytes. In conclusion, a single osteocyte can disseminate a mechanical stimulus to its surrounding osteocytes via extracellular soluble signalling factors. This reinforces the putative mechanosensory role of osteocytes, and demonstrates a possible mechanism by which a single mechanically stimulated osteocyte can communicate with other cells in a BMU, which might help to better understand the intricacies of intercellular interactions in BMUs and thus bone remodelling.

  18. Extracellular DNA in single- and multiple-species unsaturated biofilms.

    PubMed

    Steinberger, R E; Holden, P A

    2005-09-01

    The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.

  19. Effects of extracellular matrix on the malignant phenotype.

    PubMed Central

    Luikart, S. D.

    1988-01-01

    Extracellular matrix molecules, including collagen, glycosaminoglycans (usually linked to a protein core as proteoglycan), elastin, and glycoproteins, influence the initiation and maintenance of differentiation of a variety of cell types. These molecules bind to the cell surface at specific sites and nonspecifically by electrostatic forces. Such interactions may alter the cell's response to growth and differentiation factors. After neoplastic transformation, most cells retain some dependence on these factors. This paper reviews the influence of matrix components on the phenotype of a variety of malignant cells and concludes that in vitro studies of malignant cell behavior require the utilization of an appropriate microenvironment. PMID:3284211

  20. Transfer of extracellular vesicles during immune cell-cell interactions

    PubMed Central

    Gutiérrez-Vázquez, Cristina; Villarroya-Beltri, Carolina; Mittelbrunn, María; Sánchez-Madrid, Francisco

    2013-01-01

    SUMMARY The transfer of molecules between cells during cognate immune cell interactions has been reported, and recently a novel mechanism of transfer of proteins and genetic material such as small RNA between T cells and APCs has been described, involving exchange of extracellular vesicles (EVs) during the formation of the immunological synapse (IS). EVs – a term that encompasses exosomes and microvesicles – have been implicated in cell-cell communication during immune responses associated with tumors, pathogens, allergies and autoimmune diseases. This review focuses on EV transfer as a mechanism for the exchange of molecules during immune cell-cell interactions. PMID:23278745

  1. Extracellular Proteins Limit the Dispersal of BiogenicNanoparticles

    SciTech Connect

    Moreau, John W.; Weber, Peter K.; Martin, Michael C.; Gilbert,Benjamin; Hutcheon, Ian D.; Banfield, Jillian F.

    2007-04-27

    High spatial-resolution secondaryion microprobespectrometry, synchrotron radiation Fourier-transform infraredspectroscopy and polyacrylamide gel analysis demonstrate the intimateassociation of proteins with spheroidal aggregates of biogenic zincsulfide nanocrystals, an example of extracellular biomineralization.Experiments involving synthetic ZnS nanoparticles and representativeamino acids indicate a driving role for cysteine in rapid nanoparticleaggregation. These findings suggest that microbially-derivedextracellular proteins can limit dispersal of nanoparticulatemetal-bearing phases, such as the mineral products of bioremediation,that may otherwise be transported away from their source by subsurfacefluid flow.

  2. Structure and function of the skeletal muscle extracellular matrix.

    PubMed

    Gillies, Allison R; Lieber, Richard L

    2011-09-01

    The skeletal muscle extracellular matrix (ECM) plays an important role in muscle fiber force transmission, maintenance, and repair. In both injured and diseased states, ECM adapts dramatically, a property that has clinical manifestations and alters muscle function. Here we review the structure, composition, and mechanical properties of skeletal muscle ECM; describe the cells that contribute to the maintenance of the ECM; and, finally, overview changes that occur with pathology. New scanning electron micrographs of ECM structure are also presented with hypotheses about ECM structure–function relationships. Detailed structure–function relationships of the ECM have yet to be defined and, as a result, we propose areas for future study.

  3. Extracellular enzyme activity and biogeochemical cycling in restored prairies

    NASA Astrophysics Data System (ADS)

    Lynch, L.; Hernandez, D.; Schade, J. D.

    2011-12-01

    Winter microbial activity in mid-latitude prairie ecosystems is thermally sensitive and significantly influenced by snow depth. Snow insulates the soil column facilitating microbial processing of complex organic substrates. Previous studies in forests and tundra ecosystems suggest patterns of substrate utilization and limitation are seasonal; above freezing, soil microbes access fresh litter inputs and sugar exudates from plant roots, while under frozen condition they recycle nutrients incorporated in microbial biomass. In order to liberate nutrients required for carbon degradation, soil microbes invest energy in the production of extracellular enzymes that cleave monomers from polymer bonds. The inverse relationship between relative enzyme abundance and substrate availability makes enzyme assays a useful proxy to assess changes in resources over time. Our objective in this study was to assess patterns in microbial biomass, nutrient availability, and extracellular enzyme activity in four snow exclosure sites over a seven-month period. Over the past three years, we have maintained a snow removal experiment on two restored prairies in central Minnesota. In each prairie, snow was continuously removed annually from two 4 x 4 m plots by shoveling after each snow event. Extractable C, N and P, and microbial C, N and P in soil samples were measured in samples collected from these snow removal plots, as well as in adjacent unmanipulated prairie control plots. Pools of C, N, and P were estimated using standard extraction protocols, and microbial pools were estimated using chloroform fumigation direct extraction (CFDE). We conducted fluorometric extracellular enzyme assays (EEA) to assess how the degradation potential of cellulose (cellobiohydrolase, CBH), protein (leucine aminopeptidase, LAP), and phosphate esters (phosphatase, PHOS) changed seasonally. Microbial C and N declined between October and June, while microbial P declined during the fall and winter, but increased

  4. Locally optimal extracellular stimulation for chaotic desynchronization of neural populations.

    PubMed

    Wilson, Dan; Moehlis, Jeff

    2014-10-01

    We use optimal control theory to design a methodology to find locally optimal stimuli for desynchronization of a model of neurons with extracellular stimulation. This methodology yields stimuli which lead to positive Lyapunov exponents, and hence desynchronizes a neural population. We analyze this methodology in the presence of interneuron coupling to make predictions about the strength of stimulation required to overcome synchronizing effects of coupling. This methodology suggests a powerful alternative to pulsatile stimuli for deep brain stimulation as it uses less energy than pulsatile stimuli, and could eliminate the time consuming tuning process.

  5. Effect of solar irradiation on extracellular enzymes of Aeromonas proteolytica

    NASA Technical Reports Server (NTRS)

    Foster, B. G.

    1973-01-01

    The bacterium Aeromonas proteolytica was selected for studying the effects of solar irradiation on extracellular enzymes because it produces an endopeptidase that is capable of degrading proteins and a hemolysin that is active in lysing human erythrocytes. Possible alterations in the rate of enzyme production in response to the test conditions are currently underway and are not available for this preliminary report. Completed viability studies are indicative that little difference exists among the survival curves derived for cells exposed to various components of ultraviolet irradiation in space.

  6. Potential Roles of Fungal Extracellular Vesicles during Infection

    PubMed Central

    Joffe, Luna S.; Nimrichter, Leonardo

    2016-01-01

    ABSTRACT Extracellular vesicles (EVs) are produced by virtually all cell types. Within the past few years, work in this field has revealed more information about fungal EVs. Fungal EVs have been shown to carry proteins, lipids, pigments, polysaccharides, and RNA; these components are known virulence factors, a fact which supports the hypothesis that fungal EVs concentrate pathogenic determinants. Additionally, recent studies have demonstrated that fungal EVs stimulate the host immune system. In this review, putative roles of fungal EVs are discussed, including their potential as vaccination tools and their possible contribution to pathogenesis in invasive fungal diseases. PMID:27390779

  7. Potentials and capabilities of the Extracellular Vesicle (EV) Array.

    PubMed

    Jørgensen, Malene Møller; Bæk, Rikke; Varming, Kim

    2015-01-01

    Extracellular vesicles (EVs) and exosomes are difficult to enrich or purify from biofluids, hence quantification and phenotyping of these are tedious and inaccurate. The multiplexed, highly sensitive and high-throughput platform of the EV Array presented by Jørgensen et al., (J Extracell Vesicles, 2013; 2: 10) has been refined regarding the capabilities of the method for characterization and molecular profiling of EV surface markers. Here, we present an extended microarray platform to detect and phenotype plasma-derived EVs (optimized for exosomes) for up to 60 antigens without any enrichment or purification prior to analysis.

  8. Imaging cardiac extracellular matrices: a blueprint for regeneration

    PubMed Central

    Jung, Jangwook P.; Squirrell, Jayne M.; Lyons, Gary E.; Eliceiri, Kevin W.; Ogle, Brenda M.

    2013-01-01

    Once damaged, cardiac tissue does not readily repair and is therefore a primary target of regenerative therapies. One regenerative approach is the development of scaffolds that functionally mimic the cardiac extracellular matrix (ECM) to deliver stem cells or cardiac precursor populations to the heart. Technological advances in micro/nanotechnology, stem cell biology, biomaterials and tissue decellularization have propelled this promising approach forward. Surprisingly, technological advances in optical imaging methods have not been fully utilized in the field of cardiac regeneration. Here, we describe and provide examples to demonstrate how advanced imaging techniques could revolutionize how ECM-mimicking cardiac tissues are informed and evaluated. PMID:22209562

  9. A fast and mild decellularization protocol for obtaining extracellular matrix.

    PubMed

    Mirzarafie, Ariana; Grainger, Rhian K; Thomas, Ben; Bains, William; Ustok, Fatma I; Lowe, Chris R

    2014-04-01

    Degradation of extracellular matrix (ECM) function with age is a major cause of loss of tissue function with age that we would wish to reverse. Tissue engineering to provide replacement tissue requires an ECM-mimicking scaffold for cell organization. The standard protocols for achieving this take 10 days and include steps that may change the protein structure of the ECM. Here we describe a much shorter protocol for decellularizing chicken muscle, skin, and tendon samples that achieves the same efficiency as the original protocol without protein cross-link interference. Our protocol can be completed in 72 hr.

  10. Structure and Function of the Skeletal Muscle Extracellular Matrix

    PubMed Central

    Gillies, Allison R.; Lieber, Richard L.

    2011-01-01

    The skeletal muscle extracellular matrix (ECM) plays an important role in muscle fiber force transmission, maintenance, and repair. In both injured and diseased states, ECM adapts dramatically, a property thathas clinical manifestations and alters muscle function. Here, we review the structure, composition, and mechanical properties of skeletal muscle ECM, describe the cells that contribute to the maintenance of the ECM and, finally, overview changes that occur with pathology. New scanning electron micrographs of ECM structure are also presented with hypotheses about ECM structure-function relationships. Detailed structure-function relationships of the ECM have yet to be defined and, as a result, we propose areas for future studies. PMID:21949456

  11. [Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis].

    PubMed

    Dunaevskiĭ, Ia E; Gruban', T N; Beliakova, G A; Belozerskiĭ, M A

    2006-01-01

    The presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).

  12. An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans.

    PubMed

    Byrd, Angel S; O'Brien, Xian M; Johnson, Courtney M; Lavigne, Liz M; Reichner, Jonathan S

    2013-04-15

    The armament of neutrophil-mediated host defense against pathogens includes the extrusion of a lattice of DNA and microbicidal enzymes known as neutrophil extracellular traps (NETs). The receptor/ligand interactions and intracellular signaling mechanisms responsible for elaborating NETs were determined for the response to Candida albicans. Because the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with extracellular matrix, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of purified fungal pathogen-associated molecular pattern β-glucan by human neutrophils causes rapid (≤ 30 min) homotypic aggregation and NET release by a mechanism that requires Fn. Alone, immobilized β-glucan induces reactive oxygen species (ROS) production but not NET release, whereas in the context of Fn, ROS production is suppressed and NETs are extruded. NET release to Fn with β-glucan is robust, accounting for 17.2 ± 3.4% of total DNA in the cell population. Release is dependent on β-glucan recognition by complement receptor 3 (CD11b/CD18), but not Dectin-1, or ROS. The process of NET release included filling of intracellular vesicles with nuclear material that was eventually extruded. We identify a role for ERK in homotypic aggregation and NET release. NET formation to C. albicans hyphae was also found to depend on β-glucan recognition by complement receptor 3, require Fn and ERK but not ROS, and result in hyphal destruction. We report a new regulatory mechanism of NETosis in which the extracellular matrix is a key component of the rapid antifungal response.

  13. TRP channels activated by extracellular hypo-osmoticity in epithelia.

    PubMed

    Harteneck, C; Reiter, B

    2007-02-01

    TRP (transient receptor potential) channels comprise a superfamily of non-selective cation channels with at least seven subfamilies. The variety of subfamilies corresponds to the differences in the activation mechanisms and functions. TRPM3 (TRP melastatin 3) and TRPV4 (TRP vanilloid 3) have been characterized as cation channels activated by extracellular hypo-osmoticity. In addition, TRPV4 is activated by metabolites of arachidonic acid as well as alpha-isomers of phorbol esters known to be ineffective in stimulating proteins of the protein kinase C family. TRPM3 is responsive to sphingosine derivatives. The detection of splice variants with probably different activation mechanisms supports the idea that TRPM3 may have diverse cellular functions depending on the expression of a particular variant. The expression of TRPV4 in many epithelial cell types raised the question of the role of TRPV4 in epithelial physiology. Single-cell experiments as well as approaches using epithelial layers show that multiple cellular responses are triggered by TRPV4 activation and subsequent elevation of intracellular calcium. The TRPV4-induced responses increasing transcellular ion flux as well as paracellular permeability may allow the cells to adjust to changes in extracellular osmolarity. In summary, TRPV4 plays a central role in epithelial homoeostasis by modulating epithelial barrier function.

  14. Tendon Extracellular Matrix Alterations in Ullrich Congenital Muscular Dystrophy

    PubMed Central

    Sardone, Francesca; Traina, Francesco; Bondi, Alice; Merlini, Luciano; Santi, Spartaco; Maraldi, Nadir Mario; Faldini, Cesare; Sabatelli, Patrizia

    2016-01-01

    Collagen VI (COLVI) is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in COLVI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by COLVI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient’s tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of COLVI and altered distribution of collagen I (COLI) and fibronectin (FBN). In patient’s tenocyte culture, COLVI web formation and cell surface association were severely impaired; large aggregates of COLVI, which matched with COLI labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient’s tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that COLVI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of COLVI related myopathies. PMID:27375477

  15. Extracellular mycosynthesis of gold nanoparticles using Fusarium solani

    NASA Astrophysics Data System (ADS)

    Gopinath, K.; Arumugam, A.

    2014-08-01

    The development of eco-friendly methods for the synthesis of nanomaterial shape and size is an important area of research in the field of nanotechnology. The present investigation deals with the extracellular rapid biosynthesis of gold nanoparticles using Fusarium solani culture filtrate. The UV-vis spectra of the fungal culture filtrate medium containing gold ion showed peak at 527 nm corresponding to the plasmon absorbance of gold nanoparticles. FTIR spectra provide an evidence for the presence of heterocyclic compound in the culture filtrate, which increases the stability of the synthesized gold nanoparticles. The X-ray analysis respects the Bragg's law and confirmed the crystalline nature of the gold nanoparticles. AFM analysis showed the results of particle sizes (41 nm). Transmission electron microscopy (TEM) showed that the gold nanoparticles are spherical in shape with the size range from 20 to 50 nm. The use of F. solani will offer several advantages since it is considered as a non-human pathogenic organism. The fungus F. solani has a fast growth rate, rapid capacity of metallic ions reduction, NPs stabilization and facile and economical biomass handling. Extracellular biosynthesis of gold nanoparticles could be highly advantageous from the point of view of synthesis in large quantities, time consumption, eco-friendly, non-toxic and easy downstream processing.

  16. Modeling biofilms with dual extracellular electron transfer mechanisms

    SciTech Connect

    Renslow, Ryan S.; Babauta, Jerome T.; Kuprat, Andrew P.; Schenk, Jim; Ivory, Cornelius; Fredrickson, Jim K.; Beyenal, Haluk

    2013-11-28

    Electrochemically active biofilms have a unique form of respiration in which they utilize solid external materials as their terminal electron acceptor for metabolism. Currently, two primary mechanisms have been identified for long-range extracellular electron transfer (EET): a diffusion- and a conduction-based mechanism. Evidence in the literature suggests that some biofilms, particularly Shewanella oneidensis, produce components requisite for both mechanisms. In this study, a generic model is presented that incorporates both diffusion- and conduction-based mechanisms and allows electrochemically active biofilms to utilize both simultaneously. The model was applied to Shewanella oneidensis and Geobacter sulfurreducens biofilms using experimentally generated data found the literature. Our simulation results showed that 1) biofilms having both mechanisms available, especially if they can interact, may have metabolic advantage over biofilms that can use only a single mechanism; 2) the thickness of Geobacter sulfurreducens biofilms is likely not limited by conductivity; 3) accurate intrabiofilm diffusion coefficient values are critical for current generation predictions; and 4) the local biofilm potential and redox potential are two distinct measurements and cannot be assumed to have identical values. Finally, we determined that cyclic and squarewave voltammetry are currently not good tools to determine the specific percentage of extracellular electron transfer mechanisms used by biofilms. The developed model will be a critical tool in designing experiments to explain EET mechanisms.

  17. Wolbachia endosymbionts induce neutrophil extracellular trap formation in human onchocerciasis

    PubMed Central

    Tamarozzi, Francesca; Turner, Joseph D.; Pionnier, Nicolas; Midgley, Angela; Guimaraes, Ana F.; Johnston, Kelly L.; Edwards, Steven W.; Taylor, Mark J.

    2016-01-01

    The endosymbiotic bacteria, Wolbachia, induce neutrophilic responses to the human helminth pathogen Onchocerca volvulus. The formation of Neutrophil Extracellular Traps (NETs), has been implicated in anti-microbial defence, but has not been identified in human helminth infection. Here, we demonstrate NETs formation in human onchocerciasis. Extracellular NETs and neutrophils were visualised around O. volvulus in nodules excised from untreated patients but not in nodules from patients treated with the anti-Wolbachia drug, doxycycline. Whole Wolbachia or microspheres coated with a synthetic Wolbachia lipopeptide (WoLP) of the major nematode Wolbachia TLR2/6 ligand, peptidoglycan associated lipoprotein, induced NETosis in human neutrophils in vitro. TLR6 dependency of Wolbachia and WoLP NETosis was demonstrated using purified neutrophils from TLR6 deficient mice. Thus, we demonstrate for the first time that NETosis occurs during natural human helminth infection and demonstrate a mechanism of NETosis induction via Wolbachia endobacteria and direct ligation of Wolbachia lipoprotein by neutrophil TLR2/6. PMID:27752109

  18. Overview of microalgal extracellular polymeric substances (EPS) and their applications.

    PubMed

    Xiao, Rui; Zheng, Yi

    2016-11-15

    Microalgae have been studied as natural resources for a number of applications, most particularly food, animal feed, biofuels, pharmaceuticals, and nutraceuticals. In addition to the intracellular compounds of interest, microalgae can also excrete various extracellular polymeric substances (EPS) into their immediate living environment during their life cycle to form a hydrated biofilm matrix. These microalgal EPS mainly consist of polysaccharides, proteins, nucleic acids and lipids. Most notably, EPS retain their stable matrix structure and form a 3-D polymer network for cells to interact with each other, and mediate their adhesion to surfaces. EPS also play a role as extracellular energy and carbon sinks. They are also abundant source of structurally and compositionally diverse biopolymers which possess unique bioactivities for special high-value applications, specifically as antivirals, antitumor agents, antioxidants, anticoagulants and anti-inflammatories. Their superior rheological properties also make microalgal EPS particularly useful in mechanical engineering (e.g., biolubricants and drag reducers) and food science/engineering (e.g., thickener and preservatives) applications. The chemical composition and structure of EPS appear to correlate with their applications, but the fundamentals of such relationship are not well understood. This article summarizes previous research on microalgal EPS derived from green algae, diatoms and red algae, including compositions/functions/structure, production, and potential applications. The importance of exopolysaccharides and EPS proteins, with their particular metabolic characteristics, are also described because of their potential high-value applications. This review concludes with potential future research areas of microalgal EPS.

  19. Acute uptake inhibition increases extracellular serotonin in the rat forebrain.

    PubMed

    Rutter, J J; Auerbach, S B

    1993-06-01

    The effect of acute uptake inhibition on serotonin (5-HT) in the rat central nervous system was monitored by using in vivo dialysis. Peripheral administration of the selective 5-HT uptake blocker, fluoxetine, caused a dose-dependent increase in extracellular 5-HT in both the diencephalon and the striatum. Administration of fluoxetine or sertraline, another selective 5-HT uptake inhibitor, caused a prolonged (24 hr) increase in 5-HT and decrease in 5-hydroxyindoleacetic acid. In addition, fluoxetine and sertraline attenuated the 5-HT releasing effect of fenfluramine administered 24 hr later. Local infusion of fluoxetine into the diencephalon caused an increase in 5-HT that was twice as large as the effect of peripheral injection. Peripheral fluoxetine, by enhancing extracellular 5-HT in the raphe, probably resulted in activation of somatodendritic autoreceptors and inhibition of 5-HT neuronal discharge. Thus, the increase in 5-HT in the diencephalon after peripheral fluoxetine presumably reflected a balance between decreased release and inhibition of reuptake. In support of this, after first infusing fluoxetine into the diencephalon to maximally block reuptake, peripheral injection of the uptake inhibitor caused a decrease in 5-HT.

  20. Tendon Extracellular Matrix Alterations in Ullrich Congenital Muscular Dystrophy.

    PubMed

    Sardone, Francesca; Traina, Francesco; Bondi, Alice; Merlini, Luciano; Santi, Spartaco; Maraldi, Nadir Mario; Faldini, Cesare; Sabatelli, Patrizia

    2016-01-01

    Collagen VI (COLVI) is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in COLVI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by COLVI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient's tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of COLVI and altered distribution of collagen I (COLI) and fibronectin (FBN). In patient's tenocyte culture, COLVI web formation and cell surface association were severely impaired; large aggregates of COLVI, which matched with COLI labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient's tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that COLVI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of COLVI related myopathies.

  1. Modeling biofilms with dual extracellular electron transfer mechanisms

    PubMed Central

    Renslow, Ryan; Babauta, Jerome; Kuprat, Andrew; Schenk, Jim; Ivory, Cornelius; Fredrickson, Jim; Beyenal, Haluk

    2013-01-01

    Electrochemically active biofilms have a unique form of respiration in which they utilize solid external materials as terminal electron acceptors for their metabolism. Currently, two primary mechanisms have been identified for long-range extracellular electron transfer (EET): a diffusion- and a conduction-based mechanism. Evidence in the literature suggests that some biofilms, particularly Shewanella oneidensis, produce the requisite components for both mechanisms. In this study, a generic model is presented that incorporates the diffusion- and the conduction-based mechanisms and allows electrochemically active biofilms to utilize both simultaneously. The model was applied to S. oneidensis and Geobacter sulfurreducens biofilms using experimentally generated data found in the literature. Our simulation results show that 1) biofilms having both mechanisms available, especially if they can interact, may have a metabolic advantage over biofilms that can use only a single mechanism; 2) the thickness of G. sulfurreducens biofilms is likely not limited by conductivity; 3) accurate intrabiofilm diffusion coefficient values are critical for current generation predictions; and 4) the local biofilm potential and redox potential are two distinct parameters and cannot be assumed to have identical values. Finally, we determined that simulated cyclic and squarewave voltammetry based on our model are currently not capable of determining the specific percentages of extracellular electron transfer mechanisms in a biofilm. The developed model will be a critical tool for designing experiments to explain EET mechanisms. PMID:24113651

  2. An Extracellular Subtilase Switch for Immune Priming in Arabidopsis

    PubMed Central

    Mauch-Mani, Brigitte; Gil, Ma José; Vera, Pablo

    2013-01-01

    In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA)-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity. PMID:23818851

  3. Focus on Extracellular Vesicles: Introducing the Next Small Big Thing.

    PubMed

    Kalra, Hina; Drummen, Gregor P C; Mathivanan, Suresh

    2016-02-06

    Intercellular communication was long thought to be regulated exclusively through direct contact between cells or via release of soluble molecules that transmit the signal by binding to a suitable receptor on the target cell, and/or via uptake into that cell. With the discovery of small secreted vesicular structures that contain complex cargo, both in their lumen and the lipid membrane that surrounds them, a new frontier of signal transduction was discovered. These "extracellular vesicles" (EV) were initially thought to be garbage bags through which the cell ejected its waste. Whilst this is a major function of one type of EV, i.e., apoptotic bodies, many EVs have intricate functions in intercellular communication and compound exchange; although their physiological roles are still ill-defined. Additionally, it is now becoming increasingly clear that EVs mediate disease progression and therefore studying EVs has ignited significant interests among researchers from various fields of life sciences. Consequently, the research effort into the pathogenic roles of EVs is significantly higher even though their protective roles are not well established. The "Focus on extracellular vesicles" series of reviews highlights the current state of the art regarding various topics in EV research, whilst this review serves as an introductory overview of EVs, their biogenesis and molecular composition.

