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Sample records for pasteurella hyaluronan synthase

  1. Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

    SciTech Connect

    Nishida, Yoshihiro . E-mail: ynishida@med.nagoya-u.ac.jp; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    2005-07-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.

  2. Tissue distribution and subcellular localization of hyaluronan synthase isoenzymes.

    PubMed

    Törrönen, Kari; Nikunen, Kaisa; Kärnä, Riikka; Tammi, Markku; Tammi, Raija; Rilla, Kirsi

    2014-01-01

    Hyaluronan synthases (HAS) are unique plasma membrane glycosyltransferases secreting this glycosaminoglycan directly to the extracellular space. The three HAS isoenzymes (HAS1, HAS2, and HAS3) expressed in mammalian cells differ in their enzymatic properties and regulation by external stimuli, but clearly distinct functions have not been established. To overview the expression of different HAS isoenzymes during embryonic development and their subcellular localization, we immunostained mouse embryonic samples and cultured cells with HAS antibodies, correlating their distribution to hyaluronan staining. Their subcellular localization was further studied by GFP-HAS fusion proteins. Intense hyaluronan staining was observed throughout the development in the tissues of mesodermal origin, like heart and cartilages, but also for example during the maturation of kidneys and stratified epithelia. In general, staining for one or several HASs correlated with hyaluronan staining. The staining of HAS2 was most widespread, both spatially and temporally, correlating with hyaluronan staining especially in early mesenchymal tissues and heart. While epithelial cells were mostly negative for HASs, stratified epithelia became HAS positive during differentiation. All HAS isoenzymes showed cytoplasmic immunoreactivity, both in tissue sections and cultured cells, while plasma membrane staining was also detected, often in cellular extensions. HAS1 had brightest signal in Golgi, HAS3 in Golgi and microvillous protrusions, whereas most of the endogenous HAS2 immunoreactivity was localized in the ER. This differential pattern was also observed with transfected GFP-HASs. The large proportion of intracellular HASs suggests that HAS forms a reserve that is transported to the plasma membrane for rapid activation of hyaluronan synthesis.

  3. Expression of human hyaluronan synthases in response to external stimuli.

    PubMed Central

    Jacobson, A; Brinck, J; Briskin, M J; Spicer, A P; Heldin, P

    2000-01-01

    In the present study we have investigated the expression of mRNAs for hyaluronan synthase isoforms (HAS1, HAS2 and HAS3) in different cells in response to various stimuli. Human mesothelial cells, which synthesize large amounts of hyaluronan, express mRNAs encoding all three HAS isoforms, whereas their transformed counterparts, mesothelioma cells, which produce only minute amounts of hyaluronan, express only HAS3 mRNA. Human lung fibroblasts and the glioma cell line U-118 MG express only the HAS2 and HAS3 genes. The expression of the transcripts was higher in subconfluent than in confluent cultures and was well correlated with the production of hyaluronan by the cells. Stimulation of mesothelial cells with platelet-derived growth factor-BB induced an up-regulation of mRNA for HAS2 to a maximum after 6 h of stimulation; HAS1 and HAS3 genes were only induced slightly. Transforming growth factor-beta1 reduced HAS2 mRNA slightly, and hydrocortisone reduced it strongly, within 6 h of stimulation in mesothelial cell cultures but did not significantly affect the expression of mRNAs for HAS1 and HAS3. Induction of HAS1 and HAS2 protein levels in response to the stimuli above correlated with HAS transcript levels. Thus the expression of the three HAS isoforms is more prominent in growing cells than in resting cells and is differentially regulated by various stimuli suggesting distinct functional roles of the three proteins. PMID:10794710

  4. Modulation of hyaluronan synthase activity in cellular membrane fractions.

    PubMed

    Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

    2009-10-30

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

  5. Chromosomal localization of the human and mouse hyaluronan synthase genes

    SciTech Connect

    Spicer, A.P.; McDonald, J.A.; Seldin, M.F.

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  6. SEC-MALLS ANALYSIS OF HYALURONAN SIZE DISTRIBUTIONS MADE BY MEMBRANE-BOUND HYALURONAN SYNTHASE

    PubMed Central

    Baggenstoss, Bruce A.; Weigel, Paul H.

    2006-01-01

    SEC-MALLS analyses of E. coli membranes expressing Streptococcus equisimilis hyaluronan synthase (seHAS) demonstrated an inherent artifact (10–100 MDa) that co-eluted with HA, and skewed the apparent weight-average mass of HA to erroneously high values. Briefly heating samples to 65–75°C eliminated this artifact and increased the yield of recovered HA, due to the release of HA chains that were attached to membrane-bound HAS. Inclusion of alkaline phosphatase, which removed UDP produced during the reaction, improved the linearity of HA synthesis - even at high substrate utilization. Surprisingly, addition of EDTA, to chelate Mg+2 ions, did not completely stop the HAS reaction at 30°C or at 4°C. The best conditions for stopping the reaction without altering SEC-MALLS profiles of the product HA were to chill samples on ice in the presence of both EDTA and UDP. Even with excess substrate, the maximum size of product HA decreased as the enzyme concentration increased. Therefore, the maximum HA size made by HAS was determined by extrapolation to zero enzyme concentration. Using the above conditions, membrane-bound seHAS synthesized a cohort of HA products that steadily increased in weight-average molar mass, reaching a final maximal steady-state size of 4–6 MDa within 2–4 hours. PMID:16476403

  7. Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis.

    PubMed

    Jokela, Tiina; Kärnä, Riikka; Rauhala, Leena; Bart, Genevieve; Pasonen-Seppänen, Sanna; Oikari, Sanna; Tammi, Markku I; Tammi, Raija H

    2017-03-24

    The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 μm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

  8. CHARACTERIZATION OF THE PURIFIED HYALURONAN SYNTHASE FROM STREPTOCOCCUS EQUISIMILIS*

    PubMed Central

    Tlapak-Simmons, Valarie L.; Baron, Christina A.; Weigel, Paul H.

    2006-01-01

    Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg2+ to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nucleotides and UDP-sugars inhibited enzyme activity, including UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GalUA, UMP, UDP and UTP. Purified seHAS was ~40% more active in 25 mM, compared to 50 mM, PO4 in the presence of either 50 mM NaCl or KCl, and displayed a slight preference for KCl over NaCl. The pH profile was surprisingly broad, with an effective range of pH 6.5–11.5 and the optimum between pH 9 and 10. SeHAS displayed two apparent pKa values at pH 6.6 and 11.8. As the pH was increased from ~6.5, both Km and Vmax increased until pH ~10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/sec) was essentially unchanged from pH 6.5 to pH 10.5. The enzyme is temperature labile, but more stable in the presence of substrate and cardiolipin. Purified seHAS requires exogenous cardiolipin for activity and is very sensitive to the fatty acyl composition of the phospholipid. The enzyme was inactive or highly activated by synthetic cardiolipins containing, respectively, C14:0 or C18:1(Δ9) fatty acids. The apparent Ea for HA synthesis is 40 kJ (9.5 kcal/mol) disaccharide. Increasing the viscosity by increasing concentrations of PEG, ethylene glycol, glycerol, or sucrose inhibited seHAS activity. For PEGs, the extent of inhibition was proportional to their molecular mass. PEGs with average masses of 2.7, 11.7, and 20 Kg/mol caused 50% inhibition of Vmax at 21, 6.5, and 3.5 mM, respectively. The apparent Ki values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3 and 1.2 mM. PMID:15248781

  9. Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

    SciTech Connect

    Takabe, Piia; Bart, Geneviève; Ropponen, Antti; Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

    2015-09-10

    Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma.

  10. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

    SciTech Connect

    Kultti, Anne; Pasonen-Seppaenen, Sanna; Jauhiainen, Marjo; Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I.

    2009-07-01

    Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

  11. Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion.

    PubMed

    Takabe, Piia; Bart, Geneviève; Ropponen, Antti; Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

    2015-09-10

    Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells.

  12. Size exclusion chromatography-multiangle laser light scattering analysis of hyaluronan size distributions made by membrane-bound hyaluronan synthase.

    PubMed

    Baggenstoss, Bruce A; Weigel, Paul H

    2006-05-15

    Size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analyses of Escherichia coli membranes expressing Streptococcus equisimilis hyaluronan synthase (seHAS) demonstrated an inherent artifact (10-100 MDa) that coeluted with hyaluronan (HA) and skewed the apparent weight-average mass of HA to erroneously high values. Briefly heating samples to 65-75 degrees C eliminated this artifact and increased the yield of recovered HA due to the release of HA chains that were attached to membrane-bound HAS. Inclusion of alkaline phosphatase, which removed uridine 5'-diphosphate (UDP) produced during the reaction, improved the linearity of HA synthesis-even at high substrate use. Surprisingly, the addition of EDTA, to chelate Mg(2+) ions, did not completely stop the HAS reaction at 30 degrees C or at 4 degrees C. The best conditions for stopping the reaction without altering SEC-MALLS profiles of the product HA were to chill samples on ice in the presence of both EDTA and UDP. Even with excess substrate, the maximum size of product HA decreased as the enzyme concentration increased. Therefore, the maximum HA size made by HAS was determined by extrapolation to zero enzyme concentration. Using the above conditions, membrane-bound seHAS synthesized a cohort of HA products that steadily increased in weight-average molar mass, reaching a final maximal steady-state size of 4 to 6 MDa within 2-4 h.

  13. 4-Methylumbelliferone inhibits the phosphorylation of hyaluronan synthase 2 induced by 12-O-tetradecanoyl-phorbol-13-acetate.

    PubMed

    Kuroda, Yoshiyuki; Kasai, Kosuke; Nanashima, Naoki; Nozaka, Hiroyuki; Nakano, Manabu; Chiba, Mitsuru; Yoneda, Masahiko; Nakamura, Toshiya

    2013-04-01

    The effect of 4-methylumbelliferone (MU), a hyaluronan synthase-suppressor, on O-linked β-Nacetylglucosaminylation (O-GlcNAcylation) was investigated in cultured human skin fibroblasts, and we found that MU stimulated O-GlcNAcylation of the cellular proteins. Since O-GlcNAcylation affects protein phosphorylation via Ser/Thr kinases, we examined the effect of MU on both the phosphorylation of hyaluronan synthase 2 (HAS2) and hyaluronan production. The cells were cultured in the presence or absence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and MU independently or in combination. The protein fraction of each cell culture was extracted and divided into 2 parts-phosphorylated and non-phosphorylated fractions-by immobilized metal-affinity chromatography. The hyaluronan level in the medium was determined by an ELISA-like assay. Addition of MU decreased the level of hyaluronan in the medium and that of HAS2 in the phosphorylated protein fraction. On the contrary, the addition of TPA increased the levels of both of them. Interestingly, the combination of TPA and MU lowered the levels of them in treated cells as compared to those in untreated control cells. These results suggest that TPA activated protein kinase C (PKC), which stimulates the phosphorylation of HAS2, and increased hyaluronan production. Further, MU may inhibit the phosphorylation of HAS2 by PKC through the stimulation of O-GlcNAcylation.

  14. Low Dose Ultraviolet B Irradiation Increases Hyaluronan Synthesis in Epidermal Keratinocytes via Sequential Induction of Hyaluronan Synthases Has1–3 Mediated by p38 and Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Signaling*

    PubMed Central

    Rauhala, Leena; Hämäläinen, Lasse; Salonen, Pauliina; Bart, Geneviève; Tammi, Markku; Pasonen-Seppänen, Sanna; Tammi, Raija

    2013-01-01

    Hyaluronan, a major epidermal extracellular matrix component, responds strongly to different kinds of injuries. This also occurs by UV radiation, but the mechanisms involved are poorly understood. The effects of a single ultraviolet B (UVB) exposure on hyaluronan content and molecular mass, and expression of genes involved in hyaluronan metabolism were defined in monolayer and differentiated, organotypic three-dimensional cultures of rat epidermal keratinocytes. The signals regulating the response were characterized using specific inhibitors and Western blotting. In monolayer cultures, UVB increased hyaluronan synthase Has1 mRNA already 4 h postexposure, with a return to control level by 24 h. In contrast, Has2 and Has3 were persistently elevated from 8 h onward. Silencing of Has2 and especially Has3 decreased the UVB-induced accumulation of hyaluronan. p38 and Ca2+/calmodulin-dependent protein kinase II pathways were found to be involved in the UVB-induced up-regulation of Has2 and Has3 expression, respectively, and their inhibition reduced hyaluronan deposition. However, the expressions of the hyaluronan-degrading enzymes Hyal1 and Hyal2 and the hyaluronan receptor Cd44 were also up-regulated by UVB. In organotypic cultures, UVB treatment also resulted in increased expression of both Has and Hyal genes and shifted hyaluronan toward a smaller size range. Histochemical stainings indicated localized losses of hyaluronan in the epidermis. The data show that exposure of keratinocytes to acute, low dose UVB increases hyaluronan synthesis via up-regulation of Has2 and Has3. The simultaneously enhanced catabolism of hyaluronan demonstrates the complexity of the UVB-induced changes. Nevertheless, enhanced hyaluronan metabolism is an important part of the adaptation of keratinocytes to radiation injury. PMID:23645665

  15. Hyaluronan in human malignancies

    SciTech Connect

    Sironen, R.K.; Tammi, M.; Tammi, R.; Auvinen, P.K.; Anttila, M.; Kosma, V-M.

    2011-02-15

    Hyaluronan, a major macropolysaccharide in the extracellular matrix of connective tissues, is intimately involved in the biology of cancer. Hyaluronan accumulates into the stroma of various human tumors and modulates intracellular signaling pathways, cell proliferation, motility and invasive properties of malignant cells. Experimental and clinicopathological evidence highlights the importance of hyaluronan in tumor growth and metastasis. A high stromal hyaluronan content is associated with poorly differentiated tumors and aggressive clinical behavior in human adenocarcinomas. Instead, the squamous cell carcinomas and malignant melanomas tend to have a reduced hyaluronan content. In addition to the stroma-cancer cell interaction, hyaluronan can influence stromal cell recruitment, tumor angiogenesis and epithelial-mesenchymal transition. Hyaluronan receptors, hyaluronan synthases and hyaluronan degrading enzymes, hyaluronidases, are involved in the modulation of cancer progression, depending on the tumor type. Furthermore, intracellular signaling and angiogenesis are affected by the degradation products of hyaluronan. Hyaluronan has also therapeutic implications since it is involved in multidrug resistance.

  16. Amelioration of osteoarthritis by intra-articular hyaluronan synthase 2 gene therapy.

    PubMed

    Zhang, Da-Wei; Yang, Quan-Sheng; Zhu, Jin-Yu; Cao, Xiao-Rui; Li, Li-Wen; Zhu, Qing-Sheng

    2007-01-01

    Osteoarthritis (OA) is a chronic, degenerative disorder of multifactorial aetiology, characterized by loss of articular cartilage and periarticular bone remodelling. Goals of managing OA include controlling pain, maintaining and improving function and health-related quality of life, and limiting functional impairment. Although several managements had been proved to ameliorate the symptoms of osteoarthritis, no methods could cure it thoroughly. High-molecular-weight hyaluronan (HMW-HA) is a major component of synovial joint fluids which physically acts as a viscous lubricant for slow joint movements and as an elastic shock absorber during rapid movements. It also has a variety of biologic effects in vivo, such as inhibiting the release of inflammatory factors and suppressing the degradation of cartilage matrix. Intra-articular injection of synthetic HMW-HA has been used as viscosupplement for knee OA and its therapeutic efficacy has been verified. However, repeated injections of HMW-HA which is needed to control symptoms increase the probability of infection and sometimes there will have acute joint pain with effusion, which requires aspiration to exclude sepsis. In order to overcome the disadvantages of repeated injections of HMW-HA, novel strategies should be developed. As HMW-HA is synthesized by hyaluronan synthase-2 (HAS2), we postulate that HAS2 gene could be delivered into intra-articular cells by methods of gene therapy to achieve long-lasting synthesis of HMW-HA. In our opinion, this strategy seems to hold interesting future prospects for the treatment of OA.

  17. Potential impact of a single nucleotide polymorphism in the hyaluronan synthase 1 gene in Waldenstrom's macroglobulinemia.

    PubMed

    Adamia, Sophia; Treon, Steven P; Reiman, Tony; Tournilhac, Olivier; McQuarrie, Carrie; Mant, Michael J; Belch, Andrew R; Pilarski, Linda M

    2005-03-01

    The hyaluronan synthase 1 (HAS1) gene encodes a plasma membrane protein that synthesizes hyaluronan, an extracellular matrix molecule. Previously, in patients with Waldenstrom's macroglobulinemia (WM), we detected upregulation of HAS1 transcripts and identified aberrant splice variants of this gene. Aberrant splicing of HAS1 results from activation of cryptic splice sites. In turn, activation of cryptic donor and acceptor splice sites can be promoted by mutations occurring upstream of these sites and/or at the branch point of slicing. We measured the frequency of the HAS1 833A/G polymorphism (ie, single-nucleotide polymorphism; SNP) in patients with WM and healthy donors. Additionally, HAS1 gene expression was evaluated in the same group of patients. Our observations so far suggest that HAS1 833A/G SNPs contribute to aberrant splicing of this gene; this idea is supported by the fact that 833A/G SNP is located on an exonic splicing enhancer motif. Based on the results obtained thus far, we speculate that individuals with HAS1 833G/G genotype are predisposed toward aberrant HAS1 splicing and expression of HAS1 variants, resulting in an enhanced risk of developing WM. Study of a larger group of patients and healthy donors is needed to confirm these speculations and to evaluate the prognostic significance of these findings.

  18. Hyaluronan Synthase 3 Variant and Anthracycline-Related Cardiomyopathy: A Report From the Children's Oncology Group

    PubMed Central

    Wang, Xuexia; Liu, Wei; Sun, Can-Lan; Armenian, Saro H.; Hakonarson, Hakon; Hageman, Lindsey; Ding, Yan; Landier, Wendy; Blanco, Javier G.; Chen, Lu; Quiñones, Adolfo; Ferguson, Daniel; Winick, Naomi; Ginsberg, Jill P.; Keller, Frank; Neglia, Joseph P.; Desai, Sunil; Sklar, Charles A.; Castellino, Sharon M.; Cherrick, Irene; Dreyer, ZoAnn E.; Hudson, Melissa M.; Robison, Leslie L.; Yasui, Yutaka; Relling, Mary V.; Bhatia, Smita

    2014-01-01

    Purpose The strong dose-dependent association between anthracyclines and cardiomyopathy is further exacerbated by the co-occurrence of cardiovascular risk factors (diabetes and hypertension). The high morbidity associated with cardiomyopathy necessitates an understanding of the underlying pathogenesis so that targeted interventions can be developed. Patients and Methods By using a two-stage design, we investigated host susceptibility to anthracycline-related cardiomyopathy by using the ITMAT/Broad CARe cardiovascular single nucleotide polymorphism (SNP) array to profile common SNPs in 2,100 genes considered relevant to de novo cardiovascular disease. Results By using a matched case-control design (93 cases, 194 controls), we identified a common SNP, rs2232228, in the hyaluronan synthase 3 (HAS3) gene that exerts a modifying effect on anthracycline dose-dependent cardiomyopathy risk (P = 5.3 × 10−7). Among individuals with rs2232228 GG genotype, cardiomyopathy was infrequent and not dose related. However, in individuals exposed to high-dose (> 250 mg/m2) anthracyclines, the rs2232228 AA genotype conferred an 8.9-fold (95% CI, 2.1- to 37.5-fold; P = .003) increased cardiomyopathy risk compared with the GG genotype. This gene-environment interaction was successfully replicated in an independent set of 76 patients with anthracycline-related cardiomyopathy. Relative HAS3 mRNA levels measured in healthy hearts tended to be lower among individuals with AA compared with GA genotypes (P = .09). Conclusion Hyaluronan (HA) produced by HAS3 is a ubiquitous component of the extracellular matrix and plays an active role in tissue remodeling. In addition, HA is known to reduce reactive oxygen species (ROS) –induced cardiac injury. The high cardiomyopathy risk associated with AA genotype could be due to inadequate remodeling and/or inadequate protection of the heart from ROS-mediated injury on high anthracycline exposure. PMID:24470002

  19. Hyaluronan Synthase 3 Null Mice Exhibit Decreased Intestinal Inflammation and Tissue Damage in the DSS-Induced Colitis Model

    PubMed Central

    Kessler, Sean P.; Obery, Dana R.; de la Motte, Carol

    2015-01-01

    Hyaluronan (HA) overproduction is a hallmark of multiple inflammatory diseases, including inflammatory bowel disease (IBD). Hyaluronan can act as a leukocyte recruitment molecule and in the most common mouse model of intestinal inflammation, the chemically induced dextran sodium sulfate (DSS) experimental colitis model, we previously determined that changes in colon distribution of HA occur before inflammation. Therefore, we hypothesized that, during a pathologic challenge, HA promotes inflammation. In this study, we tested the progression of inflammation in mice null for the hyaluronan synthase genes (HAS1, HAS3, or both HAS1 and HAS3) in the DSS-colitis model. Our data demonstrate that both the HAS1/HAS3 double and the HAS3 null mice are protected from colitis, compared to wild-type and HAS1 null mice, as determined by measurement of weight loss, disease activity, serum IL-6 levels, histologic scoring, and immunohistochemistry. Most notable is the dramatic increase in submucosal microvasculature, hyaluronan deposition, and leukocyte infiltration in the inflamed colon tissue of wild-type and HAS1 null mice. Our data suggest, HAS3 plays a crucial role in driving gut inflammation. Developing a temporary targeted therapeutic intervention of HAS3 expression or function in the microcirculation may emerge as a desirable strategy toward tempering colitis in patients undergoing flares of IBD. PMID:26448758

  20. Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts*

    PubMed Central

    Nagaoka, Aya; Yoshida, Hiroyuki; Nakamura, Sachiko; Morikawa, Tomohiko; Kawabata, Keigo; Kobayashi, Masaki; Sakai, Shingo; Takahashi, Yoshito; Okada, Yasunori; Inoue, Shintaro

    2015-01-01

    Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients. PMID:26518873

  1. Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts.

    PubMed

    Nagaoka, Aya; Yoshida, Hiroyuki; Nakamura, Sachiko; Morikawa, Tomohiko; Kawabata, Keigo; Kobayashi, Masaki; Sakai, Shingo; Takahashi, Yoshito; Okada, Yasunori; Inoue, Shintaro

    2015-12-25

    Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients.

  2. Differential regulation and expression of hyaluronan synthases in human articular chondrocytes, synovial cells and osteosarcoma cells.

    PubMed Central

    Recklies, A D; White, C; Melching, L; Roughley, P J

    2001-01-01

    Recently three isoforms of hyaluronan synthase (HAS), the enzyme responsible for hyaluronate/hyaluronan (HA) biosynthesis, have been cloned, allowing us to study their expression pattern. Our objective was to determine which of the HAS isoenzymes were expressed in human articular chondrocytes, synovial fibroblasts and osteosarcoma cells, whether their expression could be modulated by growth factors (insulin-like growth factor-1, basic fibroblast growth factor and transforming growth factor (TGF-beta1) and cytokines [interleukin 1beta1 (IL-1beta)], and whether changes in the rate of HA synthesis by the cells correlated with changes in mRNA levels for one or more of the HAS isoforms. All three HAS isoforms were found to be expressed in the cultured cells analysed in this study, although the relative proportions varied for each cell type. HAS2 mRNA was usually predominant in chondrocytes, whereas synovial cells contained increased amounts of HAS1. HAS3 was always the least abundant message. The rapidly growing osteosarcoma cells contained almost exclusively HAS2 message. HAS usage in uncultured cartilage and synovial tissues was similar to that in the cultured cells, with HAS2 message being the predominant species in cartilage and HAS1 usually being the predominant species in synovium. HA synthesis was stimulated by the growth factors, but the extent of the response was cell-type specific. Synovial cells responded particularly well to IL-1beta, and showed a unique synergistic response when IL-1beta was used in combination with TGF-beta1. This response was much reduced in articular chondrocytes and absent in the osteosarcoma cells. Analysis of changes in HAS message levels indicated that there was often no correlation with the changes in HA secretion following exposure to growth factors. Although HAS-1 mRNA was increased in synovial cells after exposure to TGF-beta1/IL-1beta, the magnitude of the change was far less than the effect on HA synthesis. Our data thus

  3. Molecular evolution of the hyaluronan synthase 2 gene in mammals: implications for adaptations to the subterranean niche and cancer resistance

    PubMed Central

    Faulkes, Christopher G.; Davies, Kalina T. J.; Rossiter, Stephen J.; Bennett, Nigel C.

    2015-01-01

    The naked mole-rat (NMR) Heterocephalus glaber is a unique and fascinating mammal exhibiting many unusual adaptations to a subterranean lifestyle. The recent discovery of their resistance to cancer and exceptional longevity has opened up new and important avenues of research. Part of this resistance to cancer has been attributed to the fact that NMRs produce a modified form of hyaluronan—a key constituent of the extracellular matrix—that is thought to confer increased elasticity of the skin as an adaptation for living in narrow tunnels. This so-called high molecular mass hyaluronan (HMM-HA) stems from two apparently unique substitutions in the hyaluronan synthase 2 enzyme (HAS2). To test whether other subterranean mammals with similar selection pressures also show molecular adaptation in their HAS2 gene, we sequenced the HAS2 gene for 11 subterranean mammals and closely related species, and combined these with data from 57 other mammals. Comparative screening revealed that one of the two putatively important HAS2 substitutions in the NMR predicted to have a significant effect on hyaluronan synthase function was uniquely shared by all African mole-rats. Interestingly, we also identified multiple other amino acid substitutions in key domains of the HAS2 molecule, although the biological consequences of these for hyaluronan synthesis remain to be determined. Despite these results, we found evidence of strong purifying selection acting on the HAS2 gene across all mammals, and the NMR remains unique in its particular HAS2 sequence. Our results indicate that more work is needed to determine whether the apparent cancer resistance seen in NMR is shared by other members of the African mole-rat clade. PMID:25948568

  4. Recombinant synthesis of hyaluronan by Agrobacterium sp.

    PubMed

    Mao, Zichao; Chen, Rachel Ruizhen

    2007-01-01

    Hyaluronan (HA) is a sugar polymer of a repeating disaccharide, beta1-3 D-N-acetylglucosamine (GlcNAc) beta1-4 D-glucuronic acid (GlcA). It finds applications in numerous biomedical procedures such as ophthalmic surgery and osteoarthritis treatment. Until recently, the only commercial sources were extraction of rooster combs and from fermentation of pathogenic Streptococcus. In this work, we demonstrate that metabolic engineering strategies enable the recombinant synthesis of hyaluronan in a safe microorganism. Agrobacterium sp. ATCC 31749 is a commercial production strain for a food polymer, Curdlan. A broad host range expression vector was successfully developed to express the 3 kb HA synthase gene from Pasteurella multocida, along with a kfiD gene encoding UDP-glucose dehydrogenase from Escherichia coli K5 strain. Coexpression of these two heterologous enzymes enables Agrobacterium to produce HA. Hyaluronan was accumulated up to 0.3 g/L in shaker flask cultivation. The molecular weight of the polymer from various Agrobacterium strains is in the range of 0.7-2 MD. To our knowledge, this is the first successful recombinant hyaluronan synthesis in a Gram-negative bacterium that naturally produces a food product. The ease of genetic modifications provides future opportunities to tailor properties of polymers for specific applications.

  5. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

    SciTech Connect

    Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  6. Hyaluronan: A Matrix Component

    NASA Astrophysics Data System (ADS)

    Rügheimer, Louise

    2008-09-01

    The glucosaminoglycan hyaluronan is a key component of the extracellular matrix. It is a large, negatively charged molecule that can act as an ion exchange reservoir for positive ions. Hyaluronan is involved in renomedullary water handling through its water-binding capacity. In the renal medulla, the main source for hyaluronan production is the renomedullary interstitial cells. Hyaluronan synthases are found in the inner part of the plasma membrane and polymerize hyaluronan chains which are extruded into the extracellular space. Hyaluronidases are a family of enzymes involved in the degradation of hyaluronan. They have a wide range of properties, including differences in size, inhibitor sensitivities, catalytic mechanisms, substrate specificities and pH optima.

  7. Altered expression of hyaluronan synthase and hyaluronidase mRNA may affect hyaluronic acid distribution in keloid disease compared with normal skin.

    PubMed

    Sidgwick, Gary P; Iqbal, Syed A; Bayat, Ardeshir

    2013-05-01

    Keloid disease (KD) is a fibroproliferative disorder characterised partly by an altered extracellular matrix (ECM) profile. In fetal scarring, hyaluronic acid (HA) expression is increased, but is reduced in KD tissue compared with normal skin (NS). The expression of Hyaluronan Synthase (HAS) and hyaluronidase (HYAL) in KD and NS tissue were investigated for the first time using a range of techniques. Hyaluronan synthase and HYAL mRNA expression were significantly increased in NS tissue compared with KD tissue (P < 0.05). Immunohistological analysis of tissue indicated an accumulation of HAS and HYAL protein expression in KD compared with NS due to the thicker epidermis. No differences were observed in mRNA or protein expression in KD and NS fibroblasts. Reduced expression of HAS and HYAL may alter HA synthesis, degradation and accumulation in KD. Better understanding of the role of HA in KD may lead to novel therapeutic approaches to address the resulting ECM imbalance.

  8. Hyaluronan synthase HAS2 promotes tumor progression in bone by stimulating the interaction of breast cancer stem-like cells with macrophages and stromal cells

    PubMed Central

    Okuda, Hiroshi; Kobayashi, Aya; Xia, Bo; Watabe, Misako; Pai, Sudha K; Hirota, Shigeru; Xing, Fei; Liu, Wen; Pandey, Puspa R; Fukuda, Koji; Modur, Vishnu; Ghosh, Arnab; Wilber, Andrew; Watabe, Kounosuke

    2012-01-01

    The molecular mechanisms that operate within the organ microenvironment to support metastatic progression remain unclear. Here we report that upregulation of the hyaluronan synthase HAS2 occurs in highly metastatic breast stem-like cancer cells (CSCs) defined by CD44+/CD24−/ESA+ phenotype, where it plays a critical role in the generation of a pro-metastatic microenvironment in breast cancer. HAS2 was critical for interaction of CSCs with tumor associated macrophages (TAMs), leading to enhanced secretion of PDGF-BB from TAMs which then activated stromal cells and enhanced CSC self-renewal. Loss of HAS2 in CSCs or treatment with 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan synthases which blocks hyaluronan production, drastically reduced the incidence and growth of metastatic lesions in vitro or in vivo, respectively. Taken together, our findings demonstrate a critical role for HAS2 in the development of a pro-metastatic microenvironment and suggest that HAS2 inhibitors can act as anti-metastatic agents that disrupt a paracrine growth factor loop within this microenvironment. PMID:22113945

  9. Inherited Polymorphisms in Hyaluronan Synthase 1 Predict Risk of Systemic B-Cell Malignancies but Not of Breast Cancer

    PubMed Central

    Kuppusamy, Hemalatha; Ogmundsdottir, Helga M.; Baigorri, Eva; Warkentin, Amanda; Steingrimsdottir, Hlif; Haraldsdottir, Vilhelmina; Mant, Michael J.; Mackey, John; Johnston, James B.; Adamia, Sophia; Belch, Andrew R.; Pilarski, Linda M.

    2014-01-01

    Genetic variations in the hyaluronan synthase 1 gene (HAS1) influence HAS1 aberrant splicing. HAS1 is aberrantly spliced in malignant cells from multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM), but not in their counterparts from healthy donors. The presence of aberrant HAS1 splice variants predicts for poor survival in multiple myeloma (MM). We evaluated the influence of inherited HAS1 single nucleotide polymorphisms (SNP) on the risk of having a systemic B cell malignancy in 1414 individuals compromising 832 patients and 582 healthy controls, including familial analysis of an Icelandic kindred. We sequenced HAS1 gene segments from 181 patients with MM, 98 with monoclonal gammopathy of undetermined significance (MGUS), 72 with Waldenstrom macroglobulinemia (WM), 169 with chronic lymphocytic leukemia (CLL), as well as 34 members of a monoclonal gammopathy-prone Icelandic family, 212 age-matched healthy donors and a case-control cohort of 295 breast cancer patients with 353 healthy controls. Three linked single nucleotide polymorphisms (SNP) in HAS1 intron3 are significantly associated with B-cell malignancies (range p = 0.007 to p = 10−5), but not MGUS or breast cancer, and predict risk in a 34 member Icelandic family (p = 0.005, Odds Ratio = 5.8 (OR)), a relatively homogeneous cohort. In contrast, exon3 SNPs were not significantly different among the study groups. Pooled analyses showed a strong association between the linked HAS1 intron3 SNPs and B-cell malignancies (OR = 1.78), but not for sporadic MGUS or for breast cancer (OR<1.0). The minor allele genotypes of HAS1 SNPs are significantly more frequent in MM, WM, CLL and in affected members of a monoclonal gammopathy-prone family than they are in breast cancer, sporadic MGUS or healthy donors. These inherited changes may increase the risk for systemic B-cell malignancies but not for solid tumors. PMID:24950197

  10. IDENTIFICATION OF A MEMBRANE-LOCALIZED CYSTEINE CLUSTER NEAR THE SUBSTRATE BINDING SITES OF THE STREPTOCOCCUS EQUISIMILIS HYALURONAN SYNTHASE

    PubMed Central

    Kumari, Kshama; Weigel, Paul H.

    2005-01-01

    The membrane-bound hyaluronan synthase (HAS) from Streptococcus equisimilis (seHAS), which is the smallest Class I HAS, has four cysteine residues (positions 226, 262, 281, and 367) that are generally conserved within this family. Although Cys-null seHAS is still active, chemical modification of cysteine residues causes inhibition of wildtype enzyme (Kumari et al., J. Biol. Chem. 277, 13943, 2002). Here we studied the effects of N-ethylmaleimide (NEM) treatment on a panel of seHAS Cys-mutants to examine the structural and functional roles of the four cysteine residues in the activity of the enzyme. We found that Cys226, Cys262, and Cys281 are reactive with NEM, but that Cys367 is not. Substrate protection studies of wildtype seHAS and a variety of Cys-mutants revealed that binding of UDP-GlcUA, UDP-GlcNAc or UDP can protect Cys226 and Cys262 from NEM inhibition. Inhibition of the six double Cys-mutants of seHAS by sodium arsenite, which can crosslink vicinyl sulfhydryl groups, also supported the conclusion that Cys262 and Cys281 are close enough to be crosslinked. Similar results indicated that Cys281 and Cys367 are also very close in the active enzyme. We conclude that three of the four Cys residues in seHAS (Cys262, Cys281, and Cys367 ) are clustered very close together, that these Cys residues and Cys226 are located at the inner surface of the cell membrane, and that Cys226 and Cys262 are located in or near a UDP binding site. PMID:15616126

  11. Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein

    PubMed Central

    Ghosh, Anirban; Kuppusamy, Hemalatha; Pilarski, Linda M.

    2009-01-01

    Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression. PMID:19451652

  12. Cell protrusions induced by hyaluronan synthase 3 (HAS3) resemble mesothelial microvilli and share cytoskeletal features of filopodia.

    PubMed

    Koistinen, Ville; Kärnä, Riikka; Koistinen, Arto; Arjonen, Antti; Tammi, Markku; Rilla, Kirsi

    2015-10-01

    Previous studies have shown that overexpression of enzymatically active GFP-HAS induces the growth of long, slender protrusions that share many features of both filopodia and microvilli. These protrusions are dependent on continuing hyaluronan synthesis, and disrupt upon digestion of hyaluronan by hyaluronidase. However, complete understanding of their nature is still missing. This work shows that the protrusions on rat peritoneal surface are ultrastructurally indistinguishable from those induced by GFP-HAS3 in MCF-7 cells. Analysis of the actin-associated proteins villin, ezrin, espin, fascin, and Myo10 indicated that the HAS3-induced protrusions share most cytoskeletal features with filopodia, but they do not require adherence to the substratum like traditional filopodia. GFP-HAS3 overexpression was found to markedly enhance filamentous actin in the protrusions and their cortical basis. Analysis of the protrusion dynamics after enzymatic digestion of hyaluronan revealed that while GFP-HAS3 escape from the protrusions and the protrusion collapse takes place immediately, the complete retraction of the protrusions occurs more slowly. This finding also suggests that hyaluronan chain maintains HAS3 in the plasma membrane. The results of this work suggest that protrusions similar to those of HAS3 overexpressing cells in vitro exist also in cells with active hyaluronan synthesis in vivo. These protrusions are similar to common filopodia but are independent of substratum attachment due to the extracellular scaffolding by the hyaluronan coat that accounts for the growth and maintenance of these structures, previously associated to invasion, adhesion and multidrug resistance.

  13. Hyaluronan synthase assembles chitin oligomers with -GlcNAc(α1→)UDP at the reducing end.

    PubMed

    Weigel, Paul H; West, Christopher M; Zhao, Peng; Wells, Lance; Baggenstoss, Bruce A; Washburn, Jennifer L

    2015-06-01

    Class I hyaluronan synthases (HASs) assemble a polysaccharide containing the repeating disaccharide [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP and vertebrate HASs also assemble (GlcNAc-β1,4)n homo-oligomers (chitin) in the absence of GlcUA-UDP. This multi-membrane domain CAZy GT2 family glycosyltransferase, which couples HA synthesis and translocation across the cell membrane, is atypical in that monosaccharides are incrementally assembled at the reducing, rather than the non-reducing, end of the growing polymer. Using Escherichia coli membranes containing recombinant Streptococcus equisimilis HAS, we demonstrate that a prokaryotic Class I HAS also synthesizes chitin oligomers (up to 15-mers, based on MS and MS/MS analyses of permethylated products). Furthermore, chitin oligomers were found attached at their reducing end to -4GlcNAc(α1→)UDP [i.e. (GlcNAcβ1,4)nGlcNAc(α1→)UDP]. These oligomers, which contained up to at least seven HexNAc residues, consisted of β4-linked GlcNAc residues, based on the sensitivity of the native products to jack bean β-N-acetylhexosaminidase. Interestingly, these oligomers exhibited mass defects of -2, or -4 for longer oligomers, that strictly depended on conjugation to UDP, but MS/MS analyses indicate that these species result from chemical dehydrogenations occurring in the gas phase. Identification of (GlcNAc-β1,4)n-GlcNAc(α1→)UDP as HAS reaction products, made in the presence of GlcNAc(α1→)UDP only, provides strong independent confirmation for the reducing terminal addition mechanism. We conclude that chitin oligomer products made by HAS are derived from the cleavage of these novel activated oligo-chitosyl-UDP oligomers. Furthermore, it is possible that these UDP-activated chitin oligomers could serve as self-assembled primers for initiating HA synthesis and ultimately modify the non-reducing terminus of HA with a chitin cap.

  14. Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior

    PubMed Central

    Weigel, Paul H.

    2015-01-01

    Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers. PMID:26472958

  15. Hereditary cutaneous mucinosis in shar pei dogs is associated with increased hyaluronan synthase-2 mRNA transcription by cultured dermal fibroblasts.

    PubMed

    Zanna, Giordana; Docampo, María J; Fondevila, Dolors; Bardagí, Mar; Bassols, Anna; Ferrer, Lluís

    2009-10-01

    Shar pei dogs are known for the distinctive feature of thick, wrinkled skin as a consequence of high dermal mucin content. Excessive dermal deposition of mucinous substance leading to severe skin folding, and/or to the more severe vesicular form characterized by dermal vesicles or bullae, is highly prevalent in this breed and is known as idiopathic mucinosis. Hyaluronic acid (HA) is the main component that accumulates in the dermis, and high levels of HA have also been detected in the serum of shar pei dogs. In this study, the cellular and molecular mechanisms underlying cutaneous mucinosis of shar pei dogs were investigated. Thirteen shar pei dogs and four control dogs of other breeds were included. In primary dermal fibroblast cultures, transcription of the family of hyaluronan synthases (HAS) involved in HA synthesis, and of hyaluronidases (HYAL) involved in HA degradation, were studied by reverse transcriptase polymerase chain reaction. The location of HA in cell cultures was studied by immunofluorescence and confocal laser microscopy. Dermal fibroblasts transcribed HAS2, HAS3, HYAL1 and HYAL2, but no amplification for HAS1 was found. A higher transcription of HAS2 was demonstrated in shar pei dogs compared with control dogs. By confocal microscopy, HA was detected as a more diffuse and intense network-like pattern of green fluorescence in the fibroblast cells of shar pei dogs in comparison with control dogs. Together, these results provide additional evidence that hereditary cutaneous mucinosis in shar pei dogs may be a consequence of over-transcription or increased activity of HAS2.

  16. Overexpression of hyaluronan synthase 2 and gonadotropin receptors in cumulus cells of goats subjected to one-shot eCG/FSH hormonal treatment for ovarian stimulation.

    PubMed

    Santos, Juliana D R; Batista, Ribrio I T P; Magalhães, Livia C; Paula, Alexandre R; Souza, Samara S; Salamone, Daniel F; Bhat, Maajid H; Teixeira, Dárcio I A; Freitas, Vicente J F; Melo, Luciana M

    2016-07-01

    Hormonal ovarian stimulation may affect transcripts in somatic cells of cumulus-oocyte complexes (COCs) and affect the resulting oocyte quality. Here, in parallel with morphological classification and in vitro maturation (IVM) rate analysis, we investigated the expression of hyaluronan synthase 2 (HAS2), gonadotropic receptors (FSHR and LHR) and connexin 43 (GJA1) in cumulus cells (CCs) from goat COCs after multi-dose FSH (MD) or one-shot FSH/eCG (OS) treatments, using bovine COCs as control groups. The MD treatment produced more large follicles, and the resulting COCs had a better morphology and IVM rate than were obtained with OS. The OS treatment produced COCs with increased HAS2, FSHR, LHR and GJA1 expression. This gene expression pattern was also observed in the CCs of COCs that showed poor morphological characteristics. On the other hand, the mRNA levels were more similar between groups after IVM; FSHR and LHR were the main genes that showed decreased expression. Some events that occurred in bovine CCs during IVM, such as cell expansion, increased HAS2 expression and decreased GJA1 expression, were less evident or did not occur in goat COCs. In conclusion, increasing HAS2, FSHR, LHR and GJA1 expression in goat COCs does not confer greater meiotic competence to oocytes. Instead, it may result from poor regulation of gene expression in CCs by lower quality oocytes. Finally, cumulus expansion, together with HAS2 upregulation and GJA1 downregulation, seems to be more important for bovine COCs than for goat COCs. Additional studies are needed to investigate the importance of other HAS isoforms and connexins in goat COCs.

  17. Hyaluronidases and hyaluronan synthases expression is inversely correlated with malignancy in lung/bronchial pre-neoplastic and neoplastic lesions, affecting prognosis

    PubMed Central

    de Sá, V.K.; Rocha, T.P.; Moreira, AL.; Soares, F.A.; Takagaki, T.; Carvalho, L.; Nicholson, A.G.; Capelozzi, V.L.

    2015-01-01

    We collected a series of 136 lung/bronchial and 56 matched lung parenchyma tissue samples from patients who underwent lung/bronchial biopsies and presented invasive carcinoma after lung surgery. The lung/bronchial samples included basal cell hyperplasia, squamous metaplasia, moderate dysplasia, adenomatous hyperplasia, severe dysplasia, squamous cell carcinoma and adenocarcinoma. Matched lung parenchyma tissue samples included 25 squamous cell carcinomas and 31 adenocarcinomas. Immunohistochemistry was performed to analyze for the distribution of hyaluronidase (Hyal)-1 and −3, and hyaluronan synthases (HAS)-1, −2, and −3. Hyal-1 showed significantly higher expression in basal cell hyperplasia than in moderate dysplasia (P=0.01), atypical adenomatous hyperplasia (P=0.0001), or severe dysplasia (P=0.03). Lower expression of Hyal-3 was found in atypical adenomatous hyperplasia than in basal cell hyperplasia (P=0.01) or moderate dysplasia (P=0.02). HAS-2 was significantly higher in severe dysplasia (P=0.002) and in squamous metaplasia (P=0.04) compared with basal cell hyperplasia. HAS-3 was significantly expressed in basal cell hyperplasia compared with atypical adenomatous hyperplasia (P=0.05) and severe dysplasia (P=0.02). Lower expression of HAS-3 was found in severe dysplasia compared with squamous metaplasia (P=0.01) and moderate dysplasia (P=0.01). Epithelial Hyal-1 and −3 and HAS-1, −2, and −3 expressions were significantly higher in pre-neoplastic lesions than in neoplastic lesions. Comparative Cox multivariate analysis controlled by N stage and histologic tumor type showed that patients with high HAS-3 expression in pre-neoplastic cells obtained by lung/bronchial biopsy presented a significantly higher risk of death (HR=1.19; P=0.04). We concluded that localization of Hyal and HAS in lung/bronchial pre-neoplastic and neoplastic lesions was inversely related to malignancy, which implied that visualizing these factors could be a useful diagnostic

  18. Hyaluronan synthase-2 upregulation protects smpd3-deficient fibroblasts against cell death induced by nutrient deprivation, but not against apoptosis evoked by oxidized LDL

    PubMed Central

    Garoby-Salom, Sandra; Rouahi, Myriam; Mucher, Elodie; Auge, Nathalie; Salvayre, Robert; Negre-Salvayre, Anne

    2014-01-01

    The neutral type 2 sphingomyelinase (nSMase2) hydrolyzes sphingomyelin and generates ceramide, a major bioactive sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (oxidized LDL, hydrogen peroxide and nutrient starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient starvation (fetal calf serum/glucose/pyruvate-free DMEM), whereas wt fibroblasts die after 48 h incubation in this medium. In contrast, oxidized LDL and hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor GW4869 were more resistant to starvation-induced apoptosis. The resistance of fro/fro cells to starvation-induced apoptosis is associated with an increased expression of hyaluronan synthase 2 (HAS2) mRNAs and protein, which is inhibited by ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against starvation-induced apoptosis. The protective mechanism of HAS2 involves an increased expression of the heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient starvation that could

  19. Synthesis and shedding of hyaluronan from plasma membranes of human fibroblasts and metastatic and non-metastatic melanoma cells.

    PubMed Central

    Lüke, H J; Prehm, P

    1999-01-01

    The regulation of hyaluronan synthesis and shedding was analysed in human fibroblasts and in two melanoma cells that differed in the metastatic potential and proteolysis of the hyaluronan receptor CD44. Dissociation of nascent hyaluronan from plasma membranes isolated from fibroblasts by high salt concentrations led to activation of hyaluronan synthase. Hyaluronan synthesis was also enhanced in plasma membranes from fibroblasts that had been treated with hyaluronidase or trypsin. Hyaluronan oligosaccharides stimulated hyaluronan production in fibroblast cultures. These results indicated that nascent high-molecular-mass hyaluronan inhibited its own chain elongation, if it was retained in the vicinity of the synthase by cell-surface receptors. The results also indicated that increased hyaluronan synthesis and shedding correlated with proteolysis of CD44 on the melanoma cell lines, which has been observed by others. PMID:10493913

  20. Hyaluronan production increases the malignant properties of mesothelioma cells

    PubMed Central

    Li, Y; Heldin, P

    2001-01-01

    Malignant pleural mesotheliomas is in most cases associated with elevated amounts of hyaluronan. To investigate the importance of hyaluronan for the malignant properties of mesotheliomas, we have expressed murine hyaluronan synthase 2 (HAS2) in the non-hyaluronan producing mesothelioma cell line, Mero-25. We found that upon hyaluronan overproduction the mesothelioma cells changed their epitheloid character to a fibroblastic phenotype and were surrounded by pericellular matrices, the size of which correlated to the amount of synthesized hyaluronan. HAS2-transfected cells with the ability to synthesize about 520 ng hyaluronan/5 × 104cells/24 h exhibited about a 2-fold increase in the expression of the cell surface hyaluronan receptor CD44 and their locomotion increased compared to that of mock-transfected Mero-25 cells. Furthermore, the malignant properties of mesothelioma cell clones as determined by the ability to grow in a soft agar assay correlated to their hyaluronan production. These results provide evidence for an important role of hyaluronan in the aggressive spread of mesotheliomas in adjacent non-cancerous stromal tissues. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11506502

  1. Hyaluronan synthesis by developing cortical neurons in vitro

    PubMed Central

    Fowke, Tania M.; Karunasinghe, Rashika N.; Bai, Ji-Zhong; Jordan, Shawn; Gunn, Alistair J.; Dean, Justin M.