  4. Synthetic extracellular matrices for in situ tissue engineering.

    PubMed

    Pratt, Alison B; Weber, Franz E; Schmoekel, Hugo G; Müller, Ralph; Hubbell, Jeffrey A

    2004-04-05

    Cell interactions with the extracellular matrix play important roles in guiding tissue morphogenesis. The matrix stimulates cells to influence such things as differentiation and the cells actively remodel the matrix via local proteolytic activity. We have designed synthetic hydrogel networks that participate in this interplay: They signal cells via bound adhesion and growth factors, and they also respond to the remodeling influence of cell-associated proteases. Poly(ethylene glycol)-bis-vinylsulfone was crosslinked by a Michael-type addition reaction with a peptide containing three cysteine residues, the peptide sequence being cleavable between each cysteine residue by the cell-associated protease plasmin. Cells were able to invade gel networks that contained adhesion peptides and were crosslinked by plasmin-sensitive peptides, while materials lacking either of these two characteristics resisted cell infiltration. Incorporated bone morphogenetic protein-2 (BMP-2) induced bone healing in a rat model in materials that were both adhesive and plasmin-sensitive, while materials lacking plasmin sensitivity resisted formation of bone within the material. Furthermore, when a heparin bridge was incorporated as a BMP-2 affinity site, mimicking yet another characteristic of the extracellular matrix, statistically improved bone regeneration was observed.

  5. Facile preparation of salivary extracellular vesicles for cancer proteomics

    NASA Astrophysics Data System (ADS)

    Sun, Yan; Xia, Zhijun; Shang, Zhi; Sun, Kaibo; Niu, Xiaomin; Qian, Liqiang; Fan, Liu-Yin; Cao, Cheng-Xi; Xiao, Hua

    2016-04-01

    Extracellular vesicles (EVs) are membrane surrounded structures released by cells, which have been increasingly recognized as mediators of intercellular communication. Recent reports indicate that EVs participate in important biological processes and could serve as potential source for cancer biomarkers. As an attractive EVs source with merit of non-invasiveness, human saliva is a unique medium for clinical diagnostics. Thus, we proposed a facile approach to prepare salivary extracellular vesicles (SEVs). Affinity chromatography column combined with filter system (ACCF) was developed to efficiently remove the high abundant proteins and viscous interferences of saliva. Protein profiling in the SEVs obtained by this strategy was compared with conventional centrifugation method, which demonstrated that about 70% more SEVs proteins could be revealed. To explore its utility for cancer proteomics, we analyzed the proteome of SEVs in lung cancer patients and normal controls. Shotgun proteomic analysis illustrated that 113 and 95 proteins have been identified in cancer group and control group, respectively. Among those 63 proteins that have been consistently discovered only in cancer group, 12 proteins are lung cancer related. Our results demonstrated that SEVs prepared through the developed strategy are valuable samples for proteomics and could serve as a promising liquid biopsy for cancer.

  6. Nanostructured cavity devices for extracellular stimulation of HL-1 cells.

    PubMed

    Czeschik, Anna; Rinklin, Philipp; Derra, Ulrike; Ullmann, Sabrina; Holik, Peter; Steltenkamp, Siegfried; Offenhäusser, Andreas; Wolfrum, Bernhard

    2015-01-01

    Microelectrode arrays (MEAs) are state-of-the-art devices for extracellular recording and stimulation on biological tissue. Furthermore, they are a relevant tool for the development of biomedical applications like retina, cochlear and motor prostheses, cardiac pacemakers and drug screening. Hence, research on functional cell-sensor interfaces, as well as the development of new surface structures and modifications for improved electrode characteristics, is a vivid and well established field. However, combining single-cell resolution with sufficient signal coupling remains challenging due to poor cell-electrode sealing. Furthermore, electrodes with diameters below 20 µm often suffer from a high electrical impedance affecting the noise during voltage recordings. In this study, we report on a nanocavity sensor array for voltage-controlled stimulation and extracellular action potential recordings on cellular networks. Nanocavity devices combine the advantages of low-impedance electrodes with small cell-chip interfaces, preserving a high spatial resolution for recording and stimulation. A reservoir between opening aperture and electrode is provided, allowing the cell to access the structure for a tight cell-sensor sealing. We present the well-controlled fabrication process and the effect of cavity formation and electrode patterning on the sensor's impedance. Further, we demonstrate reliable voltage-controlled stimulation using nanostructured cavity devices by capturing the pacemaker of an HL-1 cell network.

  7. Specific Extracellular Matrix Remodeling Signature of Colon Hepatic Metastases

    PubMed Central

    Vezzio-Vie, Nadia; Bibeau, Frédéric; Ychou, Marc; Martineau, Pierre

    2013-01-01

    To identify genes implicated in metastatic colonization of the liver in colorectal cancer, we collected pairs of primary tumors and hepatic metastases before chemotherapy in 13 patients. We compared mRNA expression in the pairs of patients to identify genes deregulated during metastatic evolution. We then validated the identified genes using data obtained by different groups. The 33-gene signature was able to classify 87% of hepatic metastases, 98% of primary tumors, 97% of normal colon mucosa, and 95% of normal liver tissues in six datasets obtained using five different microarray platforms. The identified genes are specific to colon cancer and hepatic metastases since other metastatic locations and hepatic metastases originating from breast cancer were not classified by the signature. Gene Ontology term analysis showed that 50% of the genes are implicated in extracellular matrix remodeling, and more precisely in cell adhesion, extracellular matrix organization and angiogenesis. Because of the high efficiency of the signature to classify colon hepatic metastases, the identified genes represent promising targets to develop new therapies that will specifically affect hepatic metastasis microenvironment. PMID:24023955

  8. Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

    PubMed Central

    Felz, Simon; Al-Zuhairy, Salah; Aarstad, Olav Andreas; van Loosdrecht, Mark C.M.; Lin, Yue Mei

    2016-01-01

    To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na2CO3) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca2+). From the six extraction methods used, only the Na2CO3 extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca2+. The Ca2+-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS. PMID:27768085

  9. Extracellular nucleotide and nucleoside signaling in vascular and blood disease

    PubMed Central

    Idzko, Marco; Ferrari, Davide; Riegel, Ann-Kathrin

    2014-01-01

    Nucleotides and nucleosides—such as adenosine triphosphate (ATP) and adenosine—are famous for their intracellular roles as building blocks for the genetic code or cellular energy currencies. In contrast, their function in the extracellular space is different. Here, they are primarily known as signaling molecules via activation of purinergic receptors, classified as P1 receptors for adenosine or P2 receptors for ATP. Because extracellular ATP is rapidly converted to adenosine by ectonucleotidase, nucleotide-phosphohydrolysis is important for controlling the balance between P2 and P1 signaling. Gene-targeted mice for P1, P2 receptors, or ectonucleotidase exhibit only very mild phenotypic manifestations at baseline. However, they demonstrate alterations in disease susceptibilities when exposed to a variety of vascular or blood diseases. Examples of phenotypic manifestations include vascular barrier dysfunction, graft-vs-host disease, platelet activation, ischemia, and reperfusion injury or sickle cell disease. Many of these studies highlight that purinergic signaling events can be targeted therapeutically. PMID:25001468

  10. Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy.

    PubMed

    Edginton, Ryan S; Mattana, Sara; Caponi, Silvia; Fioretto, Daniele; Green, Ellen; Winlove, C Peter; Palombo, Francesca

    2016-09-15

    Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

  11. Spatial buffering of potassium ions in brain extracellular space.

    PubMed Central

    Chen, K C; Nicholson, C

    2000-01-01

    It has long been assumed that one important mechanism for the dissipation of local potassium gradients in the brain extracellular space is the so-called spatial buffer, generally associated with glial cells. To date, however, there has been no analytical description of the characteristic patterns of K(+) clearance mediated by such a mechanism. This study reanalyzed a mathematical model of Gardner-Medwin (1983, J. Physiol. (Lond.). 335:393-426) that had previously been solved numerically. Under suitable approximations, the transient solutions for the potassium concentrations and the corresponding membrane potentials of glial cells in a finite, parallel domain were derived. The analytic results were substantiated by numerical simulations of a detailed two-compartment model. This simulation explored the dependence of spatial buffer current and extracellular K(+) on the distribution of inward rectifier K(+) channels in the glial endfoot and nonendfoot membranes, the glial geometric length, and the effect of passive KCl uptake. Regarding the glial cells as an equivalent leaky cable, the analyses indicated that a maximum endfoot current occurs when the glial geometric length is equal to the corresponding electrotonic space constant. Consequently, a long glial process is unsuitable for spatial buffering, unless the axial space constant can match the length of the process. Finally, this study discussed whether the spatial buffer mechanism is able to efficiently transport K(+) over distances of more than several glial space constants. PMID:10827962

  12. Diverse human extracellular RNAs are widely detected in human plasma

    PubMed Central

    Freedman, Jane E.; Gerstein, Mark; Mick, Eric; Rozowsky, Joel; Levy, Daniel; Kitchen, Robert; Das, Saumya; Shah, Ravi; Danielson, Kirsty; Beaulieu, Lea; Navarro, Fabio C. P.; Wang, Yaoyu; Galeev, Timur R.; Holman, Alex; Kwong, Raymond Y.; Murthy, Venkatesh; Tanriverdi, Selim E.; Koupenova, Milka; Mikhalev, Ekaterina; Tanriverdi, Kahraman

    2016-01-01

    There is growing appreciation for the importance of non-protein-coding genes in development and disease. Although much is known about microRNAs, limitations in bioinformatic analyses of RNA sequencing have precluded broad assessment of other forms of small-RNAs in humans. By analysing sequencing data from plasma-derived RNA from 40 individuals, here we identified over a thousand human extracellular RNAs including microRNAs, piwi-interacting RNA (piRNA), and small nucleolar RNAs. Using a targeted quantitative PCR with reverse transcription approach in an additional 2,763 individuals, we characterized almost 500 of the most abundant extracellular transcripts including microRNAs, piRNAs and small nucleolar RNAs. The presence in plasma of many non-microRNA small-RNAs was confirmed in an independent cohort. We present comprehensive data to demonstrate the broad and consistent detection of diverse classes of circulating non-cellular small-RNAs from a large population. PMID:27112789

  13. Focus on Extracellular Vesicles: Introducing the Next Small Big Thing

    PubMed Central

    Kalra, Hina; Drummen, Gregor P. C.; Mathivanan, Suresh

    2016-01-01

    Intercellular communication was long thought to be regulated exclusively through direct contact between cells or via release of soluble molecules that transmit the signal by binding to a suitable receptor on the target cell, and/or via uptake into that cell. With the discovery of small secreted vesicular structures that contain complex cargo, both in their lumen and the lipid membrane that surrounds them, a new frontier of signal transduction was discovered. These “extracellular vesicles” (EV) were initially thought to be garbage bags through which the cell ejected its waste. Whilst this is a major function of one type of EV, i.e., apoptotic bodies, many EVs have intricate functions in intercellular communication and compound exchange; although their physiological roles are still ill-defined. Additionally, it is now becoming increasingly clear that EVs mediate disease progression and therefore studying EVs has ignited significant interests among researchers from various fields of life sciences. Consequently, the research effort into the pathogenic roles of EVs is significantly higher even though their protective roles are not well established. The “Focus on extracellular vesicles” series of reviews highlights the current state of the art regarding various topics in EV research, whilst this review serves as an introductory overview of EVs, their biogenesis and molecular composition. PMID:26861301

  14. Influence of extracellular calcium on allyl alcohol-induced hepatotoxicity.

    PubMed

    Strubelt, O; Younes, M; Pentz, R

    1986-07-01

    The role of calcium in allyl alcohol-induced hepatotoxicity was investigated in the isolated haemoglobin-free perfused rat liver. At a Ca++ concentration of 2.5 mmol/l in the perfusate, allyl alcohol (initial concentration 1.17 mmol/l) produced an enhanced release of GPT and SDH from the liver, an increase in the lactate/pyruvate ratio of the perfusate, a decrease in hepatic oxygen consumption and an increase of both hepatic calcium and malondialdehyde content. In the absence of Ca++ in the perfusate, no hepatic calcium accumulation occurred with allyl alcohol, but all other signs of hepatic damage were as severe as with 2.5 mmol/l Ca++. On the other hand, high extracellular Ca++ (5 mmol/l) alone led to a threefold increase of liver calcium but produced only marginal hepatotoxicity and only slightly enhanced the hepatotoxic effects of allyl alcohol. The concentrations of allyl alcohol in the perfusate were not altered at different Ca++ concentrations. In conclusion, the primary allyl alcohol-induced hepatotoxic injury does not appear to depend upon an influx of extracellular calcium.

  15. Microfluidic partitioning of the extracellular space around single cardiac myocytes.

    PubMed

    Klauke, Norbert; Smith, Godfrey L; Cooper, Jonathan M

    2007-02-01

    This paper describes the partitioning of the extracellular space around an electrically activated single cardiac myocyte, constrained within a microfluidic device. Central to this new method is the production of a hydrophobic gap-structure, which divides the extracellular space into two distinct microfluidic pools. The content of these pools was controlled using a pair of concentric automated pipets (subsequently called "dual superfusion pipet"), each providing the ability to dispense (i.e., the source, inner pipet) and aspirate (the sink, outer pipet) a buffer solution (perfusate) into each of the two pools. For rapid solution switching around the cell, additional dual superfusion pipets were inserted into the microchannel for defined time periods using a piezostepper, enabling us to add a test solution, such as a drug. Three distinct areas of the cell were manipulated, namely, the microfluidic environment, the cellular membrane, and the intracellular space. Planar integrated microelectrodes enabled the electrical stimulation of the cardiomyocyte and the recording of the evoked action potential. The device was mounted on an inverted microscope to allow simultaneous sarcomere length and epifluorescence measurements during evoked electrical activity, including, for example, the response of the stimulated end of the cardiac myocyte in comparison with the untreated cell end.

  16. The role of extracellular matrix in spinal cord development.

    PubMed

    Wiese, Stefan; Faissner, Andreas

    2015-12-01

    The development of the spinal cord represents one of the most complex structure developments of the central nervous system (CNS) as it has to unfold along the longitudinal axis and within segmental cues. There it has to cope with on the one hand connection to the periphery (skeletal muscle, dermomyotome, smooth muscles) and connect it to the higher midbrain and cortical regions of the CNS. Major studies have been performed to analyze the specific subset of transcription factors of the different types of cells within the different segments of the spinal cord. But transcription factor expression is always a result of cellular positioning as the environment defines the intracellular changes during differentiation and in adulthood. The surrounding composed of mainly extracellular matrix does not only provide a "glue" to attach cells to each other but also provides signals with special domains docking to cell surface receptors and presents soluble molecules such as basic fibroblast growth factors (bFGFs) or Wnt-proteins. The availability of these molecules depends on the matrix composition and influences the transcription factor code of each cell. Recent research has also provided strong evidence that depletion of single matrix molecules like Tenascin C (TnC) can lead to developmental changes within the progenitor pools. Therefore beyond the transcription factor code that defines cellular properties we want to focus on the role of the extracellular matrix in the development of the spinal cord.

  17. Extracellular Cysteine in Connexins: Role as Redox Sensors

    PubMed Central

    Retamal, Mauricio A.; García, Isaac E.; Pinto, Bernardo I.; Pupo, Amaury; Báez, David; Stehberg, Jimmy; Del Rio, Rodrigo; González, Carlos

    2016-01-01

    Connexin-based channels comprise hemichannels and gap junction channels. The opening of hemichannels allow for the flux of ions and molecules from the extracellular space into the cell and vice versa. Similarly, the opening of gap junction channels permits the diffusional exchange of ions and molecules between the cytoplasm and contacting cells. The controlled opening of hemichannels has been associated with several physiological cellular processes; thereby unregulated hemichannel activity may induce loss of cellular homeostasis and cell death. Hemichannel activity can be regulated through several mechanisms, such as phosphorylation, divalent cations and changes in membrane potential. Additionally, it was recently postulated that redox molecules could modify hemichannels properties in vitro. However, the molecular mechanism by which redox molecules interact with hemichannels is poorly understood. In this work, we discuss the current knowledge on connexin redox regulation and we propose the hypothesis that extracellular cysteines could be important for sensing changes in redox potential. Future studies on this topic will offer new insight into hemichannel function, thereby expanding the understanding of the contribution of hemichannels to disease progression. PMID:26858649

  18. Aging-associated changes in renal extracellular matrix.

    PubMed Central

    Abrass, C. K.; Adcox, M. J.; Raugi, G. J.

    1995-01-01

    The composition of renal extracellular matrices was examined in 6-, 12-, 18-, and 24-month-old rats by immunofluorescence microscopy. No change in composition of tubular basement membrane was detected. Increased immunostaining for laminin chains B1 and s-laminin and thrombospondin characterized the thickened glomerular basement membrane. Interstitial collagens I and III were not detected in globally sclerotic glomeruli. The major change noted in the aged rat kidney at 24 months was generalized expansion of the interstitium by thrombospondin and fibronectin. In areas of tubular atrophy there was new expression of extra domain A (EDA)+ fibronectin. Collagens I and III were detected focally in the interstitium adjacent to areas of tubular atrophy, but otherwise collagens I, III, and IV and laminin did not contribute to the interstitial fibrosis. Interstitial fibrosis was detectable at 18 months of age and preceded the development of sclerotic glomeruli, tubular atrophy, or accumulations of interstitial collagen. These changes in extracellular matrix composition distinguish the aging kidney from other sclerotic forms of renal disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7887455

  19. Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

    PubMed Central

    Edginton, Ryan S.; Mattana, Sara; Caponi, Silvia; Fioretto, Daniele; Green, Ellen; Winlove, C. Peter; Palombo, Francesca

    2016-01-01

    Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis. PMID:27684584

  20. Urinary extracellular vesicles and the kidney: biomarkers and beyond.

    PubMed

    Salih, Mahdi; Zietse, Robert; Hoorn, Ewout J

    2014-06-01

    Extracellular vesicles have been isolated in various body fluids, including urine. The cargo of urinary extracellular vesicles (uEVs) is composed of proteins and nucleic acids reflecting the physiological and possibly pathophysiological state of cells lining the nephron. Because urine is a noninvasive and readily available biofluid, the discovery of uEVs has opened a new field of biomarker research. Their potential use as diagnostic, prognostic, or therapeutic biomarkers for various kidney diseases, including glomerulonephritis, acute kidney injury, tubular disorders, and polycystic kidney disease, is currently being explored. Some challenges, however, remain. These challenges include the need to standardize isolation methods, normalization between samples, and validation of candidate biomarkers. Also, the development of a high-throughput platform to isolate and analyze uEVs, for example, an enzyme-linked immunosorbent assay, is desirable. Here, we review recent studies on uEVs dealing with kidney physiology and pathophysiology. Furthermore, we discuss new and exciting developments regarding vesicles, including their role in cell-to-cell communication and the possibility of using vesicles as a therapy for kidney disorders.

  1. Mechanical model for a collagen fibril pair in extracellular matrix.

    PubMed

    Chan, Yue; Cox, Grant M; Haverkamp, Richard G; Hill, James M

    2009-04-01

    In this paper, we model the mechanics of a collagen pair in the connective tissue extracellular matrix that exists in abundance throughout animals, including the human body. This connective tissue comprises repeated units of two main structures, namely collagens as well as axial, parallel and regular anionic glycosaminoglycan between collagens. The collagen fibril can be modeled by Hooke's law whereas anionic glycosaminoglycan behaves more like a rubber-band rod and as such can be better modeled by the worm-like chain model. While both computer simulations and continuum mechanics models have been investigated for the behavior of this connective tissue typically, authors either assume a simple form of the molecular potential energy or entirely ignore the microscopic structure of the connective tissue. Here, we apply basic physical methodologies and simple applied mathematical modeling techniques to describe the collagen pair quantitatively. We found that the growth of fibrils was intimately related to the maximum length of the anionic glycosaminoglycan and the relative displacement of two adjacent fibrils, which in return was closely related to the effectiveness of anionic glycosaminoglycan in transmitting forces between fibrils. These reveal the importance of the anionic glycosaminoglycan in maintaining the structural shape of the connective tissue extracellular matrix and eventually the shape modulus of human tissues. We also found that some macroscopic properties, like the maximum molecular energy and the breaking fraction of the collagen, were also related to the microscopic characteristics of the anionic glycosaminoglycan.

  2. Aluminum adjuvants elicit fibrin-dependent extracellular traps in vivo

    PubMed Central

    Munks, Michael W.; McKee, Amy S.; MacLeod, Megan K.; Powell, Roger L.; Degen, Jay L.; Reisdorph, Nichole A.; Kappler, John W.

    2010-01-01

    It has been recognized for nearly 80 years that insoluble aluminum salts are good immunologic adjuvants and that they form long-lived nodules in vivo. Nodule formation has long been presumed to be central for adjuvant activity by providing an antigen depot, but the composition and function of these nodules is poorly understood. We show here that aluminum salt nodules formed within hours of injection and contained the clotting protein fibrinogen. Fibrinogen was critical for nodule formation and required processing to insoluble fibrin by thrombin. DNase treatment partially disrupted the nodules, and the nodules contained histone H3 and citrullinated H3, features consistent with extracellular traps. Although neutrophils were not essential for nodule formation, CD11b+ cells were implicated. Vaccination of fibrinogen-deficient mice resulted in normal CD4 T-cell and antibody responses and enhanced CD8 T-cell responses, indicating that nodules are not required for aluminum's adjuvant effect. Moreover, the ability of aluminum salts to retain antigen in the body, the well-known depot effect, was unaffected by the absence of nodules. We conclude that aluminum adjuvants form fibrin-dependent nodules in vivo, that these nodules have properties of extracellular traps, and the nodules are not required for aluminum salts to act as adjuvants. PMID:20876456

  3. Myeloid Extracellular Vesicles: Messengers from the Demented Brain

    PubMed Central

    Nigro, Annamaria; Colombo, Federico; Casella, Giacomo; Finardi, Annamaria; Verderio, Claudia; Furlan, Roberto

    2016-01-01

    Blood-borne monocyte derived cells play a pivotal, initially unrecognized, role in most central nervous system disorders, including diseases initially classified as purely neurodegenerative (i.e., Alzheimer’s disease, Parkinson’s disease, and ALS). Their trafficking to the brain and spinal cord has been extensively studied in classical neuroinflammatory disorders such as multiple sclerosis. Central nervous system resident myeloid cells, namely microglia and perivascular macrophages, also are in the spotlight of investigations on neurological disorders. Myeloid cells, such as infiltrating macrophages and microglia, have been described as having both protective and destructive features in neurological disorders, thus identification of their functional phenotype during disease evolution would be of paramount importance. Extracellular vesicles, namely exosomes and shed vesicles, are released by virtually any cell type and can be detected and identified in terms of cell origin in biological fluids. They therefore constitute an ideal tool to access information on cells residing in an inaccessible site such as the brain. We will review here available information on extracellular vesicles detection in neurological disorders with special emphasis on neurodegenerative diseases. PMID:26858720

  4. Role of extracellular histones in the cardiomyopathy of sepsis.

    PubMed

    Kalbitz, Miriam; Grailer, Jamison J; Fattahi, Fatemeh; Jajou, Lawrence; Herron, Todd J; Campbell, Katherine F; Zetoune, Firas S; Bosmann, Markus; Sarma, J Vidya; Huber-Lang, Markus; Gebhard, Florian; Loaiza, Randall; Valdivia, Hector H; Jalife, José; Russell, Mark W; Ward, Peter A

    2015-05-01

    The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca(2+)]i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nacht-, LRR-, and PYD-domains-containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca(2+)]i, as well as defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction.

  5. MATHEMATICAL MODELING OF EXTRACELLULAR ELECTRON TRANSFER IN BIOFILMS

    SciTech Connect

    Renslow, Ryan S.; Babauta, Jerome T.; Kuprat, Andrew P.; Schenk, Jim; Ivory, Cornelius; Fredrickson, Jim K.; Beyenal, Haluk

    2015-09-12

    Electrochemically active biofilms have a unique form of respiration in which they utilize solid external materials as terminal electron acceptors for their metabolism. Currently, two primary mechanisms have been identified for long-range extracellular electron transfer (EET): a diffusion- and a conduction-based mechanism. Evidence in the literature suggests that some biofilms, particularly Shewanella oneidensis, produce the requisite components for both mechanisms. In this study, a generic model is presented that incorporates the diffusion- and the conduction-based mechanisms and allows electrochemically active biofilms to utilize both simultaneously. The model was applied to S. oneidensis and Geobacter sulfurreducens biofilms using experimentally generated data found in the literature. Our simulation results show that 1) biofilms having both mechanisms available, especially if they can interact, may have a metabolic advantage over biofilms that can use only a single mechanism; 2) the thickness of G. sulfurreducens biofilms is likely not limited by conductivity; 3) accurate intrabiofilm diffusion coefficient values are critical for current generation predictions; and 4) the local biofilm potential and redox potential are two distinct parameters and cannot be assumed to have identical values. Finally, we determined that simulated cyclic and squarewave voltammetry based on our model are currently not capable of determining the specific percentages of extracellular electron transfer mechanisms in a biofilm. The developed model will be a critical tool for designing experiments to explain EET mechanisms.

  6. Extracellular Vesicles Exploit Viral Entry Routes for Cargo Delivery

    PubMed Central

    van Dongen, Helena M.; Masoumi, Niala

    2016-01-01

    SUMMARY Extracellular vesicles (EVs) have emerged as crucial mediators of intercellular communication, being involved in a wide array of key biological processes. Eukaryotic cells, and also bacteria, actively release heterogeneous subtypes of EVs into the extracellular space, where their contents reflect their (sub)cellular origin and the physiologic state of the parent cell. Within the past 20 years, presumed subtypes of EVs have been given a rather confusing diversity of names, including exosomes, microvesicles, ectosomes, microparticles, virosomes, virus-like particles, and oncosomes, and these names are variously defined by biogenesis, physical characteristics, or function. The latter category, functions, in particular the transmission of biological signals between cells in vivo and how EVs control biological processes, has garnered much interest. EVs have pathophysiological properties in cancer, neurodegenerative disorders, infectious disease, and cardiovascular disease, highlighting possibilities not only for minimally invasive diagnostic applications but also for therapeutic interventions, like macromolecular drug delivery. Yet, in order to pursue therapies involving EVs and delivering their cargo, a better grasp of EV targeting is needed. Here, we review recent progress in understanding the molecular mechanisms underpinning EV uptake by receptor-ligand interactions with recipient cells, highlighting once again the overlap of EVs and viruses. Despite their highly heterogeneous nature, EVs require common viral entry pathways, and an unanticipated specificity for cargo delivery is being revealed. We discuss the challenges ahead in delineating specific roles for EV-associated ligands and cellular receptors. PMID:26935137

  7. Extracellular Electron Transport (EET): Metal Cycling in Extreme Places

    NASA Astrophysics Data System (ADS)

    Nealson, K. H.

    2014-12-01

    Extracellular electron transport, or EET, is the process whereby bacteria either donate electrons to an electron acceptor (usually insoluble), or take up electrons from and electron donor (usually insoluble) that is located outside the cell. Iron cycling is inherently linked to EET, as both reduced iron (electron donors), and oxidized iron (electron acceptors) can be found as insoluble minerals, and require specialized molecular machines to accomplish these extracellular geobiological reactions. Bacteria in the group Shewanella are able to catalyze EET in both directions, and are involved with a number of different iron conversions, but are not good role models for extreme conditions - to our knowledge there are no shewanellae that are tolerant to extremes of temperature or pH, the two usual. This being said, when cells are energy starved via limitation for electron acceptors, they respond by turning on the system(s) for EET. Thus, in this presentation the known mechanism(s) of EET will be discussed, along with recent findings and reports of EET-capable organisms from a variety of extreme environments. From these data, I put forward the hypothesis that there are many microbes (many of them from extreme environments) that will be resistant to cultivation by "standard microbiological methods", yet lend themselves well to cultivation via electrochemical methods.

  8. Facile preparation of salivary extracellular vesicles for cancer proteomics

    PubMed Central

    Sun, Yan; Xia, Zhijun; Shang, Zhi; Sun, Kaibo; Niu, Xiaomin; Qian, Liqiang; Fan, Liu-Yin; Cao, Cheng-Xi; Xiao, Hua

    2016-01-01

    Extracellular vesicles (EVs) are membrane surrounded structures released by cells, which have been increasingly recognized as mediators of intercellular communication. Recent reports indicate that EVs participate in important biological processes and could serve as potential source for cancer biomarkers. As an attractive EVs source with merit of non-invasiveness, human saliva is a unique medium for clinical diagnostics. Thus, we proposed a facile approach to prepare salivary extracellular vesicles (SEVs). Affinity chromatography column combined with filter system (ACCF) was developed to efficiently remove the high abundant proteins and viscous interferences of saliva. Protein profiling in the SEVs obtained by this strategy was compared with conventional centrifugation method, which demonstrated that about 70% more SEVs proteins could be revealed. To explore its utility for cancer proteomics, we analyzed the proteome of SEVs in lung cancer patients and normal controls. Shotgun proteomic analysis illustrated that 113 and 95 proteins have been identified in cancer group and control group, respectively. Among those 63 proteins that have been consistently discovered only in cancer group, 12 proteins are lung cancer related. Our results demonstrated that SEVs prepared through the developed strategy are valuable samples for proteomics and could serve as a promising liquid biopsy for cancer. PMID:27091080

  9. Extracellular cleavage of E-cadherin promotes epithelial cell extrusion.

    PubMed

    Grieve, Adam G; Rabouille, Catherine

    2014-08-01

    Epithelial cell extrusion and subsequent apoptosis is a key mechanism to prevent the accumulation of excess cells. By contrast, when driven by oncogene expression, apical cell extrusion is followed by proliferation and represents an initial step of tumorigenesis. E-cadherin (E-cad), the main component of adherens junctions, has been shown to be essential for epithelial cell extrusion, but its mechanistic contribution remains unclear. Here, we provide clear evidence that cell extrusion can be driven by the cleavage of E-cad, both in a wild-type and an oncogenic environment. We first show that CDC42 activation in a single epithelial cell results in its efficient matrix metalloproteinase (MMP)-sensitive extrusion through MEK signalling activation and this is supported by E-cad cleavage. Second, using an engineered cleavable form of E-cad, we demonstrate that, by itself, truncation of extracellular E-cad at the plasma membrane promotes apical extrusion. We propose that extracellular cleavage of E-cad generates a rapid change in cell-cell adhesion that is sufficient to drive apical cell extrusion. Whereas in normal epithelia, extrusion is followed by apoptosis, when combined with active oncogenic signalling, it is coupled to cell proliferation.

  10. Hypochlorous acid regulates neutrophil extracellular trap release in humans.

    PubMed

    Palmer, L J; Cooper, P R; Ling, M R; Wright, H J; Huissoon, A; Chapple, I L C

    2012-02-01

    Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti-microbial strategy regulated non-exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine.

  11. Microbial extracellular electron transfer and its relevance to iron corrosion.

    PubMed

    Kato, Souichiro

    2016-03-01

    Extracellular electron transfer (EET) is a microbial metabolism that enables efficient electron transfer between microbial cells and extracellular solid materials. Microorganisms harbouring EET abilities have received considerable attention for their various biotechnological applications, including bioleaching and bioelectrochemical systems. On the other hand, recent research revealed that microbial EET potentially induces corrosion of iron structures. It has been well known that corrosion of iron occurring under anoxic conditions is mostly caused by microbial activities, which is termed as microbiologically influenced corrosion (MIC). Among diverse MIC mechanisms, microbial EET activity that enhances corrosion via direct uptake of electrons from metallic iron, specifically termed as electrical MIC (EMIC), has been regarded as one of the major causative factors. The EMIC-inducing microorganisms initially identified were certain sulfate-reducing bacteria and methanogenic archaea isolated from marine environments. Subsequently, abilities to induce EMIC were also demonstrated in diverse anaerobic microorganisms in freshwater environments and oil fields, including acetogenic bacteria and nitrate-reducing bacteria. Abilities of EET and EMIC are now regarded as microbial traits more widespread among diverse microbial clades than was thought previously. In this review, basic understandings of microbial EET and recent progresses in the EMIC research are introduced.

  12. Uptake of extracellular double-stranded RNA by SID-2

    PubMed Central

    McEwan, Deborah L; Weisman, Alexandra S; Hunter, Craig P

    2012-01-01

    Summary Ingested dsRNAs trigger RNA interference (RNAi) in many invertebrates including the nematode Caenorhabditis elegans. Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C. elegans for the import of ingested dsRNA and, when expressed in Drosophila S2 cells, SID-2 enables the uptake of dsRNAs. SID-2-dependent dsRNA transport requires an acidic extracellular environment and is selective for dsRNAs with at least 50 base pairs. Through structure-function analysis, we identify several SID-2 regions required for this activity including three extracellular, positively-charged, histidines. Finally, we find that SID-2-dependent transport is inhibited by drugs that interfere with vesicle transport. Therefore, we propose that environmental dsRNAs are imported from the acidic intestinal lumen by SID-2 via endocytosis and are released from internalized vesicles in a secondary step mediated by the dsRNA-channel SID-1. Similar multistep mechanisms may underlie the widespread observations of environmental RNAi. PMID:22902558

  13. Extracellular enzyme activity in a willow sewage treatment system.

    PubMed

    Brzezinska, Maria Swiontek; Lalke-Porczyk, Elżbieta; Kalwasińska, Agnieszka

    2012-12-01

    This paper presents the results of studies on the activity of extra-cellular enzymes in soil-willow vegetation filter soil which is used in the post-treatment of household sewage in an onsite wastewater treatment system located in central Poland. Wastewater is discharged from the detached house by gravity into the onsite wastewater treatment system. It flows through a connecting pipe into a single-chamber septic tank and is directed by the connecting pipe to a control well to be further channelled in the soil-willow filter by means of a subsurface leaching system. Soil samples for the studies were collected from two depths of 5 cm and 1 m from three plots: close to the wastewater inflow, at mid-length of the plot and close to its terminal part. Soil samples were collected from May to October 2009. The activity of the extra-cellular enzymes was assayed by the fluorometric method using 4-methylumbelliferyl and 7-amido-4-methylcoumarin substrate. The ranking of potential activity of the assayed enzymes was the same at 5 cm and 1 m soil depths, i.e. esterase > phosphmomoesterase > leucine-aminopeptidase > β-glucosidase > α-glucosidase. The highest values of enzymatic activity were recorded in the surface layer of the soil at the wastewater inflow and decreased with increasing distance from that point.

  14. Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

    SciTech Connect

    O'Day, Danton H.; Huber, Robert J.; Suarez, Andres

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Extracellular calmodulin is present throughout growth and development in Dictyostelium. Black-Right-Pointing-Pointer Extracellular calmodulin localizes within the ECM during development. Black-Right-Pointing-Pointer Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. Black-Right-Pointing-Pointer Extracellular calmodulin exists in eukaryotic microbes. Black-Right-Pointing-Pointer Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca{sup 2+}/CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.

  15. Circulating extracellular vesicles in cancer diagnosis and monitoring: an appraisal of clinical potential.

    PubMed

    Choi, Dong-Sic; Lee, Jaewook; Go, Gyeongyun; Kim, Yoon-Keun; Gho, Yong Song

    2013-10-01

    Mammalian cells, including cancer cells, secrete extracellular vesicles. These vesicles are nanosized, bilayered proteolipids with diameters of 50-1,000 nm. It has been suggested that cancer cell-derived extracellular vesicles play diverse roles in cancer progression, which involve invasion, immune modulation, neovascularization, and metastasis. Moreover, their serum levels are significantly elevated in cancer patients compared with normal controls. Recent high-throughput proteomic and transcriptomic studies of these complex extracellular organelles have accelerated the discovery of cancer-specific biomarkers and the development of novel diagnostic tools based on extracellular vesicles. Although many vesicle-associated biomarker candidates have been reported for various types of cancer, few have been validated for clinical use due to preanalytical, technical, temporal, and financial problems. Here, we discuss the potential of extracellular vesicles as sources of biomarkers for cancer diagnosis and monitoring, as well as the limitations and obstacles to adoption of extracellular vesicle-based diagnosis.

  16. Sensitivity of temporal excitation properties to the neuronal element activated by extracellular stimulation.

    PubMed

    Miocinovic, Svjetlana; Grill, Warren M

    2004-01-15

    Measurements of the chronaxies and refractory periods with extracellular stimuli have been used to conclude that large diameter axons are responsible for the effects of deep brain stimulation (DBS). We hypothesized that because action potential initiation by extracellular stimulation occurs in the axons of central nervous system (CNS) neurons, the chronaxies and refractory periods determined using extracellular stimulation would be similar for cells and axons. Computer simulation was used to determine the sensitivity of chronaxie and refractory period to the neural element stimulated. The results demonstrate that chronaxies and refractory periods were dependent on the polarity of the extracellular stimulus and the electrode-to-neuron distance, and indicate that there is little systematic difference in either chronaxies or refractory periods between local cells or axons of passage with extracellular stimulation. This finding points out the difficulty in drawing conclusions regarding which neuronal elements are activated based on extracellular measurements of temporal excitation properties.

  17. Extracellular calcium and cholinergic stimulation of isolated canine parietal cells.

    PubMed Central

    Soll, A H

    1981-01-01

    The role of calcium gating in cholinergic stimulation of the function of parietal cells was studied using cells isolated from canine fundic mucosa by treatment with collagenase and EDTA and enriched by velocity separation in an elutriator rotor. Monitoring the accumulation of [14C[ aminopyrine as an index of parietal cell response, stimulation by carbachol, but not by histamine, was highly dependent upon the concentration of extracellular calcium. Incubation of parietal cells in 0-.1 mM calcium, rather than the usual 1.8 mM concentration, reduced the response to 100 microM carbachol by 92 +/- 2%, whereas histamine stimulation was impaired by 28 +/- 5%. A similar reduction in extracellular calcium suppressed the response to gastrin (100 nM) by 67 +/- 7%. The impairment of cholinergic stimulation found at low extracellular calcium concentrations was rapidly reversed with the readdition of calcium. Lanthanum, which blocks calcium movement across membranes, caused a similar pattern of effects on secretagogue stimulation of aminopyrine accumulation, with 100 microM lanthanum suppressing carbachol stimulation by 83 +/- 2%. This concentration of lanthanum suppressed gastrin stimulation by 40 +/- 7% and histamine stimulation by only 12 +/- 9%. Carbachol, but not histamine nor gastrin, stimulated 45Ca++ uptake. The magnitude of carbachol-stimulated calcium uptake correlated with the parietal cell content of the fractions examined (r = 0.88), and was dose responsive over carbachol concentrations from 1 microM to 1 mM. Atropine (100 nM) caused surmountable inhibition, and these effects of carbachol and atropine on calcium uptake correlated with their effects on oxygen consumption (r = 0.93) and [14C]-aminopyrine accumulation (r = 0.90). Cells preloaded with 45Ca++ lost cellular calcium in a time-dependent fashion; however, this rate of egress was not accelerated by treatment with histamine, gastrin, or carbachol, thus failing to implicate mobilization of intracellular calcium

  18. Extracellular pH modulates GABAergic neurotransmission in rat hypothalamus.

    PubMed

    Chen, Z L; Huang, R Q

    2014-06-20

    Changes in extracellular pH have a modulatory effect on GABAA receptor function. It has been reported that pH sensitivity of the GABA receptor is dependent on subunit composition and GABA concentration. Most of previous investigations focused on GABA-evoked currents, which only reflect the postsynaptic receptors. The physiological relevance of pH modulation of GABAergic neurotransmission is not fully elucidated. In the present studies, we examined the influence of extracellular pH on the GABAA receptor-mediated inhibitory neurotransmission in rat hypothalamic neurons. The inhibitory postsynaptic currents (IPSCs), tonic currents, and the GABA-evoked currents were recorded with whole-cell patch techniques on the hypothalamic slices from Sprague-Dawley rats at 15-26 postnatal days. The amplitude and frequency of spontaneous GABA IPSCs were significantly increased while the external pH was changed from 7.3 to 8.4. In the acidic pH (6.4), the spontaneous GABA IPSCs were reduced in amplitude and frequency. The pH induced changes in miniature GABA IPSCs (mIPSCs) similar to that in spontaneous IPSCs. The pH effect on the postsynaptic GABA receptors was assessed with exogenously applied varying concentrations of GABA. The tonic currents and the currents evoked by sub-saturating concentration of GABA ([GABA]) (10 μM) were inhibited by acidic pH and potentiated by alkaline pH. In contrast, the currents evoked by saturating [GABA] (1mM) were not affected by pH changes. We also investigated the influence of pH buffers and buffering capacity on pH sensitivity of GABAA receptors on human recombinant α1β2γ2 GABAA receptors stably expressed in HEK 293 cells. The pH influence on GABAA receptors was similar in HEPES- and MES-buffered media, and not dependent on protonated buffers, suggesting that the observed pH effect on GABA response is a specific consequence of changes in extracellular protons. Our data suggest that the hydrogen ions suppress the GABAergic neurotransmission

  19. The tetrapartite synapse: extracellular matrix remodeling contributes to corticoaccumbens plasticity underlying drug addiction

    PubMed Central

    Smith, Alexander C.W.; Scofield, Michael D.; Kalivas, Peter W.

    2015-01-01

    Synaptic plasticity has long been known to involve three key elements of neuropil, the presynapse, the postsynapse and adjacent glia. Here we review the role of the extracellular matrix in synaptic plasticity as a necessary component forming the tetrapartite synapse. We describe the role of matrix metalloproteinases as enzymes sculpting extracellular proteins and thereby creating an extracellular signaling domain required for synaptic plasticity. Specifically we focus on the role of the tetrapartite synapse in mediating the effects of addictive drugs at corticostriatal synapses, and conclude that the extracellular signaling domain and its regulation by matrix metalloproteinases is critical for developing and expressing drug seeking behaviors. PMID:25838241

  20. Redox properties of extracellular polymeric substances (EPS) from electroactive bacteria

    NASA Astrophysics Data System (ADS)

    Li, Shan-Wei; Sheng, Guo-Ping; Cheng, Yuan-Yuan; Yu, Han-Qing

    2016-12-01

    Although the capacity for electroactive bacteria to convert environmental metallic minerals and organic pollutants is well known, the role of the redox properties of microbial extracellular polymeric substances (EPS) in this process is poorly understood. In this work, the redox properties of EPS from two widely present electroactive bacterial strains (Shewanella oneidensis and Pseudomonas putida) were explored. Electrochemical analysis demonstrates that the EPS extracted from the two strains exhibited redox properties. Spectroelectrochemical and protein electrophoresis analyses indicate that the extracted EPS from S. oneidensis and P. putida contained heme-binding proteins, which were identified as the possible redox components in the EPS. The results of heme-mediated behavior of EPS may provide an insight into the important roles of EPS in electroactive bacteria to maximize their redox capability for biogeochemical cycling, environmental bioremediation and wastewater treatment.

  1. Extracellular Matrix and Integrins in Embryonic Stem Cell Differentiation

    PubMed Central

    Wang, Han; Luo, Xie; Leighton, Jake

    2015-01-01

    Embryonic stem cells (ESCs) are pluripotent cells with great therapeutic potentials. The in vitro differentiation of ESC was designed by recapitulating embryogenesis. Significant progress has been made to improve the in vitro differentiation protocols by toning soluble maintenance factors. However, more robust methods for lineage-specific differentiation and maturation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) cues should be considered besides growth factor cues. ECM proteins bind to cells and act as ligands of integrin receptors on cell surfaces. Here, we summarize the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM–integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuroectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. In the future, ECM combinations for the optimal ESC differentiation environment will require substantial study. PMID:26462244

  2. Extracellular matrix bioscaffolds in tissue remodeling and morphogenesis

    PubMed Central

    Swinehart, Ilea T.; Badylak, Stephen F.

    2016-01-01

    During normal morphogenesis the extracellular matrix (ECM) influences cell motility, proliferation, apoptosis, and differentiation. Tissue engineers have attempted to harness the cell signaling potential of ECM to promote the functional reconstruction, if not regeneration, of injured or missing adult tissues that otherwise heal by the formation of scar tissue. ECM bioscaffolds, derived from decellularized tissues, have been used to promote the formation of site appropriate, functional tissues in many clinical applications including skeletal muscle, fibrocartilage, lower urinary tract, and esophageal reconstruction, among others. These scaffolds function by the release or exposure of growth factors and cryptic peptides, modulation of the immune response, and recruitment of progenitor cells. Herein, we describe this process of ECM induced constructive remodeling and examine similarities to normal tissue morphogenesis. PMID:26699796

  3. Extracellular miRNA: A Collision of Two Paradigms.

    PubMed

    Turchinovich, Andrey; Tonevitsky, Alexander G; Burwinkel, Barbara

    2016-10-01

    Since their discovery in 2008, extracellular miRNAs (ex-miRNAs) have persisted as one of the major themes of molecular and cellular biology. The main reason for this remarkable interest is the increasing number of research papers reporting that cell-free circulating miRNA mediates both short-range and distant communication between various cells, and could impact on diverse physiological and pathological processes. However, there are also multiple conflicting lines of evidence that challenge the biological significance of circulating ex-miRNA, suggesting that they are merely byproducts of cell activity and cell death without any particular function. This review aims to summarize these contrasting opinions and to foster further experimental validation of both paradigms.

  4. Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis.

    PubMed

    Zou, Chun; Duan, Xuguo; Wu, Jing

    2016-04-01

    In this study, the pullulanase gene from Bacillus deramificans was efficiently expressed in Brevibacillus choshinensis. The optimal medium for protein expression was determined through a combination of single-factor experiments and response surface methodology. The initial pH of the medium and the culture temperature were optimized. The pullulanase yield increased 10.8-fold through medium and condition optimization at the shake-flask level. From the results of these experiments, the dissolved oxygen level was optimized in a 3-L fermentor. Under these optimized conditions, the pullulanase activity and the specific pullulanase productivity reached 1005.8 U/mL and 110.5 × 10(3) U/g dry cell weight, respectively, with negligible intracellular expression. The Brevibacillus choshinensis expression system has proven to be valuable for the extracellular production of pullulanase.

  5. Extracellular matrix production in vitro in cartilage tissue engineering.

    PubMed

    Chen, Jie-Lin; Duan, Li; Zhu, Weimin; Xiong, Jianyi; Wang, Daping

    2014-04-05

    Cartilage tissue engineering is arising as a technique for the repair of cartilage lesions in clinical applications. However, fibrocartilage formation weakened the mechanical functions of the articular, which compromises the clinical outcomes. Due to the low proliferation ability, dedifferentiation property and low production of cartilage-specific extracellular matrix (ECM) of the chondrocytes, the cartilage synthesis in vitro has been one of the major limitations for obtaining high-quality engineered cartilage constructs. This review discusses cells, biomaterial scaffolds and stimulating factors that can facilitate the cartilage-specific ECM production and accumulation in the in vitro culture system. Special emphasis has been put on the factors that affect the production of ECM macromolecules such as collagen type II and proteoglycans in the review, aiming at providing new strategies to improve the quality of tissue-engineered cartilage.