    2017-01-01

    Hyaluronan is a linear glycosaminoglycan that forms the backbone of perineuronal nets around neurons in the cerebral cortex. However, it remains controversial whether neurons are capable of independent hyaluronan synthesis. Herein, we examined the expression of hyaluronan and hyaluronan synthases (HASs) throughout cortical neuron development in vitro. Enriched cultures of cortical neurons were established from E16 rats. Neurons were collected at days in vitro (DIV) 0 (4 h), 1, 3, 7, 14, and 21 for qPCR or immunocytochemistry. In the relative absence of glia, neurons exhibited HAS1–3 mRNA at all time-points. By immunocytochemistry, puncta of HAS2–3 protein and hyaluronan were located on neuronal cell bodies, neurites, and lamellipodia/growth cones from as early as 4 h in culture. As neurons matured, hyaluronan was also detected on dendrites, filopodia, and axons, and around synapses. Percentages of hyaluronan-positive neurons increased with culture time to ~93% by DIV21, while only half of neurons at DIV21 expressed the perineuronal net marker Wisteria floribunda agglutinin. These data clearly demonstrate that neurons in vitro can independently synthesise hyaluronan throughout all maturational stages, and that hyaluronan production is not limited to neurons expressing perineuronal nets. The specific structural localisation of hyaluronan suggests potential roles in neuronal development and function. PMID:28287145

  2. Pets and Pasteurella Infections

    MedlinePlus

    ... Ear Nose & Throat Emotional Problems Eyes Fever From Insects or Animals Genitals and Urinary Tract Glands & Growth ... Preventable Diseases Healthy Children > Health Issues > Conditions > From Insects or Animals > Pets and Pasteurella Infections Health Issues ...

  3. Hyaluronan production enhances shedding of plasma membrane-derived microvesicles.

    PubMed

    Rilla, Kirsi; Pasonen-Seppänen, Sanna; Deen, Ashik J; Koistinen, Ville V T; Wojciechowski, Sara; Oikari, Sanna; Kärnä, Riikka; Bart, Genevieve; Törrönen, Kari; Tammi, Raija H; Tammi, Markku I

    2013-08-01

    Many cell types secrete plasma membrane-bound microvesicles, suggested to play an important role in tissue morphogenesis, wound healing, and cancer spreading. However, the mechanisms of their formation have remained largely unknown. It was found that the tips of long microvilli induced in cells by overexpression of hyaluronan synthase 3 (HAS3) were detach into the culture medium as microvesicles. Moreover, several cell types with naturally active hyaluronan synthesis released high numbers of plasma membrane-derived vesicles, and inhibition of hyaluronan synthesis reduced their formation. The vesicles contained HAS, and were covered with a thick hyaluronan coat, a part of which was retained even after purification with high-speed centrifugation. HAS3 overexpressing MDCK cells cultured in a 3-D matrix as epithelial cysts released large amounts of HAS- and hyaluronan-positive vesicles from their basal surfaces into the extracellular matrix. As far as we know, hyaluronan synthesis is one of the first molecular mechanisms shown to stimulate the production of microvesicles. The microvesicles have a potential to deliver the hyaluronan synthase machinery and membrane and cytoplasmic materials to other cells, influencing tissue regeneration, inflammation and tumor progression.

  4. The ubiquitous hyaluronan: Functionally implicated in the oviduct?

    PubMed

    Rodriguez-Martinez, H; Tienthai, P; Atikuzzaman, M; Vicente-Carrillo, A; Rubér, M; Alvarez-Rodriguez, M

    2016-07-01

    Hyaluronan (hyaluronic acid) is a simple, nonantigenic, nonsulfated glycosaminoglycan present everywhere in the extracellular compartments of the body. Noteworthy, it is highly conserved phylogenetically, from sauropsida to mammals; and plays a plethora of roles from embryonic/fetal development to adult physiological and pathological events, including tumor development. In reproduction, hyaluronan has proven related to initial events as sperm survival, buildup of the sperm reservoir in the oviduct, regulation of sperm capacitation, and prefertilization to later participate in embryo, fetal, and placental development. Synthesis, binding (via the CD44 membrane receptor), and degradation of hyaluronan occur in male and female genital organs, the oviduct being no exception. This review discusses our current knowledge on roles of this ubiquitous glycosaminoglycan on the survival of immunologically foreign spermatozoa in the pig oviduct, a relevant event for fertility. During preovulatory storage in the functional tubal sperm reservoir, spermatozoa are entrapped in a mucus-like tubal fluid. This fluid contains fluctuating levels of hyaluronan, which is synthesized by the lining epithelium by hyaluronan synthase 3. Both hyaluronan and its CD44 receptor are particularly evident in the deep mucosal furrows of the sperm reservoir, in which most spermatozoa are embedded in; kept alive, uncapacitated but also undetected by the immune system of the female. Hyaluronan is also present in the seminal plasma, and evidence points toward an involvement of hyaluronan and its receptor in the local (tubal and possibly uterine) production of antiinflammatory cytokines, such as interleukin-10, pertaining maternal immune tolerance of these foreign cells.

  5. Hyaluronan synthesis induces microvillus-like cell surface protrusions.

    PubMed

    Kultti, Anne; Rilla, Kirsi; Tiihonen, Riikka; Spicer, Andrew P; Tammi, Raija H; Tammi, Markku I

    2006-06-09

    Hyaluronan synthases (HASs) are plasma membrane enzymes that simultaneously elongate, bind, and extrude the growing hyaluronan chain directly into extracellular space. In cells transfected with green fluorescent protein (GFP)-tagged Has3, the dorsal surface was decorated by up to 150 slender, 3-20-microm-long microvillus-type plasma membrane protrusions, which also contained filamentous actin, the hyaluronan receptor CD44, and lipid raft microdomains. Enzymatic activity of HAS was required for the growth of the microvilli, which were not present in cells transfected with other GFP proteins or inactive GFP-Has3 mutants or in cells incubated with exogenous soluble hyaluronan. The microvilli induced by HAS3 were gradually withered by introduction of an inhibitor of hyaluronan synthesis and rapidly retracted by hyaluronidase digestion, whereas they were not affected by competition with hyaluronan oligosaccharides and disruption of the CD44 gene, suggesting independence of hyaluronan receptors. The data bring out the novel concept that the glycocalyx created by dense arrays of hyaluronan chains, tethered to HAS during biosynthesis, can induce and maintain prominent microvilli.

  6. Pericellular hyaluronan coat visualized in live cells with a fluorescent probe is scaffolded by plasma membrane protrusions.

    PubMed

    Rilla, Kirsi; Tiihonen, Riikka; Kultti, Anne; Tammi, Markku; Tammi, Raija

    2008-10-01

    Many cell types wear up to 20-mum-wide hyaluronidase-sensitive surface coats, detected by exclusion of sedimenting particles like fixed erythrocytes. The structure of the coat is enigmatic, being apparently too thick to be accounted by random coils or even extended chains of just hyaluronan attached to cell surface. We have shown that hyaluronan synthesis enforced by green fluorescent protein-hyaluronan synthase transfection creates microvillous protrusions. The idea that the plasma membrane protrusions rather than hyaluronan alone is responsible for the exclusion space was studied with a fluorescent probe for hyaluronan and a dye with membrane affinity, applied to live cell cultures. Mesothelial and smooth muscle cells, fibroblasts, and chondrocytes, all known for their endogenously active hyaluronan synthesis, showed hyaluronan-coated plasma membrane protrusions, barely visible in phase contrast microscopy. Treatment with hyaluronidase and inhibition of hyaluronan synthesis caused retraction of the protrusions unless they were attached to substratum. Hyaluronan and the exclusion space were reduced, but did not disappear, by purified hyaluronan hexasaccharides that compete with hyaluronan attached to CD44. The results suggest that slender plasma membrane protrusions are an inherent feature of hyaluronan coats, form their scaffold, and largely result from ongoing hyaluronan synthesis in their plasma membrane. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

  7. Hyaluronan synthesis in virus PBCV-1-infected chlorella-like green algae.

    PubMed

    Graves, M V; Burbank, D E; Roth, R; Heuser, J; DeAngelis, P L; Van Etten, J L

    1999-04-25

    We previously reported that the chlorella virus PBCV-1 genome encodes an authentic, membrane-associated glycosyltransferase, hyaluronan synthase (HAS). Hyaluronan, a linear polysaccharide chain composed of alternating beta1,4-glucuronic acid and beta1, 3-N-acetylglucosamine groups, is present in vertebrates as well as a few pathogenic bacteria. Studies of infected cells show that the transcription of the PBCV-1 has gene begins within 10 min of virus infection and ends at 60-90 min postinfection. The hyaluronan polysaccharide begins to accumulate as hyaluronan-lyase sensitive, hair-like fibers on the outside of the chlorella cell wall by 15-30 min postinfection; by 240 min postinfection, the infected cells are coated with a dense fibrous network. This hyaluronan slightly reduces attachment of a second chlorella virus to the infected algae. An analysis of 41 additional chlorella viruses indicates that many, but not all, produce hyaluronan during infection.

  8. Hyaluronan in limb morphogenesis.

    PubMed

    Li, Yingcui; Toole, Bryan P; Dealy, Caroline N; Kosher, Robert A

    2007-05-15

    Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself. Has2 expression and HA production are downregulated in the proximal central core of the limb bud during the formation of the precartilage condensations of the skeletal elements, suggesting that downregulation of HA may be necessary for the close juxtaposition of cells and the resulting cell-cell interactions that trigger cartilage differentiation during condensation. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements that do form in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately. We also demonstrate that sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that downregulation of HA is indeed necessary for formation of the precartilage condensations that trigger cartilage differentiation. Taken together these results suggest involvement of HA in various aspects of limb morphogenesis.

  9. Selective expression of hyaluronan and receptor for hyaluronan mediated motility (Rhamm) in the adult mouse subventricular zone and rostral migratory stream and in ischemic cortex.

    PubMed

    Lindwall, Charlotta; Olsson, Martina; Osman, Ahmed M; Kuhn, H Georg; Curtis, Maurice A

    2013-03-29

    Hyaluronan is a large glycosaminoglycan, which is abundant in the extracellular matrix of the developing rodent brain. In the adult brain however, levels of hyaluronan are significantly reduced. In this study, we used neurocan-GFP as a histochemical probe to analyze the distribution of hyaluronan in the adult mouse subventricular zone (SVZ), as well as in the rostral migratory stream (RMS). Interestingly, we observed that hyaluronan is generally downregulated in the adult brain, but notably remains at high levels in the SVZ and RMS; areas in which neural stem/progenitor cells (NSPCs) persist, proliferate and migrate throughout life. In addition, we found that the receptor for hyaluronan-mediated motility (Rhamm) was expressed in migrating neuroblasts in these areas, indicating that Rhamm could be involved in regulating hyaluronan-mediated cell migration. Hyaluronan levels are balanced by synthesis through hyaluronan synthases (Has) and degradation by hyaluronidases (Hyal). We found that Has1 and Has2, as well as Hyal1 and Hyal2 were expressed in GFAP positive cells in the adult rodent SVZ and RMS, indicating that astrocytes could be regulating hyaluronan-mediated functions in these areas. We also demonstrate that hyaluronan levels are substantially increased at six weeks following a photothrombotic stroke lesion to the adult mouse cortex. Furthermore, GFAP positive cells in the peri-infarct area express Rhamm. Thus, hyaluronan may be involved in regulating cell migration in the normal SVZ and RMS and could also be responsible for priming the peri-infarct area following an ischemic lesion for cell migration.

  10. A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease.

    PubMed

    Chang, Mary Y; Tanino, Yoshinori; Vidova, Veronika; Kinsella, Michael G; Chan, Christina K; Johnson, Pamela Y; Wight, Thomas N; Frevert, Charles W

    2014-02-01

    The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4(-/-) mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.

  11. Reprint of: A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease.

    PubMed

    Chang, Mary Y; Tanino, Yoshinori; Vidova, Veronika; Kinsella, Michael G; Chan, Christina K; Johnson, Pamela Y; Wight, Thomas N; Frevert, Charles W

    2014-04-01

    The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4(-/-) mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.

  12. A Rapid Increase in Macrophage-Derived Versican and Hyaluronan in Infectious Lung Disease

    PubMed Central

    Chang, Mary Y.; Tanino, Yoshinori; Vidova, Veronika; Kinsella, Michael G.; Chan, Christina K.; Johnson, Pamela Y.; Wight, Thomas N.; Frevert, Charles W.

    2014-01-01

    The goals of this study were to characterize the changes to chondroitin sulfate proteoglycans and hyaluronan in lungs in the acute response to gram-negative bacterial infection, and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4−/− mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection. PMID:24472738

  13. A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease

    PubMed Central

    Chang, Mary Y.; Tanino, Yoshinori; Vidova, Veronika; Kinsella, Michael G.; Chan, Christina K.; Johnson, Pamela Y.; Wight, Thomas N.; Frevert, Charles W.

    2014-01-01

    The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4−/− mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection. PMID:24727035

  14. Actinobacillus rossii sp. nov., Actinobacillus seminis sp. nov., nom. rev., Pasteurella bettii sp. nov., Pasteurella lymphangitidis sp. nov., Pasteurella mairi sp. nov., and Pasteurella trehalosi sp. nov.

    PubMed

    Sneath, P H; Stevens, M

    1990-04-01

    Evidence from numerical taxonomic analysis and DNA-DNA hybridization supports the proposal of new species in the genera Actinobacillus and Pasteurella. The following new species are proposed: Actinobacillus rossii sp. nov., from the vaginas of postparturient sows; Actinobacillus seminis sp. nov., nom. rev., associated with epididymitis of sheep; Pasteurella bettii sp. nov., associated with human Bartholin gland abscess and finger infections; Pasteurella lymphangitidis sp. nov. (the BLG group), which causes bovine lymphangitis; Pasteurella mairi sp. nov., which causes abortion in sows; and Pasteurella trehalosi sp. nov., formerly biovar T of Pasteurella haemolytica, which causes septicemia in older lambs.

  15. Hyaluronan and Stone Disease

    NASA Astrophysics Data System (ADS)

    Asselman, Marino

    2008-09-01

    Kidney stones cannot be formed as long as crystals are passed in the urine. However, when crystals are retained it becomes possible for them to aggregate and form a stone. Crystals are expected to be formed not earlier than the distal tubules and collecting ducts. Studies both in vitro and in vivo demonstrate that calcium oxalate monohydrate crystals do not adhere to intact distal epithelium, but only when the epithelium is proliferating or regenerating, so that it possesses dedifferentiated cells expressing hyaluronan, osteopontin (OPN) and their mutual receptor CD44 at the apical cell membrane. The polysaccharide hyaluronan is an excellent crystal binding molecule because of its negative ionic charge. We hypothesized that the risk for crystal retention in the human kidney would be increased when tubular cells express hyaluronan at their apical cell membrane. Two different patient categories in which nephrocalcinosis frequently occurs were studied to test this hypothesis (preterm neonates and kidney transplant patients). Hyaluronan (and OPN) expression at the luminal membrane of tubular cells indeed was observed, which preceded subsequent retention of crystals in the distal tubules. Tubular nephrocalcinosis has been reported to be associated with decline of renal function and thus further studies to extend our knowledge of the mechanisms of retention and accumulation of crystals in the kidney are warranted. Ultimately, this may allow the design of new strategies for the prevention and treatment of both nephrocalcinosis and nephrolithiasis in patients.

  16. The Content and Size of Hyaluronan in Biological Fluids and Tissues

    PubMed Central

    Cowman, Mary K.; Lee, Hong-Gee; Schwertfeger, Kathryn L.; McCarthy, James B.; Turley, Eva A.

    2015-01-01

    Hyaluronan is a simple repeating disaccharide polymer, synthesized at the cell surface by integral membrane synthases. The repeating sequence is perfectly homogeneous, and is the same in all vertebrate tissues and fluids. The polymer molecular mass is more variable. Most commonly, hyaluronan is synthesized as a high-molecular mass polymer, with an average molecular mass of approximately 1000–8000 kDa. There are a number of studies showing increased hyaluronan content, but reduced average molecular mass with a broader range of sizes present, in tissues or fluids when inflammatory or tissue-remodeling processes occur. In parallel studies, exogenous hyaluronan fragments of low-molecular mass (generally, <200 kDa) have been shown to affect cell behavior through binding to receptor proteins such as CD44 and RHAMM (gene name HMMR), and to signal either directly or indirectly through toll-like receptors. These data suggest that receptor sensitivity to hyaluronan size provides a biosensor of the state of the microenvironment surrounding the cell. Sensitive methods for isolation and characterization of hyaluronan and its fragments have been developed and continue to improve. This review provides an overview of the methods and our current state of knowledge of hyaluronan content and size distribution in biological fluids and tissues. PMID:26082778

  17. The Content and Size of Hyaluronan in Biological Fluids and Tissues.

    PubMed

    Cowman, Mary K; Lee, Hong-Gee; Schwertfeger, Kathryn L; McCarthy, James B; Turley, Eva A

    2015-01-01

    Hyaluronan is a simple repeating disaccharide polymer, synthesized at the cell surface by integral membrane synthases. The repeating sequence is perfectly homogeneous, and is the same in all vertebrate tissues and fluids. The polymer molecular mass is more variable. Most commonly, hyaluronan is synthesized as a high-molecular mass polymer, with an average molecular mass of approximately 1000-8000 kDa. There are a number of studies showing increased hyaluronan content, but reduced average molecular mass with a broader range of sizes present, in tissues or fluids when inflammatory or tissue-remodeling processes occur. In parallel studies, exogenous hyaluronan fragments of low-molecular mass (generally, <200 kDa) have been shown to affect cell behavior through binding to receptor proteins such as CD44 and RHAMM (gene name HMMR), and to signal either directly or indirectly through toll-like receptors. These data suggest that receptor sensitivity to hyaluronan size provides a biosensor of the state of the microenvironment surrounding the cell. Sensitive methods for isolation and characterization of hyaluronan and its fragments have been developed and continue to improve. This review provides an overview of the methods and our current state of knowledge of hyaluronan content and size distribution in biological fluids and tissues.

  18. Hyaluronan-mediated cellular adhesion

    NASA Astrophysics Data System (ADS)

    Curtis, Jennifer

    2005-03-01

    Many cells surround themselves with a cushioning halo of polysaccharides that is further strengthened and organized by proteins. In fibroblasts and chrondrocytes, the primary component of this pericellular matrix is hyaluronan, a large linear polyanion. Hyaluronan production is linked to a variety of disease, developmental, and physiological processes. Cells manipulate the concentration of hyaluronan and hyaluronan receptors for numerous activities including modulation of cell adhesion, cell motility, and differentiation. Recent investigations by identify hyaluronan's role in mediating early-stage cell adhesion. An open question is how the cell removes the 0.5-10 micron thick pericellular matrix to allow for further mature adhesion events requiring nanometer scale separations. In this investigation, holographic optical tweezers are used to study the adhesion and viscoelastic properties of chondrocytes' pericellular matrix. Ultimately, we aim to shed further light on the spatial and temporal details of the dramatic transition from micron to nanometer gaps between the cell and its adhesive substrate.

  19. Classification of Pasteurella species B as Pasteurella oralis sp. nov.

    PubMed

    Christensen, Henrik; Bertelsen, Mads F; Bojesen, Anders Miki; Bisgaard, Magne

    2012-06-01

    Pasteurella species B has so far only been reported from the oral cavity of dogs, cats and a ferret. In the present study, information from 15 recent isolates from different sources, including African hedgehogs (Atelerix albiventris), banded mongoose (Mungos mungo), Moholi bushbabies (Galago moholi) and pneumonia of a cat, were compared to five strains investigated previously from bite wounds in humans inflicted by a cat and dog and from gingiva of a cat. rpoB gene sequence comparison showed that 17 isolates, including the reference strain (CCUG 19794(T)), had identical sequences, whereas two were closely related and demonstrated 97.9 and 99.6 % similarity to strain CCUG 19794(T), respectively; the type strain of Pasteurella stomatis was the most closely related strain, with 92.3 % similarity. This is within the mean range (76-100 %) of rpoB gene sequence similarity between species of the same genus within the family Pasteurellaceae. 16S rRNA gene sequencing of four strains selected based on rpoB sequence comparison showed at least 99.7 % similarity between strains of Pasteurella species B, with 96.2 % similarity to the type strain of the closest related species (Pasteurella canis), indicating that Pasteurella species B should have separate species status. Separate species status was also documented when recN sequence comparisons were converted to a genome similarity of 93.7 % within Pasteurella species B and 59.0 % to the type strain of the closest related species (P. canis). Based on analysis of the phylogenetic and phenotypic data, and since most isolates originate from the oral cavities of a diverse group of animals, it is suggested that these bacteria be classified as Pasteurella oralis sp. nov.; the type strain is P683(T) ( = CCUG 19794(T) = CCM 7950(T) = strain 23193(T) = MCCM 00102(T)), obtained from a cat. Previous reports of the type strain have shown ubiquinone-8, demethylmenaquinone-8 and menaquinone-8 as the major quinones. Polyamines in the type

  20. Hyaluronan stimulates pancreatic cancer cell motility

    PubMed Central

    Cheng, Xiao-Bo; Kohi, Shiro; Koga, Atsuhiro; Hirata, Keiji; Sato, Norihiro

    2016-01-01

    Hyaluronan (HA) accumulates in pancreatic ductal adenocarcinoma (PDAC), but functional significance of HA in the aggressive phenotype remains unknown. We used different models to investigate the effect of HA on PDAC cell motility by wound healing and transwell migration assay. Changes in cell motility were examined in 8 PDAC cell lines in response to inhibition of HA production by treatment with 4-methylumbelliferone (4-MU) and to promotion by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or by co-culture with tumor-derived stromal fibroblasts. We also investigated changes in cell motility by adding exogenous HA. Additionally, mRNA expressions of hyaluronan synthases and hyaluronidases were examined using real time RT-PCR. Inhibition of HA by 4-MU significantly decreased the migration, whereas promotion of HA by TPA or co-culture with tumor-derived fibroblasts significantly increased the migration of PDAC cells. The changes in HA production by these treatments tended to be associated with changes in HAS3 mRNA expression. Furthermore, addition of exogenous HA, especially low-molecular-weight HA, significantly increased the migration of PDAC cells. These findings suggest that HA stimulates PDAC cell migration and thus represents an ideal therapeutic target to prevent invasion and metastasis. PMID:26684359

  1. Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

    PubMed Central

    Evanko, Stephen P.; Potter-Perigo, Susan; Petty, Loreen J.; Workman, Gail A.; Wight, Thomas N.