  6. Redox properties of extracellular polymeric substances (EPS) from electroactive bacteria

    PubMed Central

    Li, Shan-Wei; Sheng, Guo-Ping; Cheng, Yuan-Yuan; Yu, Han-Qing

    2016-01-01

    Although the capacity for electroactive bacteria to convert environmental metallic minerals and organic pollutants is well known, the role of the redox properties of microbial extracellular polymeric substances (EPS) in this process is poorly understood. In this work, the redox properties of EPS from two widely present electroactive bacterial strains (Shewanella oneidensis and Pseudomonas putida) were explored. Electrochemical analysis demonstrates that the EPS extracted from the two strains exhibited redox properties. Spectroelectrochemical and protein electrophoresis analyses indicate that the extracted EPS from S. oneidensis and P. putida contained heme-binding proteins, which were identified as the possible redox components in the EPS. The results of heme-mediated behavior of EPS may provide an insight into the important roles of EPS in electroactive bacteria to maximize their redox capability for biogeochemical cycling, environmental bioremediation and wastewater treatment. PMID:27991531

  7. Neutrophil extracellular traps in dermatology: Caught in the NET.

    PubMed

    Hoffmann, Jochen H O; Enk, Alexander H

    2016-10-01

    Neutrophil, or polymorphonuclear granulocytes (PMN) constitute the most abundant type of leucocytes in peripheral human blood. One of the major advances in the last decade was the discovery of neutrophil extracellular trap (NET) formation: a process by which neutrophils externalize web-like chromatin strands decorated with antimicrobial peptides. These structures were soon implicated in immune defense and auto-immunity alike and now link neutrophils to the pathogenesis of a variety of diseases of dermatological relevance. Currently, NET formation is mainly subdivided into suicidal and vital NETosis. Controversy exists regarding the capacity of NETs to kill pathogens, and little is known about the way NETs are formed in vivo. Here, we discuss the current terminology, methods for NET quantification, pathways leading to NET formation, and the role of NETs in systemic and cutaneous immune defense and auto-immunity, with a focus on psoriasis and systemic lupus erythematosus.

  8. The extracellular matrix: A dynamic niche in cancer progression

    PubMed Central

    Lu, Pengfei; Weaver, Valerie M.

    2012-01-01

    The local microenvironment, or niche, of a cancer cell plays important roles in cancer development. A major component of the niche is the extracellular matrix (ECM), a complex network of macromolecules with distinctive physical, biochemical, and biomechanical properties. Although tightly controlled during embryonic development and organ homeostasis, the ECM is commonly deregulated and becomes disorganized in diseases such as cancer. Abnormal ECM affects cancer progression by directly promoting cellular transformation and metastasis. Importantly, however, ECM anomalies also deregulate behavior of stromal cells, facilitate tumor-associated angiogenesis and inflammation, and thus lead to generation of a tumorigenic microenvironment. Understanding how ECM composition and topography are maintained and how their deregulation influences cancer progression may help develop new therapeutic interventions by targeting the tumor niche. PMID:22351925

  9. Extracellular Adenosine Mediates a Systemic Metabolic Switch during Immune Response

    PubMed Central

    Bajgar, Adam; Kucerova, Katerina; Jonatova, Lucie; Tomcala, Ales; Schneedorferova, Ivana; Okrouhlik, Jan; Dolezal, Tomas

    2015-01-01

    Immune defense is energetically costly, and thus an effective response requires metabolic adaptation of the organism to reallocate energy from storage, growth, and development towards the immune system. We employ the natural infection of Drosophila with a parasitoid wasp to study energy regulation during immune response. To combat the invasion, the host must produce specialized immune cells (lamellocytes) that destroy the parasitoid egg. We show that a significant portion of nutrients are allocated to differentiating lamellocytes when they would otherwise be used for development. This systemic metabolic switch is mediated by extracellular adenosine released from immune cells. The switch is crucial for an effective immune response. Preventing adenosine transport from immune cells or blocking adenosine receptor precludes the metabolic switch and the deceleration of development, dramatically reducing host resistance. Adenosine thus serves as a signal that the “selfish” immune cells send during infection to secure more energy at the expense of other tissues. PMID:25915062

  10. Extracellular vesicles and viruses: Are they close relatives?

    PubMed Central

    Nolte-‘t Hoen, Esther; Cremer, Tom; Gallo, Robert C.; Margolis, Leonid B.

    2016-01-01

    Extracellular vesicles (EVs) released by various cells are small phospholipid membrane-enclosed entities that can carry miRNA. They are now central to research in many fields of biology because they seem to constitute a new system of cell–cell communication. Physical and chemical characteristics of many EVs, as well as their biogenesis pathways, resemble those of retroviruses. Moreover, EVs generated by virus-infected cells can incorporate viral proteins and fragments of viral RNA, being thus indistinguishable from defective (noninfectious) retroviruses. EVs, depending on the proteins and genetic material incorporated in them, play a significant role in viral infection, both facilitating and suppressing it. Deciphering the mechanisms of EV-cell interactions may facilitate the design of EVs that inhibit viral infection and can be used as vehicles for targeted drug delivery. PMID:27432966

  11. Extracellular vesicles shuffling intercellular messages: for good or for bad.

    PubMed

    Lo Cicero, Alessandra; Stahl, Philip D; Raposo, Graça

    2015-08-01

    The release of extracellular vesicles (EVs) is a highly conserved process exploited by diverse organisms as a mode of intercellular communication. Vesicles of sizes ranging from 30 to 1000nm, or even larger, are generated by blebbing of the plasma membrane (microvesicles) or formed in multivesicular endosomes (MVEs) to be secreted by exocytosis as exosomes. Exosomes, microvesicles and other EVs contain membrane and cytosolic components that include proteins, lipids and RNAs, a composition that differs related to their site of biogenesis. Several mechanisms are involved in vesicle formation at the plasma membrane or in endosomes, which is reflected in their heterogeneity, size and composition. EVs have significant promise for therapeutics and diagnostics and for understanding physiological and pathological processes all of which have boosted research to find modulators of their composition, secretion and targeting.

  12. [Specificity of extracellular alkaline RNAase from Penicillium chrysogenum 152A].

    PubMed

    Bezborodova, S I; Markelova, N Y; Gulayeva, V I

    1975-01-01

    Specificity of chromatographically homogenous extracellular alkaline RNAase from Pen. crysogenum 152A on RNA, synthetic polynucleotides, dinucleosidemonophosphates and nucleoside-2',3'-cyclophosphates is studied. The enzyme is found to release from RNA guanosine-3'-monophosphate and guanosine-2',3'-cyclophosphate only. Guanylic acid is a 3'-terminal nucleotide of oligonucleotides of different length. The enzyme readily hydrolyses poly-I and practically do not splits poly-G. GpN is demonstrated to be a good substrate for the RNase, while G greater than p hydrolyses with a low rate. The RNAase catalyses the synthesis of GpC (47.7 per cent yield) and GpU (38.8 per cent yield). Thus, the RNAase from Pen. chrysogenum 152A is considered to be guanyl-RNAase.

  13. Association Mechanisms of Sand with Anionic Extracellular Polysaccharides (EPS)

    NASA Astrophysics Data System (ADS)

    Albalasmeh, Ammar; Berhe, Asmeret; Ghezzehei, Teamrat

    2013-04-01

    The configuration, molecular weight and the type and charge of functional groups often have a strong effect on adhesion of Extracellular Polysaccharides (EPS) to solid surfaces. Our hypothesis is that the EPS with higher molecular weight will be more associated with sand as compared to those with a low molecular weight. In this study, we investigated the association of anionic EPS by sand of six size fractions. The association experiment was carried out at different time, initial concentrations, and sorbent concentrations. EPS concentrations were determined by Sulfuric Acid-UV method. Preliminary results indicate that increasing the concentration will lead to the formation of longer molecules. Therefore, more association with sand will be occurred until critical length is reached where increasing the length of the molecules will adversely affect the association.

  14. Extracellular vesicles of the blood-brain barrier

    PubMed Central

    András, Ibolya E; Toborek, Michal

    2016-01-01

    Extracellular vesicles (ECV), like exosomes, gained recently a lot of attention as potentially playing a significant role in neurodegenerative diseases, particularly in Aβ pathology. While there are a lot of reports on ECV/exosomes derived from a variety of cell types, there is limited information on ECV/exosomes originated from brain microvascular endothelial cells forming the blood-brain barrier (BBB). In this review, we summarize the literature data on brain endothelial ECV/exosomes and present our own data on BBB-derived ECV and their possible involvement in the brain's Aβ pathology. We propose that ECV/exosome release from brain endothelial cells associated with Aβ affects different cells of the neurovascular unit and may be an important contributor to the Aβ deposition in the central nervous system. PMID:27141419

  15. Extracellular vesicles: small bricks for tissue repair/regeneration

    PubMed Central

    Taverna, Simona; Pucci, Marzia

    2017-01-01

    Extracellular vesicles (EVs) are nano-sized membrane vesicles involved in intercellular communication. EVs have pleiotropic actions in physiological and pathological conditions. The ability of EVs to transports proteins, drugs and nucleic acid, to target specific cells and to increase the stability of therapeutic cargo, make EVs interesting as new devices for the treatment of human disease. In a recently published issue of European journal of pharmaceutical sciences, Silva and colleagues reviewed the ability of EVs to modulate tissue repair and regeneration, focusing on their roles and therapeutic potential as immunomodulatory messengers. In this perspective, we discussed the open questions regarding the dual role of EVs in immune system, as well as the technical limitation of the procedure for EVs isolation and administration in clinical practices. EV-based therapies require further studies to consider EVs as promising candidate for a novel cell-free therapy in the context of regeneration medicine. PMID:28275628

  16. Extracellular matrix structure governs invasion resistance in bacterial biofilms.

    PubMed

    Nadell, Carey D; Drescher, Knut; Wingreen, Ned S; Bassler, Bonnie L

    2015-08-01

    Many bacteria are highly adapted for life in communities, or biofilms. A defining feature of biofilms is the production of extracellular matrix that binds cells together. The biofilm matrix provides numerous fitness benefits, including protection from environmental stresses and enhanced nutrient availability. Here we investigate defense against biofilm invasion using the model bacterium Vibrio cholerae. We demonstrate that immotile cells, including those identical to the biofilm resident strain, are completely excluded from entry into resident biofilms. Motile cells can colonize and grow on the biofilm exterior, but are readily removed by shear forces. Protection from invasion into the biofilm interior is mediated by the secreted protein RbmA, which binds mother-daughter cell pairs to each other and to polysaccharide components of the matrix. RbmA, and the invasion protection it confers, strongly localize to the cell lineages that produce it.

  17. Extracellular matrix hydrogels from decellularized tissues: Structure and function.

    PubMed

    Saldin, Lindsey T; Cramer, Madeline C; Velankar, Sachin S; White, Lisa J; Badylak, Stephen F

    2017-02-01

    Extracellular matrix (ECM) bioscaffolds prepared from decellularized tissues have been used to facilitate constructive and functional tissue remodeling in a variety of clinical applications. The discovery that these ECM materials could be solubilized and subsequently manipulated to form hydrogels expanded their potential in vitro and in vivo utility; i.e. as culture substrates comparable to collagen or Matrigel, and as injectable materials that fill irregularly-shaped defects. The mechanisms by which ECM hydrogels direct cell behavior and influence remodeling outcomes are only partially understood, but likely include structural and biological signals retained from the native source tissue. The present review describes the utility, formation, and physical and biological characterization of ECM hydrogels. Two examples of clinical application are presented to demonstrate in vivo utility of ECM hydrogels in different organ systems. Finally, new research directions and clinical translation of ECM hydrogels are discussed.

  18. Using extracellular action potential recordings to constrain compartmental models.

    PubMed

    Gold, Carl; Henze, Darrell A; Koch, Christof

    2007-08-01

    We investigate the use of extracellular action potential (EAP) recordings for biophysically faithful compartmental models. We ask whether constraining a model to fit the EAP is superior to matching the intracellular action potential (IAP). In agreement with previous studies, we find that the IAP method under-constrains the parameters. As a result, significantly different sets of parameters can have virtually identical IAP's. In contrast, the EAP method results in a much tighter constraint. We find that the distinguishing characteristics of the waveform--but not its amplitude-resulting from the distribution of active conductances are fairly invariant to changes of electrode position and detailed cellular morphology. Based on these results, we conclude that EAP recordings are an excellent source of data for the purpose of constraining compartmental models.

  19. The design of reversible hydrogels to capture extracellular matrix dynamics

    NASA Astrophysics Data System (ADS)

    Rosales, Adrianne M.; Anseth, Kristi S.

    2016-02-01

    The extracellular matrix (ECM) is a dynamic environment that constantly provides physical and chemical cues to embedded cells. Much progress has been made in engineering hydrogels that can mimic the ECM, but hydrogel properties are, in general, static. To recapitulate the dynamic nature of the ECM, many reversible chemistries have been incorporated into hydrogels to regulate cell spreading, biochemical ligand presentation and matrix mechanics. For example, emerging trends include the use of molecular photoswitches or biomolecule hybridization to control polymer chain conformation, thereby enabling the modulation of the hydrogel between two states on demand. In addition, many non-covalent, dynamic chemical bonds have found increasing use as hydrogel crosslinkers or tethers for cell signalling molecules. These reversible chemistries will provide greater temporal control of adhered cell behaviour, and they allow for more advanced in vitro models and tissue-engineering scaffolds to direct cell fate.

  20. Nanostructured cavity devices for extracellular stimulation of HL-1 cells

    NASA Astrophysics Data System (ADS)

    Czeschik, Anna; Rinklin, Philipp; Derra, Ulrike; Ullmann, Sabrina; Holik, Peter; Steltenkamp, Siegfried; Offenhäusser, Andreas; Wolfrum, Bernhard

    2015-05-01

    Microelectrode arrays (MEAs) are state-of-the-art devices for extracellular recording and stimulation on biological tissue. Furthermore, they are a relevant tool for the development of biomedical applications like retina, cochlear and motor prostheses, cardiac pacemakers and drug screening. Hence, research on functional cell-sensor interfaces, as well as the development of new surface structures and modifications for improved electrode characteristics, is a vivid and well established field. However, combining single-cell resolution with sufficient signal coupling remains challenging due to poor cell-electrode sealing. Furthermore, electrodes with diameters below 20 µm often suffer from a high electrical impedance affecting the noise during voltage recordings. In this study, we report on a nanocavity sensor array for voltage-controlled stimulation and extracellular action potential recordings on cellular networks. Nanocavity devices combine the advantages of low-impedance electrodes with small cell-chip interfaces, preserving a high spatial resolution for recording and stimulation. A reservoir between opening aperture and electrode is provided, allowing the cell to access the structure for a tight cell-sensor sealing. We present the well-controlled fabrication process and the effect of cavity formation and electrode patterning on the sensor's impedance. Further, we demonstrate reliable voltage-controlled stimulation using nanostructured cavity devices by capturing the pacemaker of an HL-1 cell network.Microelectrode arrays (MEAs) are state-of-the-art devices for extracellular recording and stimulation on biological tissue. Furthermore, they are a relevant tool for the development of biomedical applications like retina, cochlear and motor prostheses, cardiac pacemakers and drug screening. Hence, research on functional cell-sensor interfaces, as well as the development of new surface structures and modifications for improved electrode characteristics, is a vivid and

  1. Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13

    SciTech Connect

    Sakurai, Atsuo; Okahashi, Nobuo; Maruyama, Fumito; Ooshima, Takashi; Hamada, Shigeyuki; Nakagawa, Ichiro

    2008-08-29

    Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assay revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis.

  2. Insight On Colorectal Carcinoma Infiltration by Studying Perilesional Extracellular Matrix.

    PubMed

    Nebuloni, Manuela; Albarello, Luca; Andolfo, Annapaola; Magagnotti, Cinzia; Genovese, Luca; Locatelli, Irene; Tonon, Giovanni; Longhi, Erika; Zerbi, Pietro; Allevi, Raffaele; Podestà, Alessandro; Puricelli, Luca; Milani, Paolo; Soldarini, Armando; Salonia, Andrea; Alfano, Massimo

    2016-03-04

    The extracellular matrix (ECM) from perilesional and colorectal carcinoma (CRC), but not healthy colon, sustains proliferation and invasion of tumor cells. We investigated the biochemical and physical diversity of ECM in pair-wised comparisons of healthy, perilesional and CRC specimens. Progressive linearization and degree of organization of fibrils was observed from healthy to perilesional and CRC ECM, and was associated with a steady increase of stiffness and collagen crosslinking. In the perilesional ECM these modifications coincided with increased vascularization, whereas in the neoplastic ECM they were associated with altered modulation of matrisome proteins, increased content of hydroxylated lysine and lysyl oxidase. This study identifies the increased stiffness and crosslinking of the perilesional ECM predisposing an environment suitable for CRC invasion as a phenomenon associated with vascularization. The increased stiffness of colon areas may represent a new predictive marker of desmoplastic region predisposing to invasion, thus offering new potential application for monitoring adenoma with invasive potential.

  3. Insight On Colorectal Carcinoma Infiltration by Studying Perilesional Extracellular Matrix

    PubMed Central

    Nebuloni, Manuela; Albarello, Luca; Andolfo, Annapaola; Magagnotti, Cinzia; Genovese, Luca; Locatelli, Irene; Tonon, Giovanni; Longhi, Erika; Zerbi, Pietro; Allevi, Raffaele; Podestà, Alessandro; Puricelli, Luca; Milani, Paolo; Soldarini, Armando; Salonia, Andrea; Alfano, Massimo

    2016-01-01

    The extracellular matrix (ECM) from perilesional and colorectal carcinoma (CRC), but not healthy colon, sustains proliferation and invasion of tumor cells. We investigated the biochemical and physical diversity of ECM in pair-wised comparisons of healthy, perilesional and CRC specimens. Progressive linearization and degree of organization of fibrils was observed from healthy to perilesional and CRC ECM, and was associated with a steady increase of stiffness and collagen crosslinking. In the perilesional ECM these modifications coincided with increased vascularization, whereas in the neoplastic ECM they were associated with altered modulation of matrisome proteins, increased content of hydroxylated lysine and lysyl oxidase. This study identifies the increased stiffness and crosslinking of the perilesional ECM predisposing an environment suitable for CRC invasion as a phenomenon associated with vascularization. The increased stiffness of colon areas may represent a new predictive marker of desmoplastic region predisposing to invasion, thus offering new potential application for monitoring adenoma with invasive potential. PMID:26940881

  4. Extracellular Superoxide Dismutase: Growth Promoter or Tumor Suppressor?

    PubMed Central

    Laukkanen, Mikko O.

    2016-01-01

    Extracellular superoxide dismutase (SOD3) gene transfer to tissue damage results in increased healing, increased cell proliferation, decreased apoptosis, and decreased inflammatory cell infiltration. At molecular level, in vivo SOD3 overexpression reduces superoxide anion (O2−) concentration and increases mitogen kinase activation suggesting that SOD3 could have life-supporting characteristics. The hypothesis is further strengthened by the observations showing significantly increased mortality in conditional knockout mice. However, in cancer SOD3 has been shown to either increase or decrease cell proliferation and survival depending on the model system used, indicating that SOD3-derived growth mechanisms are not completely understood. In this paper, the author reviews the main discoveries in SOD3-dependent growth regulation and signal transduction. PMID:27293512

  5. Preparation of Cardiac Extracellular Matrix from an Intact Porcine Heart

    PubMed Central

    Wainwright, John M.; Czajka, Caitlin A.; Patel, Urvi B.; Freytes, Donald O.; Tobita, Kimimasa; Gilbert, Thomas W.

    2010-01-01

    Whole organ engineering would benefit from a three-dimensional scaffold produced from intact organ-specific extracellular matrix (ECM). The microenvironment and architecture provided by such a scaffold would likely support site-appropriate cell differentiation and spatial organization. The methods to produce such scaffolds from intact organs require customized decellularization protocols. In the present study, intact adult porcine hearts were successfully decellularized in less than 10 h using pulsatile retrograde aortic perfusion. Serial perfusion of an enzymatic, nonionic detergent, ionic detergent, and acid solution with hypotonic and hypertonic rinses was used to systematically remove cellular content. The resultant cardiac ECM retained collagen, elastin, and glycosaminoglycans, and mechanical integrity. Cardiac ECM supported the formation of organized chicken cardiomyocyte sarcomere structure in vitro. The intact decellularized porcine heart provides a tissue engineering template that may be beneficial for future preclinical studies and eventual clinical applications. PMID:19702513

  6. Preparation of cardiac extracellular matrix from an intact porcine heart.

    PubMed

    Wainwright, John M; Czajka, Caitlin A; Patel, Urvi B; Freytes, Donald O; Tobita, Kimimasa; Gilbert, Thomas W; Badylak, Stephen F

    2010-06-01

    Whole organ engineering would benefit from a three-dimensional scaffold produced from intact organ-specific extracellular matrix (ECM). The microenvironment and architecture provided by such a scaffold would likely support site-appropriate cell differentiation and spatial organization. The methods to produce such scaffolds from intact organs require customized decellularization protocols. In the present study, intact adult porcine hearts were successfully decellularized in less than 10 h using pulsatile retrograde aortic perfusion. Serial perfusion of an enzymatic, nonionic detergent, ionic detergent, and acid solution with hypotonic and hypertonic rinses was used to systematically remove cellular content. The resultant cardiac ECM retained collagen, elastin, and glycosaminoglycans, and mechanical integrity. Cardiac ECM supported the formation of organized chicken cardiomyocyte sarcomere structure in vitro. The intact decellularized porcine heart provides a tissue engineering template that may be beneficial for future preclinical studies and eventual clinical applications.

  7. Extracellular matrix and its receptors in Drosophila neural development

    PubMed Central

    Broadie, Kendal; Baumgartner, Stefan; Prokop, Andreas

    2011-01-01

    Extracellular matrix (ECM) and matrix receptors are intimately involved in most biological processes. The ECM plays fundamental developmental and physiological roles in health and disease, including processes underlying the development, maintenance and regeneration of the nervous system. To understand the principles of ECM-mediated functions in the nervous system, genetic model organisms like Drosophila provide simple, malleable and powerful experimental platforms. This article provides an overview of ECM proteins and receptors in Drosophila. It then focuses on their roles during three progressive phases of neural development: 1) neural progenitor proliferation, 2) axonal growth and pathfinding and 3) synapse formation and function. Each section highlights known ECM and ECM-receptor components and recent studies done in mutant conditions to reveal their in vivo functions, all illustrating the enormous opportunities provided when merging work on the nervous system with systematic research into ECM-related gene functions. PMID:21688401

  8. Decellularization of Rat Kidneys to Produce Extracellular Matrix Scaffolds.

    PubMed

    Jin, Mei; Yaling, Yu; Zhibin, Wang; Jianse, Zhang

    2016-01-01

    The extracellular matrix (ECM) retains three-dimensional structures for the stimulation of cell growth, with components of the ECM relatively conserved between species. Interest in the use of decellularized scaffold-based strategies for organ regeneration is increasing rapidly. Decellularized scaffolds derived from animal organs are a promising material for organ engineering, with a number of prominent advances having been reported in the past few years.In this article we describe a simple and robust methodology for generating decellularized rat kidneys. To obtain these scaffolds, we perfuse rat kidneys with detergents through the abdominal aorta. After decellularization, kidney scaffolds are harvested for evaluation of vascular structure and histology. Qualitative evaluation involves vascular corrosion casting, transmission electron microscopy, and several different histological and immunofluorescent methods. SDS residue levels are assessed by ultraviolet-visible spectrophotometer (UV-VIS).

  9. Extracellular matrix structure governs invasion resistance in bacterial biofilms

    PubMed Central

    Nadell, Carey D; Drescher, Knut; Wingreen, Ned S; Bassler, Bonnie L

    2015-01-01

    Many bacteria are highly adapted for life in communities, or biofilms. A defining feature of biofilms is the production of extracellular matrix that binds cells together. The biofilm matrix provides numerous fitness benefits, including protection from environmental stresses and enhanced nutrient availability. Here we investigate defense against biofilm invasion using the model bacterium Vibrio cholerae. We demonstrate that immotile cells, including those identical to the biofilm resident strain, are completely excluded from entry into resident biofilms. Motile cells can colonize and grow on the biofilm exterior, but are readily removed by shear forces. Protection from invasion into the biofilm interior is mediated by the secreted protein RbmA, which binds mother–daughter cell pairs to each other and to polysaccharide components of the matrix. RbmA, and the invasion protection it confers, strongly localize to the cell lineages that produce it. PMID:25603396

  10. Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium semitectum

    SciTech Connect

    Basavaraja, S.; Balaji, S.D.; Lagashetty, Arunkumar; Rajasab, A.H.; Venkataraman, A.

    2008-05-06

    Development of environmental friendly procedures for the synthesis of metal nanoparticles through biological processes is evolving into an important branch of nanobiotechnology. In this paper, we report on the use of fungus 'Fusarium semitectum' for the extracellular synthesis of silver nanoparticles from silver nitrate solution (i.e. through the reduction of Ag{sup +} to Ag{sup 0}). Highly stable and crystalline silver nanoparticles are produced in solution by treating the filtrate of the fungus F. semitectum with the aqueous silver nitrate solution. The formations of nanoparticles are understood from the UV-vis and X-ray diffraction studies. Transmission electron microscopy of the silver particles indicated that they ranged in size from 10 to 60 nm and are mostly spherical in shape. Interestingly the colloidal suspensions of silver nanoparticles are stable for many weeks. Possible medicinal applications of these silver nanoparticles are envisaged.