    2015-01-01

    The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4 day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccarides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein

  2. Hyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemicus.

    PubMed

    Chen, Wendy Yiting; Marcellin, Esteban; Hung, Jacky; Nielsen, Lars Keld

    2009-07-03

    The molecular weight of hyaluronan is important for its rheological and biological function. The molecular mechanisms underlying chain termination and hence molecular weight control remain poorly understood, not only for hyaluronan synthases but also for other beta-polysaccharide synthases, e.g. cellulose, chitin, and 1,3-betaglucan synthases. In this work, we manipulated metabolite concentrations in the hyaluronan pathway by overexpressing the five genes of the hyaluronan synthesis operon in Streptococcus equi subsp. zooepidemicus. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased molecular weight, whereas overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased molecular weight. The highest molecular mass observed was at 3.4 +/- 0.1 MDa twice that observed in the wild-type strain, 1.8 +/- 0.1 MDa. The data indicate that (a) high molecular weight is achieved when an appropriate balance of UDP-N-acetylglucosamine and UDP-glucuronic acid is achieved, (b) UDP-N-acetylglucosamine exerts the dominant effect on molecular weight, and (c) the wild-type strain has suboptimal levels of UDP-N-acetylglucosamine. Consistent herewith molecular weight correlated strongly (rho = 0.84, p = 3 x 10(-5)) with the concentration of UDP-N-acetylglucosamine. Data presented in this paper represent the first model for hyaluronan molecular weight control based on the concentration of activated sugar precursors. These results can be used to engineer strains producing high molecular weight hyaluronan and may provide insight into similar polymerization mechanisms in other polysaccharides.

  3. Hyaluronan in Tubular and Interstitial Nephrocalcinosis

    NASA Astrophysics Data System (ADS)

    Verkoelen, Carl F.

    2007-04-01

    Hyaluronan (HA) is the major glycosaminoglycan (GAG) component of the renal medullary interstitium. HA is extremely large (up to 104 kDa) and composed of thousands repeating disaccharides of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). HA is synthesized by hyaluronan synthases (HASs) and degraded by hyaluronidases (Hyals). The production of HA by renomedullary interstitial cells is mediated by local osmolality. When excess water needs to be excreted, increased interstitial HA seems to antagonize water reabsorption, while the opposite occurs during water conservation. Hence, papillary interstitial HA is low and Hyal high during anti-diuresis, whereas during diuresis HA is high and Hyal low. The polyanion HA plays a role in the reabsorption of hypotonic fluid by immobilizing cations (Na+) via the carboxylate (COO-) groups of GlcUA. The binding of Ca2+ to anionic HA is probably also responsible for the fact that the papilla does not become a stone despite the extremely high interstitial phosphate and oxalate. HA is also an excellent crystal binding molecule. The expression of HA at the luminal surface of renal tubular cells leads to tubular nephrocalcinosis (tubular NC). Calcium staining methods (Von Kossa, Yasue) demonstrated that crystallization inhibitors cannot avoid the occasional precipitation of calcium phosphate in the papillary interstitium (interstitial NC). These crystals are probably immediately immobilized by the gel-like HA matrix. After ulcerating through the pelvic wall the calcified matrix becomes a Randall's plaque. The attachment of calcium oxalate crystals from the primary urine to plaque may ultimately lead to the development of clinical stones in the renal calyces (nephrolithiasis).

  4. Hyaluronan: towards novel anti-cancer therapeutics.

    PubMed

    Karbownik, Michał S; Nowak, Jerzy Z

    2013-01-01

    The understanding of the role of hyaluronan in physiology and various pathological conditions has changed since the complex nature of its synthesis, degradation and interactions with diverse binding proteins was revealed. Initially perceived only as an inert component of connective tissue, it is now known to be involved in multiple signaling pathways, including those involved in cancer pathogenesis and progression. Hyaluronan presents a mixture of various length polymer molecules from finely fragmented oligosaccharides, polymers intermediate in size, to huge aggregates of high molecular weight hyaluronan. While large molecules promote tissue integrity and quiescence, the generation of breakdown products enhances signaling transduction, contributing to the pro-oncogenic behavior of cancer cells. Low molecular weight hyaluronan has well-established angiogenic properties, while the smallest hyaluronan oligomers may counteract tumor development. These equivocal properties make the role of hyaluronan in cancer biology very complex. This review surveys recent data on hyaluronan biosynthesis, metabolism, and interactions with its binding proteins called hyaladherins (CD44, RHAMM), providing themolecular background underlying its differentiated biological activity. In particular, the article critically presents current ideas on actual role of hyaluronan in cancer. The paper additionally maps a path towards promising novel anti-cancer therapeutics which target hyaluronan metabolic enzymes and hyaladherins, and constitute hyaluronan-based drug delivery systems.

  5. Hyaluronan and hyaluronidase in genitourinary tumors

    PubMed Central

    Simpson, Melanie A.; Lokeshwar, Vinata B.

    2008-01-01

    Genitourinary cancers are the most frequently diagnosed cancers in men and the fifth most common in women. Management of disease through accurate and cost effective early diagnostic markers, as well as identification of valid prognostic indicators, has contributed significantly to improved treatment outcomes. In this review, we will discuss the function, regulation and clinical utility of hyaluronan (HA), genes encoding its metabolic enzymes and receptors that mediate its cellular effects. Specific HA synthase (HAS) and hyaluronidase (HAase) genes encode the enzymes that produce HA polymers and oligosaccharides, respectively. Differential effects of these enzymes in progression of genitourinary tumors are determined by the relative balance between HAS and HAase levels, as well as the distribution of receptors. The genes are regulated in a complex fashion at the transcriptional and post-translational levels, but also by epigenetic events, alternative mRNA splicing, and subcellular localization. Importantly, the major tumor-derived HAase enzyme, HYAL-1, either alone or together with HA, is an accurate diagnostic and prognostic marker for genitourinary tumors. PMID:18508614

  6. 9 CFR 113.69 - Pasteurella Multocida Vaccine, Bovine.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Pasteurella Multocida Vaccine, Bovine... REQUIREMENTS Live Bacterial Vaccines § 113.69 Pasteurella Multocida Vaccine, Bovine. Pasteurella Multocida Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent...

  7. 9 CFR 113.68 - Pasteurella Haemolytica Vaccine, Bovine.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Pasteurella Haemolytica Vaccine... REQUIREMENTS Live Bacterial Vaccines § 113.68 Pasteurella Haemolytica Vaccine, Bovine. Pasteurella Haemolytica Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent...

  8. 9 CFR 113.69 - Pasteurella Multocida Vaccine, Bovine.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Pasteurella Multocida Vaccine, Bovine... REQUIREMENTS Live Bacterial Vaccines § 113.69 Pasteurella Multocida Vaccine, Bovine. Pasteurella Multocida Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent...

  9. 9 CFR 113.69 - Pasteurella Multocida Vaccine, Bovine.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Pasteurella Multocida Vaccine, Bovine... REQUIREMENTS Live Bacterial Vaccines § 113.69 Pasteurella Multocida Vaccine, Bovine. Pasteurella Multocida Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent...

  10. 9 CFR 113.70 - Pasteurella Multocida Vaccine, Avian Isolate.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Pasteurella Multocida Vaccine, Avian... REQUIREMENTS Live Bacterial Vaccines § 113.70 Pasteurella Multocida Vaccine, Avian Isolate. Pasteurella Multocida Vaccine, Avian Isolate, shall be prepared as a desiccated live culture of an avirulent or...

  11. 9 CFR 113.70 - Pasteurella Multocida Vaccine, Avian Isolate.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Pasteurella Multocida Vaccine, Avian... REQUIREMENTS Live Bacterial Vaccines § 113.70 Pasteurella Multocida Vaccine, Avian Isolate. Pasteurella Multocida Vaccine, Avian Isolate, shall be prepared as a desiccated live culture of an avirulent or...

  12. 9 CFR 113.68 - Pasteurella Haemolytica Vaccine, Bovine.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Pasteurella Haemolytica Vaccine... REQUIREMENTS Live Bacterial Vaccines § 113.68 Pasteurella Haemolytica Vaccine, Bovine. Pasteurella Haemolytica Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent...

  13. 9 CFR 113.69 - Pasteurella Multocida Vaccine, Bovine.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Pasteurella Multocida Vaccine, Bovine... REQUIREMENTS Live Bacterial Vaccines § 113.69 Pasteurella Multocida Vaccine, Bovine. Pasteurella Multocida Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent...

  14. 9 CFR 113.68 - Pasteurella Haemolytica Vaccine, Bovine.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Pasteurella Haemolytica Vaccine... REQUIREMENTS Live Bacterial Vaccines § 113.68 Pasteurella Haemolytica Vaccine, Bovine. Pasteurella Haemolytica Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent...

  15. 9 CFR 113.69 - Pasteurella Multocida Vaccine, Bovine.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Pasteurella Multocida Vaccine, Bovine... REQUIREMENTS Live Bacterial Vaccines § 113.69 Pasteurella Multocida Vaccine, Bovine. Pasteurella Multocida Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent...

  16. 9 CFR 113.70 - Pasteurella Multocida Vaccine, Avian Isolate.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Pasteurella Multocida Vaccine, Avian... REQUIREMENTS Live Bacterial Vaccines § 113.70 Pasteurella Multocida Vaccine, Avian Isolate. Pasteurella Multocida Vaccine, Avian Isolate, shall be prepared as a desiccated live culture of an avirulent or...

  17. 9 CFR 113.68 - Pasteurella Haemolytica Vaccine, Bovine.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Pasteurella Haemolytica Vaccine... REQUIREMENTS Live Bacterial Vaccines § 113.68 Pasteurella Haemolytica Vaccine, Bovine. Pasteurella Haemolytica Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent...

  18. 9 CFR 113.70 - Pasteurella Multocida Vaccine, Avian Isolate.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Pasteurella Multocida Vaccine, Avian... REQUIREMENTS Live Bacterial Vaccines § 113.70 Pasteurella Multocida Vaccine, Avian Isolate. Pasteurella Multocida Vaccine, Avian Isolate, shall be prepared as a desiccated live culture of an avirulent or...

  19. 9 CFR 113.68 - Pasteurella Haemolytica Vaccine, Bovine.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Pasteurella Haemolytica Vaccine... REQUIREMENTS Live Bacterial Vaccines § 113.68 Pasteurella Haemolytica Vaccine, Bovine. Pasteurella Haemolytica Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent...

  20. 9 CFR 113.70 - Pasteurella Multocida Vaccine, Avian Isolate.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Pasteurella Multocida Vaccine, Avian... REQUIREMENTS Live Bacterial Vaccines § 113.70 Pasteurella Multocida Vaccine, Avian Isolate. Pasteurella Multocida Vaccine, Avian Isolate, shall be prepared as a desiccated live culture of an avirulent or...

  1. Synthesis and Organization of Hyaluronan and Versican by Embryonic Stem Cells Undergoing Embryoid Body Differentiation

    PubMed Central

    Shukla, Shreya; Nair, Rekha; Rolle, Marsha W.; Braun, Kathleen R.; Chan, Christina K.; Johnson, Pamela Y.; Wight, Thomas N.; McDevitt, Todd C.

    2010-01-01

    Embryonic stem cells (ESCs) provide a convenient model to probe the molecular and cellular dynamics of developmental cell morphogenesis. ESC differentiation in vitro via embryoid bodies (EBs) recapitulates many aspects of early stages of development, including the epithelial–mesenchymal transition (EMT) of pluripotent cells into more differentiated progeny. Hyaluronan and versican are important extracellular mediators of EMT processes, yet the temporal expression and spatial distribution of these extracellular matrix (ECM) molecules during EB differentiation remains undefined. Thus, the objective of this study was to evaluate the synthesis and organization of hyaluronan and versican by using murine ESCs during EB differentiation. Hyaluronan and versican (V0 and V1 isoforms), visualized by immunohistochemistry and evaluated biochemically, accumulated within EBs during the course of differentiation. Interestingly, increasing amounts of a 70-kDa proteolytic fragment of versican were also detected over time, along with ADAMTS-1 and -5 protein expression. ESCs expressed each of the hyaluronan synthases (HAS) -1, -2, and -3 and versican splice variants (V0, V1, V2, and V3) throughout EB differentiation, but HAS-2, V0, and V1 were expressed at significantly increased levels at each time point examined. Hyaluronan and versican exhibited overlapping expression patterns within EBs in regions of low cell density, and versican expression was excluded from clusters of epithelial (cytokeratin-positive) cells but was enriched within the vicinity of mesenchymal (N-cadherin-positive) cells. These results indicate that hyaluronan and versican synthesized by ESCs within EB microenvironments are associated with EMT processes and furthermore suggest that endogenously produced ECM molecules play a role in ESC differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 58

  2. Activation of the FGFR-STAT3 pathway in breast cancer cells induces a hyaluronan-rich microenvironment that licenses tumor formation.

    PubMed

    Bohrer, Laura R; Chuntova, Pavlina; Bade, Lindsey K; Beadnell, Thomas C; Leon, Ronald P; Brady, Nicholas J; Ryu, Yungil; Goldberg, Jodi E; Schmechel, Stephen C; Koopmeiners, Joseph S; McCarthy, James B; Schwertfeger, Kathryn L

    2014-01-01

    Aberrant activation of fibroblast growth factor receptors (FGFR) contributes to breast cancer growth, progression, and therapeutic resistance. Because of the complex nature of the FGF/FGFR axis, and the numerous effects of FGFR activation on tumor cells and the surrounding microenvironment, the specific mechanisms through which aberrant FGFR activity contributes to breast cancer are not completely understood. We show here that FGFR activation induces accumulation of hyaluronan within the extracellular matrix and that blocking hyaluronan synthesis decreases proliferation, migration, and therapeutic resistance. Furthermore, FGFR-mediated hyaluronan accumulation requires activation of the STAT3 pathway, which regulates expression of hyaluronan synthase 2 (HAS2) and subsequent hyaluronan synthesis. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that STAT3 inhibition decreases both FGFR-driven tumor growth and hyaluronan levels within the tumor. Finally, our results suggest that combinatorial therapies inhibiting both FGFR activity and hyaluronan synthesis is more effective than targeting either pathway alone and may be a relevant therapeutic approach for breast cancers associated with high levels of FGFR activity. In conclusion, these studies indicate a novel targetable mechanism through which FGFR activation in breast cancer cells induces a protumorigenic microenvironment.

  3. Catabolism of hyaluronan: involvement of transition metals

    PubMed Central

    Šoltés, Ladislav; Kogan, Grigorij

    2009-01-01

    One of the very complex structures in the vertebrates is the joint. The main component of the joint is the synovial fluid with its high-molar-mass glycosaminoglycan hyaluronan, which turnover is approximately twelve hours. Since the synovial fluid does not contain any hyaluronidases, the fast hyaluronan catabolism is caused primarily by reductive-oxidative processes. Eight transition metals – V23, Mn25, Fe26, Co27, Ni28, Cu29, Zn30, and Mo42 – naturally occurring in living organism are essential for the control of various metabolic and signaling pathways. They are also the key elements in catabolism of hyaluronan in the joint. In this overview, the role of these metals in physiological and pathophysiological catabolism of hyaluronan is described. The participation of these metals in the initiation and propagation of the radical degradation hyaluronan is critically reviewed. PMID:21217859

  4. Layer-by-layer films from hyaluronan and amine modified hyaluronan

    PubMed Central

    Schneider, Aurore; Senger, Bernard; Schaaf, Pierre; Voegel, Jean-Claude; Frisch, Benoit

    2008-01-01

    Hyaluronan is a polysaccharide that is increasingly investigated for its role in cellular adhesion and for the preparation of biomimetic matrices for tissue engineering. Hyaluronan gels are prepared for application as space fillers whereas hyaluronan films are usually obtained by adsorbing or grafting a single hyaluronan layer onto a biomaterial surface. Here, we examine the possibility to employ the layer-by-layer technique to deposit thin films of cationic modified hyaluronan (HA+) and hyaluronan (HA) of controlled thicknesses. The buildup conditions are investigated and growth is compared to that of other polyelectrolyte multilayer films containing either HA as polyanion or HA+ as polycation. The films could be formed in a low ionic strength medium but required to be cross-linked prior to be put in contact with physiological medium. NIH3T3 fibroblasts were perfectly viable on self-assembled hyaluronan films with however a preference for hyaluronan ending films. These findings point out the possibility to tune the thickness of thin hyaluronan films at the nanometer scale. Such architectures could be employed for investigating cell/substrate interactions or for functionalizing biomaterial surfaces. PMID:17309215

  5. [Biochemical tests for identifying Pasteurella multocida].

    PubMed

    Karaivanov, L

    1984-01-01

    Studied was the biochemical activity of a total of 168 strains of Pasteurella--73 isolated from birds (48 from cases of acute fowl cholera, and 25--of chronic cholera), and 95 isolated from mammals (3 from lambs, 24 from pigs, 36 from cattle, and 32 from rabbits) with regard to the tests determining the hemolytic activity, production of indol, reduction of nitrates, breakdown of urea, beta galactosidase activity, production of hydrogen sulfide, ornitin-, arginine-, lysine-decarboxylase-, and phosphatase activity, and the fermentation of substrates such as manite, glucose, galactose, saccharose, manose, levulose, dulcite, lactose, maltose, rafinose, trechalose, salicin, melobiose, icelobiose, arabinose, xylose, and sorbite. To differentiate Pasteurella multocida strains isolated from mamals from those isolated from birds the phosphatase activity test on solid media with sodium phenolphtalein diphosphate had to be employed Pasteurella organisms isolated from mammals showed positive phosphatase activity, while those isolated from birds exhibited a negative one. Arabinose and xylose fermentation tests could simultaneously be used. Pasteurellae isolated in cases of acute fowl cholera showed positive reaction for arabinose and a negative one for xylose, while the strains isolated from mammals showed the reverse activity. The strains isolated in cases of chronic fowl cholera were shown to belong to this group.

  6. Pasteurella multocida: from Zoonosis to Cellular Microbiology

    PubMed Central

    Ho, Mengfei

    2013-01-01

    SUMMARY In a world where most emerging and reemerging infectious diseases are zoonotic in nature and our contacts with both domestic and wild animals abound, there is growing awareness of the potential for human acquisition of animal diseases. Like other Pasteurellaceae, Pasteurella species are highly prevalent among animal populations, where they are often found as part of the normal microbiota of the oral, nasopharyngeal, and upper respiratory tracts. Many Pasteurella species are opportunistic pathogens that can cause endemic disease and are associated increasingly with epizootic outbreaks. Zoonotic transmission to humans usually occurs through animal bites or contact with nasal secretions, with P. multocida being the most prevalent isolate observed in human infections. Here we review recent comparative genomics and molecular pathogenesis studies that have advanced our understanding of the multiple virulence mechanisms employed by Pasteurella species to establish acute and chronic infections. We also summarize efforts being explored to enhance our ability to rapidly and accurately identify and distinguish among clinical isolates and to control pasteurellosis by improved development of new vaccines and treatment regimens. PMID:23824375

  7. Modulation of TGFβ1-Dependent Myofibroblast Differentiation by Hyaluronan

    PubMed Central

    Webber, Jason; Jenkins, Robert H.; Meran, Soma; Phillips, Aled; Steadman, Robert

    2009-01-01

    Myofibroblasts are contractile cells that are characterized by the expression of α-smooth muscle actin and mediate the closure of wounds and the formation of collagen-rich scars. Their presence in organs such as lungs, liver, and kidney has long been established as a marker of progressive fibrosis. The transforming growth factor beta1-driven differentiation of fibroblasts is a major source of myofibroblasts, and recent data have shown that hyaluronan is a major modulator of this process. This study examines this differentiation mechanism in more detail. Transforming growth factor beta1-dependent differentiation to the myofibroblastic phenotype was antagonized by the inhibition of hyaluronan synthesis, confirming that hyaluronan was necessary for differentiation. This response, however, was not reproduced by simply adding hyaluronan to fibroblasts, as the results implicated hyaladherins, as well as the macromolecular assembly of de novo hyaluronan, as essential in this process. We previously suggested that there is a relocalization of lipid-raft components during myofibroblastic differentiation. The present study demonstrates that the hyaluronan receptor CD44, the hyaluronidase HYAL 2, and the transforming growth factor beta1-receptor ALK5 all relocalized from raft to non-raft locations, which was reversed by the addition of exogenous hyaluronan. These data highlight a role for endogenous hyaluronan in the mediation of myofibroblastic differentiation. While hyaluronan synthesis was both essential and necessary for differentiation, exogenously provided hyaluronan antagonized differentiation, underscoring a pathological role for hyaluronan in such cell fate processes. PMID:19541937

  8. Ingested hyaluronan moisturizes dry skin

    PubMed Central

    2014-01-01

    Hyaluronan (HA) is present in many tissues of the body and is essential to maintain moistness in the skin tissues, which contain approximately half the body’s HA mass. Due to its viscosity and moisturizing effect, HA is widely distributed as a medicine, cosmetic, food, and, recently marketed in Japan as a popular dietary supplement to promote skin moisture. In a randomized, double-blind, placebo-controlled clinical study it was found that ingested HA increased skin moisture and improved treatment outcomes for patients with dry skin. HA is also reported to be absorbed by the body distributed, in part, to the skin. Ingested HA contributes to the increased synthesis of HA and promotes cell proliferation in fibroblasts. These effects show that ingestion of HA moisturizes the skin and is expected to improve the quality of life for people who suffer from dry skin. This review examines the moisturizing effects of dry skin by ingested HA and summarizes the series of mechanisms from absorption to pharmacological action. PMID:25014997

  9. Hyaluronan as an Immune Regulator in Human Diseases

    PubMed Central

    NOBLE, PAUL W.; LIANG, JIURONG; JIANG, DIANHUA

    2010-01-01

    Accumulation and turnover of extracellular matrix components are the hallmarks of tissue injury. Fragmented hyaluronan stimulates the expression of inflammatory genes by a variety of immune cells at the injury site. Hyaluronan binds to a number of cell surface proteins on a variety of cell types. Hyaluronan fragments signal through both Toll-like receptor (TLR) 4 and TLR2 as well as CD44 to stimulate inflammatory genes in inflammatory cells. Hyaluronan is also present on the cell surface of epithelial cells and provides protection against tissue damage by interacting with TLR2 and TLR4 on these parenchymal cells. Hyaluronan and hyaluronan-binding proteins regulate inflammation, tissue injury and repair through regulating inflammatory cell recruitment, release of inflammatory cytokines, and stem cell migration. This review focuses on the role of hyaluronan as an immune regulator in human diseases. PMID:21248167

  10. Hyaluronan-CD44 Interactions in Cancer: Paradoxes and Possibilities

    PubMed Central

    Toole, Bryan P.

    2009-01-01

    Hyaluronan is a prominent component of the micro-environment in most malignant tumors and can be prognostic for tumor progression. Extensive experimental evidence in animal models implicates hyaluronan interactions in tumor growth and metastasis, but it is also evident that a balance of synthesis and turnover by hyaluronidases is critical. CD44, a major hyaluronan receptor, is commonly but not uniformly associated with malignancy, and is frequently used as a marker for cancer stem cells in human carcinomas. Multivalent interactions of hyaluronan with CD44 collaborate in driving numerous tumor-promoting signaling pathways and transporter activities. It is widely accepted that hyaluronan-CD44 interactions are crucial in both malignancy and resistance to therapy, but major challenges for future research in the field are the mechanism of activation of hyaluronan-CD44 signaling in cancer cells, the relative importance of variant forms of CD44 and other hyaluronan receptors, e.g. Rhamm, in different tumor contexts, and the role of stromal versus tumor cell production and turnover of hyaluronan. Despite these caveats, it is clear that hyaluronan-CD44 interactions are an important target for translation into the clinic. Among the approaches that show promise are antibodies and vaccines to specific variants of CD44 that are uniquely expressed at critical stages of progression of a particular cancer, hyaluronidase-mediated reduction of barriers to drug access, and small hyaluronan oligosaccharides that attenuate constitutive hyaluronan-receptor signaling and enhance chemosensitivity. In addition, hyaluronan is being used to tag drugs and delivery vehicles for targeting of anti-cancer agents to CD44-expressing tumor cells. PMID:20008845

  11. Hyaluronan Synthesis, Catabolism, and Signaling in Neurodegenerative Diseases

    PubMed Central

    Sherman, Larry S.; Matsumoto, Steven; Su, Weiping; Srivastava, Taasin; Back, Stephen A.

    2015-01-01

    The glycosaminoglycan hyaluronan (HA), a component of the extracellular matrix, has been implicated in regulating neural differentiation, survival, proliferation, migration, and cell signaling in the mammalian central nervous system (CNS). HA is found throughout the CNS as a constituent of proteoglycans, especially within perineuronal nets that have been implicated in regulating neuronal activity. HA is also found in the white matter where it is diffusely distributed around astrocytes and oligodendrocytes. Insults to the CNS lead to long-term elevation of HA within damaged tissues, which is linked at least in part to increased transcription of HA synthases. HA accumulation is often accompanied by elevated expression of at least some transmembrane HA receptors including CD44. Hyaluronidases that digest high molecular weight HA into smaller fragments are also elevated following CNS insults and can generate HA digestion products that have unique biological activities. A number of studies, for example, suggest that both the removal of high molecular weight HA and the accumulation of hyaluronidase-generated HA digestion products can impact CNS injuries through mechanisms that include the regulation of progenitor cell differentiation and proliferation. These studies, reviewed here, suggest that targeting HA synthesis, catabolism, and signaling are all potential strategies to promote CNS repair. PMID:26448752

  12. Regulation and roles of the hyaluronan system in mammalian reproduction.

    PubMed

    Fouladi-Nashta, Ali A; Raheem, Kabir A; Marei, Waleed F; Ghafari, Fataneh; Hartshorne, Geraldine M

    2017-02-01

    Hyaluronan (HA) is a non-sulphated glycosaminoglycan polymer naturally occurring in many tissues and fluids of mammals, including the reproductive system. Its biosynthesis by HA synthase (HAS1-3) and catabolism by hyaluronidases (HYALs) are affected by ovarian steroid hormones. Depending upon its molecular size, HA functions both as a structural component of tissues in the form of high-molecular-weight HA or as a signalling molecule in the form of small HA molecules or HA fragments with effects mediated through interaction with its specific cell-membrane receptors. HA is produced by oocytes and embryos and in various segments of the reproductive system. This review provides information about the expression and function of members of the HA system, including HAS, HYALs and HA receptors. We examine their role in various processes from folliculogenesis through oocyte maturation, fertilisation and early embryo development, to pregnancy and cervical dilation, as well as its application in assisted reproduction technologies. Particular emphasis has been placed upon the role of the HA system in pre-implantation embryo development and embryo implantation, for which we propose a hypothetical sequential model.

  13. Pasteurella multocida Bacteremia in an Immunocompromised Patient

    PubMed Central

    Parekh, Jai; Townley, Theresa

    2016-01-01

    We present the case of a 61-year-old Caucasian gentleman who presented with a one-day history of fever, chills, and altered mental status. His symptoms were initially thought to be secondary to cellulitis. Blood cultures grew Pasteurella multocida, a rare pathogen to cause bacteremia. Our patient was treated with ciprofloxacin for two weeks and made a complete and uneventful recovery. Our patient's uncontrolled diabetes mellitus and chronic kidney disease put him at a higher risk for developing serious P. multocida infection. The patient's dog licking the wounds on his legs was considered as the possible source of infection. As P. multicoda bacteremia is rare, but severe with a high mortality rate, it is imperative to have a high index of suspicion for this infection especially in the vulnerable immunocompromised population. PMID:27847521

  14. Intranasal immunization of mice against Pasteurella multocida.

    PubMed Central

    Smith, R H; Babiuk, L A; Stockdale, P H

    1981-01-01

    A potassium thiocyanate (KSCN) extract of Pasteurella multocida serotype III:A was shown to protect mice from an intranasal challenge with up to 300 50% lethal doses of P. multocida. In addition to preventing death, bacteria were rapidly cleared from the lungs of immunized mice so that by 72 to 96 h postchallenge no bacteria were present in the lungs of immunized mice, whereas up to 10(9) bacteria were present in lungs of nonimmunized mice. Immunization by the intranasal route was slightly better than that by the intramuscular route. Protection was considered specific, since immunization with P. multocida protected only against P. multocida and not against Salmonella agona. Furthermore, a similar KSCN extract from P. haemolytica did not protect against P. multocida challenge. A comparison of the KSCN extract with a Formalin-killed bacterin suggested that the KSCN extract may be superior to the bacterin. PMID:7216441

  15. The role of hyaluronan in wound healing.

    PubMed

    Frenkel, Joseph S

    2014-04-01

    The polysaccharide hyaluronan (HA) (synonyms - hyaluronic acid, hyaluronate) is a versatile, polymorphic, glycosoaminoglycan with vast biological functions. HA is found throughout the body, primarily residing in skin, thus playing an important role in wound healing. Research regarding HA's function has changed over the years, primarily focussing on a particular aspect or function. The contribution of HA in each stage of normal wound healing as well as its clinical wound dressing applications will be examined.

  16. Characteristics and biotypes of Pasteurella multocida isolated from humans.

    PubMed Central

    Oberhofer, T R

    1981-01-01

    Fifty-two isolates of Pasteurella (48 strains of Pasteurella multocida and 4 strains of atypical Pasteurella) were identified by conventional and commercial test systems. All strains fermented glucose, sucrose, and fructose in purple broth base (Difco Laboratories) with bromocresol purple as indicator, although the atypical Pasteurella produced fermentation reactions that were barely perceptible. Eleven different biotypes were identified by fermentation reactions in maltose, mannitol, xylose, sorbitol, and trehalose media. There was a correlation of biotypes to cat bites, with 61% of cat bite isolates falling into biotype A and B. A correlation of biotype and dog bite isolates was not seen. The choice of medium used for fermentation tests was critical as evidenced by the inability of the organisms to grow in a second commercially purchased preparation of purple broth base. The reliability of commercial test systems in identifying Pasteurella was 81% for Oxi/Ferm (Roche Diagnostics, Div. Hoffmann-La Roche, Inc., Nutley, N.J.), 68% for API (Analytab Products, Plainview, N.Y.), and 11% for Minitek (BBL Microbiology Systems, Cockeysville, MD.). PMID:7240390

  17. The effect of Pasteurella haemolytica and the leukotoxin of Pasteurella haemolytica on bovine lung explants.

    PubMed Central

    Wilkie, I W; Fallding, M H; Shewen, P E; Yager, J A

    1990-01-01

    Bovine lung explants were used in a study designed to compare the pathogenic effects of Pasteurella haemolytica type 1, a nonpathogenic organism Neisseria subflava, or the crude leukotoxin of P. haemolytica on alveolar macrophages and lung parenchymal cells. Concentrated, purified peripheral blood neutrophil suspensions were added with the bacteria to some explants. Duplicate pairs of cultures from each treatment group were fixed at regular intervals up to 24 hours after seeding and morphological changes were assessed by light and electron microscopy. Pasteurella haemolytica caused deterioration of alveolar macrophages within one hour but did not affect parenchymal cells for more than 12 hours. Neisseria subflava did not affect alveolar macrophages initially, but caused an accelerated deterioration after four hours. After 24 hours, bacterial overgrowth caused similar deterioration of all cells in explants seeded with either bacterium. Alveolar macrophages phagocytosed large numbers of N. subflava but rarely ingested P. haemolytica. Added neutrophils did not have any discernible effect on any of the explants and did not potentiate bacterial effects. Addition of crude leukotoxin of P. haemolytica to the culture medium significantly accelerated alveolar macrophage deterioration without apparent effect on parenchymal cell survival. These results support the hypothesis that the severe tissue destruction of fulminant pneumonic pasteurellosis is not a direct result of bacterial infection. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:2306666

  18. Pasteurella caballi, a new species from equine clinical specimens.

    PubMed Central

    Schlater, L K; Brenner, D J; Steigerwalt, A G; Moss, C W; Lambert, M A; Packer, R A

    1989-01-01

    The name Pasteurella caballi is proposed for a group of organisms represented by 29 strains isolated from respiratory and other infections in horses. P. caballi strains are gram-negative, oxidase-positive, nonmotile, fermentative rods with the key characteristics of the genus Pasteurella. These strains differed from other Pasteurella species in that all were aerogenic and catalase negative, and some strains produced acid from myo-inositol and L-rhamnose. The levels of DNA relatedness of 28 P. caballi strains with labeled DNA from the proposed type strain averaged 91 and 85% (hydroxyapatite method at 55 and 70 degrees C). P. caballi was 13 to 53% related to strains representing 22 other species of the family Pasteurellaceae. The guanine-plus-cytosine content of the DNA of four strains was 41 to 42 mol%. The type strain is 83851 (=ATCC 49197). PMID:2584369

  19. A numerical taxonomic study of Actinobacillus, Pasteurella and Yersinia.

    PubMed

    Sneath, P H; Stevens, M

    1985-10-01

    A numerical taxonomic study of strains of Actinobacillus, Pasteurella and Yersinia, with some allied bacteria, showed 23 reasonably distinct groups. These fell into three major areas. Area A contained species of Actinobacillus and Pasteurella: A. suis, A. equuli, A. lignieresii, P. haemolytica biovar A, P. haemolytica biovar T, P. multocida, A. actinomycetemcomitans, 'P. bettii', 'A. seminis', P. ureae and P. aerogenes. Also included in A was a composite group of Pasteurella pneumotropica and P. gallinarum, together with unnamed groups referred to as 'BLG', 'Mair', 'Ross' and 'aer-2'. Area B contained species of Yersinia: Y. enterocolitica, Y. pseudotuberculosis, Y. pestis and a group 'ent-b' similar to Y. enterocolitica. Area C contained non-fermenting strains: Y. philomiragia, Moraxella anatipestifer and a miscellaneous group 'past-b'. There were also a small number of unnamed single strains.

  20. Hyaluronan is not elevated in urine or serum in Hutchinson-Gilford Progeria Syndrome.

    PubMed

    Gordon, Leslie B; Harten, Ingrid A; Calabro, Anthony; Sugumaran, Geetha; Csoka, Antonei B; Brown, W Ted; Hascall, Vincent; Toole, Bryan P

    2003-07-01

    Elevations in urinary hyaluronan have been used as the principal laboratory indicator for diagnosis of Hutchinson-Gilford Progeria Syndrome (HGPS). Previous reports have provided evidence suggesting that children with HGPS have altered hyaluronan metabolism as indicated by a mean 17-fold increase in urinary hyaluronan over normal values. In addition, adults with Werner's syndrome have elevated urinary hyaluronan and even more prominent elevations in serum hyaluronan over age-matched controls. It is not known whether serum hyaluronan is elevated or whether serum hyaluronan levels correlate with urinary hyaluronan levels in children with HGPS. In a large cohort of 19 HGPS patients, we sought to confirm elevations in urinary hyaluronan concentration, to establish whether serum hyaluronan is elevated, to measure the size of urinary hyaluronan, and to determine whether serum or urine hyaluronidase levels are altered. We have analyzed urinary and serum hyaluronan levels in patients with HGPS and control patients (1) by using an enzyme-linked immunosorbent assay (ELISA)-like method in which sample hyaluronan in solution and hyaluronan in solid phase compete for a solution of biotinylated hyaluronan-binding protein, and (2) by fluorophore-assisted carbohydrate electrophoresis. The size of urinary hyaluronan was measured by using Sepharose CL-6B size exclusion chromatography. Serum and urinary hyaluronidases were evaluated quantitatively, by using ELISA, and qualitatively, by using a gel detection method. HGPS patients did not show a significant elevation in either urinary or serum hyaluronan. We detected no difference in the size of urinary hyaluronan between HGPS children and age-matched controls. Serum and urinary hyaluronidase levels were not significantly different in normal and HGPS patients. These studies indicate that neither serum nor urinary hyaluronan concentration is a reliable diagnostic or prognostic marker for HGPS and underscore a difference between adult

  1. Isolation of Pasteurella haemolytica leukotoxin mutants.

    PubMed Central

    Chidambaram, M; Sharma, B; Petras, S F; Reese, C P; Froshauer, S; Weinstock, G M

    1995-01-01

    Two mutants of Pasteurella haemolytica A1 that do not produce leukotoxin were isolated. Following mutagenesis, colonies were screened with antiserum by a filter assay for absence of the secreted leukotoxin. The two mutants both appeared to produce normal amounts of other antigens, as judged by reactivity with polyclonal serum from an animal with pasteurellosis, and were not altered in beta-hemolytic activity as seen on blood agar plates. There was no evidence of either cell-associated or secreted leukotoxin protein when Western blots (immunoblots) were carried out with the polyclonal serum or with a monoclonal antibody directed against the leukotoxin. Southern blots revealed that both mutants show the wild-type restriction pattern at the leukotoxin locus, although the strain with the lktA2 mutation showed differences in other regions of the chromosome on analysis by pulsed-field gel electrophoresis. The strain with the lktA2 mutation grew more slowly than did the wild-type strain, while the strain with the lktA1 mutation was indistinguishable from the wild-type strain in its growth properties. The strain with the lktA1 mutation should be valuable in determining the role of the leukotoxin in virulence as well as in identifying other virulence factors of P. haemolytica. PMID:7868223

  2. Single-molecule imaging of hyaluronan in human synovial fluid

    NASA Astrophysics Data System (ADS)

    Kappler, Joachim; Kaminski, Tim P.; Gieselmann, Volkmar; Kubitscheck, Ulrich; Jerosch, Jörg

    2010-11-01

    Human synovial fluid contains a high concentration of hyaluronan, a high molecular weight glycosaminoglycan that provides viscoelasticity and contributes to joint lubrication. In osteoarthritis synovial fluid, the concentration and molecular weight of hyaluronan decrease, thus impairing shock absorption and lubrication. Consistently, substitution of hyaluronan (viscosupplementation) is a widely used treatment for osteoarthritis. So far, the organization and dynamics of hyaluronan in native human synovial fluid and its action mechanism in viscosupplementation are poorly characterized at the molecular level. Here, we introduce highly sensitive single molecule microscopy to analyze the conformation and interactions of fluorescently labeled hyaluronan molecules in native human synovial fluid. Our findings are consistent with a random coil conformation of hyaluronan in human synovial fluid, and point to specific interactions of hyaluronan molecules with the synovial fluid matrix. Furthermore, single molecule microscopy is capable of detecting the breakdown of the synovial fluid matrix in osteoarthritis. Thus, single molecule microscopy is a useful new method to probe the structure of human synovial fluid and its changes in disease states like osteoarthritis.

  3. A transport medium for specimens containing Pasteurella pestis.

    PubMed

    Cavanaugh, D C; Vivona, S; Do-Van-Quy; Gibson, F L; Deuber, G L; Rust, J H

    1967-01-01

    A medium, originally designed by Stuart and co-workers and later modified by Cary & Blair, for the maintenance and transport, without multiplication, of pathogenic bacteria contained in bacteriological specimens was tested in the laboratory and in the field in Viet-Nam to determine its effectiveness in preserving specimens known to contain Pasteurella pestis.The results indicate that this medium should be useful in diagnostic plague studies in areas where transport facilities are inadequate. Properly collected clinical specimens, sent to a central laboratory by any means and under any climatic conditions likely to be encountered in the hot tropics, should yield viable Pasteurella pestis for at least 30 days.

  4. A Case of Polyarticular Pasteurella multocida Septic Arthritis

    PubMed Central

    Nitoslawski, Sarah; McConnell, Todd M.; Semret, Makeda; Stein, Michael A.

    2016-01-01

    A 76-year-old man with a history of osteoarthritis presents with right leg erythema and inability to weight-bear and pain in his right shoulder. Synovial fluid cell count of the knee and shoulder showed abundant neutrophils, and cultures of the knee showed growth of Pasteurella multocida. The patient owned four cats with which he had frequent contact, but history and physical examination elicited no evidence of scratches or bites. This case highlights the invasive potential of Pasteurella multocida in an immunocompetent individual and its capacity to cause septic arthritis in the setting of frequent animal contact. PMID:27366169

  5. Pasteurella multocida meningitis in an adult: case report

    PubMed Central

    Armstrong, G; Sen, R; Wilkinson, J

    2000-01-01

    Pasteurella multocida is known to form part of the normal flora in the nasopharynx or gastrointestinal tract in many domestic and wild animals. Most human P multocida infections are soft tissue infections caused by dog or cat bites. Less commonly this bacterium is associated with infections affecting other organ systems of man. A case of fatal P multocida meningitis discovered at the necropsy of a 52 year old man is described. P multocida is an unusual causative agent of meningitis which tends to affect those at the extremes of age. Key Words: Pasteurella multocida • meningitis PMID:10823146

  6. Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation.

    PubMed

    Malaisse, Jérémy; Pendaries, Valérie; Hontoir, Fanny; De Glas, Valérie; Van Vlaender, Daniel; Simon, Michel; Lambert de Rouvroit, Catherine; Poumay, Yves; Flamion, Bruno

    2016-03-18

    Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal.

  7. Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

    PubMed Central

    Kashima, Yuichiro; Takahashi, Masafumi; Shiba, Yuji; Itano, Naoki; Izawa, Atsushi; Koyama, Jun; Nakayama, Jun; Taniguchi, Shun'ichiro; Kimata, Koji; Ikeda, Uichi

    2013-01-01

    Background Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy. Methods and Results HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS. Conclusion VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease. PMID:23484050

  8. Antitumor effects of hyaluronan inhibition in desmoid tumors.

    PubMed

    Briggs, Alexandra; Rosenberg, Laura; Buie, Justin D; Rizvi, Hira; Bertagnolli, Monica M; Cho, Nancy L

    2015-02-01

    Desmoid tumors (DTs) are rare, mesenchymal tumors that exhibit features of an abundant wound healing process. Previously, we showed that mesenchymal stem cells (MSCs) are constituents of DTs and may contribute to desmoid tumorigenesis via activities associated with wound healing. Hyaluronan (HA) is a long-charged chain of repeating glucuronate and N-acetylglucosamine disaccharides that is synthesized by HA synthases (HAS) and degraded by hyaluronidases (HYAL). HA is secreted into the extracellular matrix by injured stroma and is important for normal tissue repair and neoplastic progression. Here, we investigated the presence of HA in DTs and the antitumor effects of the HA inhibitor, 4-methylumbelliferone (4-MU), on DT-derived mesenchymal cells. By immunohistochemistry and enzyme-linked immunosorbent assay, we found abundant expression of HA in 29/30 DTs as well as >5-fold increased HA levels in DT-derived cell lines relative to controls. Immunohistochemistry also demonstrated high expression of HAS2 in DTs, and quantitative PCR analysis showed increased HAS2 upregulation in frozen DTs and DT-derived cells. 4-MU treatment of DT-derived cells significantly decreased proliferation as well as HA and HAS2 levels. Fluorescent immunohistochemistry showed that MSCs in DTs coexpressed HA, HAS2, HYAL2, as well as the major HA receptor CD44 and HA coreceptor TLR4. Taken together, our results suggest that paracrine regulation of HA signaling in DTs may contribute to MSC recruitment and tumor proliferation. Future studies investigating the role of HA in tumor-stroma crosstalk and inhibition of HA-MSC interactions as a novel therapeutic target in DTs and other solid tumors are warranted.

  9. Endotoxin free hyaluronan and hyaluronan fragments do not stimulate TNF-α, interleukin-12 or upregulate co-stimulatory molecules in dendritic cells or macrophages

    PubMed Central

    Dong, Yifei; Arif , Arif; Olsson, Mia; Cali, Valbona; Hardman, Blair; Dosanjh, Manisha; Lauer, Mark; Midura, Ronald J.; Hascall, Vincent C.; Brown, Kelly L.; Johnson, Pauline

    2016-01-01

    The extracellular matrix glycosaminoglycan, hyaluronan, has been described as a regulator of tissue inflammation, with hyaluronan fragments reported to stimulate innate immune cells. High molecular mass hyaluronan is normally present in tissues, but upon inflammation lower molecular mass fragments are generated. It is unclear if these hyaluronan fragments induce an inflammatory response or are a consequence of inflammation. In this study, mouse bone marrow derived macrophages and dendritic cells (DCs) were stimulated with various sizes of hyaluronan from different sources, fragmented hyaluronan, hyaluronidases and heavy chain modified-hyaluronan (HA-HC). Key pro-inflammatory molecules, tumour necrosis factor alpha, interleukin-1 beta, interleukin-12, CCL3, and the co-stimulatory molecules, CD40 and CD86 were measured. Only human umbilical cord hyaluronan, bovine testes and Streptomyces hyaluronlyticus hyaluronidase stimulated macrophages and DCs, however, these reagents were found to be contaminated with endotoxin, which was not fully removed by polymyxin B treatment. In contrast, pharmaceutical grade hyaluronan and hyaluronan fragments failed to stimulate in vitro-derived or ex vivo macrophages and DCs, and did not induce leukocyte recruitment after intratracheal instillation into mouse lungs. Hence, endotoxin-free pharmaceutical grade hyaluronan does not stimulate macrophages and DCs in our inflammatory models. These results emphasize the importance of ensuring hyaluronan preparations are endotoxin free. PMID:27869206

  10. Mannose reduces hyaluronan and leukocytes in wound granulation tissue and inhibits migration and hyaluronan-dependent monocyte binding.

    PubMed

    Jokela, Tiina A; Kuokkanen, Jukka; Kärnä, Riikka; Pasonen-Seppänen, Sanna; Rilla, Kirsi; Kössi, Jyrki; Laato, Matti; Tammi, Raija H; Tammi, Markku I

    2013-01-01

    Wound healing is a highly regulated process starting from coagulation and ending in tissue remodeling. The end result varies from perfectly restored tissue, such as in early fetal skin, to scars in adults. The balanced repair process is frequently disturbed by local or systemic factors, like infections and diabetes. A rapid increase of hyaluronan is an inherent feature of wounds and is associated with tissue swelling, epithelial and mesenchymal cell migration and proliferation, and induction of cytokine signaling. Hyaluronan extending from cell surface into structures called cables can trap leukocytes and platelets and change their functions. All these features of hyaluronan modulate inflammation. The present data show that mannose, a recently described inhibitor of hyaluronan synthesis, inhibits dermal fibroblast invasion and prevents the enhanced leukocyte binding to hyaluronan that takes place in cells treated with an inflammatory mediator interleukin-1β. Mannose also reduced hyaluronan in subcutaneous sponge granulation tissue, a model of skin wound, and suppressed its leukocyte recruitment and tissue growth. Mannose thus seems to suppress wounding-induced inflammation in skin by attenuating hyaluronan synthesis.

  11. Polypeptide Grafted Hyaluronan: Synthesis and Characterization

    SciTech Connect

    Wang, Xiaojun; Messman, Jamie M; Mays, Jimmy; Baskaran, Durairaj

    2010-01-01

    Poly(L-leucine) grafted hyaluronan (HA-g-PLeu) has been synthesized via a Michael addition reaction between primary amine terminated poly(L-leucine) and acrylate-functionalized HA (TBAHA-acrylate). The precursor hyaluronan was first functionalized with acrylate groups by reaction with acryloyl chloride in the presence of triethylamine in N,N-dimethylformamide. 1H NMR analysis of the resulting product indicated that an increase in the concentration of acryloylchoride with respect to hydroxyl groups on HA has only a moderate effect on functionalization efficiency, f. A precise control of stoichiometry was not achieved, which could be attributed to partial solubility of intermolecular aggregates and the hygroscopic nature of HA. Michael addition at high [PLeu- NH2]/[acrylate]TBAHA ratios gave a molar grafting ratio of only 0.20 with respect to the repeat unit of HA, indicating grafting limitation due to insolubility of the grafted HA-g-PLeu. Soluble HA-g-PLeu graft copolymers were obtained for low grafting ratios (<0.039) with <8.6% by mass of PLeu and were characterized thoroughly using light scattering, 1H NMR, FT-IR, and AFM techniques. Light scattering experiments showed a strong hydrophobic interaction between PLeu chains, resulting in aggregates with segregated nongrafted HA segments. This yields local networks of aggregates, as demonstrated by atomic force microscopy. Circular dichroism spectroscopy showed a -sheet conformation for aggregates of poly(L-leucine).