  11. Hypoxia and the extracellular matrix: drivers of tumour metastasis

    PubMed Central

    Gilkes, Daniele M.; Semenza, Gregg L.; Wirtz, Denis

    2014-01-01

    Of the deaths attributed to cancer, 90% are due to metastasis, and treatments that prevent or cure metastasis remain elusive. Emerging data indicate that hypoxia and the extracellular matrix (ECM) might have crucial roles in metastasis. During tumour evolution, changes in the composition and the overall content of the ECM reflect both its biophysical and biological properties and these strongly influence tumour and stromal cell properties, such as proliferation and motility. Originally thought of as independent contributors to metastatic spread, recent studies have established a direct link between hypoxia and the composition and the organization of the ECM, which suggests a new model in which multiple microenvironmental signals might converge to synergistically influence metastatic outcome. PMID:24827502

  12. Extracellular Matrix and Fibroblast Communication Following Myocardial Infarction

    PubMed Central

    Ma, Yonggang; Halade, Ganesh V.; Lindsey, Merry L.

    2012-01-01

    The extracellular matrix (ECM) provides structural support by serving as a scaffold for cells, and as such the ECM maintains normal tissue homeostasis and mediates the repair response following injury. In response to myocardial infarction (MI), ECM expression is generally upregulated in the left ventricle (LV), which regulates LV remodeling by modulating scar formation. The ECM directly affects scar formation by regulating growth factor release and cell adhesion, and indirectly affects scar formation by regulating the inflammatory, angiogenic, and fibroblast responses. This review summarizes the current literature on ECM expression patterns and fibroblast mechanisms in the myocardium, focusing on the ECM response to MI. In addition, we discuss future research areas that are needed to better understand the molecular mechanisms of ECM action, both in general and as a means to optimize infarct healing. PMID:22926488

  13. Therapeutic applications of extracellular vesicles: clinical promise and open questions.

    PubMed

    György, Bence; Hung, Michelle E; Breakefield, Xandra O; Leonard, Joshua N

    2015-01-01

    This review provides an updated perspective on rapidly proliferating efforts to harness extracellular vesicles (EVs) for therapeutic applications. We summarize current knowledge, emerging strategies, and open questions pertaining to clinical potential and translation. Potentially useful EVs comprise diverse products of various cell types and species. EV components may also be combined with liposomes and nanoparticles to facilitate manufacturing as well as product safety and evaluation. Potential therapeutic cargoes include RNA, proteins, and drugs. Strategic issues considered herein include choice of therapeutic agent, means of loading cargoes into EVs, promotion of EV stability, tissue targeting, and functional delivery of cargo to recipient cells. Some applications may harness natural EV properties, such as immune modulation, regeneration promotion, and pathogen suppression. These properties can be enhanced or customized to enable a wide range of therapeutic applications, including vaccination, improvement of pregnancy outcome, and treatment of autoimmune disease, cancer, and tissue injury.

  14. Emerging Roles of Extracellular Vesicles in the Nervous System

    PubMed Central

    Bali, Jitin; Barr, Maureen M.; Court, Felipe A.; Krämer-Albers, Eva-Maria; Picou, Frederic; Raposo, Graça; van der Vos, Kristan E.; van Niel, Guillaume; Wang, Juan

    2014-01-01

    Information exchange executed by extracellular vesicles, including exosomes, is a newly described form of intercellular communication important in the development and physiology of neural systems. These vesicles can be released from cells, are packed with information including signaling proteins and both coding and regulatory RNAs, and can be taken up by target cells, thereby facilitating the transfer of multilevel information. Recent studies demonstrate their critical role in physiological processes, including nerve regeneration, synaptic function, and behavior. These vesicles also have a sinister role in the propagation of toxic amyloid proteins in neurodegenerative conditions, including prion diseases and Alzheimer's and Parkinson's diseases, in inducing neuroinflammation by exchange of information between the neurons and glia, as well as in aiding tumor progression in the brain by subversion of normal cells. This article provides a summary of topics covered in a symposium and is not meant to be a comprehensive review of the subject. PMID:25392515

  15. Secretion and extracellular space travel of Wnt proteins.

    PubMed

    Gross, Julia Christina; Boutros, Michael

    2013-08-01

    Wnt signaling pathways control many processes during development, stem cell maintenance and homeostasis, and their aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer. Wnts are hydrophobic proteins, however, quite paradoxically, they can travel over distances to induce cell-type specific responses. While there has been an initial focus on elucidating the intracellular signaling cascade, discoveries in the past few years have shed light on a highly complex, and regulated secretory process that guides Wnt proteins through the exocytic pathway. Wnt proteins are at least in portion packaged onto extracellular carriers such as exosomes. Similar to dysregulation of components in the Wnt receiving cell, failure to regulate Wnt secretion has been linked to cancer. Here, we review recent discoveries on factors and processes implicated in Wnt secretion.

  16. Microbial extracellular polymeric substances (EPS) in fresh water sediments.

    PubMed

    Gerbersdorf, Sabine Ulrike; Westrich, Bernhard; Paterson, David M

    2009-08-01

    Microbially produced extracellular polymeric substances (EPS) have been linked with many important ecological functions in natural sediments; yet, most information has been derived from marine systems. The present paper is the first comprehensive study on EPS (i.e., carbohydrates and proteins) dynamics in riverine sediments addressing spatial (six reservoirs and four groyne fields across three European rivers), temporal (all seasons in 2003-2005), and vertical (over a 50-cm sediment depth transect) pattern. The variation in hydrodynamic regime found in the reservoirs and groyne fields was reflected in the biomass and composition of the benthic microorganisms that produce EPS. The microphytobenthic communities consisted mainly of diatoms and a higher algal biomass (up to 248 microg g(-1) dry weight, DW) seemed to be indicative for higher amounts of secreted colloidal carbohydrates. Consequently, the model proposed by Underwood and Smith (1998) for the relation chlorophyll-colloidal carbohydrates was also applicable for upper riverine sediment layers. The close relation between algal biomass and bacterial cell counts (10(8)-10(9) cells g(-1) DW) supports the idea of bacterial use of the secreted EPS. However, the data also suggest a contribution to the EPS pool through bacterial secretion of proteins/extracellular enzymes and possibly carbohydrates. Over depth, the relationships between microorganisms and EPS became increasingly decoupled along with increasing ratios of bound (refractory) to colloidal (labile) EPS. These data suggest fresh production of polymeric substances in upper sediment layers and mainly accumulation of refractory, biodegraded material in deeper layers. The high contents of EPS colloidal and bound carbohydrates (0.1-1.8 and 1.3-6.7 mg g(-1) DW, respectively) and EPS proteins (0.4-12.9 mg g(-1) DW) at the freshwater study sites might indicate an important role in sediment ecology.

  17. High yield production of extracellular recombinant levansucrase by Bacillus megaterium.

    PubMed

    Korneli, Claudia; Biedendieck, Rebekka; David, Florian; Jahn, Dieter; Wittmann, Christoph

    2013-04-01

    In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH 6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg L(-1)) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U mL(-1) on fructose and 17.2 U mL(-1) on glycerol). This was further increased in high cell density fed-batch processes up to 55 U mL(-1), reflecting a levansucrase concentration of 0.52 g L(-1). This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.

  18. Cells involved in extracellular matrix remodeling after acute myocardial infarction

    PubMed Central

    Garcia, Larissa Ferraz; Mataveli, Fábio D’Aguiar; Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell; Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva

    2015-01-01

    Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. PMID:25993074

  19. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

    PubMed

    Watson, Steve P

    2009-01-01

    The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of

  20. Extracellular entrapment and degradation of single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Farrera, Consol; Bhattacharya, Kunal; Lazzaretto, Beatrice; Andón, Fernando T.; Hultenby, Kjell; Kotchey, Gregg P.; Star, Alexander; Fadeel, Bengt

    2014-05-01

    Neutrophils extrude neutrophil extracellular traps (NETs) consisting of a network of chromatin decorated with antimicrobial proteins to enable non-phagocytic killing of microorganisms. Here, utilizing a model of ex vivo activated human neutrophils, we present evidence of entrapment and degradation of carboxylated single-walled carbon nanotubes (SWCNTs) in NETs. The degradation of SWCNTs was catalyzed by myeloperoxidase (MPO) present in purified NETs and the reaction was facilitated by the addition of H2O2 and NaBr. These results show that SWCNTs can undergo acellular, MPO-mediated biodegradation and imply that the immune system may deploy similar strategies to rid the body of offending microorganisms and engineered nanomaterials.Neutrophils extrude neutrophil extracellular traps (NETs) consisting of a network of chromatin decorated with antimicrobial proteins to enable non-phagocytic killing of microorganisms. Here, utilizing a model of ex vivo activated human neutrophils, we present evidence of entrapment and degradation of carboxylated single-walled carbon nanotubes (SWCNTs) in NETs. The degradation of SWCNTs was catalyzed by myeloperoxidase (MPO) present in purified NETs and the reaction was facilitated by the addition of H2O2 and NaBr. These results show that SWCNTs can undergo acellular, MPO-mediated biodegradation and imply that the immune system may deploy similar strategies to rid the body of offending microorganisms and engineered nanomaterials. Electronic supplementary information (ESI) available: Suppl. Fig. 1 - length distribution of SWCNTs; suppl. Fig. 2 - characterization of pristine vs. oxidized SWCNTs; suppl. Fig. 3 - endotoxin evaluation; suppl. Fig. 4 - NET characterization; suppl. Fig. 5 - UV-Vis/NIR analysis of biodegradation of oxidized SWCNTs; suppl. Fig. 6 - cytotoxicity of partially degraded SWCNTs. See DOI: 10.1039/c3nr06047k

  1. NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

    PubMed Central

    Röhm, Marc; Grimm, Melissa J.; D'Auria, Anthony C.; Almyroudis, Nikolaos G.

    2014-01-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47phox−/−) mice which had resolved in wild-type mice by day 5 but progressed in p47phox−/− mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47phox−/− mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  2. Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development

    PubMed Central

    Stanic, Karen; Saldivia, Natalia; Förstera, Benjamín; Torrejón, Marcela; Montecinos, Hernán; Caprile, Teresa

    2016-01-01

    Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons. PMID:27733818

  3. The Extracellular Matrix in Photosynthetic Mats: A Cyanobacterial Gingerbread House

    NASA Astrophysics Data System (ADS)

    Stuart, R.; Stannard, W.; Bebout, B.; Pett-Ridge, J.; Mayali, X.; Weber, P. K.; Lipton, M. S.; Lee, J.; Everroad, R. C.; Thelen, M.

    2014-12-01

    Hypersaline laminated cyanobacterial mats are excellent model systems for investigating photoautotrophic contributions to biogeochemical cycling on a millimeter scale. These self-sustaining ecosystems are characterized by steep physiochemical gradients that fluctuate dramatically on hour timescales, providing a dynamic environment to study microbial response. However, elucidating the distribution of energy from light absorption into biomass requires a complete understanding of the various constituents of the mat. Extracellular polymeric substances (EPS), which can be composed of proteins, polysaccharides, lipids and DNA are a major component of these mats and may function in the redistribution of nutrients and metabolites within the community. To test this notion, we established a model mat-building culture for comparison with the phylogenetically diverse natural mat communities. In these two systems we determined how proteins and glycans in the matrix changed as a function of light and tracked nutrient flow from the matrix. Using mass spectrometry metaproteomics analysis, we found homologous proteins in both field and culture extracellular matrix that point to cyanobacterial turnover of amino acids, inorganic nutrients, carbohydrates and nucleic acids from the EPS. Other abundant functions identified included oxidative stress response from both the cyanobacteria and heterotrophs and cyanobacterial structural proteins that may play a role in mat cohesion. Several degradative enzymes also varied in abundance in the EPS in response to light availability, suggesting active secretion. To further test cyanobacterial EPS turnover, we generated isotopically-labeled EPS and used NanoSIMS to trace uptake of this labeled EPS. Our findings suggest Cyanobacteria may facilitate nutrient transfer to other groups, as well as uptake of their own products through degradation of EPS components. This work provides evidence for the essential roles of EPS for storage, structural

  4. Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

    PubMed Central

    Francisco, Romeu; Verissimo, Paula; Santos, Susana S.; Fonseca, Luís; Abrantes, Isabel M. O.; Morais, Paula V.

    2013-01-01

    Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion. PMID:24244546

  5. Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis.

    PubMed

    Hielscher, Abigail; Ellis, Kim; Qiu, Connie; Porterfield, Josh; Gerecht, Sharon

    2016-01-01

    The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5β1 and αvβ3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis.

  6. Extracellular N-Acetylaspartate in Human Traumatic Brain Injury.

    PubMed

    Shannon, Richard J; van der Heide, Susan; Carter, Eleanor L; Jalloh, Ibrahim; Menon, David K; Hutchinson, Peter J; Carpenter, Keri L H

    2016-02-15

    N-acetylaspartate (NAA) is an amino acid derivative primarily located in the neurons of the adult brain. The function of NAA is incompletely understood. Decrease in brain tissue NAA is presently considered symptomatic and a potential biomarker of acute and chronic neuropathological conditions. The aim of this study was to use microdialysis to investigate the behavior of extracellular NAA (eNAA) levels after traumatic brain injury (TBI). Sampling for this study was performed using cerebral microdialysis catheters (M Dialysis 71) perfused at 0.3 μL/min. Extracellular NAA was measured in microdialysates by high-performance liquid chromatography in 30 patients with severe TBI and for comparison, in radiographically "normal" areas of brain in six non-TBI neurosurgical patients. We established a detailed temporal eNAA profile in eight of the severe TBI patients. Microdialysate concentrations of glucose, lactate, pyruvate, glutamate, and glycerol were measured on an ISCUS clinical microdialysis analyzer. Here, we show that the temporal profile of microdialysate eNAA was characterized by highest levels in the earliest time-points post-injury, followed by a steady decline; beyond 70 h post-injury, average levels were 40% lower than those measured in non-TBI patients. There was a significant inverse correlation between concentrations of eNAA and pyruvate; eNAA showed significant positive correlations with glycerol and the lactate/pyruvate (L/P) ratio measured in microdialysates. The results of this on-going study suggest that changes in eNAA after TBI relate to the release of intracellular components, possibly due to neuronal death or injury, as well as to adverse brain energy metabolism.

  7. Role of Extracellular Matrix-Mediated Interactions in Thymocyte Migration

    PubMed Central

    Dalmau, Sérgio Ranto; Dealmeida, Vinícius Cotta

    2000-01-01

    Cell adhesion, migration, differentiation and survival or death is amongst a large spectrum of biological responses that can be elicited by ligation of extracellular matrix components to their corresponding receptors. As regards the physiology of the thymus, cell migration is a crucial event in the general process of T cell differentiation. Studies on the intrathymic distribution of ECM components revealed that fibronectin, laminin and type IV collagen, are not restrictedly located at typical basement membrane sites, also forming a thick network in the medullary region of the thymic lobules, whereas very thin ECM fibers are found within the cortex. These ECM components are essentially produced by thymic microenvironmental cells, which also drive thymocyte differentiation. Signals triggered by ECM are conveyed into thymocytes or microenvironmental cells through specific membrane receptors, and most of them belong to the integrin type, such as the VLA-3, VLA-4, VLA-5 and VLA-6. In vitro studies revealed that adhesion of thymocytes to thymic microenvironmental cells is mediated by extracellular matrix. Such an adhesion is preferentially done by immature thymocytes. Importantly, ECM-mediated interactions also govern the entrance and exit of thymocytes in the lymphoepithelial complexes named thymic nurse cells. Lastly, pathological conditions, including infectious and autoimmune diseases, in which changes of ECM ligands and receptors are observed, course with alterations in thymocyte migration and death. In conclusion, the fact that ECM can modulate traffic, differentiation, death and survival of normal thymocytes adds clues for understanding how ECM-mediated interactions behave in the thymus, not only in normal, but also in pathological conditions. PMID:11097218

  8. Comparative Study of Extracellular Vesicles from the Urine of Healthy Individuals and Prostate Cancer Patients

    PubMed Central

    Bryzgunova, Olga E.; Zaripov, Marat M.; Skvortsova, Tatyana E.; Lekchnov, Evgeny A.; Grigor’eva, Alina E.; Morozkin, Evgeny S.; Ryabchikova, Elena I.; Yurchenko, Yuri B.; Voitsitskiy, Vladimir E.; Laktionov, Pavel P.

    2016-01-01

    Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90–95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles. PMID:27305142

  9. Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation.

    PubMed

    Kunadt, Marcel; Eckermann, Katrin; Stuendl, Anne; Gong, Jing; Russo, Belisa; Strauss, Katrin; Rai, Surya; Kügler, Sebastian; Falomir Lockhart, Lisandro; Schwalbe, Martin; Krumova, Petranka; Oliveira, Luis M A; Bähr, Mathias; Möbius, Wiebke; Levin, Johannes; Giese, Armin; Kruse, Niels; Mollenhauer, Brit; Geiss-Friedlander, Ruth; Ludolph, Albert C; Freischmidt, Axel; Feiler, Marisa S; Danzer, Karin M; Zweckstetter, Markus; Jovin, Thomas M; Simons, Mikael; Weishaupt, Jochen H; Schneider, Anja

    2015-05-01

    Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson's Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson's disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.

  10. Increased dependency of cardiac pacemaker activity on extracellular Ca after adrenergic blockade in the frog heart.

    PubMed

    Fukuda, Y

    1986-01-01

    The frog sinus venosus shows spontaneous regular pacemaker activity, even in the absence of extracellular Ca2+. When an alpha-adrenergic blocking agent (phentolamine) is applied, the rate of pacemaker activity, height of action potential, rate of slow diastolic depolarization, and the maximum diastolic potential become strongly dependent upon the extracellular Ca2+ concentration.

  11. Unfolded protein response-induced ERdj3 secretion links ER stress to extracellular proteostasis

    PubMed Central

    Genereux, Joseph C; Qu, Song; Zhou, Minghai; Ryno, Lisa M; Wang, Shiyu; Shoulders, Matthew D; Kaufman, Randal J; Lasmézas, Corinne I; Kelly, Jeffery W; Wiseman, R Luke

    2015-01-01

    The Unfolded Protein Response (UPR) indirectly regulates extracellular proteostasis through transcriptional remodeling of endoplasmic reticulum (ER) proteostasis pathways. This remodeling attenuates secretion of misfolded, aggregation-prone proteins during ER stress. Through these activities, the UPR has a critical role in preventing the extracellular protein aggregation associated with numerous human diseases. Here, we demonstrate that UPR activation also directly influences extracellular proteostasis through the upregulation and secretion of the ER HSP40 ERdj3/DNAJB11. Secreted ERdj3 binds misfolded proteins in the extracellular space, substoichiometrically inhibits protein aggregation, and attenuates proteotoxicity of disease-associated toxic prion protein. Moreover, ERdj3 can co-secrete with destabilized, aggregation-prone proteins in a stable complex under conditions where ER chaperoning capacity is overwhelmed, preemptively providing extracellular chaperoning of proteotoxic misfolded proteins that evade ER quality control. This regulated co-secretion of ERdj3 with misfolded clients directly links ER and extracellular proteostasis during conditions of ER stress. ERdj3 is, to our knowledge, the first metazoan chaperone whose secretion into the extracellular space is regulated by the UPR, revealing a new mechanism by which UPR activation regulates extracellular proteostasis. PMID:25361606

  12. Adaptation of cat motoneurons to sustained and intermittent extracellular activation.

    PubMed Central

    Spielmann, J M; Laouris, Y; Nordstrom, M A; Robinson, G A; Reinking, R M; Stuart, D G

    1993-01-01

    1. The main purpose of this study was to quantify the adaptation of spinal motoneurons to sustained and intermittent activation, using an extracellular route of stimulating current application to single test cells, in contrast to an intracellular route, as has been used previously. In addition, associations were tested between firing rate properties of the tested cells and other type (size)-related properties of these cells and their motor units. 2. Motoneurons supplying the medial gastrocnemius muscle of the deeply anaesthetized cat were stimulated for 240 s with microelectrodes which passed sustained extracellular current at 1.25 times the threshold for repetitive firing. Many cells were also tested following a rest period with intermittent 1 s current pulses (duration 600 ms) at the same relative stimulus strength. Cell discharge was assessed from the EMG of the motor unit innervated by the test neuron. The motoneurons and their motor units were assigned to four categories (i.e. types FF, FR, S and F; where F = FF + FR) based on conventional criteria. In all, twenty F (16 FF, 4 FR) and fourteen S cells were studied with sustained stimulation. Thirty of these cells (17 F, 13 S) and an additional two cells (1 F, 1 S) were studied with intermittent stimulation. 3. The mean threshold current required for sustained firing for a period of > or = 2 s was not significantly different for F and S cells. However, most of the other measured parameters of motoneuron firing differed significantly for these two cell groups. For example, at 1.25 times the threshold current for repetitive firing, the mean firing duration in response to 240 s of sustained activation was 123 +/- 88 s (+/- S.D.) for F cells vs. 233 +/- 19 s for S cells. These values were significantly longer than those from a comparable, previously reported study that employed intracellular stimulation. With intermittent stimulation, the firing durations of F and S cells were not significantly different from each

  13. The 2nd United Kingdom Extracellular Vesicle Forum Meeting Abstracts

    PubMed Central

    Clayton, Aled; Lawson, Charlotte; Gardiner, Chris; Harrison, Paul; Carter, David

    2016-01-01

    The UK Extracellular Vesicles (UKEV) Forum meetings were born of the realization that there were a number of UK laboratories studying extracellular vesicle biology and using similar techniques but without a regular national meeting dedicated to EVs at which to share their findings. This was compounded by the fact that many of these labs were working in different fields and thus networking and sharing of ideas and best practice was sometimes difficult. The first workshop was organized in 2013 by Dr Charlotte Lawson, under the auspices of the Society for Endocrinology, led to the founding of the UKEV Forum and the organization of a British Heart Foundation sponsored 1-day conference held in London in December 2014. Although growing in size every year, the central aims of these workshops have remained the same: to provide a forum for discussion and exchange of ideas, to allow young scientists to present their data in the form of short talks and poster presentations and to discuss their work with more established scientists in the field. Here we include the presented abstracts for the 2015 1-day conference hosted by Cardiff University. This meeting was attended by approximately 130 delegates throughout the United Kingdom, but also attended by delegates from Belgium, Netherlands, France, Ireland and other nations. The day composed of plenary presentations from Prof Matthias Belting, Lund University, Sweden and Dr Guillaume van Niel, Institut Curie, Paris together with 10 short presentations from submitted abstracts. The topics covered were broad, with sessions on Mechanisms of EV production, EVs in Infection, EVs in Cancer and in Blood and Characterizing EVs in Biological fluids. This hopefully gives a reflection of the range of EV-related studies being conducted currently in the UK. There were also 33 poster presentations equally broad in subject matter. The organizers are grateful to the Life Science Research Network Wales – a Welsh government-funding scheme that

  14. High Throughput Sequencing of Extracellular RNA from Human Plasma

    PubMed Central

    Danielson, Kirsty M.; Rubio, Renee; Abderazzaq, Fieda; Das, Saumya; Wang, Yaoyu E.