  12. Polypeptide Grafted Hyaluronan: Synthesis and Characterization

    PubMed Central

    Wang, Xiaojun; Messman, Jamie; Mays, Jimmy W.; Baskaran, Durairaj

    2010-01-01

    Poly(L-leucine) grafted hyaluronan (HA-g-PLeu) has been synthesized via a Michael addition reaction between primary amine terminated poly(L-leucine) and acrylate functionalized HA (TBAHA-acrylate). The precursor hyaluronan was first functionalized with acrylate groups by reaction with acryloyl chloride in the presence of triethylamine in N,N-dimethylformamide. 1H NMR analysis of the resulting product indicated that an increase in the concentration of acryloylchoride with respect to hydroxyl groups on HA has only a moderate effect on functionalization efficiency, f. A precise control of stoichiometry was not achieved, which could be attributed to partial solubility of intermolecular aggregates and the hygroscopic nature of HA. Michael addition at high [PLeu-NH2]/[acrylate]TBAHA ratios gave a molar grafting ratio of only 0.20 with respect to the repeat unit of HA, indicating grafting limitation due to insolubility of the grafted HA-g-PLeu. Soluble HA-g-PLeu graft copolymers were obtained for low grafting ratios (< 0.039) with < 8.6 % by mass of PLeu and were characterized thoroughly using light scattering, 1H NMR, FT-IR and AFM techniques. Light scattering experiments showed a strong hydrophobic interaction between PLeu chains, resulting in aggregates with segregated non-grafted HA segments. This yields local networks of aggregates as demonstrated by atomic force microscopy. Circular dichroism spectroscopy showed a β-sheet conformation for aggregates of poly(L-leucine). PMID:20690642

  13. Aortic Endograft Infection by Pasteurella multocida: A Rare Case.

    PubMed

    Jayakrishnan, Thejus T; Keyashian, Brian; Amene, Juliet; Malinowski, Michael

    2016-08-01

    Infection of an aortic endograft is a rare complication following endovascular aneurysm repair. These patients have been treated with explantation of the graft to obtain source control followed by an extra-anatomic bypass to restore circulation. The present case study describes an interesting case of Pasteurella infection involving an aortic endograft managed nonoperatively by percutaneous drainage and graft preservation.

  14. Genome Sequence of Pasteurella multocida Strain Razi_Pm0001

    PubMed Central

    Tadayon, Keyvan

    2017-01-01

    ABSTRACT We report here the genome sequence of Pasteurella multocida Razi_Pm0001 from bovine origin, isolated in Iran in 1936. The genome has a size of 2,360,663 bp, a G+C content of 40.4%, and is predicted to contain 2,052 coding sequences. PMID:28153892

  15. Inhibition of Oesophageal Squamous Cell Carcinoma Progression by in vivo Targeting of Hyaluronan Synthesis

    PubMed Central

    2011-01-01

    Background Oesophageal cancer is a highly aggressive tumour entity with at present poor prognosis. Therefore, novel treatment options are urgently needed. Hyaluronan (HA) is a polysaccharide present in the matrix of human oesophageal squamous cell carcinoma (ESCC). Importantly, in vitro ESCC cells critically depend on HA synthesis to maintain the proliferative phenotype. The aim of the present study is (1) to study HA-synthase (HAS) expression and regulation in human ESCC, and (2) to translate the in vitro results into a mouse xenograft model of human ESCC to study the effects of systemic versus tumour targeted HAS inhibition on proliferation and distribution of tumour-bound and stromal hyaluronan. Methods mRNA expression was investigated in human ESCC biopsies by semiquantitative real-time RT PCR. Furthermore, human ESCC were xenografted into NMRI nu/nu mice. The effects on tumour progression and morphology of 4-methylumbelliferone (4-MU), an inhibitor of HA-synthesis, and of lentiviral knock down of HA-synthase 3 (HAS3), the main HAS isoform in the human ESCC tissues and the human ESCC cell line used in this study, were determined. Tumour progression was monitored by calliper measurements and by flat-panel detector volume computed tomography (fpVCT). HA content, cellular composition and proliferation (Ki67) were determined histologically. Results mRNA of HAS isoform 3 (HAS3) was upregulated in human ESCC biopsies and HAS3 mRNA was positively correlated to expression of the epidermal growth factor (EGF) receptor. EGF was also proven to be a strong inductor of HAS3 mRNA expression in vitro. During the course of seven weeks, 4-MU inhibited progression of xenograft tumours. Interestingly, remodelling of the tumour into a more differentiated phenotype and inhibition of cell proliferation were observed. Lentiviral knockdown of HAS3 in human ESCC cells prior to xenografting mimicked all effects of 4-MU treatment suggesting that hyaluronan produced by ESCC is accountable

  16. Hyaluronan, a Crucial Regulator of Inflammation

    PubMed Central

    Petrey, Aaron C.; de la Motte, Carol A.

    2014-01-01

    Hyaluronan (HA), a major component of the extracellular matrix (ECM), plays a key role in regulating inflammation. Inflammation is associated with accumulation and turnover of HA polymers by multiple cell types. Increasingly through the years, HA has become recognized as an active participant in inflammatory, angiogenic, fibrotic, and cancer promoting processes. HA and its binding proteins regulate the expression of inflammatory genes, the recruitment of inflammatory cells, the release of inflammatory cytokines, and can attenuate the course of inflammation, providing protection against tissue damage. A growing body of evidence suggests the cell responses are HA molecular weight dependent. HA fragments generated by multiple mechanisms throughout the course of inflammatory pathologies, elicit cellular responses distinct from intact HA. This review focuses on the role of HA in the promotion and resolution of inflammation. PMID:24653726

  17. Pasteurella Multocida Peritonitis After Cat Scratch in a Patient with Cirrhotic Ascites

    PubMed Central

    Gunathilake, Roshan; Verma, Ajay; Caffery, Michael; Sowden, Sowden

    2015-01-01

    Pasteurella multocida, a zoonotic agent transmitted by canines and felines, has been very rarely reported to cause bacterial peritonitis in humans. Pasteurella multocida peritonitis is associated with high mortality even with appropriate treatment, therefore its early recognition is essential. We report a case of Pasteurella multocida peritonitis following cat scratch in a patient with Child Pugh Class C alcoholic cirrhosis, culminating in multiple organ failure and death PMID:26294953

  18. The Where, When, How, and Why of Hyaluronan Binding by Immune Cells

    PubMed Central

    Lee-Sayer, Sally S. M.; Dong, Yifei; Arif, Arif A.; Olsson, Mia; Brown, Kelly L.; Johnson, Pauline

    2015-01-01

    Hyaluronan is made and extruded from cells to form a pericellular or extracellular matrix (ECM) and is present in virtually all tissues in the body. The size and form of hyaluronan present in tissues are indicative of a healthy or inflamed tissue, and the interactions of hyaluronan with immune cells can influence their response. Thus, in order to understand how inflammation is regulated, it is necessary to understand these interactions and their consequences. Although there is a large turnover of hyaluronan in our bodies, the large molecular mass form of hyaluronan predominates in healthy tissues. Upon tissue damage and/or infection, the ECM and hyaluronan are broken down and an inflammatory response ensues. As inflammation is resolved, the ECM is restored, and high molecular mass hyaluronan predominates again. Immune cells encounter hyaluronan in the tissues and lymphoid organs and respond differently to high and low molecular mass forms. Immune cells differ in their ability to bind hyaluronan and this can vary with the cell type and their activation state. For example, peritoneal macrophages do not bind soluble hyaluronan but can be induced to bind after exposure to inflammatory stimuli. Likewise, naïve T cells, which typically express low levels of the hyaluronan receptor, CD44, do not bind hyaluronan until they undergo antigen-stimulated T cell proliferation and upregulate CD44. Despite substantial knowledge of where and when immune cells bind hyaluronan, why immune cells bind hyaluronan remains a major outstanding question. Here, we review what is currently known about the interactions of hyaluronan with immune cells in both healthy and inflamed tissues and discuss how hyaluronan binding by immune cells influences the inflammatory response. PMID:25926830

  19. Reclassification of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov.

    PubMed

    Blackall, Patrick J; Christensen, Henrik; Beckenham, Tim; Blackall, Linda L; Bisgaard, Magne

    2005-01-01

    This paper describes a phenotypic and genotypic investigation of the taxonomy of [Haemophilus] paragallinarum, Pasteurella gallinarum, Pasteurella avium and Pasteurella volantium, a major subcluster within the avian 16S rRNA cluster 18 of the family Pasteurellaceae. An extended phenotypic characterization was performed of the type strain of [Haemophilus] paragallinarum, which is NAD-dependent, and eight NAD-independent strains of [Haemophilus] paragallinarum. Complete 16S rRNA gene sequences were obtained for one NAD-independent and four NAD-dependent [Haemophilus] paragallinarum strains. These five sequences along with existing 16S rRNA gene sequences for 11 other taxa within avian 16S rRNA cluster 18 as well as seven other taxa from the Pasteurellaceae were subjected to phylogenetic analysis. The analysis demonstrated that [Haemophilus] paragallinarum, Pasteurella gallinarum, Pasteurella avium and Pasteurella volantium formed a monophyletic group with a minimum of 96.8 % sequence similarity. This group can also be separated by phenotypic testing from all other recognized and named taxa within the Pasteurellaceae. As both genotypic and phenotypic testing support the separate and distinct nature of this subcluster, the transfer is proposed of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium to a new genus Avibacterium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov. The type strains are NCTC 1118T (Avibacterium gallinarum), NCTC 11296T (Avibacterium paragallinarum), NCTC 11297T (Avibacterium avium) and NCTC 3438T (Avibacterium volantium). Key characteristics that separate these four species are catalase activity (absent only in Avibacterium paragallinarum) and production of acid from galactose (negative only in Avibacterium paragallinarum), maltose (negative only in Avibacterium avium) and mannitol (negative

  20. Hyaluronan is essential for the expansion of the cranial base growth plates.

    PubMed

    Gakunga, P T; Kuboki, Y; Opperman, L A

    2000-01-01

    Exquisite control of chondrocyte function in the zone of hypertrophy results in expansive growth of cartilaginous growth plates, and is a prerequisite for normal skeletal lengthening. We hypothesize that hyaluronan-mediated hydrostatic pressure causes lacunae expansion in the zone of hypertrophy; an important mechanism in cartilaginous growth plate and associated skeletal expansion. The role of hyaluronan and CD44 in this mechanism was studied using organ culture of the bipolar cranial base synchondroses. Hyaluronan was present in the hypertrophic zones, pericellular to the hypertrophic chondrocytes, while no hyaluronan was detected in the resting, proliferating and maturing zones. This localization of hyaluronan was associated with increased lacunae size, suggesting that chondrocytes deposit and retain pericellular hyaluronan as they mature. In comparison, Toluidine Blue staining was associated with the territorial matrix. Hyaluronidase, the hyaluronan-degrading enzyme, and CD44, the receptor for hyaluronan which also participates in the uptake and degradation of hyaluronan, were co-localized within the zone of ossification. This pattern of expression suggests that cells in the early zone of ossification internalize and degrade hyaluronan through a CD44-mediated mechanism. Treatment of the cultured segments with either Streptomyces hyaluronidase or hyaluronan hexasaccharides inhibited lacunae expansion. These observations demonstrate that hyaluronan-mediated mechanisms play an important role in controlling normal skeletal lengthening.

  1. Pasteurella multocida bacterial meningitis caused by contact with pigs

    PubMed Central

    López, C.; Sanchez-Rubio, P.; Betrán, A.; Terré, R.

    2013-01-01

    Pasteurella multocida belongs to the normal flora of the respiratory and digestive tract of many animals. Animal exposure is a considerable risk factor for Pasteurella infection. P. multocida is the most common cause of local infection after an animal bite but is an unusual cause of meningitis. We present a case of bacterial meningitis by P. multocida in a 37-year-old man who worked in a pig farm and was bitten by a pig. The patient had a defect located in the lamina cribosa and this lesion could be the gateway of the infection, although in this case the infection could also be acquired through the pig bite. The bacteria was identified as P. multocida with the biochemical test API 20E (bioMérieux). In agreement with findings in the literature, the strain was susceptible in vitro to penicillin, ampicillin, cefotaxime, ceftriaxone ciprofloxacin, levofloxacin, imipenem and tetracycline. PMID:24294240

  2. Sepsis-induced purpura fulminans caused by Pasteurella multocida

    PubMed Central

    Borges, Lisa; Oliveira, Nelson; Cássio, Isabel; Costa, Humberto

    2014-01-01

    A 52-year-old man was admitted with a cutaneous rash associated with septic shock and multiorganic failure, 6 days after a dog bite. He was started on empiric antibiotherapy and supportive measures. The patient's condition aggravated, with need for invasive mechanical ventilation and intermittent haemodialysis, and evolution from a petechiae-like rash to purpura and gangrene, culminating in bilateral lower limb amputation. The blood cultures revealed only Pasteurella multocida, after 10 days of incubation. P multocida infection is a rare cause of soft tissue infection that subsides with oral antibiotherapy. Infections causing sepsis are rare and appear in immunocompromised patients. Purpura fulminans induced by sepsis is a rare, life-threatening disorder. This syndrome should be recognised promptly, so early treatment is instituted. We found no case reports of purpura fulminans caused by Pasteurella infections in our literature review. PMID:24554680

  3. VIRULENCE AND CITRULLINE UREIDASE ACTIVITY OF PASTEURELLA TULARENSIS12

    PubMed Central

    Marchette, Nyven J.; Nicholes, Paul S.

    1961-01-01

    Marchette, Nyven J. (University of Utah, Salt Lake City), and Paul S. Nicholes. Virulence and citrulline ureidase activity of Pasteurella tularensis. J. Bacteriol. 82:26–32. 1961.—The presence of a citrulline ureidase system in Pasteurella tularensis strains of high virulence, and its absence in avirulent strains and strains of low virulence was confirmed. The presence of this system, however, was shown to be not directly related to virulence. The only wild strain of P. tularensis tested that lacked a citrulline ureidase system was isolated from a rodent. All the strains, isolated from rabbits, rabbit ticks, a human being, and a horse, that were tested possessed this system. The existence of two North American varieties of P. tularensis was postulated on the basis of virulence and citrulline ureidase activity. PMID:13766500

  4. Virulence and citrulline ureidase activity of Pasteurella tularensis.

    PubMed

    MARCHETTE, N J; NICHOLES, P S

    1961-07-01

    Marchette, Nyven J. (University of Utah, Salt Lake City), and Paul S. Nicholes. Virulence and citrulline ureidase activity of Pasteurella tularensis. J. Bacteriol. 82:26-32. 1961.-The presence of a citrulline ureidase system in Pasteurella tularensis strains of high virulence, and its absence in avirulent strains and strains of low virulence was confirmed. The presence of this system, however, was shown to be not directly related to virulence. The only wild strain of P. tularensis tested that lacked a citrulline ureidase system was isolated from a rodent. All the strains, isolated from rabbits, rabbit ticks, a human being, and a horse, that were tested possessed this system. The existence of two North American varieties of P. tularensis was postulated on the basis of virulence and citrulline ureidase activity.

  5. A cryopreservation method for Pasteurella multocida from wetland samples

    USGS Publications Warehouse

    Moore, Melody K.; Shadduck, D.J.; Goldberg, D.R.; Samuel, M.D.

    1998-01-01

    A cryopreservation method and improved isolation techniques for detection of Pasteurella multocida from wetland samples were developed. Wetland water samples were collected in the field, diluted in dimethyl sulfoxide (DMSO, final concentration 10%), and frozen at -180 C in a liquid nitrogen vapor shipper. Frozen samples were transported to the laboratory where they were subsequently thawed and processed in Pasteurella multocida selective broth (PMSB) to isolate P. multocida. This method allowed for consistent isolation of 2 to 18 organisms/ml from water seeded with known concentrations of P. multocida. The method compared favorably with the standard mouse inoculation method and allowed for preservation of the samples until they could be processed in the laboratory.

  6. Systemic infection by Pasteurella canis biotype 1 in newborn puppies.

    PubMed

    de la Puente Redondo, V A; Gutiérrez Martín, C B; García del Blanco, N; Antolín Ayala, M I; Alonso Alonso, P; Rodríguez Ferri, E F

    2000-01-01

    Pasteurella canis biotype 1, usually associated with the oral cavity of dogs and cats, or with human wound infections following dog bites, was isolated from newborn puppies with a fatal systemic infection. The identity of P. canis was confirmed by arbitrarily primed polymerase chain reaction and the organism was susceptible to all the penicillins, cephalosporins, tetracyclines and fluoroquinolones tested and to most of the aminoglycosides tested. This represents the first report of systemic pasteurellosis caused by P. canis in dogs.

  7. Community-acquired pneumonia due to Pasteurella multocida.

    PubMed

    Marinella, Mark A

    2004-12-01

    Most cases of community-acquired pneumonia result from infection with predictable common pathogens. However, rare patients develop pneumonia from unusual bacterial species such as Pasteurella multocida, a Gram-negative oral commensal of most dogs and cats. The majority of P. multocida infections involve skin and soft tissue and complicate a bite or scratch. I report the case of an elderly man who owned 16 cats and developed bacteremic pneumonia with P. multocida. .

  8. The porcine sperm reservoir in relation to the function of hyaluronan

    PubMed Central

    TIENTHAI, Paisan

    2015-01-01

    The oviduct plays a role in successful animal reproduction not only in spermatozoa and ova transport to the fertilization site but also by affording a microenvironment for fertilization and early embryonic development. The sperm reservoir (SR) is restricted in the uterotubal junction (UTJ) and caudal isthmus. Billions of porcine spermatozoa are distributed to the female reproductive tract during/after insemination, and small amounts of them are stored for about 36–40 hours in the SR, which maintains sperm viability in the pre-ovulation period through its surface epithelium and production of fluid. The SR regulates the release of spermatozoa so that only a small population moves towards the fertilization site (ampulla) to decrease polyspermy. This review attempts to provide information about the structure and function of the porcine SR, its intraluminal content (hyaluronan, HA), and the influences of HA on porcine spermatozoa in vivo. In pigs, the spermatozoa are stored in a mucous-like fluid within the UTJ and caudal isthmus in the pre-ovulation period. The oviduct fluid contains sulfated glycosaminoglycans (GAGs) and non-sulfated GAGs, i.e., HA. It is interesting to note that HA is synthesized by hyaluronan synthase-3 (HAS-3), and its receptor, CD44, is found in the epithelium of the porcine SR site. Additionally, sperm capacitation does not occur in vivo in the SR during the pre- and peri-ovulation periods, but spermatozoa in the SR will attempt to capacitate if exposed to bicarbonate. However, capacitation in the SR will rise in the post-ovulation period, indicating the role of HA in modulating sperm capacitation after ovulation. All data support the understanding that the porcine SR ensures the viability of fertile spermatozoa and maintains the non-capacitated status during the pre-ovulation period. This basic knowledge about the SR is believed to be useful to advance sperm preparation procedures for in vitro fertilization (IVF) and improve the preservation

  9. Spontaneous Metastasis of Prostate Cancer Is Promoted by Excess Hyaluronan Synthesis and Processing

    PubMed Central

    Bharadwaj, Alamelu G.; Kovar, Joy L.; Loughman, Eileen; Elowsky, Christian; Oakley, Gregory G.; Simpson, Melanie A.

    2009-01-01

    Accumulation of extracellular hyaluronan (HA) and its processing enzyme, the hyaluronidase Hyal1, predicts invasive, metastatic progression of human prostate cancer. To dissect the roles of hyaluronan synthases (HAS) and Hyal1 in tumorigenesis and metastasis, we selected nonmetastatic 22Rv1 prostate tumor cells that overexpress HAS2, HAS3, or Hyal1 individually, and compared these cells with co-transfectants expressing Hyal1 + HAS2 or Hyal1 + HAS3. Cells expressing only HAS were less tumorigenic than vector control transfectants on orthotopic injection into mice. In contrast, cells co-expressing Hyal1 + HAS2 or Hyal1 + HAS3 showed greater than sixfold and twofold increases in tumorigenesis, respectively. Fluorescence and histological quantification revealed spontaneous lymph node metastasis in all Hyal1 transfectant-implanted mice, and node burden increased an additional twofold when Hyal1 and HAS were co-expressed. Cells only expressing HAS were not metastatic. Thus, excess HA synthesis and processing in concert accelerate the acquisition of a metastatic phenotype by prostate tumor cells. Intratumoral vascularity did not correlate with either tumor size or metastatic potential. Analysis of cell cycle progression revealed shortened doubling times of Hyal1-expressing cells. Both adhesion and motility on extracellular matrix were diminished in HA-overproducing cells; however, motility was increased twofold by Hyal1 expression and fourfold to sixfold by Hyal1/HAS co-expression, in close agreement with observed metastatic potential. This is the first comprehensive examination of these enzymes in a relevant prostate cancer microenvironment. PMID:19218337

  10. Hyaluronan Expressed by the Hematopoietic Microenvironment Is Required for Bone Marrow Hematopoiesis*

    PubMed Central

    Goncharova, Valentina; Serobyan, Naira; Iizuka, Shinji; Schraufstatter, Ingrid; de Ridder, Audrey; Povaliy, Tatiana; Wacker, Valentina; Itano, Naoki; Kimata, Koji; Orlovskaja, Irina A.; Yamaguchi, Yu; Khaldoyanidi, Sophia

    2012-01-01

    The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2flox/flox;Has1−/−;Has3−/− triple knock-out (tKO) mice as compared with wild type (WT) and Has1−/−;Has3−/− double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. Collectively, the results suggest that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment. PMID:22654110

  11. High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat.

    PubMed

    Tian, Xiao; Azpurua, Jorge; Hine, Christopher; Vaidya, Amita; Myakishev-Rempel, Max; Ablaeva, Julia; Mao, Zhiyong; Nevo, Eviatar; Gorbunova, Vera; Seluanov, Andrei

    2013-07-18

    The naked mole rat (Heterocephalus glaber) displays exceptional longevity, with a maximum lifespan exceeding 30 years. This is the longest reported lifespan for a rodent species and is especially striking considering the small body mass of the naked mole rat. In comparison, a similarly sized house mouse has a maximum lifespan of 4 years. In addition to their longevity, naked mole rats show an unusual resistance to cancer. Multi-year observations of large naked mole-rat colonies did not detect a single incidence of cancer. Here we identify a mechanism responsible for the naked mole rat's cancer resistance. We found that naked mole-rat fibroblasts secrete extremely high-molecular-mass hyaluronan (HA), which is over five times larger than human or mouse HA. This high-molecular-mass HA accumulates abundantly in naked mole-rat tissues owing to the decreased activity of HA-degrading enzymes and a unique sequence of hyaluronan synthase 2 (HAS2). Furthermore, the naked mole-rat cells are more sensitive to HA signalling, as they have a higher affinity to HA compared with mouse or human cells. Perturbation of the signalling pathways sufficient for malignant transformation of mouse fibroblasts fails to transform naked mole-rat cells. However, once high-molecular-mass HA is removed by either knocking down HAS2 or overexpressing the HA-degrading enzyme, HYAL2, naked mole-rat cells become susceptible to malignant transformation and readily form tumours in mice. We speculate that naked mole rats have evolved a higher concentration of HA in the skin to provide skin elasticity needed for life in underground tunnels. This trait may have then been co-opted to provide cancer resistance and longevity to this species.