    2017-01-01

    The presence and relative stability of extracellular RNAs (exRNAs) in biofluids has led to an emerging recognition of their promise as ‘liquid biopsies’ for diseases. Most prior studies on discovery of exRNAs as disease-specific biomarkers have focused on microRNAs (miRNAs) using technologies such as qRT-PCR and microarrays. The recent application of next-generation sequencing to discovery of exRNA biomarkers has revealed the presence of potential novel miRNAs as well as other RNA species such as tRNAs, snoRNAs, piRNAs and lncRNAs in biofluids. At the same time, the use of RNA sequencing for biofluids poses unique challenges, including low amounts of input RNAs, the presence of exRNAs in different compartments with varying degrees of vulnerability to isolation techniques, and the high abundance of specific RNA species (thereby limiting the sensitivity of detection of less abundant species). Moreover, discovery in human diseases often relies on archival biospecimens of varying age and limiting amounts of samples. In this study, we have tested RNA isolation methods to optimize profiling exRNAs by RNA sequencing in individuals without any known diseases. Our findings are consistent with other recent studies that detect microRNAs and ribosomal RNAs as the major exRNA species in plasma. Similar to other recent studies, we found that the landscape of biofluid microRNA transcriptome is dominated by several abundant microRNAs that appear to comprise conserved extracellular miRNAs. There is reasonable correlation of sets of conserved miRNAs across biological replicates, and even across other data sets obtained at different investigative sites. Conversely, the detection of less abundant miRNAs is far more dependent on the exact methodology of RNA isolation and profiling. This study highlights the challenges in detecting and quantifying less abundant plasma miRNAs in health and disease using RNA sequencing platforms. PMID:28060806

  15. Formation of Neutrophil Extracellular Traps under Low Oxygen Level

    PubMed Central

    Branitzki-Heinemann, Katja; Möllerherm, Helene; Völlger, Lena; Husein, Diab M.; de Buhr, Nicole; Blodkamp, Stefanie; Reuner, Friederike; Brogden, Graham; Naim, Hassan Y.; von Köckritz-Blickwede, Maren

    2016-01-01

    Since their discovery, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET-release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia-inducible factor 1α (HIF-1α) by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21%) oxygen level and compared to hypoxic (1%) conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA) or Staphylococcus (S.) aureus wild-type and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of hif-1α as well as hif-1α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils

  16. The origin, function, and diagnostic potential of RNA within extracellular vesicles present in human biological fluids.

    PubMed

    Taylor, Douglas D; Gercel-Taylor, Cicek

    2013-01-01

    We have previously demonstrated that tumor cells release membranous structures into their extracellular environment, which are termed exosomes, microvesicles or extracellular vesicles depending on specific characteristics, including size, composition and biogenesis pathway. These cell-derived vesicles can exhibit an array of proteins, lipids and nucleic acids derived from the originating tumor. This review focuses of the transcriptome (RNA) of these extracellular vesicles. Based on current data, these vesicular components play essential roles as conveyers of intercellular communication and mediators of many of the pathological conditions associated with cancer development, progression and therapeutic failures. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, signal pathway activation through growth factor/receptor transfer, chemoresistance, and genetic exchange. These tumor-derived extracellular vesicles not only to represent a central mediator of the tumor microenvironment, but their presence in the peripheral circulation may serve as a surrogate for tumor biopsies, enabling real-time diagnosis and disease monitoring.

  17. Extracellular proteins secreted by probiotic bacteria as mediators of effects that promote mucosa-bacteria interactions.

    PubMed

    Sánchez, Borja; Urdaci, María C; Margolles, Abelardo

    2010-11-01

    During the last few years, a substantial body of scientific evidence has accumulated suggesting that certain surface-associated and extracellular components produced by probiotic bacteria could be responsible for some of their mechanisms of action. These bacterial components would be able to directly interact with the host mucosal cells; they include exopolysaccharides, bacteriocins, lipoteichoic acids and surface-associated and extracellular proteins. Extracellular proteins include proteins that are actively transported to the bacterial surroundings through the cytoplasmic membrane, as well as those that are simply shed from the bacterial surface. Compared to the other bacterial components, the interactive ability of extracellular proteins/peptides has been less extensively studied. In this review, current findings supporting an interaction between extracellular proteins/peptides produced by probiotic bacteria (strains of the genera Bifidobacterium, Lactobacillus and Escherichia) and host mucosal cells are discussed. Research needs and future trends are also considered.

  18. Structural basis for phosphatidylinositol-phosphate biosynthesis

    PubMed Central

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-01-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis. PMID:26510127

  19. Augmented Fish Health Monitoring in Idaho, 1989-1990 Annual Report.

    SciTech Connect

    Hauck, A. K.

    1990-10-01

    The Idaho augmented fish health monitoring contract DE-A179-87BP65903 was awarded in June 1987 and fully implemented in January 1988. The third annual report of activities serviced under this contract is presented. The prevailing fish health problems in 1989 include persistent infections caused by infectious pancreatic necrosis virus (IPNV), by Myxobolus (Myxosoma) cerebralis, Renibacterium salmoninarum and drug resistant Aeromonas salmonicida at select hatcheries on Idaho's upper Columbia River tributaries. Administrative focus during the year was to fill vacant positions and still maintain the monitoring effort at levels agreed on under contract. Complete diagnostic and inspection services were provided to eleven Idaho anadromous facilities. The present report describes work done to meet contract agreements and summarizes the fish health findings of anadromous stocks reared at and returning to Idaho's facilities during 1989.

  20. Work package 4 report: Broodfish testing for bacterial infections

    USGS Publications Warehouse

    Michel, Christian; Elliott, Diane G.; Jansson, Eva; Urdaci, Maria; Midtlyng, Paul J.

    2005-01-01

    This report summarises current scientific information and experience obtained with various methods for testing of salmonid broodfish or spawn for bacterial kidney disease (BKD - Renibacterium salmoninarum infection) in order to prevent vertical transmission of the organism to the offspring. Assessment is also being performed for Flavobacterium psychrophilum infections causing rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (CWD), and for Piscirickettsia salmonis infection causing salmon rickettsial syndrome (SRS) in salmonid fish species. Methods for screening to document the absence of BKD in fish populations are well established. Some of them have also proven successful for testing individual fish from infected populations in order to avoid vertical transmission of the infectious agent. Several diagnostic methods for flavobacteriosis and piscirickettsiosis have also been established but none of them, as yet, has been validated for use in programmes to prevent vertical transmission of disease. Priority subjects for further research in order to improve the management and control of these vertically transmissible fish diseases are suggested.

  1. Structural basis for phosphatidylinositol-phosphate biosynthesis

    NASA Astrophysics Data System (ADS)

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-10-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

  2. Some potentials and limits of the leucocrit test as a fish health assessment method

    USGS Publications Warehouse

    Wedemeyer, G.A.; Gould, R.W.; Yasutake, W.T.

    1983-01-01

    The sensitivity of the leucocrit as a stress tolerance and fish health assessment method was evaluated by subjecting juvenile coho salmon, Oncorhynchus kisutch, or steelhead trout, Salmo gairdneri, to standardized crowding, handling, temperature and disease challenges. The leucocrit was a sensitive indicator of the physiological stress resulting from crowding at population densities of 0·2–0·4 kg l−1, and to the stress of handling and to temperature changes. It was relatively insensitive to physiological sampling procedures which supports its continued development as a stress assessment method.In the case of fish diseases, subclinical or active Renibacterium salmoninarum and Yersinia ruckeriinfections had essentially no effect on leucocrit values. In contrast, active Aeromonas salmonicidainfections significantly depressed the leucocrit. However, no change was seen during the subclinical (incubation) phase prior to the development of an epizootic. Thus, the potential of the leucocrit as a fish health assessment method appears limited.

  3. Conditional switches for extracellular matrix patterning in Drosophila melanogaster.

    PubMed

    Khokhar, Arvinder; Chen, Nan; Yuan, Ji-Ping; Li, Yishi; Landis, Gary N; Beaulieu, Gregory; Kaur, Harminder; Tower, John

    2008-03-01

    An F(1) mutagenesis strategy was developed to identify conditional mutations affecting extracellular matrix (ECM) patterning. Tubulogenesis requires coordinated movement of epithelial cells and deposition of a multilayered ECM. In the Drosophila ovary, an epithelium of follicle cells creates the eggshells, including the paired tubular dorsal appendages (DAs) that act as breathing tubes for the embryo. A P-element mutagenesis strategy allowed for conditional overexpression of hundreds of genes in follicle cells. Conditional phenotypes were scored at the level of individual mutant (F(1)) female flies. ECM pattern regulators were readily identified including MAPK signaling gene ets domain lacking (fused DAs), Wnt pathway genes frizzled 3 and osa (long DAs), Hh pathway gene debra (branched DAs), and transcription factor genes sima/HIF-1alpha, ush, lilli, Tfb1, broad, and foxo. In moving cells the [Ca(2+)]/calcineurin pathway can regulate adhesion to ECM while adherens junctions link cells together. Accordingly, thin eggshell and DA phenotypes were identified for the calcineurin regulator calreticulin and the adherens junction component arc. Finally a tubulogenesis defect phenotype was identified for the gene pterodactyl, homologous to the mammalian serine/threonine receptor-associated protein (STRAP) that integrates the TGF-beta and PI3K/AKT signaling pathways. Because phenotypes can be scored in each mutant fly before and after gene induction, this F(1) conditional mutagenesis strategy should allow for increased scale in screens for mutations affecting repeated (reiterated) events in adult animals, including gametogenesis, movement, behavior, and learning.

  4. The potential for targeting extracellular LOX proteins in human malignancy.

    PubMed

    Mayorca-Guiliani, Alejandro; Erler, Janine T

    2013-11-25

    The extracellular matrix (ECM) is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX) enzymes cross-links collagens and elastin and, therefore, is a central player in ECM deposition and maturation. Extensive research has revealed how the LOX proteins participate in every stage of cancer progression, and two family members, LOX and LOX-like 2, have been linked to metastasis, the final stage of cancer responsible for over 90% of cancer patient deaths. However, LOX biosynthesis results in by-product with antiproliferative properties in certain cancers, and LOX enzymes may have different effects depending on the molecular network in which they are active. Therefore, the design of therapies targeting the LOX family needs to be guided by the molecular makeup of the individual disease and will probably require other agents to act on both the LOX enzymes and their associated network.

  5. The potential for targeting extracellular LOX proteins in human malignancy

    PubMed Central

    Mayorca-Guiliani, Alejandro; Erler, Janine T

    2013-01-01

    The extracellular matrix (ECM) is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX) enzymes cross-links collagens and elastin and, therefore, is a central player in ECM deposition and maturation. Extensive research has revealed how the LOX proteins participate in every stage of cancer progression, and two family members, LOX and LOX-like 2, have been linked to metastasis, the final stage of cancer responsible for over 90% of cancer patient deaths. However, LOX biosynthesis results in by-product with antiproliferative properties in certain cancers, and LOX enzymes may have different effects depending on the molecular network in which they are active. Therefore, the design of therapies targeting the LOX family needs to be guided by the molecular makeup of the individual disease and will probably require other agents to act on both the LOX enzymes and their associated network. PMID:24348049

  6. Functions of Cancer-Derived Extracellular Vesicles in Immunosuppression.

    PubMed

    Czernek, Liliana; Düchler, Markus

    2017-01-18

    Extracellular vesicles, including exosomes, constitute an important element of intercellular communication by carrying a variety of molecules from producer to target cells. The transport of mRNA and miRNA can directly modulate gene expression in the target cells. The miRNA content in exosomes is characteristic for the cell from which the vesicles were derived enabling the usage of exosomes as biomarkers for the diagnosis various diseases, including cancer. Cancer-derived exosomes support the survival and progression of tumors in many ways and also contribute to the neutralization of the anti-cancer immune response. Exosomes participate in all known mechanisms by which cancer evades the immune system. They influence the differentiation and activation of immune suppressor cells, they modulate antigen presentation, and are able to induce T-cell apoptosis. Although cancer-derived exosomes mainly suppress the immune system and facilitate tumor progression, they are also important sources of tumor antigens with potential clinical application in stimulating immune responses. This review summarizes how exosomes assist cancer to escape immune recognition and to acquire control over the immune system.

  7. Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis.

    PubMed

    Mejía, Susana P; Cano, Luz E; López, Juan A; Hernandez, Orville; González, Ángel

    2015-05-01

    Neutrophils play an important role as effector cells and contribute to the resistance of the host against microbial pathogens. Neutrophils are able to produce extracellular traps (NETs) in response to medically important fungi, including Aspergillus spp., Candida albicans and Cryptococcus gattii. However, NET production in response to Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) did not alter NET production against conidia, it partially suppressed NET formation against P. brasiliensis yeasts. Cytochalasin D or IFN-γ did not affect the production of NETs against the fungus. Additionally, a mutant strain of P. brasiliensis with reduced expression of an alternative oxidase induced significantly higher levels of NETs in comparison with the WT strain. Finally, c.f.u. quantification of P. brasiliensis showed no significant differences when neutrophils were treated with DPI, DNase I or cytochalasin D as compared with untreated cells. These data establish that NET formation by human neutrophils appears to be either dependent or independent of reactive oxygen species production, correlating with the fungal morphotype used for stimulation. However, this mechanism was ineffective in killing the fungus.

  8. Tissue-Specific Effects of Esophageal Extracellular Matrix

    PubMed Central

    Keane, Timothy J.; DeWard, Aaron; Londono, Ricardo; Saldin, Lindsey T.; Castleton, Arthur A.; Carey, Lisa; Nieponice, Alejandro; Lagasse, Eric

    2015-01-01

    Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate repair or remodeling of numerous tissues, including the esophagus. The theoretically ideal scaffold for tissue repair is the ECM derived from the particular tissue to be treated, that is, site-specific or homologous ECM. The preference or potential advantage for the use of site-specific ECM remains unknown in the esophageal location. The objective of the present study was to characterize the in vitro cellular response and in vivo host response to a homologous esophageal ECM (eECM) versus nonhomologous ECMs derived from small intestinal submucosa and urinary bladder. The in vitro response of esophageal stem cells was characterized by migration, proliferation, and three-dimensional (3D) organoid formation assays. The in vivo remodeling response was evaluated in a rat model of esophageal mucosal resection. Results of the study showed that the eECM retains favorable tissue-specific characteristics that enhance the migration of esophageal stem cells and supports the formation of 3D organoids to a greater extent than heterologous ECMs. Implantation of eECM facilitates the remodeling of esophageal mucosa following mucosal resection, but no distinct advantage versus heterologous ECM could be identified. PMID:26192009

  9. Cardiac Extracellular Vesicles in Normal and Infarcted Heart

    PubMed Central

    Chistiakov, Dimitry A.; Orekhov, Alexander N.; Bobryshev, Yuri V.

    2016-01-01

    Heart is a complex assembly of many cell types constituting myocardium, endocardium and epicardium that intensively communicate to each other in order to maintain the proper cardiac function. There are many types of intercellular intracardiac signals, with a prominent role of extracellular vesicles (EVs), such as exosomes and microvesicles, for long-distant delivering of complex messages. Cardiomyocytes release EVs, whose content could significantly vary depending on the stimulus. In stress, such as hypoxia, inflammation or injury, cardiomyocytes increase secretion of EVs. In hypoxic conditions, cardiac EVs are enriched with angiogenic and prosurvival factors. In acute myocardial infarction (AMI), damaged cardiac muscle cells produce EVs with increased content of angiogenic, anti-apoptotic, mitogenic and growth factors in order to induce repair and healing of the infarcted myocardium. Exosomal microRNAs play a central role in cardiac regeneration. In AMI, circulating cardiac EVs abundantly contain cardiac-specific miRNAs that serve as indicators of cardiac damage and have a big diagnostic potential as AMI biomarkers. Cardioprotective and regenerative properties of exosomes derived from cardiac and non-cardiac stem/progenitor cells are very helpful to be used in cell-free cardiotherapy and regeneration of post-infarct myocardium. PMID:26742038

  10. Expansion of extracellular volume in early polycystic kidney disease.

    PubMed

    Danielsen, H; Pedersen, E B; Nielsen, A H; Herlevsen, P; Kornerup, H J; Posborg, V

    1986-01-01

    Blood volume (BV), extracellular volume (ECV), blood pressure (BP), creatinine clearance (CCr), plasma levels of angiotensin II (AII), aldosterone (Aldo) and arginine vasopressin (AVP), and serum osmolality (Sosm) were determined in 18 patients with adult polycystic kidney disease, 8 normotensive (group I), 10 hypertensive (group II), and in 11 control subjects (group III). ECV but not BV was increased in group I compared with group III, whereas BV and ECV did not differ significantly between groups II and III. In group II, Aldo and AVP were increased and AII tended to be increased, while in group I the hormone levels did not differ significantly from those in group III. Sosm did not differ significantly between the groups. In the combined patient group, CCr correlated positively with BV and ECV and negatively with BP. In the patients, AII and AVP were positively correlated with BP but not with CCr. The results suggest that both the renin-angiotensin system and AVP might be involved in the BP elevation, whereas expansion of ECV can be found without an increase in BP.

  11. Extracellular vesicles as novel carriers for therapeutic molecules.

    PubMed

    Yim, Nambin; Choi, Chulhee

    2016-11-01

    Extracellular vesicles (EVs) are natural carriers of biomolecules that play central roles in cell-to-cell communications. Based on this, there have been various attempts to use EVs as therapeutic drug carriers. From chemical reagents to nucleic acids, various macromolecules were successfully loaded into EVs; however, loading of proteins with high molecular weight has been huddled with several problems. Purification of recombinant proteins is expensive and time consuming, and easily results in modification of proteins due to physical or chemical forces. Also, the loading efficiency of conventional methods is too low for most proteins. We have recently proposed a new method, the so-called exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs), to overcome the limitations. Since EXPLORs are produced by actively loading of intracellular proteins into EVs using blue light without protein purification steps, we demonstrated that the EXPLOR technique significantly improves the loading and delivery efficiency of therapeutic proteins. In further in vitro and in vivo experiments, we demonstrate the potential of EXPLOR technology as a novel platform for biopharmaceuticals, by successful delivery of several functional proteins such as Cre recombinase, into the target cells. [BMB Reports 2016; 49(11): 585-586].

  12. Origin of life: LUCA and extracellular membrane vesicles (EMVs)

    NASA Astrophysics Data System (ADS)

    Gill, S.; Forterre, P.

    2016-01-01

    Cells from the three domains of life produce extracellular membrane vesicles (EMVs), suggesting that EMV production is an important aspect of cellular physiology. EMVs have been implicated in many aspects of cellular life in all domains, including stress response, toxicity against competing strains, pathogenicity, detoxification and resistance against viral attack. These EMVs represent an important mode of inter-cellular communication by serving as vehicles for transfer of DNA, RNA, proteins and lipids between cells. Here, we review recent progress in the understanding of EMV biology and their various roles. We focus on the role of membrane vesicles in early cellular evolution and how they would have helped shape the nature of the last universal common ancestor. A membrane-protected micro-environment would have been a key to the survival of spontaneous molecular systems and efficient metabolic reactions. Interestingly, the morphology of EMVs is strongly reminiscent of the morphology of some virions. It is thus tempting to make a link between the origin of the first protocell via the formation of vesicles and the origin of viruses.

  13. Extracellular matrix: A dynamic microenvironment for stem cell niche☆

    PubMed Central

    Gattazzo, Francesca; Urciuolo, Anna; Bonaldo, Paolo

    2014-01-01

    Background Extracellular matrix (ECM) is a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties specific for each tissue and able to regulate cell behavior. Stem cells have a key role in the maintenance and regeneration of tissues and they are located in a specific microenvironment, defined as niche. Scope of review We overview the progresses that have been made in elucidating stem cell niches and discuss the mechanisms by which ECM affects stem cell behavior. We also summarize the current tools and experimental models for studying ECM–stem cell interactions. Major conclusions ECM represents an essential player in stem cell niche, since it can directly or indirectly modulate the maintenance, proliferation, self-renewal and differentiation of stem cells. Several ECM molecules play regulatory functions for different types of stem cells, and based on its molecular composition the ECM can be deposited and finely tuned for providing the most appropriate niche for stem cells in the various tissues. Engineered biomaterials able to mimic the in vivo characteristics of stem cell niche provide suitable in vitro tools for dissecting the different roles exerted by the ECM and its molecular components on stem cell behavior. General significance ECM is a key component of stem cell niches and is involved in various aspects of stem cell behavior, thus having a major impact on tissue homeostasis and regeneration under physiological and pathological conditions. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. PMID:24418517

  14. Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps

    PubMed Central

    Ávila, Eva E.; Rodríguez, Mayra C.; Díaz-Godínez, César; Laclette, Juan P.; Becker, Ingeborg; Carrero, Julio C.

    2016-01-01

    Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica. PMID:27415627

  15. Extracellular peptidases of the cereal pathogen Fusarium graminearum

    PubMed Central

    Lowe, Rohan G. T.; McCorkelle, Owen; Bleackley, Mark; Collins, Christine; Faou, Pierre; Mathivanan, Suresh; Anderson, Marilyn

    2015-01-01

    The plant pathogenic fungus Fusarium graminearum (Fgr) creates economic and health risks in cereals agriculture. Fgr causes head blight (or scab) of wheat and stalk rot of corn, reducing yield, degrading grain quality, and polluting downstream food products with mycotoxins. Fungal plant pathogens must secrete proteases to access nutrition and to breakdown the structural protein component of the plant cell wall. Research into the proteolytic activity of Fgr is hindered by the complex nature of the suite of proteases secreted. We used a systems biology approach comprising genome analysis, transcriptomics and label-free quantitative proteomics to characterize the peptidases deployed by Fgr during growth. A combined analysis of published microarray transcriptome datasets revealed seven transcriptional groupings of peptidases based on in vitro growth, in planta growth, and sporulation behaviors. A high resolution mass spectrometry-based proteomics analysis defined the extracellular proteases secreted by F. graminearum. A meta-classification based on sequence characters and transcriptional/translational activity in planta and in vitro provides a platform to develop control strategies that target Fgr peptidases. PMID:26635820

  16. Tailoring material properties of a nanofibrous extracellular matrix derived hydrogel

    NASA Astrophysics Data System (ADS)

    Johnson, Todd D.; Lin, Stephen Y.; Christman, Karen L.

    2011-12-01

    In the native tissue, the interaction between cells and the extracellular matrix (ECM) is essential for cell migration, proliferation, differentiation, mechanical stability, and signaling. It has been shown that decellularized ECMs can be processed into injectable formulations, thereby allowing for minimally invasive delivery. Upon injection and increase in temperature, these materials self-assemble into porous gels forming a complex network of fibers with nanoscale structure. In this study we aimed to examine and tailor the material properties of a self-assembling ECM hydrogel derived from porcine myocardial tissue, which was developed as a tissue specific injectable scaffold for cardiac tissue engineering. The impact of gelation parameters on ECM hydrogels has not previously been explored. We examined how modulating pH, temperature, ionic strength, and concentration affected the nanoscale architecture, mechanical properties, and gelation kinetics. These material characteristics were assessed using scanning electron microscopy, rheometry, and spectrophotometry, respectively. Since the main component of the myocardial matrix is collagen, many similarities between the ECM hydrogel and collagen gels were observed in terms of the nanofibrous structure and modulation of properties by altering ionic strength. However, variation from collagen gels was noted for the gelation temperature along with varied times and rates of gelation. These discrepancies when compared to collagen are likely due to the presence of other ECM components in the decellularized ECM based hydrogel. These results demonstrate how the material properties of ECM hydrogels could be tailored for future in vitro and in vivo applications.

  17. [Extracellular polymeric substances disintegration by Fenton oxidation of excess sludge].

    PubMed

    Li, Juan; Zhang, Pan-Yue; Zeng, Guang-Ming; Song, Xiang-Guo; Dong, Jin-Hua; Zhagn, Hai-Yan; Wu, Zhen; Jia, Xiao-Lei

    2009-02-15

    Fenton oxidation was used to disintegrate extracellular polymeric substances (EPS) of excess sludge with its strong oxidation ability. The concentration of polysaccharide, protein and the change of soluble chemical oxygen demand (SCOD) disintegrated from EPS represent the EPS disintegration degree. The objective of this study is to optimize the operational conditions for EPS disintegration with Fenton oxidation. It is shown that the optimal operational condition is as following: pH = 2.5, reaction time = 90 min, H2O2/Fe2+ (weight dosage ratio) = 8:1 and reaction temperature is about 65-70 degrees C. Under this condition after the Fenton oxidation, SCOD, concentration of polysaccharide, protein in the supernate increase from 45.88, 10.96 and 11.99 mg x L(-1) to 684.93, 382.17 and 302.62 mg x L(-1), respectively; the average diameter and the median diameter of sludge particulates reduce from 838.89 microm and 859.20 microm to 137.22 microm and 148.69 microm, respectively. As a result, EPS is effectively disintegrated by Fenton oxidation and the sludge is greatly mineralized, which benefits the further sludge reduction and utilization.