  12. Hyaluronan in cytosol--microinjection-based probing of its existence and suggested functions.

    PubMed

    Siiskonen, Hanna; Rilla, Kirsi; Kärnä, Riikka; Bart, Genevieve; Jing, Wei; Haller, Michael F; DeAngelis, Paul L; Tammi, Raija H; Tammi, Markku I

    2013-02-01

    Hyaluronan (HA) is a large glycosaminoglycan produced by hyaluronan synthases (HAS), enzymes normally active at plasma membrane. While HA is delivered into the extracellular space, intracellular HA is also seen, mostly in vesicular structures, but there are also reports on its presence in the cytosol and specific locations and functions there. We probed the possibility of HA localization and functions in cytosol by microinjecting fluorescent HA binding complex (fHABC), HA fragments and hyaluronidase (HYAL) into cytosol. Microinjection of fHABC did not reveal HA-specific intracellular binding sites. Likewise, specific cytosolic binding sites for HA were not detected, as microinjected fluorescent HA composed of 4-8 monosaccharide units (HA4-HA8) were evenly distributed throughout the cells, including the nucleus, but excluded from membrane-bound organelles. The largest HA tested (∼HA120 or ∼25 kDa) did not enter the nucleus, and HA10-HA28 were progressively excluded from parts of nuclei resembling nucleoli. In contrast, HA oligosaccharides endocytosed from medium remained in vesicular compartments. The activity of HA synthesis was estimated by measuring the HA coat on green fluorescent protein (GFP)-HAS3-transfected MCF-7 cells. Microinjection of HA4 reduced coat size at 4 h, but increased at 24 h after injection, while larger HA-oligosaccharides and HYAL had no influence. As a positive control, microinjection of glucose increased coat size. In summary, no evidence for the presence or function of HA in cytosol was obtained. Also, the synthesis of HA and the active site of HAS were not accessible to competition, binding and degradation by cytosolic effectors, while synthesis responded to increased substrate supply.

  13. Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions.

    PubMed

    Minsky, Burcu Baykal; Antoni, Christiane H; Boehm, Heike

    2016-02-17

    Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end.

  14. Rheology of mixed alginate-hyaluronan aqueous solutions.

    PubMed

    Travan, Andrea; Fiorentino, Simona; Grassi, Mario; Borgogna, Massimiliano; Marsich, Eleonora; Paoletti, Sergio; Donati, Ivan

    2015-01-01

    The present manuscript addresses the description of binary systems of hyaluronan (HA) and alginate (Alg) in semi-concentrated solution. The two polysaccharides were completely miscible in the entire range of relative weight fraction explored at a total polymer concentration of up to 3% (w/V). The rheological study encompassed steady flow and mechanical spectra for HA/Alg systems at different weight fractions with hyaluronan at different molecular weights. These extensive analyses allowed us to propose a model for the molecular arrangement in solution that envisages a mutual exclusion between the two polysaccharides even though a clear phase separation does not occur. This result may have profound implications when combinations of alginate and hyaluronan are proposed in the field of biomedical materials.

  15. Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions

    PubMed Central

    Minsky, Burcu Baykal; Antoni, Christiane H.; Boehm, Heike

    2016-01-01

    Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end. PMID:26883791

  16. Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions

    NASA Astrophysics Data System (ADS)

    Minsky, Burcu Baykal; Antoni, Christiane H.; Boehm, Heike

    2016-02-01

    Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end.

  17. Interaction of Hyaluronan with Cationic Nanoparticles.

    PubMed

    Bano, Fouzia; Carril, Mónica; Di Gianvincenzo, Paolo; Richter, Ralf P

    2015-08-04

    The polysaccharide hyaluronan (HA) is a main component of peri- and extracellular matrix, and an attractive molecule for materials design in tissue engineering and nanomedicine. Here, we study the morphology of complexes that form upon interaction of nanometer-sized amine-coated gold particles with this anionic, linear, and regular biopolymer in solution and grafted to a surface. We find that cationic nanoparticles (NPs) have profound effects on HA morphology on the molecular and supramolecular scale. Quartz crystal microbalance (QCM-D) shows that depending on their relative abundance, cationic NPs promote either strong compaction or swelling of films of surface-grafted HA polymers (HA brushes). Transmission electron and atomic force microscopy reveal that the NPs do also give rise to complexes of distinct morphologies-compact nanoscopic spheres and extended microscopic fibers-upon interaction with HA polymers in solution. In particular, stable and hydrated spherical complexes of single HA polymers with NPs can be prepared when balancing the ionizable groups on HA and NPs. The observed self-assembly phenomena could be useful for the design of drug delivery vehicles and a better understanding of the reorganization of HA-rich synthetic or biological matrices.

  18. Hyaluronan, Inflammation, and Breast Cancer Progression

    PubMed Central

    Schwertfeger, Kathryn L.; Cowman, Mary K.; Telmer, Patrick G.; Turley, Eva A.; McCarthy, James B.

    2015-01-01

    Breast cancer-induced inflammation in the tumor reactive stroma supports invasion and malignant progression and is contributed to by a variety of host cells including macrophages and fibroblasts. Inflammation appears to be initiated by tumor cells and surrounding host fibroblasts that secrete pro-inflammatory cytokines and chemokines and remodel the extracellular matrix (ECM) to create a pro-inflammatory “cancerized” or tumor reactive microenvironment that supports tumor expansion and invasion. The tissue polysaccharide hyaluronan (HA) is an example of an ECM component within the cancerized microenvironment that promotes breast cancer progression. Like many ECM molecules, the function of native high-molecular weight HA is altered by fragmentation, which is promoted by oxygen/nitrogen free radicals and release of hyaluronidases within the tumor microenvironment. HA fragments are pro-inflammatory and activate signaling pathways that promote survival, migration, and invasion within both tumor and host cells through binding to HA receptors such as CD44 and RHAMM/HMMR. In breast cancer, elevated HA in the peri-tumor stroma and increased HA receptor expression are prognostic for poor outcome and are associated with disease recurrence. This review addresses the critical issues regarding tumor-induced inflammation and its role in breast cancer progression focusing specifically on the changes in HA metabolism within tumor reactive stroma as a key factor in malignant progression. PMID:26106384

  19. Genome integrity, stem cells and hyaluronan

    PubMed Central

    Darzynkiewicz, Zbigniew; Balazs, Endre A.

    2012-01-01

    Faithful preservation of genome integrity is the critical mission of stem cells as well as of germ cells. Reviewed are the following mechanisms involved in protecting DNA in these cells: (a) The efflux machinery that can pump out variety of genotoxins in ATP-dependent manner; (b) the mechanisms maintaining minimal metabolic activity which reduces generation of reactive oxidants, by-products of aerobic respiration; (c) the role of hypoxic niche of stem cells providing a gradient of variable oxygen tension; (d) (e) the presence of hyaluronan (HA) and HA receptors on stem cells and in the niche; (f) the role of HA in protecting DNA from oxidative damage; (g) the specific function of HA in protecting DNA in stem cells; (h) the interactions of HA with sperm cells and oocytes that also may shield their DNA from oxidative damage, and (e) mechanisms by which HA exerts the anti-oxidant activity. While HA has multitude of functions its anti-oxidant capabilities are often overlooked but may be of significance in preservation of integrity of stem and germ cells genome. PMID:22383371

  20. Host Response in Rabbits to Infection with Pasteurella multocida Serogroup F Strains Originating from Fowl Cholera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of two avian Pasteurella multocida serogroup F strains to induce disease in rabbits was investigated in this study. Two groups of 18 Pasteurella-free rabbits each were intranasally challenged with strains isolated from chicken and turkey, respectively. Half the animals in each challenge ...

  1. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

  2. Hypotheses on the evolution of hyaluronan: A highly ironic acid

    PubMed Central

    Csoka, Antonei B; Stern, Robert

    2013-01-01

    Hyaluronan is a high-molecular-weight glycosaminoglycan (GAG) prominent in the extracellular matrix. Emerging relatively late in evolution, it may have evolved to evade immune recognition. Chondroitin is a more ancient GAG and a possible hyaluronan precursor. Epimerization of a 4-hydroxyl in N-acetylgalactosamine in chondroitin to N-acetylglucosamine of hyaluronan is the only structural difference other than chain length between these two polymers. The axial 4-hydroxyl group extends out perpendicular from the equatorial plane of N-acetylgalactosamine in chondroitin. We suspect that this hydroxyl is a prime target for immune recognition. Conversion of a thumbs-up hydroxyl group into a thumbs-down position in the plane of the sugar endows hyaluronan with the ability to avoid immune recognition. Chitin is another potential precursor to hyaluronan. But regardless whether of chondroitin or of chitin origin, an ancient chondroitinase enzyme sequence seems to have been commandeered to catalyze the cleavage of the new hyaluronan substrate. The evolution of six hyaluronidase-like sequences in the human genome from a single chondroitinase as found in Caenorhabditis elegans can now be traced. Confirming our previous predictions, two duplication events occurred, with three hyaluronidase-like sequences occurring in the genome of Ciona intestinalis (sea squirt), the earliest known chordate. This was probably followed by en masse duplication, with six such genes present in the genome of zebra fish onwards. These events occurred, however, much earlier than predicted. It is also apparent on an evolutionary time scale that in several species, this gene family is continuing to evolve. PMID:23315448

  3. Transport of a hyaluronan-binding protein in brain tissue.

    PubMed

    Kappler, Joachim; Hegener, Oliver; Baader, Stephan L; Franken, Sebastian; Gieselmann, Volkmar; Häberlein, Hanns; Rauch, Uwe

    2009-09-01

    Hyaluronan is an unsulfated linear glycosaminoglycan with the ability to nucleate extracellular matrices by the formation of aggregates with lecticans. These matrices are essential during development of the central nervous system. In the prospective white matter of the developing brain hyaluronan is organized into fiber-like structures according to confocal microscopy of fixed slices which may guide the migration of neural precursor cells [Baier, C., S.L. Baader, J. Jankowski, V. Gieselmann, K. Schilling, U. Rauch, and J. Kappler. 2007. Hyaluronan is organized into fiber-like structures along migratory pathways in the developing mouse cerebellum. Matrix Biol. 26: 348-58]. By using plasmon surface resonance, microinjection into brain slices and fluorescence correlation spectroscopy, we show that the brain-specific lecticans bind to, but also dissociate rather rapidly from hyaluronan. After microinjection into native cerebellar slices a GFP-tagged hyaluronan-binding neurocan fragment was enriched at binding sites in the prospective white matter, which had a directional orientation and formed local stationary concentration gradients in areas where binding sites are abundant. Fluorescence correlation spectroscopy measurements at fixed brain slices revealed that fiber-bound neurocan-GFP was mobile with D(fiber(neurocan-GFP))=4x10(-10)cm(2)/s. Therefore, we propose that hyaluronan-rich fibers in the prospective white matter of the developing mouse cerebellum can guide the diffusion of lecticans. Since lecticans bind a variety of growth and mobility factors, their guided diffusion may contribute to the transport of these polypeptides and to the formation of concentration gradients. This mechanism could serve to encode positional information during development.

  4. Removal rate of ( sup 3 H)hyaluronan injected subcutaneously in rabbits

    SciTech Connect

    Reed, R.K.; Laurent, U.B.; Fraser, J.R.; Laurent, T.C. )

    1990-08-01

    Hyaluronan is an important constituent of the extracellular matrix in skin, and recent studies suggest that there is a pool of easily removable (free) hyaluronan drained by lymph. The removal rate of free hyaluronan in skin was measured from the elimination of ({sup 3}H)hyaluronan, injected subcutaneously in 13 rabbits. The removal of radioactivity was determined from appearance of {sup 3}H in plasma. During the first 24 h after injection, 10-87% of the tracer entered blood, less in injectates with high concentrations of hyaluronan. The removal was monoexponential with a half-life of 0.5-1 day when concentration of hyaluronan was 5 mg/ml or less. When hyaluronan concentration was 10 mg/ml or higher, the removal was slow for about 24 h and then became similar to that in experiments with low hyaluronan concentration. Free hyaluronan at physiological concentrations is thus turned over with the same rate as serum albumin, supporting the concept that hyaluronan is removed essentially by lymph flow to be degraded in lymph nodes and liver.

  5. Preparation of hyaluronan polyaldehyde-a precursor of biopolymer conjugates.

    PubMed

    Šedová, Petra; Buffa, Radovan; Kettou, Sofiane; Huerta-Angeles, Gloria; Hermannová, Martina; Leierová, Veronika; Šmejkalová, Daniela; Moravcová, Martina; Velebný, Vladimír

    2013-04-19

    Native hyaluronan (HA) has been oxidized to polyaldehyde polymers with a degree of substitution (DS) of up to 50%. Two different procedures enabling the control of the degree of substitution were followed in this study. Selective oxidation of primary hydroxyl groups of N-acetyl-D-glucosamine of hyaluronan was performed either in an aqueous solution containing AcNH-TEMPO/NaBr/NaOCl or in an aprotic solvent containing Dess-Martin periodinane (DMP). It was found that a change of reaction parameters (reaction time and temperature, type of catalyst, oxidant-to-HA ratio, presence of nitrogen, buffer type, and concentration) had an influence on the degree of substitution and molecular weight. The derivatives were characterized by MS, NMR spectroscopy, and SEC-MALLS. Degradation of hyaluronic acid by the oxidant was observed and confirmed by SEC. The effect of oxidized derivatives of hyaluronan on cells was studied by means of NIH 3T3 fibroblast viability, which indicates that prepared hyaluronan polyaldehydes are biocompatible and suitable for medical applications and tissue engineering. The function of polyaldehyde as precursor for other modification was illustrated in the reaction with lysine.

  6. Molecular characteristics of some commercial high-molecular-weight hyaluronans.

    PubMed

    Soltés, L; Mendichi, R; Lath, D; Mach, M; Bakos, D

    2002-10-01

    Commercially available hyaluronan (HA) samples were investigated by the method of size exclusion chromatography (SEC). The fractions eluted from the SEC column were on-line molecularly characterized by using a multi-angle laser light scattering (MALLS) photometer. Along with the SEC-MALLS technique, the high-molecular-weight HA biopolymers were (off-line) analyzed by capillary viscometry.

  7. Hydrogen Peroxide as an Effective Disinfectant for Pasteurella multocida

    PubMed Central

    Jung, In-Soo; Kim, Hyun-Jung; Jung, Won-Yong

    2014-01-01

    Pasteurella multocida (P. multocida) infections vary widely, from local infections resulting from animal bites and scratches to general infections. As of yet, no vaccine against P. multocida has been developed, and the most effective way to prevent pathogenic transmission is to clean the host environment using disinfectants. In this study, we identified which disinfectants most effectively inhibited environmental isolates of P. multocida. Three readily available disinfectants were compared: 3% hydrogen peroxide (HP), 70% isopropyl alcohol, and synthetic phenol. In suspension tests and zone inhibition tests, 3% HP was the most promising disinfectant against P. multocida. PMID:24954350

  8. Pasteurella multocida serotype 1 isolated from a lesser snow goose

    USGS Publications Warehouse

    Samuel, M.D.; Goldberg, D.R.; Shadduck, D.J.; Price, J.I.; Cooch, E.G.

    1997-01-01

    Pharyngeal swabs were collected from 298 lesser snow geese (Chen caerulescens caerulescens) at Banks Island (Northwest Territories. Canada) in the summer of 1994. Pasteurella multocida serotype 1 was isolated from an adult male bird and P. multocida serotype 3 was isolated from an adult female goose. Pathogenicity of the serotype 1 isolate was confirmed by inoculation in Pekin ducks (Anas platyrhynchos). The serotype 3 isolate was non-pathogenic in Pekin ducks. This is the first documented isolation of pathogenic P. multocida serotype 1 from apparently healthy wild snow geese.

  9. Controlled release of plasmid DNA from hyaluronan nanoparticles.

    PubMed

    Mahor, Sunil; Collin, Estelle; Dash, Biraja C; Pandit, Abhay

    2011-07-01

    Encapsulation of plasmid DNA (pDNA) in nanoparticulate gene delivery systems offers the possibility of control in dosing, enhanced pDNA uptake, increased resistance to nuclease degradation and sustained release of functionally active pDNA over time. Extracellular matrix based biomaterial i.e. hyaluronan (HA) was used to encapsulate pDNA (pCMV-GLuc, Gaussia Luciferase reporter plasmid DNA having CMV promoter) in submicron size particulate system. Nano size range (~400-600 nm) pDNA loaded hyaluronan nanoparticles were formulated by ionic gelation followed by the cross-linking method with high encapsulation efficiency (~75-85%). The particle preparation process was further optimized for molecular weight, cross-linking method, cross-linking time and plasmid/polymer ratio. The entrapped plasmid maintained its structural and functional integrity as revealed by agarose gel electrophoresis. The pDNA was released from the hyaluronan nanoparticles in a controlled manner over a period of one month. In vitro transfection by one-week released pDNA from nanoparticles with transfecting agent branched polyethyleneimine (bPEI) resulted in significantly higher expression levels than those in pDNA alone which demonstrated the functional bioactivity of released pDNA. For cellular localization studies, the hyaluronan nanoparticles encapsulated with FITC-dextran were incubated with adipose derived stem cells (ADSCs) and localization in the cellular environment were investigated. The results of this study illustrate that hyaluronan nanoparticles were rapidly internalized by the cells through nonspecific endocytosis and remained intact in the cytosol for up to 24 h.

  10. Molecular Analysis of Tetracycline Resistance in Pasteurella aerogenes

    PubMed Central

    Kehrenberg, Corinna; Schwarz, Stefan

    2001-01-01

    Tetracycline-resistant Pasteurella aerogenes isolates obtained from the intestinal tract of swine were investigated for their tet genes by PCR analysis and hybridization experiments. In contrast to Pasteurella isolates from the respiratory tract, tet(H) genes were detected in the chromosomal DNA of only 2 of the 24 isolates, one of which also carried two copies of a tet(B) gene. All other P. aerogenes isolates carried tet(B) genes, which are the predominant tet genes among Enterobacteriaceae. A single isolate harbored a tet(B) gene as part of a truncated Tn10 element on the 4.8-kb plasmid pPAT2. Comparative analysis of the pPAT2 sequence suggested that the Tn10 relic on plasmid pPAT2 is the result of several illegitimate recombination events. The remaining 21 P. aerogenes isolates carried one or two copies of the tet(B) gene in their chromosomal DNA. In the majority of the cases, these tet(B) genes were associated with copies of Tn10 as confirmed by their SfuI and BamHI hybridization patterns. No correlation between the number of tet gene copies and the MICs of tetracycline, doxycyline and minocycline was observed. PMID:11557485

  11. A RHAMM mimetic peptide blocks hyaluronan signaling and reduces inflammation and fibrogenesis in excisional skin wounds.

    PubMed

    Tolg, Cornelia; Hamilton, Sara R; Zalinska, Ewa; McCulloch, Lori; Amin, Ripal; Akentieva, Natalia; Winnik, Francoise; Savani, Rashmin; Bagli, Darius J; Luyt, Len G; Cowman, Mary K; McCarthy, Jim B; Turley, Eva A

    2012-10-01

    Hyaluronan is activated by fragmentation and controls inflammation and fibroplasia during wound repair and diseases (eg, cancer). Hyaluronan-binding peptides were identified that modify fibrogenesis during skin wound repair. Peptides were selected from 7- to 15mer phage display libraries by panning with hyaluronan-Sepharose beads and assayed for their ability to block fibroblast migration in response to hyaluronan oligosaccharides (10 kDa). A 15mer peptide (P15-1), with homology to receptor for hyaluronan mediated motility (RHAMM) hyaluronan binding sequences, was the most effective inhibitor. P15-1 bound to 10-kDa hyaluronan with an affinity of K(d) = 10(-7) and appeared to specifically mimic RHAMM since it significantly reduced binding of hyaluronan oligosaccharides to recombinant RHAMM but not to recombinant CD44 or TLR2,4, and altered wound repair in wild-type but not RHAMM(-/-) mice. One topical application of P15-1 to full-thickness excisional rat wounds significantly reduced wound macrophage number, fibroblast number, and blood vessel density compared to scrambled, negative control peptides. Wound collagen 1, transforming growth factor β-1, and α-smooth muscle actin were reduced, whereas tenascin C was increased, suggesting that P15-1 promoted a form of scarless healing. Signaling/microarray analyses showed that P15-1 blocks RHAMM-regulated focal adhesion kinase pathways in fibroblasts. These results identify a new class of reagents that attenuate proinflammatory, fibrotic repair by blocking hyaluronan oligosaccharide signaling.

  12. Effect of intra-articular hyaluronan on pressure-flow relation across synovium in anaesthetized rabbits.

    PubMed Central

    McDonald, J N; Levick, J R

    1995-01-01

    1. Hyaluronan is the major polysaccharide of synovial fluid, responsible for its high viscosity. The effect of hyaluronan on fluid transport across the synovial lining of the joint was investigated. Rate of fluid absorption from the joint cavity (Qs) was measured at intra-articular pressures (Pj) of up to 24 cmH2O in knees of anaesthetized rabbits, in the presence or absence of hyaluronan in intra-articular infusates. 2. Viscometry studies in vitro showed that the commercial hyaluronan used had a molecular weight of 549,000-774,000, a radius of gyration of 48-99 nm and a critical concentration for molecular overlap of 1.3 g l-1. 3. With intra-articular Krebs solution (control) or subnormal, subcritical concentrations of hyaluronan (0.5 g l-1), flow increased with pressure. Hyaluronan reduced the fluid escape rate by reducing slope dQs/dPj by 32-64% relative to Krebs solution. 4. At normal to high hyaluronan concentrations (3-6 g l-1) and low pressures, hyaluronan again reduced slope dQs/dPj, by 39-64%. The reduction in slope was slight, however, when compared with the reduction in bulk fluidity (1/relative viscosity). Fluidity at high shear rates was reduced to 6% of control values by 6 g l-1 hyaluronan. The effect on slope did not correlate significantly with the effect on fluidity. 5. At pressures above approximately 12 cmH2O, 3-6 g l-1 hyaluronan altered the shape of the pressure-flow relation: a flow plateau developed. In some joints raising pressure even reduced trans-synovial flow slightly. The pressure required to drive unit trans-synovial flow (an index of outflow resistance) increased 2.5-fold between 5 and 25 cmH2O in the presence of hyaluronan. By contrast, in the absence of hyaluronan the outflow resistance fell as pressure was raised. 6. It is suggested that the increasing resistance to flow in the presence of hyaluronan may be caused by partial molecular sieving of hyaluronan by the small porosities of the synovial interstitial matrix, leading to

  13. Hyaluronan: a critical component of epithelial-mesenchymal and epithelial-carcinoma transitions.

    PubMed

    Toole, Bryan P; Zoltan-Jones, Alexandra; Misra, Suniti; Ghatak, Shibnath

    2005-01-01

    Hyaluronan plays a central role in the transition of epithelia to mesenchyme in the embryo and in the acquisition of transformed properties in carcinoma cells. In some cases, hyaluronan is both essential and sufficient for induction of epithelial-mesenchymal transitions (EMTs). Underlying its role are the effects of hyaluronan on receptor kinase activities, cell survival pathways, and multidrug transporters. A more complete understanding of the mechanisms whereby hyaluronan exerts its influences on cell behavior will enhance our understanding of normal and pathological EMTs and may lead to improved therapies for cancer patients.

  14. Hyaluronan Export through Plasma Membranes Depends on Concurrent K+ Efflux by Kir Channels

    PubMed Central

    Hagenfeld, Daniel; Borkenhagen, Beatrice; Schulz, Tobias; Schillers, Hermann; Schumacher, Udo; Prehm, Peter

    2012-01-01

    Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K+ channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl2 which all belong to ATP-sensitive inwardly-rectifying Kir channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K+ channels Kir3.4 and Kir6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K+ efflux. PMID:22701748

  15. Hyaluronan Is Required for Generation of Hematopoietic Cells during Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Schraufstatter, Ingrid U.; Serobyan, Naira; Loring, Jeanne; Khaldoyanidi, Sophia K.