  18. An extracellular adhesion molecule complex patterns dendritic branching and morphogenesis

    PubMed Central

    Dong, Xintong; Liu, Oliver W.; Howell, Audrey S.; Shen, Kang

    2014-01-01

    Summary Robust dendrite morphogenesis is a critical step in the development of reproducible neural circuits. However, little is known about the extracellular cues that pattern complex dendrite morphologies. In the model nematode C. elegans, the sensory neuron PVD establishes stereotypical, highly-branched dendrite morphology. Here, we report the identification of a tripartite ligand-receptor complex of membrane adhesion molecules that is both necessary and sufficient to instruct spatially restricted growth and branching of PVD dendrites. The ligand complex SAX-7/L1CAM and MNR-1 function at defined locations in the surrounding hypodermal tissue, while DMA-1 acts as the cognate receptor on PVD. Mutations in this complex lead to dramatic defects in the formation, stabilization, and organization of the dendritic arbor. Ectopic expression of SAX-7 and MNR-1 generates a predictable, unnaturally patterned dendritic tree in a DMA-1 dependent manner. Both in vivo and in vitro experiments indicate that all three molecules are needed for interaction. PMID:24120131

  19. A Noninvasive Method to Study Regulation of Extracellular ...

    EPA Pesticide Factsheets

    Time-domain nuclear magnetic resonance (TD-NMR)-based measurement of body composition of rodents is an effective method to quickly and repeatedly measure proportions of fat, lean, and fluid without anesthesia. TD-NMR provides a measure of free water in a living animal, termed % fluid, and is a measure of unbound water in the vascular and extracelular spaces. We hypothesized that injecting a bolus of fluid into the peritoneal cavity would lead to an abrupt increase in %fluid and the rate of clearance monitored with TD-NMR would provide a noninvasive assessment of the free water homeostasis in an awake rat. Several strains of laboratory rats were injected intraperitoneally with 10 ml/kg isotonic or hypertonic saline and % fluid was monitored repeatedly with a Bruker "Minispec" TD-NMR body composition system.Following isotonic saline, %fluid increased immediately by 0.5% followed by a recovery over ~6h. Injecting hypertonic (3 times normal saline) resulted in a significantly greater rise in %fluid and longer recovery. lntraperitoneal and subcutaneous fluid injection led to similar rates of clearance. The Wistar-Kyoto rat strain displayed significantly slower recovery to fluid loads compared with Long-Evans and Sprague-Dawley strains. Rats exercised chronically showed significant increases in %fluid, but the rate of clearance of fluid was similar to that of sedentary animals. We conclude that this technique could be used to study vascular and extracellular volume ho

  20. Biological properties of extracellular vesicles and their physiological functions

    PubMed Central

    Yáñez-Mó, María; Siljander, Pia R.-M.; Andreu, Zoraida; Zavec, Apolonija Bedina; Borràs, Francesc E.; Buzas, Edit I.; Buzas, Krisztina; Casal, Enriqueta; Cappello, Francesco; Carvalho, Joana; Colás, Eva; Silva, Anabela Cordeiro-da; Fais, Stefano; Falcon-Perez, Juan M.; Ghobrial, Irene M.; Giebel, Bernd; Gimona, Mario; Graner, Michael; Gursel, Ihsan; Gursel, Mayda; Heegaard, Niels H. H.; Hendrix, An; Kierulf, Peter; Kokubun, Katsutoshi; Kosanovic, Maja; Kralj-Iglic, Veronika; Krämer-Albers, Eva-Maria; Laitinen, Saara; Lässer, Cecilia; Lener, Thomas; Ligeti, Erzsébet; Linē, Aija; Lipps, Georg; Llorente, Alicia; Lötvall, Jan; Manček-Keber, Mateja; Marcilla, Antonio; Mittelbrunn, Maria; Nazarenko, Irina; Hoen, Esther N.M. Nolte-‘t; Nyman, Tuula A.; O'Driscoll, Lorraine; Olivan, Mireia; Oliveira, Carla; Pállinger, Éva; del Portillo, Hernando A.; Reventós, Jaume; Rigau, Marina; Rohde, Eva; Sammar, Marei; Sánchez-Madrid, Francisco; Santarém, N.; Schallmoser, Katharina; Ostenfeld, Marie Stampe; Stoorvogel, Willem; Stukelj, Roman; Van der Grein, Susanne G.; Vasconcelos, M. Helena; Wauben, Marca H. M.; De Wever, Olivier

    2015-01-01

    In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system. PMID:25979354

  1. Extracellular matrix protein CCN1 limits oncolytic efficacy in glioma.

    PubMed

    Haseley, Amy; Boone, Sean; Wojton, Jeffrey; Yu, Lianbo; Yoo, Ji Young; Yu, Jianhua; Kurozumi, Kazuhiko; Glorioso, Joseph C; Caligiuri, Michael A; Kaur, Balveen

    2012-03-15

    Oncolytic viral therapy has been explored widely as an option for glioma treatment but its effectiveness has remained limited. Cysteine rich 61 (CCN1) is an extracellular matrix (ECM) protein elevated in cancer cells that modulates their adhesion and migration by binding cell surface receptors. In this study, we examined a hypothesized role for CCN1 in limiting the efficacy of oncolytic viral therapy for glioma, based on evidence of CCN1 induction that occurs in this setting. Strikingly, we found that exogenous CCN1 in glioma ECM orchestrated a cellular antiviral response that reduced viral replication and limited cytolytic efficacy. Gene expression profiling and real-time PCR analysis revealed a significant induction of type-I interferon responsive genes in response to CCN1 exposure. This induction was accompanied by activation of the Jak/Stat signaling pathway, consistent with induction of an innate antiviral cellular response. Both effects were mediated by the binding of CCN1 to the cell surface integrin α6β1, activating its signaling and leading to rapid secretion of interferon-α, which was essential for the innate antiviral effect. Together, our findings reveal how an integrin signaling pathway mediates activation of a type-I antiviral interferon response that can limit the efficacy of oncolytic viral therapy. Furthermore, they suggest therapeutic interventions to inhibit CCN1-integrin α6 interactions to sensitize gliomas to viral oncolysis.

  2. Kefiran antagonizes cytopathic effects of Bacillus cereus extracellular factors.

    PubMed

    Medrano, Micaela; Pérez, Pablo Fernando; Abraham, Analía Graciela

    2008-02-29

    Kefiran, the polysaccharide produced by microorganisms present in kefir grains, is a water-soluble branched glucogalactan containing equal amounts of D-glucose and D-galactose. In this study, the effect of kefiran on the biological activity of Bacillus cereus strain B10502 extracellular factors was assessed by using cultured human enterocytes (Caco-2 cells) and human erythrocytes. In the presence of kefiran concentrations ranging from 300 to 1000 mg/L, the ability of B. cereus B10502 spent culture supernatants to detach and damage cultured human enterocytes was significantly abrogated. In addition, mitochondrial dehydrogenase activity was higher when kefiran was present during the cell toxicity assays. Protection was also demonstrated in hemolysis and apoptosis/necrosis assays. Scanning electron microscopy showed the protective effect of kefiran against structural cell damages produced by factors synthesized by B. cereus strain B10502. Protective effect of kefiran depended on strain of B. cereus. Our findings demonstrate the ability of kefiran to antagonize key events of B. cereus B10502 virulence. This property, although strain-specific, gives new perspectives for the role of bacterial exopolysaccharides in functional foods.

  3. Extracellular water across the adult lifespan: reference values for adults.

    PubMed

    Silva, Analiza M; Wang, Jack; Pierson, Richard N; Wang, Zimian; Spivack, John; Allison, David B; Heymsfield, Steven B; Sardinha, Luis B; Heshka, Stanley

    2007-05-01

    Extracellular water (ECW) is a large and clinically important body compartment that varies widely in volume both in health and disease. Interpretation of ECW measurements in the clinical setting requires consideration of potential influencing factors such as age, race, sex and other variables that influence fluid status. An important gap in physiological research is a lack of normative ECW values against which to reference perturbations in fluid homeostasis. The current study's aim was to develop conditional quantile equations for ECW based on weight, height, age, sex and race using a large (n = 1538, 854 females and 684 males) healthy adult multi-ethnic (African American, Asian, European American, Hispanic) sample. ECW was derived from total body water and potassium measured by isotope dilution and whole-body 40K counting, respectively. Quantile regression methods were used to identify five percentile levels (10th, 25th, 50th, 75th, 90th). Weight and height were significant variables at each quantile in both males and females; age made a significant contribution in the male but not the female sample. These regression equations provide ECW quantile reference values based on a large multi-ethnic adult population that should not only prove useful in clinical settings and physiological research, but serve as a model approach for developing body composition normative ranges.

  4. Biological properties of extracellular vesicles and their physiological functions.

    PubMed

    Yáñez-Mó, María; Siljander, Pia R-M; Andreu, Zoraida; Zavec, Apolonija Bedina; Borràs, Francesc E; Buzas, Edit I; Buzas, Krisztina; Casal, Enriqueta; Cappello, Francesco; Carvalho, Joana; Colás, Eva; Cordeiro-da Silva, Anabela; Fais, Stefano; Falcon-Perez, Juan M; Ghobrial, Irene M; Giebel, Bernd; Gimona, Mario; Graner, Michael; Gursel, Ihsan; Gursel, Mayda; Heegaard, Niels H H; Hendrix, An; Kierulf, Peter; Kokubun, Katsutoshi; Kosanovic, Maja; Kralj-Iglic, Veronika; Krämer-Albers, Eva-Maria; Laitinen, Saara; Lässer, Cecilia; Lener, Thomas; Ligeti, Erzsébet; Linē, Aija; Lipps, Georg; Llorente, Alicia; Lötvall, Jan; Manček-Keber, Mateja; Marcilla, Antonio; Mittelbrunn, Maria; Nazarenko, Irina; Nolte-'t Hoen, Esther N M; Nyman, Tuula A; O'Driscoll, Lorraine; Olivan, Mireia; Oliveira, Carla; Pállinger, Éva; Del Portillo, Hernando A; Reventós, Jaume; Rigau, Marina; Rohde, Eva; Sammar, Marei; Sánchez-Madrid, Francisco; Santarém, N; Schallmoser, Katharina; Ostenfeld, Marie Stampe; Stoorvogel, Willem; Stukelj, Roman; Van der Grein, Susanne G; Vasconcelos, M Helena; Wauben, Marca H M; De Wever, Olivier

    2015-01-01

    In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

  5. Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae.

    PubMed

    Wu, Siva; Baum, Marc M; Kerwin, James; Guerrero, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-12-01

    Nontypeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen, can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24- and 96-h NTHi biofilms contained polysaccharides and proteinaceous components as detected by nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24-h biofilms, two were found only in 96-h biofilms, and fifteen were present in the ECM of both 24- and 96-h NTHi biofilms. All proteins identified were either associated with bacterial membranes or cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation.

  6. Comparative transcriptomic analysis of human and Drosophila extracellular vesicles

    PubMed Central

    Lefebvre, Fabio Alexis; Benoit Bouvrette, Louis Philip; Perras, Lilyanne; Blanchet-Cohen, Alexis; Garnier, Delphine; Rak, Janusz; Lécuyer, Éric

    2016-01-01

    Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing specific repertoires of genetic material. In mammals, EVs can mediate the horizontal transfer of various cargos and signaling molecules, notably miRNA and mRNA species. Whether this form of intercellular communication prevails in other metazoans remains unclear. Here, we report the first parallel comparative morphologic and transcriptomic characterization of EVs from Drosophila and human cellular models. Electronic microscopy revealed that human and Drosophila cells release similar EVs with diameters ranging from 30 to 200 nm, which contain complex populations of transcripts. RNA-seq identified abundant ribosomal RNAs, related pseudogenes and retrotransposons in human and Drosophila EVs. Vault RNAs and Y RNAs abounded in human samples, whereas small nucleolar RNAs involved in pseudouridylation were most prevalent in Drosophila EVs. Numerous mRNAs were identified, largely consisting of exonic sequences displaying full-length read coverage and enriched for translation and electronic transport chain functions. By analogy with human systems, these sizeable similarities suggest that EVs could potentially enable RNA-mediated intercellular communication in Drosophila. PMID:27282340

  7. Designing an extracellular matrix protein with enhanced mechanical stability

    PubMed Central

    Ng, Sean P.; Billings, Kate S.; Ohashi, Tomoo; Allen, Mark D.; Best, Robert B.; Randles, Lucy G.; Erickson, Harold P.; Clarke, Jane

    2007-01-01

    The extracellular matrix proteins tenascin and fibronectin experience significant mechanical forces in vivo. Both contain a number of tandem repeating homologous fibronectin type III (fnIII) domains, and atomic force microscopy experiments have demonstrated that the mechanical strength of these domains can vary significantly. Previous work has shown that mutations in the core of an fnIII domain from human tenascin (TNfn3) reduce the unfolding force of that domain significantly: The composition of the core is apparently crucial to the mechanical stability of these proteins. Based on these results, we have used rational redesign to increase the mechanical stability of the 10th fnIII domain of human fibronectin, FNfn10, which is directly involved in integrin binding. The hydrophobic core of FNfn10 was replaced with that of the homologous, mechanically stronger TNfn3 domain. Despite the extensive substitution, FNoTNc retains both the three-dimensional structure and the cell adhesion activity of FNfn10. Atomic force microscopy experiments reveal that the unfolding forces of the engineered protein FNoTNc increase by ≈20% to match those of TNfn3. Thus, we have specifically designed a protein with increased mechanical stability. Our results demonstrate that core engineering can be used to change the mechanical strength of proteins while retaining functional surface interactions. PMID:17535921

  8. Mixed Extracellular Matrix Ligands Synergistically Modulate Integrin Adhesion and Signaling

    PubMed Central

    Reyes, Catherine D.; Petrie, Timothy A.; García, Andrés J

    2008-01-01

    Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non-adhesive surfaces presenting well-defined, independent densities of two integrin-specific engineered ligands for the type I collagen (COL-I) receptor α2β1 and the fibronectin (FN) receptor α5β1 to evaluate the effects of integrin cross-talk on adhesive responses. Engineered surfaces displayed ligand density-dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL-I ligand surfaces. Moreover, surfaces presenting mixed COL-I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling. PMID:18613064

  9. The Extracellular Matrix in Bronchopulmonary Dysplasia: Target and Source

    PubMed Central

    Mižíková, Ivana; Morty, Rory E.

    2015-01-01

    Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth that contributes significantly to morbidity and mortality in neonatal intensive care units. BPD results from life-saving interventions, such as mechanical ventilation and oxygen supplementation used to manage preterm infants with acute respiratory failure, which may be complicated by pulmonary infection. The pathogenic pathways driving BPD are not well-delineated but include disturbances to the coordinated action of gene expression, cell–cell communication, physical forces, and cell interactions with the extracellular matrix (ECM), which together guide normal lung development. Efforts to further delineate these pathways have been assisted by the use of animal models of BPD, which rely on infection, injurious mechanical ventilation, or oxygen supplementation, where histopathological features of BPD can be mimicked. Notable among these are perturbations to ECM structures, namely, the organization of the elastin and collagen networks in the developing lung. Dysregulated collagen deposition and disturbed elastin fiber organization are pathological hallmarks of clinical and experimental BPD. Strides have been made in understanding the disturbances to ECM production in the developing lung, but much still remains to be discovered about how ECM maturation and turnover are dysregulated in aberrantly developing lungs. This review aims to inform the reader about the state-of-the-art concerning the ECM in BPD, to highlight the gaps in our knowledge and current controversies, and to suggest directions for future work in this exciting and complex area of lung development (patho)biology. PMID:26779482

  10. Extracellular-matrix tethering regulates stem-cell fate

    NASA Astrophysics Data System (ADS)

    Trappmann, Britta; Gautrot, Julien E.; Connelly, John T.; Strange, Daniel G. T.; Li, Yuan; Oyen, Michelle L.; Cohen Stuart, Martien A.; Boehm, Heike; Li, Bojun; Vogel, Viola; Spatz, Joachim P.; Watt, Fiona M.; Huck, Wilhelm T. S.

    2012-07-01

    To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.

  11. EVpedia: a community web portal for extracellular vesicles research

    PubMed Central

    Kim, Dae-Kyum; Lee, Jaewook; Kim, Sae Rom; Choi, Dong-Sic; Yoon, Yae Jin; Kim, Ji Hyun; Go, Gyeongyun; Nhung, Dinh; Hong, Kahye; Jang, Su Chul; Kim, Si-Hyun; Park, Kyong-Su; Kim, Oh Youn; Park, Hyun Taek; Seo, Ji Hye; Aikawa, Elena; Baj-Krzyworzeka, Monika; van Balkom, Bas W. M.; Belting, Mattias; Blanc, Lionel; Bond, Vincent; Bongiovanni, Antonella; Borràs, Francesc E.; Buée, Luc; Buzás, Edit I.; Cheng, Lesley; Clayton, Aled; Cocucci, Emanuele; Dela Cruz, Charles S.; Desiderio, Dominic M.; Di Vizio, Dolores; Ekström, Karin; Falcon-Perez, Juan M.; Gardiner, Chris; Giebel, Bernd; Greening, David W.; Gross, Julia Christina; Gupta, Dwijendra; Hendrix, An; Hill, Andrew F.; Hill, Michelle M.; Nolte-'t Hoen, Esther; Hwang, Do Won; Inal, Jameel; Jagannadham, Medicharla V.; Jayachandran, Muthuvel; Jee, Young-Koo; Jørgensen, Malene; Kim, Kwang Pyo; Kim, Yoon-Keun; Kislinger, Thomas; Lässer, Cecilia; Lee, Dong Soo; Lee, Hakmo; van Leeuwen, Johannes; Lener, Thomas; Liu, Ming-Lin; Lötvall, Jan; Marcilla, Antonio; Mathivanan, Suresh; Möller, Andreas; Morhayim, Jess; Mullier, François; Nazarenko, Irina; Nieuwland, Rienk; Nunes, Diana N.; Pang, Ken; Park, Jaesung; Patel, Tushar; Pocsfalvi, Gabriella; del Portillo, Hernando; Putz, Ulrich; Ramirez, Marcel I.; Rodrigues, Marcio L.; Roh, Tae-Young; Royo, Felix; Sahoo, Susmita; Schiffelers, Raymond; Sharma, Shivani; Siljander, Pia; Simpson, Richard J.; Soekmadji, Carolina; Stahl, Philip; Stensballe, Allan; Stępień, Ewa; Tahara, Hidetoshi; Trummer, Arne; Valadi, Hadi; Vella, Laura J.; Wai, Sun Nyunt; Witwer, Kenneth; Yáñez-Mó, María; Youn, Hyewon; Zeidler, Reinhard; Gho, Yong Song

    2015-01-01

    Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. Results: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. Availability and implementation: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info. Contact: ysgho@postech.ac.kr PMID:25388151

  12. Differential modulation by extracellular ATP of carotid chemosensory responses.

    PubMed

    Spergel, D; Lahiri, S

    1993-06-01

    The possibility that the carotid body has ATP surface receptors that mediate O2 chemoreception was tested. To distinguish between the event(s) initiating chemoreception and those at the neurotransmitter level, we also tested the chemosensory response to nicotine before and after ATP administration. Carotid bodies from cats anesthetized with pentobarbital sodium were perfused and superfused in vitro with modified Tyrode solution (PCO2 < 1 Torr, pH 7.4, 36 degrees C) equilibrated at PO2 > 400 or approximately 150 Torr while chemosensory discharge was recorded extracellularly. ATP and adenosine 5'-[gamma-thio]triphosphate stimulated discharge with similar dose dependence, whereas adenosine had little effect. ATP infusion for > or = 2 min evoked an initial stimulation of discharge followed by a decline to baseline (desensitization). Desensitization did not affect the response to hypoxia (perfusate flow interruption) but inhibited the response to nicotine (4-nmol pulse). Therefore, 1) the carotid body has surface ATP receptors that may mediate the chemosensory response to nicotine but not to hypoxia and 2) nicotinic receptors are not required for carotid body O2 chemoreception.

  13. Extracellular space preservation aids the connectomic analysis of neural circuits

    PubMed Central

    Pallotto, Marta; Watkins, Paul V; Fubara, Boma; Singer, Joshua H; Briggman, Kevin L

    2015-01-01

    Dense connectomic mapping of neuronal circuits is limited by the time and effort required to analyze 3D electron microscopy (EM) datasets. Algorithms designed to automate image segmentation suffer from substantial error rates and require significant manual error correction. Any improvement in segmentation error rates would therefore directly reduce the time required to analyze 3D EM data. We explored preserving extracellular space (ECS) during chemical tissue fixation to improve the ability to segment neurites and to identify synaptic contacts. ECS preserved tissue is easier to segment using machine learning algorithms, leading to significantly reduced error rates. In addition, we observed that electrical synapses are readily identified in ECS preserved tissue. Finally, we determined that antibodies penetrate deep into ECS preserved tissue with only minimal permeabilization, thereby enabling correlated light microscopy (LM) and EM studies. We conclude that preservation of ECS benefits multiple aspects of the connectomic analysis of neural circuits. DOI: http://dx.doi.org/10.7554/eLife.08206.001 PMID:26650352

  14. Extracellular Vesicles and Vascular Injury: New Insights for Radiation Exposure

    PubMed Central

    Flamant, Stéphane; Tamarat, Radia

    2016-01-01

    This article reviews our current knowledge about cell-derived extracellular vesicles (EVs), including microparticles and exosomes, and their emergence as mediators of a new important mechanism of cell-to-cell communication. Particular emphasis has been given to the increasing involvement of EVs in the field of radiation-induced vascular injury. Although EVs have been considered for a long time as cell “dust”, they in fact precisely reflect the physiological state of the cells. The role of microparticles and exosomes in mediating vascular dysfunction suggests that they may represent novel pathways in short- or long-distance paracrine intercellular signaling in vascular environment. In this article, the mechanisms involved in the biogenesis of microparticles and exosomes, their composition and participation in the pathogenesis of vascular dysfunction are discussed. Furthermore, this article highlights the concept of EVs as potent vectors of biological information and protagonists of an intercellular communication network. Special emphasis is made on EV-mediated microRNA transfer and on the principal consequences of such signal exchange on vascular injury and radiation-induced non-targeted effect. The recent progress in elucidating the biology of EVs has provided new insights for the field of radiation, advancing their use as diagnostic biomarkers or in therapeutic interventions. PMID:27459703

  15. Extracellular calcium sensing in rat aortic vascular smooth muscle cells

    SciTech Connect

    Smajilovic, Sanela; Hansen, Jakob Lerche; Christoffersen, Tue E.H.

    2006-10-06

    Extracellular calcium (Ca2+o) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca2+o stimulates proliferation of the cells. The effects of Ca2+o were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca2+o-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca2+o-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.

  16. Bacterial Extracellular Polysaccharides in Biofilm Formation and Function

    PubMed Central

    Limoli, Dominique H.; Jones, Christopher J.; Wozniak, Daniel J.

    2015-01-01

    Microbes produce a biofilm matrix consisting of proteins, extracellular DNA, and polysaccharides that is integral in the formation of bacterial communities. Historical studies of polysaccharides revealed that their overproduction often alters the colony morphology and can be diagnostic in identifying certain species. The polysaccharide component of the matrix can provide many diverse benefits to the cells in the biofilm, including adhesion, protection, and structure. Aggregative polysaccharides act as molecular glue, allowing the bacterial cells to adhere to each other as well as surfaces. Adhesion facilitates the colonization of both biotic and abiotic surfaces by allowing the bacteria to resist physical stresses imposed by fluid movement that could separate the cells from a nutrient source. Polysaccharides can also provide protection from a wide range of stresses, such as desiccation, immune effectors, and predators such as phagocytic cells and amoebae. Finally, polysaccharides can provide structure to biofilms, allowing stratification of the bacterial community and establishing gradients of nutrients and waste products. This can be advantageous for the bacteria by establishing a heterogeneous population that is prepared to endure stresses created by the rapidly changing environments that many bacteria encounter. The diverse range of polysaccharide structures, properties, and roles highlight the importance of this matrix constituent to the successful adaptation of bacteria to nearly every niche. Here, we present an overview of the current knowledge regarding the diversity and benefits that polysaccharide production provides to bacterial communities within biofilms. PMID:26185074

  17. The plasticity of extracellular fluid homeostasis in insects.

    PubMed

    Beyenbach, Klaus W

    2016-09-01

    In chemistry, the ratio of all dissolved solutes to the solution's volume yields the osmotic concentration. The present Review uses this chemical perspective to examine how insects deal with challenges to extracellular fluid (ECF) volume, solute content and osmotic concentration (pressure). Solute/volume plots of the ECF (hemolymph) reveal that insects tolerate large changes in all three of these ECF variables. Challenges beyond those tolerances may be 'corrected' or 'compensated'. While a correction simply reverses the challenge, compensation accommodates the challenge with changes in the other two variables. Most insects osmoregulate by keeping ECF volume and osmotic concentration within a wide range of tolerance. Other insects osmoconform, allowing the ECF osmotic concentration to match the ambient osmotic concentration. Aphids are unique in handling solute and volume loads largely outside the ECF, in the lumen of the gut. This strategy may be related to the apparent absence of Malpighian tubules in aphids. Other insects can suspend ECF homeostasis altogether in order to survive extreme temperatures. Thus, ECF homeostasis in insects is highly dynamic and plastic, which may partly explain why insects remain the most successful class of animals in terms of both species number and biomass.

  18. Substrate dependent production of extracellular biosurfactant by a marine bacterium.

    PubMed

    Das, Palashpriya; Mukherjee, Soumen; Sen, Ramkrishna

    2009-01-01

    The potential of a marine microorganism to utilize different carbon substrates for the production of an extracellular biosurfactant was evaluated. Among the several carbon substrates tested for this purpose, production of the crude biosurfactant was found to be highest with glycerol (2.9+/-0.11 g L(-1)) followed by starch (2.5+/-0.11 g L(-1)), glucose (1.16+/-0.11 g L(-1)) and sucrose (0.94+/-0.07 g L(-1)). The crude biosurfactant obtained from glycerol, starch and sucrose media had significantly higher antimicrobial action than those obtained from glucose containing medium. RP-HPLC resolved the crude biosurfactants into several fractions one of which had significant antimicrobial action. The antimicrobial fraction was found in higher concentrations in biosurfactant obtained using glycerol, starch and sucrose as compared to the biosurfactants from glucose medium, thereby explaining higher antimicrobial activity. The carbon substrate was thus found to affect biosurfactant production both in a qualitative and quantitative manner.

  19. Effects of extracellular potassium diffusion on electrically coupled neuron networks

    NASA Astrophysics Data System (ADS)

    Wu, Xing-Xing; Shuai, Jianwei

    2015-02-01

    Potassium accumulation and diffusion during neuronal epileptiform activity have been observed experimentally, and potassium lateral diffusion has been suggested to play an important role in nonsynaptic neuron networks. We adopt a hippocampal CA1 pyramidal neuron network in a zero-calcium condition to better understand the influence of extracellular potassium dynamics on the stimulus-induced activity. The potassium concentration in the interstitial space for each neuron is regulated by potassium currents, Na+-K+ pumps, glial buffering, and ion diffusion. In addition to potassium diffusion, nearby neurons are also coupled through gap junctions. Our results reveal that the latency of the first spike responding to stimulus monotonically decreases with increasing gap-junction conductance but is insensitive to potassium diffusive coupling. The duration of network oscillations shows a bell-like shape with increasing potassium diffusive coupling at weak gap-junction coupling. For modest electrical coupling, there is an optimal K+ diffusion strength, at which the flow of potassium ions among the network neurons appropriately modulates interstitial potassium concentrations in a degree that provides the most favorable environment for the generation and continuance of the action potential waves in the network.

  20. LRIG1 extracellular domain: structure and function analysis.

    PubMed

    Xu, Yibin; Soo, Priscilla; Walker, Francesca; Zhang, Hui Hua; Redpath, Nicholas; Tan, Chin Wee; Nicola, Nicos A; Adams, Timothy E; Garrett, Thomas P; Zhang, Jian-Guo; Burgess, Antony W

    2015-05-22

    We have expressed and purified three soluble fragments of the human LRIG1-ECD (extracellular domain): the LRIG1-LRR (leucine-rich repeat) domain, the LRIG1-3Ig (immunoglobulin-like) domain, and the LRIG1-LRR-1Ig fragment using baculovirus vectors in insect cells. The two LRIG1 domains crystallised so that we have been able to determine the three-dimensional structures at 2.3Å resolution. We developed a three-dimensional structure for the LRIG1-ECD using homology modelling based on the LINGO-1 structure. The LRIG1-LRR domain and the LRIG1-LRR-1Ig fragment are monomers in solution, whereas the LRIG1-3Ig domain appears to be dimeric. We could not detect any binding of the LRIG1 domains or the LRIG1-LRR-1Ig fragment to the EGF receptor (EGFR), either in solution using biosensor analysis or when the EGFR was expressed on the cell surface. The FLAG-tagged LRIG1-LRR-1Ig fragment binds weakly to colon cancer cells regardless of the presence of EGFRs. Similarly, neither the soluble LRIG1-LRR nor the LRIG1-3Ig domains nor the full-length LRIG1 co-expressed in HEK293 cells inhibited ligand-stimulated activation of cell-surface EGFR.

  1. Lung protection by inhalation of exogenous solubilized extracellular matrix

    PubMed Central

    Wu, Jinglei; Ravikumar, Priya; Nguyen, Kytai T.; Hsia, Connie C. W.

    2017-01-01

    Decellularized extracellular matrix (ECM) contains complex tissue-specific components that work in concert to promote tissue repair and constructive remodeling and has been used experimentally and clinically to accelerate epithelial wound repair, leading us to hypothesize that lung-derived ECM could mitigate acute lung injury. To explore the therapeutic potential of ECM for noninvasive delivery to the lung, we decellularized and solubilized porcine lung ECM, then characterized the composition, concentration, particle size and stability of the preparation. The ECM preparation at 3.2 mg/mL with average particle size <3 μm was tested in vitro on human A549 lung epithelial cells exposed to 95% O2 for 24 hours, and in vivo by tracheal instillation or nebulization into the lungs of rats exposed intermittently or continuously to 90% O2 for a cumulative 72 hours. Our results showed that the preparation was enriched in collagen, reduced in glycosaminoglycans, and contained various bioactive molecules. Particle size was concentration-dependent. Compared to the respective controls treated with cell culture medium in vitro or saline in vivo, ECM inhalation normalized cell survival and alveolar morphology, and reduced hyperoxia-induced apoptosis and oxidative damage. This proof-of-concept study established the methodology, feasibility and therapeutic potential of exogenous solubilized ECM for pulmonary cytoprotection, possibly as an adjunct or potentiator of conventional therapy. PMID:28151947

  2. Extracellular vesicles as novel carriers for therapeutic molecules

    PubMed Central

    Yim, Nambin; Choi, Chulhee

    2016-01-01

    Extracellular vesicles (EVs) are natural carriers of biomolecules that play central roles in cell-to-cell communications. Based on this, there have been various attempts to use EVs as therapeutic drug carriers. From chemical reagents to nucleic acids, various macromolecules were successfully loaded into EVs; however, loading of proteins with high molecular weight has been huddled with several problems. Purification of recombinant proteins is expensive and time consuming, and easily results in modification of proteins due to physical or chemical forces. Also, the loading efficiency of conventional methods is too low for most proteins. We have recently proposed a new method, the so-called exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs), to overcome the limitations. Since EXPLORs are produced by actively loading of intracellular proteins into EVs using blue light without protein purification steps, we demonstrated that the EXPLOR technique significantly improves the loading and delivery efficiency of therapeutic proteins. In further in vitro and in vivo experiments, we demonstrate the potential of EXPLOR technology as a novel platform for biopharmaceuticals, by successful delivery of several functional proteins such as Cre recombinase, into the target cells. PMID:27733233

  3. Extracellular matrix content of ruptured anterior cruciate ligament tissue.

    PubMed

    Young, Kate; Samiric, Tom; Feller, Julian; Cook, Jill

    2011-08-01

    Anterior cruciate ligaments (ACLs) can rupture with simple movements, suggesting that structural changes in the ligament may reduce the loading capacity of the ligament. We aimed to investigate if proteoglycan and collagen levels were different between ruptured and non-ruptured ACLs. We also compared changes in ruptured tissue over time. During arthroscopic knee reconstruction surgery 24 ruptured ACLs were collected from participants (10 females; 14 males; mean age 24 years). Four non-ruptured ACLs were obtained from participants undergoing total knee replacement surgery (one female, three males; mean age 66 years). Western blot analysis was used to characterise core proteins of aggrecan, versican, decorin and biglycan and glycosaminoglycan assays were also conducted. Collagen levels were measured by hydroxyproline (OHPr) assays. Significantly lower levels of collagen, were found in ruptured ACL compared to non-ruptured ACL (p=0.004). Lower levels of both small and large proteoglycans were found in ruptured than non-ruptured ACLs. No correlation was found between time since rupture and proteoglycan or collagen levels. Ruptured ACLs had less collagen and proteoglycans than non-ruptured ACLs. These changes indicate either extracellular matrix protein levels were reduced prior to rupture or levels decreased immediately after rupture. It is possible that the composition and structure of ACLs that rupture are different to normal ACLs, potentially reducing the tissue's ability to withstand loading. An enhanced understanding of the aetiology of ACL injury could help identify individuals who may be predisposed to rupture.

  4. Extracellular Streptomyces lividans vesicles: composition, biogenesis and antimicrobial activity.

    PubMed

    Schrempf, Hildgund; Merling, Philipp

    2015-07-01

    We selected Streptomyces lividans to elucidate firstly the biogenesis and antimicrobial activities of extracellular vesicles that a filamentous and highly differentiated Gram-positive bacterium produces. Vesicle types range in diameter from 110 to 230 nm and 20 to 60 nm, respectively; they assemble to clusters, and contain lipids and phospholipids allowing their in situ imaging by specific fluorescent dyes. The presence of the identified secondary metabolite undecylprodigiosin provokes red fluorescence of a portion of the heterogeneous vesicle populations facilitating in vivo monitoring. Protuberances containing vesicles generate at tips, and alongside of substrate hyphae, and enumerate during late vegetative growth to droplet-like exudates. Owing to in situ imaging in the presence and absence of a green fluorescent vancomycin derivative, we conclude that protuberances comprising vesicles arise at sites with enhanced levels of peptidoglycan subunits [pentapeptide of lipid II (C55)-linked disaccharides], and reduced levels of polymerized and cross-linked peptidoglycan within hyphae. These sites correlate with enhanced levels of anionic phospholipids and lipids. Vesicles provoke pronounced damages of Aspergillus proliferans, Verticillium dahliae and induced clumping and distortion of Escherichia coli. These harmful effects are likely attributable to the action of the identified vesicular compounds including different enzyme types, components of signal transduction cascades and undecylprodigiosin. Based on our pioneering findings, we highlight novel clues with environmental implications and application potential.

  5. Antibody-Based Assays for Phenotyping of Extracellular Vesicles

    PubMed Central

    Pugholm, Lotte Hatting; Revenfeld, Anne Louise Schacht; Søndergaard, Evo Kristina Lindersson; Jørgensen, Malene Møller

    2015-01-01

    Extracellular vesicles (EVs) are a heterogeneous population of membrane-enclosed vesicles. EVs are recognized as important players in cell-to-cell communication and are described to be involved in numerous biological and pathological processes. The fact that EVs are involved in the development and progression of several diseases has formed the basis for the use of EV analysis in a clinical setting. As the interest in EVs has increased immensely, multiple techniques have been developed aiming at characterizing these vesicles. These techniques characterize different features of EVs, like the size distribution, enumeration, protein composition, and the intravesicular cargo (e.g., RNA). This review focuses on techniques that exploit the specificity and sensitivity associated with antibody-based assays to characterize the protein phenotype of EVs. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. Thus, protein profiling of EVs holds great diagnostic and prognostic potential. PMID:26770974

  6. Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation

    PubMed Central

    Kalra, Hina; Simpson, Richard J.; Ji, Hong; Aikawa, Elena; Altevogt, Peter; Askenase, Philip; Bond, Vincent C.; Borràs, Francesc E.; Breakefield, Xandra; Budnik, Vivian; Buzas, Edit; Camussi, Giovanni; Clayton, Aled; Cocucci, Emanuele; Falcon-Perez, Juan M.; Gabrielsson, Susanne; Gho, Yong Song; Gupta, Dwijendra; Harsha, H. C.; Hendrix, An; Hill, Andrew F.; Inal, Jameel M.; Jenster, Guido; Krämer-Albers, Eva-Maria; Lim, Sai Kiang; Llorente, Alicia; Lötvall, Jan; Marcilla, Antonio; Mincheva-Nilsson, Lucia; Nazarenko, Irina; Nieuwland, Rienk; Nolte-'t Hoen, Esther N. M.; Pandey, Akhilesh; Patel, Tushar; Piper, Melissa G.; Pluchino, Stefano; Prasad, T. S. Keshava; Rajendran, Lawrence; Raposo, Graca; Record, Michel; Reid, Gavin E.; Sánchez-Madrid, Francisco; Schiffelers, Raymond M.; Siljander, Pia; Stensballe, Allan; Stoorvogel, Willem; Taylor, Douglas; Thery, Clotilde; Valadi, Hadi; van Balkom, Bas W. M.; Vázquez, Jesús; Vidal, Michel; Wauben, Marca H. M.; Yáñez-Mó, María; Zoeller, Margot; Mathivanan, Suresh

    2012-01-01

    Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field. PMID:23271954

  7. The role for neutrophil extracellular traps in cystic fibrosis autoimmunity

    PubMed Central

    Skopelja, Sladjana; Hamilton, B. JoNell; Jones, Jonathan D.; Yang, Mei-Ling; Mamula, Mark; Ashare, Alix; Gifford, Alex H.; Rigby, William F.C.

    2016-01-01

    While respiratory failure in cystic fibrosis (CF) frequently associates with chronic infection by Pseudomonas aeruginosa, no single factor predicts the extent of lung damage in CF. To elucidate other causes, we studied the autoantibody profile in CF and rheumatoid arthritis (RA) patients, given the similar association of airway inflammation and autoimmunity in RA. Even though we observed that bactericidal permeability-increasing protein (BPI), carbamylated proteins, and citrullinated proteins all localized to the neutrophil extracellular traps (NETs), which are implicated in the development of autoimmunity, our study demonstrates striking autoantibody specificity in CF. Particularly, CF patients developed anti-BPI autoantibodies but hardly any anti-citrullinated protein autoantibodies (ACPA). In contrast, ACPA-positive RA patients exhibited no reactivity with BPI. Interestingly, anti-carbamylated protein autoantibodies (ACarPA) were found in both cohorts but did not cross-react with BPI. Contrary to ACPA and ACarPA, anti-BPI autoantibodies recognized the BPI C-terminus in the absence of posttranslational modifications. In fact, we discovered that P. aeruginosa–mediated NET formation results in BPI cleavage by P. aeruginosa elastase, which suggests a novel mechanism in the development of autoimmunity to BPI. In accordance with this model, autoantibodies associated with presence of P. aeruginosa on sputum culture. Finally, our results provide a role for autoimmunity in CF disease severity, as autoantibody levels associate with diminished lung function. PMID:27777975

  8. Extracellular Cyclophilins Contribute to the Regulation of Inflammatory Responses1

    PubMed Central

    Arora, Kamalpreet; Gwinn, William M.; Bower, Molly A.; Watson, Alan; Okwumabua, Ifeanyi; MacDonald, H. Robson; Bukrinsky, Michael I.; Constant, Stephanie L.

    2010-01-01

    The main regulators of leukocyte trafficking during inflammatory responses are chemokines. However, another class of recently identified chemotactic agents is extracellular cyclophilins, the proteins mostly known as receptors for the immunosuppressive drug, cyclosporine A. Cyclophilins can induce leukocyte chemotaxis in vitro and have been detected at elevated levels in inflamed tissues, suggesting that they might contribute to inflammatory responses. We recently identified CD147 as the main signaling receptor for cyclophilin A. In the current study we examined the contribution of cyclophilin-CD147 interactions to inflammatory responses in vivo using a mouse model of acute lung injury. Blocking cyclophilin-CD147 interactions by targeting CD147 (using anti-CD147 Ab) or cyclophilin (using nonimmunosuppressive cyclosporine A analog) reduced tissue neutrophilia by up to 50%, with a concurrent decrease in tissue pathology. These findings are the first to demonstrate the significant contribution of cyclophilins to inflammatory responses and provide a potentially novel approach for reducing inflammation-mediated diseases. PMID:15972687

  9. Solubilization of leonardite by an extracellular fraction from Coriolus versicolor

    SciTech Connect

    Pyne, J.W. Jr.; Stewart, D.L.; Fredrickson, J.; Wilson, B.W.

    1987-12-01

    Coriolus versicolor has previously been shown to degrade leonardite, an oxidized form of lignite. An extracellular fraction containing protein purified from a C. versicolor culture solubilized leonardite in vitro. Expression of the activity did not require the presence of leonardite and appeared during idiophase. During ion-exchange and gel filtration column chromatography, leonardite-biosolubilizing activity eluted with syringaldazine oxidase activity and with protein, as measured by A//sub 280/ and the biuret protein assay. Syringaldazine is a substrate of the polyphenol oxidase formed by C. versicolor. Comparison of leonardite-biosolubilizing activity with the effects of chelators and surface-active agents on leonardite showed that biosolubilization was not due to either surfactant or chelating ability. Heat treatment of the preparation at 60/sup 0/C for 30 min significantly reduced both syringaldazine oxidase and leonardite-biosolubilizing activities. Cyanide, azide, and thioglycolate, which are know inhibitors of syringaldazine oxidase activity of C. versicolor, also inhibited leonardite biosolubilization. From these data, we conclude that the purified protein fraction from C. versicolor contains a syringaldazine oxidase activity that participates in leonardite biosolubilization by enzymatic action.

  10. LRP1 regulates remodeling of the extracellular matrix by fibroblasts

    PubMed Central

    Gaultier, Alban; Hollister, Margaret; Reynolds, Irene; Hsieh, En-hui; Gonias, Steven L.

    2009-01-01

    Low density lipoprotein receptor-related protein (LRP1) is an endocytic receptor for diverse proteases, protease inhibitors, and other plasma membrane proteins, including the urokinase receptor (uPAR). LRP1 also functions in cell-signaling and regulates gene expression. The goal of this study was to determine whether LRP1 regulates remodeling of provisional extracellular matrix (ECM) by fibroblasts. To address this problem, we utilized an in vitro model in which type I collagen was reconstituted and overlaid with fibronectin. Either the collagen or fibronectin was fluorescently-labeled. ECM remodeling by fibroblasts deficient in LRP1, uPAR, or MT1-MMP was studied. MT1-MMP was required for efficient remodeling of the deep collagen layer but not involved in fibronectin remodeling. Instead, fibronectin was remodeled by a system that required urokinase-type plasminogen activator (uPA), uPAR, and exogenously-added plasminogen. LRP1 markedly inhibited fibronectin remodeling by regulating cell-surface uPAR and plasminogen activation. LRP1 also regulated remodeling of the deep collagen layer but not by controlling MT1-MMP. Instead, LRP1 deficiency or inhibition de-repressed a secondary pathway for collagen remodeling, which was active in MT1-MMP-deficient cells but not in uPAR-deficient cells. These results demonstrate that LRP1 regulates ECM remodeling principally by repressing pathways that require plasminogen activation by uPA in association with uPAR. PMID:19699300

  11. Evasion of Neutrophil Extracellular Traps by Respiratory Pathogens.

    PubMed

    Storisteanu, Daniel M L; Pocock, Joanna M; Cowburn, Andrew S; Juss, Jatinder K; Nadesalingam, Angalee; Nizet, Victor; Chilvers, Edwin R

    2017-04-01

    The release of neutrophil extracellular traps (NETs) is a major immune mechanism intended to capture pathogens. These histone- and protease-coated DNA structures are released by neutrophils in response to a variety of stimuli, including respiratory pathogens, and have been identified in the airways of patients with respiratory infection, cystic fibrosis, acute lung injury, primary graft dysfunction, and chronic obstructive pulmonary disease. NET production has been demonstrated in the lungs of mice infected with Staphylococcus aureus, Klebsiella pneumoniae, and Aspergillus fumigatus. Since the discovery of NETs over a decade ago, evidence that "NET evasion" might act as an immune protection strategy among respiratory pathogens, including group A Streptococcus, Bordetella pertussis, and Haemophilus influenzae, has been growing, with the majority of these studies being published in the past 2 years. Evasion strategies fall into three main categories: inhibition of NET release by down-regulating host inflammatory responses; degradation of NETs using pathogen-derived DNases; and resistance to the microbicidal components of NETs, which involves a variety of mechanisms, including encapsulation. Hence, the evasion of NETs appears to be a widespread strategy to allow pathogen proliferation and dissemination, and is currently a topic of intense research interest. This article outlines the evidence supporting the three main strategies of NET evasion-inhibition, degradation, and resistance-with particular reference to common respiratory pathogens.

  12. Extracellular Membrane Vesicles and Immune Regulation in the Brain

    PubMed Central

    Cossetti, Chiara; Smith, Jayden A.; Iraci, Nunzio; Leonardi, Tommaso; Alfaro-Cervello, Clara; Pluchino, Stefano

    2012-01-01

    The brain is characterized by a complex and integrated network of interacting cells in which cell-to-cell communication is critical for proper development and function. Initially considered as an immune privileged site, the brain is now regarded as an immune specialized system. Accumulating evidence reveals the presence of immune components in the brain, as well as extensive bidirectional communication that takes place between the nervous and the immune system both under homeostatic and pathological conditions. In recent years the secretion of extracellular membrane vesicles (EMVs) has been described as a new and evolutionary well-conserved mechanism of cell-to-cell communication, with EMVs influencing the microenvironment through the traffic of bioactive molecules that include proteins and nucleic acids, such as DNA, protein coding, and non-coding RNAs. Increasing evidence suggests that EMVs are a promising candidate to study cross-boundary cell-to-cell communication pathways. Herein we review the role of EMVs secreted by neural cells in modulating the immune response(s) within the brain under physiological and pathological circumstances. PMID:22557978

  13. Tolerance in Organ Transplantation: From Conventional Immunosuppression to Extracellular Vesicles

    PubMed Central

    Monguió-Tortajada, Marta; Lauzurica-Valdemoros, Ricardo; Borràs, Francesc E.

    2014-01-01

    Organ transplantation is often the unique solution for organ failure. However, rejection is still an unsolved problem. Although acute rejection is well controlled, the chronic use of immunosuppressive drugs for allograft acceptance causes numerous side effects in the recipient and do not prevent chronic allograft dysfunction. Different alternative therapies have been proposed to replace the classical treatment for allograft rejection. The alternative therapies are mainly based in pre-infusions of different types of regulatory cells, including DCs, MSCs, and Tregs. Nevertheless, these approaches lack full efficiency and have many problems related to availability and applicability. In this context, the use of extracellular vesicles, and in particular exosomes, may represent a cell-free alternative approach in inducing transplant tolerance and survival. Preliminary approaches in vitro and in vivo have demonstrated the efficient alloantigen presentation and immunomodulation abilities of exosomes, leading to alloantigen-specific tolerance and allograft acceptance in rodent models. Donor exosomes have been used alone, processed by recipient antigen-presenting cells, or administered together with suboptimal doses of immunosuppressive drugs, achieving specific allograft tolerance and infinite transplant survival. In this review, we gathered the latest exosome-based strategies for graft acceptance and discuss the tolerance mechanisms involved in organ tolerance mediated by the administration of exosomes. We will also deal with the feasibility and difficulties that arise from the application of this strategy into the clinic. PMID:25278936

  14. Extracellular Vesicles as Therapeutic Tools in Cardiovascular Diseases

    PubMed Central

    Fleury, Audrey; Martinez, Maria Carmen; Le Lay, Soazig

    2014-01-01

    Extracellular vesicles (EVs), including microvesicles (MVs) and exosomes, are small vesicles secreted from a wide variety of cells. Whereas MVs are particles released by the outward budding of the plasma membrane, exosomes are derived from endocytic compartments. Secretion of EVs can be enhanced by specific stimuli, and increased plasma circulating levels of EVs have been correlated with pathophysiological situations. MVs, already present in the blood of healthy individuals, are considerably elevated in several cardiovascular diseases associated with inflammation, suggesting that they can mediate deleterious effects such as endothelial dysfunction or thrombosis. Nonetheless, very recent studies also demonstrate that MVs may act as biological information vectors transferring proteins or genetic material to maintain cell homeostasis, favor cell repair, or even promote angiogenesis. Additionally, exosomes have also been shown to have pro-angiogenic and cardio-protective properties. These beneficial effects, therefore, reveal the potential therapeutical use of EVs in the field of