    2010-01-01

    Hyaluronan (HA) is an important component of the microenvironment in bone marrow, but its role in regulation of the development of hematopoietic cells is not well understood. To address the role of HA in regulation of human embryonic stem cell (hESC) differentiation into the hematopoietic lineage, we screened for genes encoding components of the HA pathway. Using gene arrays, we found that HA synthases and HA receptors are expressed in both undifferentiated and differentiating hESCs. Enzymatic degradation of HA resulted in decreased numbers of hematopoietic progenitors and lower numbers of CD45+ cells generated in HA-deprived embryoid bodies (EBs). In addition, deprivation of HA resulted in the inhibition of generation of CD31+ cells, stromal fibroblast-like cells and contracting myocytes in EBs. RT-PCR and immunocytochemistry revealed that HA deprivation did not influence the dynamics of OCT4 expression, but decreased the expression of BRY, an early mesoderm marker, and BMP2, a later mesoderm marker in differentiating EBs. In addition, the endoderm markers α-FP and SOX17 were decreased, whereas the expression of the ectoderm markers GFAP and FGF5 was higher in HA-deprived cultures. Our findings indicate that endogenously produced HA contributes to the network that regulates the differentiation of hESC and the generation of mesodermal lineage in general and hematopoietic cells specifically. PMID:20861924

  16. Hyaluronan Stabilizes Focal Adhesions, Filopodia, and the Proliferative Phenotype in Esophageal Squamous Carcinoma Cells*

    PubMed Central

    Twarock, Sören; Tammi, Markku I.; Savani, Rashmin C.; Fischer, Jens W.

    2010-01-01

    Hyaluronan (HA) is a polysaccharide component in the parenchyma and stroma of human esophageal squamous cell carcinoma (ESCC). Clinically, esophageal cancer represents a highly aggressive tumor type with poor prognosis resulting in a 5-year survival rate of 5%. The aim of the present study was the detailed analysis of the role of HA synthesis for ESCC phenotype in vitro using the ESCC cell line OSC1. In OSC1 cells, pericellular HA-matrix surrounding extended actin-dependent filopodia was detected. The small molecule inhibitor of HA synthesis, 4-methylumbelliferone (4-MU, 0.3 mm) caused loss of these filopodia and focal adhesions and inhibited proliferation and migration. In search of the underlying mechanism cleavage of focal adhesion kinase (FAK) was detected by immunoblotting. In addition, displacing HA by an HA-binding peptide (Pep-1, 500 μg/ml) and digestion of pericellular HA by hyaluronidase resulted in cleavage of focal adhesions. Furthermore, real-time reverse transcription PCR revealed that HA synthase 3 (HAS3) > HAS2 are the predominant HA-synthases in OSC1. Lentiviral transduction with shHAS3, and to a lesser extent with shHAS2, reduced intact FAK protein and filopodia as well as proliferation and migration. Furthermore, down-regulation by lentiviral shRNA of RHAMM (receptor of HA-mediated motility) but not CD44 induced loss of filopodia and caused FAK cleavage. In contrast, knockdown of both HA receptors inhibited proliferation and migration of OSC1. In conclusion, HA synthesis and, in turn, RHAMM and CD44 signaling promoted an activated phenotype of OSC1. Because RHAMM appears to support both filopodia, FAK, and the proliferative and migratory phenotype, it may be promising to explore RHAMM as a potential therapeutic target in esophageal cancer. PMID:20463012

  17. Hyaluronan Contributes to Bronchiolitis Obliterans Syndrome and Stimulates Lung Allograft Rejection through Activation of Innate Immunity

    PubMed Central

    Wang, Xingan; Sugimoto, Seichiro; Kennedy, Vanessa E.; Zhang, Helen L.; Pavlisko, Elizabeth N.; Kelly, Fran L.; Huang, Howard; Kreisel, Daniel; Palmer, Scott M.

    2014-01-01

    Rationale: Although innate immunity is increasingly recognized to contribute to lung allograft rejection, the significance of endogenous innate ligands, such as hyaluronan (HA) fragments, in clinical or experimental lung transplantation is uncertain. Objectives: To determine if HA is associated with clinical bronchiolitis obliterans syndrome (BOS) in lung transplant recipients, and evaluate the effect of low- or high-molecular-weight HA on experimental lung allograft rejection, including dependence on innate signaling pathways or effector cells. Methods: HA concentrations were measured in bronchoalveolar lavage and plasma samples from lung recipients with or without established BOS. BOS and normal lung tissues were assessed for HA localization and expression of HA synthases. Murine orthotopic lung recipients with established tolerance were treated with low- or high-molecular-weight HA under varied experimental conditions, including Toll-like receptor (TLR) 2/4 and myeloid differentiation protein 88 deficiency and neutrophil depletion. Measurements and Main Results: HA localized within areas of intraluminal small airways fibrosis in BOS lung tissue. Moreover, transcripts for HA synthase enzymes were significantly elevated in BOS versus normal lung tissues and both lavage fluid and plasma HA concentrations were increased in recipients with BOS. Treatment with low-molecular-weight HA abrogated tolerance in murine orthotopic lung recipients in a TLR2/4- and myeloid differentiation protein 88–dependent fashion and drove expansion of alloantigen-specific T lymphocytes. Additionally, TLR-dependent signals stimulated neutrophilia that promoted rejection. In contrast, high-molecular-weight HA attenuated basal allograft inflammation. Conclusions: These data suggest that accumulation of HA could contribute to BOS by directly activating innate immune signaling pathways that promote allograft rejection and neutrophilia. PMID:24471427

  18. Pasteurella multocida urinary tract infection in a patient with cervical cancer.

    PubMed

    Singh, Manmeet B; Harrington, Amanda T

    2017-01-01

    Introduction. Infections caused by Pasteurella species are commonly associated with contact with dogs and cats, typically involving bites and scratches, but casual contact with household pets can also be a risk factor. Urinary tract infection (UTI) caused by Pasteurella species is rare and a significant majority of cases have some known risk factor associated with an underlying chronic illness or structural and/or functional urological abnormality. Case presentation. Here, we present a case of a UTI due to Pasteurella multocida in a patient with squamous cell carcinoma of the cervix who also had a household cat. Conclusion. Providers and laboratorians should be aware of risk factors associated with UTIs caused by Pasteurella species.

  19. Pasteurella multocida urinary tract infection in a patient with cervical cancer

    PubMed Central

    Singh, Manmeet B.

    2017-01-01

    Introduction. Infections caused by Pasteurella species are commonly associated with contact with dogs and cats, typically involving bites and scratches, but casual contact with household pets can also be a risk factor. Urinary tract infection (UTI) caused by Pasteurella species is rare and a significant majority of cases have some known risk factor associated with an underlying chronic illness or structural and/or functional urological abnormality. Case presentation. Here, we present a case of a UTI due to Pasteurella multocida in a patient with squamous cell carcinoma of the cervix who also had a household cat. Conclusion. Providers and laboratorians should be aware of risk factors associated with UTIs caused by Pasteurella species. PMID:28348800

  20. A Rare Case of Glossitis due to Pasteurella multocida after a Cat Scratch

    PubMed Central

    Doan, Thien; Revere, Elizabeth

    2016-01-01

    Pasteurella is one of the zoonotic pathogens that can cause variety of serious infections in animals and humans such as bacteremia, septic shock, endocarditis, meningitis, prosthetic and native valve infections, osteomyelitis, skin and soft tissue infections, abscesses, and even pneumonia with empyema. However, there have been few reports of upper respiratory involvements like tonsillitis and epiglottitis in humans. We present a case of recurrent Pasteurella glossitis after a cat scratch which has not been reported in humans. PMID:27840749

  1. Pasteurella multocida infected total knee arthroplasty: a case report and review of the literature

    PubMed Central

    Ferguson, KB; Bharadwaj, R; MacDonald, A; Syme, B

    2014-01-01

    Pasteurella multocida is a rare cause of prosthetic joint infection. This infection generally follows significant animal contact, usually licks and scratches. We report a case of P multocida infection that was treated with linezolid with salvage of the implant. Linezolid is generally active against Gram-positive organisms only with the exception of Pasteurella, which is Gram-negative. We extensively review the previous reported cases of implant infection with P multocida. PMID:24780653

  2. A Rare Case of Glossitis due to Pasteurella multocida after a Cat Scratch.

    PubMed

    Niknam, Negin; Doan, Thien; Revere, Elizabeth

    2016-01-01

    Pasteurella is one of the zoonotic pathogens that can cause variety of serious infections in animals and humans such as bacteremia, septic shock, endocarditis, meningitis, prosthetic and native valve infections, osteomyelitis, skin and soft tissue infections, abscesses, and even pneumonia with empyema. However, there have been few reports of upper respiratory involvements like tonsillitis and epiglottitis in humans. We present a case of recurrent Pasteurella glossitis after a cat scratch which has not been reported in humans.

  3. [Larval stages of Ascaris lumbricoides: hyaluronan-binding capacity].

    PubMed

    Ponce-León, Patricia; Foresto, Patricia; Valverde, Juana

    2009-03-01

    Hyaluronic acid has important functions in inflammatory and tissue reparation processes. Owing to the varied strategies of the parasites to evade the host's immune response, as well as the multiple functions and physiological importance of hyaluronic acid, the aim was to study the hyaluronan binding capacity by Ascaris lumbricoides larval stages. Larval concentrates were prepared by hatching A. lumbricoides eggs. The larvae were collected by the Baermann method. The test of serum soluble CD44 detection by Agregation Inhibition was modified. All the larval concentrates presented hyaluronan binding capacity. The obtained results allow to suppose the existence of an hyaluronic acid specific receptor in A. lumbricoides. This receptor eventually might compete with the usual receptors of the host. The parasite might use this mechanism to evade the immune response.

  4. Hyaluronan, a truly "youthful" polysaccharide. Its medical applications.

    PubMed

    Robert, L

    2015-02-01

    Hyaluronan (hyaluronic acid, HA) is a ubiquitous linear polysaccharide endowed with some exceptional physicochemical properties such as strong hydration and viscoelasticity that depend on the size of the molecule. It plays a variety of important physiological roles in tissue hydration and mechanical protection, for example in the umbilical cord, skin and most other tissues. Since its large scale preparation and the invention by E.A. Balazs of the preparation of its non-inflammatory fraction (NIF-NaHA), there have been several important medical and cosmetic applications, most notably of viscosurgery for eye operation, intra-articular injections for osteoarthritis and also for wrinkle filling on the face, as well as for drug administration. Its concentration in tissues is decreasing with age, source of loss of function and structure of tissues. The purpose of this review is to present a succinct overview of the essential properties of hyaluronan and its medical and esthetic applications.

  5. Atrophic rhinitis caused by Pasteurella multocida type D: morphometric analysis.

    PubMed Central

    Martineau-Doizé, B; Dumas, G; Larochelle, R; Frantz, J C; Martineau, G P

    1991-01-01

    In order to study the distribution and the extent of atrophy caused by Pasteurella multocida in the nasal conchae, experimental piglets were injected intramuscularly at seven days of age with either two or four 50% mouse lethal doses per kg body weight of P. multocida type D dermonecrotoxin. Experimental and control piglets were killed four, six and ten days postinjection. Serial transverse paraffin embedded sections of the noses were cut throughout the entire length of the nasal conchae. The area of the nasal ventral conchae was measured and the morphometric index of the nasal cavity was calculated. It was observed that P. multocida type D dermonecrotoxin induced severe atrophy of the nasal ventral conchae. This atrophy was present along the entire conchae. However, it was most severe at the level of the first and second premolar teeth. Images Fig. 1. Fig. 4. PMID:1889032

  6. Antibiotic sensitivity patterns among Indian strains of avian Pasteurella multocida.

    PubMed

    Shivachandra, S B; Kumar, A A; Biswas, A; Ramakrishnan, M A; Singh, Vijendra P; Srivastava, S K

    2004-11-01

    An investigation was carried out to study the antibiotic sensitivity of avian strains of Pasteurella multocida and to select an effective antimicrobial agent for control of avian pasteurellosis in India. A total of 123 strains of P. multocida recently isolated from different avian species (chicken, duck, turkey, quail, and goose), from different regions of India were subjected to antibiotic sensitivity tests using 20 different antibiotics. Absolute resistance was observed against sulfadiazine. The studies indicated that the strains were most sensitive to chloramphenicol (73.98%), followed by enrofloxacin (71.54%), lincomycin (64.23%), norfloxacin (61.79%) and doxycycline-HCl (56.91%). The majority of the strains were found to exhibit intermediate sensitivity. Chloramphenicol was selected and suggested for treatment. Antibiogram studies also revealed the emergence of multidrug-resistant strains of P. multocida among Indian poultry.

  7. Effect of Pasteurella multocida toxin on bone resorption in vitro.

    PubMed Central

    Felix, R; Fleisch, H; Frandsen, P L

    1992-01-01

    Pasteurella multocida toxin (PMT), which is the primary etiologic factor in the pathogenesis of progressive atrophic rhinitis in pigs, was found to stimulate bone resorption in vitro. This stimulation was observed both in cultures of murine calvaria by measuring the release of calcium and of the lysosomal enzyme beta-glucuronidase and in murine long bone cultures by measuring the release of calcium. Both systems showed the same dose response curve, with the maximal effect at a concentration of 5 ng/ml. The effect on calvaria was studied in more detail. PMT increased bone resorption 24 h after its addition and always had to be present to express an effect. Calcitonin was able to inhibit this increase of resorption completely, and inhibitors of prostaglandin synthesis suppressed it partially. Although the data show an effect of PMT on bone tissue, the results do not exclude an action on cells in the nasal cavity, which could indirectly stimulate bone resorption. PMID:1452328

  8. On-line separation and characterization of hyaluronan oligosaccharides derived from radical depolymerization

    PubMed Central

    Zhao, Xue; Yang, Bo; Li, Lingyun; Zhang, Fuming; Linhardt, Robert J.

    2013-01-01

    Hydroxyl radicals are widely implicated in the oxidation of carbohydrates in biological and industrial processes and are often responsible for their structural modification resulting in functional damage. In this study, the radical depolymerization of the polysaccharide hyaluronan was studied in a reaction with hydroxyl radicals generated by Fenton Chemistry. A simple method for isolation and identification of the resulting non-sulfated oligosaccharide products of oxidative depolymerization was established. Hyaluronan oligosaccharides were analyzed using ion-pairing reversed phase high performance liquid chromotography coupled with tandem electrospray mass spectrometry. The sequence of saturated hyaluronan oligosaccharides having even- and odd-numbers of saccharide units, afforded through oxidative depolymerization, were identified. This study represents a simple, effective ‘fingerprinting’ protocol for detecting the damage done to hyaluronan by oxidative radicals. This study should help reveal the potential biological outcome of reactive-oxygen radical-mediated depolymerization of hyaluronan. PMID:23768593

  9. Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity

    PubMed Central

    1994-01-01

    The molecular mechanisms whereby hyaluronan (HA) stimulates cell motility was investigated in a C-H-ras transformed 10T 1/2 fibroblast cell line (C3). A significant (p < 0.001) stimulation of C3 cell motility with HA (10 ng/ml) was accompanied by an increase in protein tyrosine phosphorylation as detected by anti-phosphotyrosine antibodies using immunoblot analysis and immunofluorescence staining of cells. Tyrosine phosphorylation of several proteins was found to be both rapid and transient with phosphorylation occurring within 1 min of HA addition and dissipating below control levels 10-15 min later. These responses were also elicited by an antibody generated against a peptide sequence within the HA receptor RHAMM. Treatment of cells with tyrosine kinase inhibitors (genistein, 10 micrograms/ml or herbimycin A, 0.5 micrograms/ml) or microinjection of anti-phosphotyrosine antibodies inhibited the transient protein tyrosine phosphorylation in response to HA as well as prevented HA stimulation of cell motility. To determine a link between HA-stimulated tyrosine phosphorylation and the resulting cell locomotion, cytoskeletal reorganization was examined in C3 cells plated on fibronectin and treated with HA or anti-RHAMM antibody. These agents caused a rapid assembly and disassembly of focal adhesions as revealed by immunofluorescent localization of vinculin. The time course with which HA and antibody induced focal adhesion turnover exactly paralleled the induction of transient protein tyrosine phosphorylation. In addition, phosphotyrosine staining colocalized with vinculin within structures in the lamellapodia of these cells. Notably, the focal adhesion kinase, pp125FAK, was rapidly phosphorylated and dephosphorylated after HA stimulation. These results suggest that HA stimulates locomotion via a rapid and transient protein tyrosine kinase signaling event mediated by RHAMM. They also provide a possible molecular basis for focal adhesion turnover, a process that is

  10. TGF-beta 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan

    PubMed Central

    1993-01-01

    TGF-beta is a potent stimulator of motility in a variety of cell types. It has recently been shown that hyaluronan (HA) can directly promote locomotion of cells through interaction with the HA receptor RHAMM. We have investigated the role of RHAMM and HA in TGF-beta-stimulated locomotion and show that TGF-beta triggers the transcription, synthesis and membrane expression of the RHAMM receptor and the secretion of HA coincident with the induction of the locomotory response. This was demonstrated by both incubating cells with exogenous TGF-beta 1 and by stimulating the production of bioactive TGF-beta 1 in tumor cells transfected with TGF-beta 1 under the control of the metallothionein promoter. TGF-beta 1-induced locomotion was suppressed by antibodies that prevented HA/RHAMM interaction, using polyclonal antibodies to either RHAMM fusion protein or RHAMM peptides, or mAbs to purified RHAMM. Peptides corresponding to the HA-binding motif of RHAMM also suppressed TGF-beta 1-induced increases in motility rate. Spontaneous locomotion of fibrosarcoma cells was blocked by neutralizing secreted TGF-beta with panspecific TGF-beta antibodies and by inhibition of TGF- beta 1 secretion with antisense oligonucleotides. Polyclonal anti-RHAMM fusion protein antibodies and peptide from the RHAMM HA-binding motif also suppressed the spontaneous motility rate of fibrosarcoma cells. These data suggest that fibrosarcoma cell locomotion requires TGF-beta, and the pathway by which TGF-beta stimulates locomotion uses the HA receptor RHAMM and HA. PMID:7693717

  11. TGF-beta 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan.

    PubMed

    Samuel, S K; Hurta, R A; Spearman, M A; Wright, J A; Turley, E A; Greenberg, A H

    1993-11-01

    TGF-beta is a potent stimulator of motility in a variety of cell types. It has recently been shown that hyaluronan (HA) can directly promote locomotion of cells through interaction with the HA receptor RHAMM. We have investigated the role of RHAMM and HA in TGF-beta-stimulated locomotion and show that TGF-beta triggers the transcription, synthesis and membrane expression of the RHAMM receptor and the secretion of HA coincident with the induction of the locomotory response. This was demonstrated by both incubating cells with exogenous TGF-beta 1 and by stimulating the production of bioactive TGF-beta 1 in tumor cells transfected with TGF-beta 1 under the control of the metallothionein promoter. TGF-beta 1-induced locomotion was suppressed by antibodies that prevented HA/RHAMM interaction, using polyclonal antibodies to either RHAMM fusion protein or RHAMM peptides, or mAbs to purified RHAMM. Peptides corresponding to the HA-binding motif of RHAMM also suppressed TGF-beta 1-induced increases in motility rate. Spontaneous locomotion of fibrosarcoma cells was blocked by neutralizing secreted TGF-beta with panspecific TGF-beta antibodies and by inhibition of TGF-beta 1 secretion with antisense oligonucleotides. Polyclonal anti-RHAMM fusion protein antibodies and peptide from the RHAMM HA-binding motif also suppressed the spontaneous motility rate of fibrosarcoma cells. These data suggest that fibrosarcoma cell locomotion requires TGF-beta, and the pathway by which TGF-beta stimulates locomotion uses the HA receptor RHAMM and HA.

  12. Pasteurella multocida Toxin Triggers RANKL-Independent Osteoclastogenesis.

    PubMed

    Chakraborty, Sushmita; Kloos, Bianca; Harre, Ulrike; Schett, Georg; Kubatzky, Katharina F

    2017-01-01

    Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While Bordetella bronchiseptica strains cause mild and reversible symptoms, infection of pigs with toxigenic Pasteurella multocida strains causes a severe and irreversible decay. The responsible virulence factor Pasteurella multocida toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL

  13. Pasteurella multocida Toxin Triggers RANKL-Independent Osteoclastogenesis

    PubMed Central

    Chakraborty, Sushmita; Kloos, Bianca; Harre, Ulrike; Schett, Georg; Kubatzky, Katharina F.

    2017-01-01

    Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While Bordetella bronchiseptica strains cause mild and reversible symptoms, infection of pigs with toxigenic Pasteurella multocida strains causes a severe and irreversible decay. The responsible virulence factor Pasteurella multocida toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL

  14. A RHAMM Mimetic Peptide Blocks Hyaluronan Signaling and Reduces Inflammation and Fibrogenesis in Excisional Skin Wounds

    PubMed Central

    Tolg, Cornelia; Hamilton, Sara R.; Zalinska, Ewa; McCulloch, Lori; Amin, Ripal; Akentieva, Natalia; Winnik, Francoise; Savani, Rashmin; Bagli, Darius J.; Luyt, Len G.; Cowman, Mary K.; McCarthy, Jim B.; Turley, Eva A.

    2013-01-01

    Hyaluronan is activated by fragmentation and controls inflammation and fibroplasia during wound repair and diseases (eg, cancer). Hyaluronan-binding peptides were identified that modify fibrogenesis during skin wound repair. Peptides were selected from 7- to 15mer phage display libraries by panning with hyaluronan-Sepharose beads and assayed for their ability to block fibroblast migration in response to hyaluronan oligosaccharides (10 kDa). A 15mer peptide (P15-1), with homology to receptor for hyaluronan mediated motility (RHAMM) hyaluronan binding sequences, was the most effective inhibitor. P15-1 bound to 10-kDa hyaluronan with an affinity of Kd = 10−7 and appeared to specifically mimic RHAMM since it significantly reduced binding of hyaluronan oligosaccharides to recombinant RHAMM but not to recombinant CD44 or TLR2,4, and altered wound repair in wild-type but not RHAMM−/− mice. One topical application of P15-1 to full-thickness excisional rat wounds significantly reduced wound macrophage number, fibroblast number, and blood vessel density compared to scrambled, negative control peptides. Wound collagen 1, transforming growth factor β-1, and α-smooth muscle actin were reduced, whereas tenascin C was increased, suggesting that P15-1 promoted a form of scarless healing. Signaling/microarray analyses showed that P15-1 blocks RHAMM-regulated focal adhesion kinase pathways in fibroblasts. These results identify a new class of reagents that attenuate proinflammatory, fibrotic repair by blocking hyaluronan oligosaccharide signaling. PMID:22889846

  15. Oxidized low density lipoprotein (LDL) affects hyaluronan synthesis in human aortic smooth muscle cells.

    PubMed

    Viola, Manuela; Bartolini, Barbara; Vigetti, Davide; Karousou, Evgenia; Moretto, Paola; Deleonibus, Sara; Sawamura, Tatsuya; Wight, Thomas N; Hascall, Vincent C; De Luca, Giancarlo; Passi, Alberto

    2013-10-11

    Thickening of the vessel in response to high low density lipoprotein(s) (LDL) levels is a hallmark of atherosclerosis, characterized by increased hyaluronan (HA) deposition in the neointima. Human native LDL trapped within the arterial wall undergoes modifications such as oxidation (oxLDL). The aim of our study is to elucidate the link between internalization of oxLDL and HA production in vitro, using human aortic smooth muscle cells. LDL were used at an effective protein concentration of 20-50 μg/ml, which allowed 80% cell viability. HA content in the medium of untreated cells was 28.9 ± 3.7 nmol HA-disaccharide/cell and increased after oxLDL treatment to 53.9 ± 5.6. OxLDL treatments doubled the transcripts of HA synthase HAS2 and HAS3. Accumulated HA stimulated migration of aortic smooth muscle cells and monocyte adhesiveness to extracellular matrix. The effects induced by oxLDL were inhibited by blocking LOX-1 scavenger receptor with a specific antibody (10 μg/ml). The cholesterol moiety of LDL has an important role in HA accumulation because cholesterol-free oxLDL failed to induce HA synthesis. Nevertheless, cholesterol-free oxLDL and unmodified cholesterol (20 μg/ml) induce only HAS3 transcription, whereas 22,oxysterol affects both HAS2 and HAS3. Moreover, HA deposition was associated with higher expression of endoplasmic reticulum stress markers (CHOP and GRP78). Our data suggest that HA synthesis can be induced in response to specific oxidized sterol-related species delivered through oxLDL.

  16. Inhibition of Hyaluronan Synthesis Reduces Versican and Fibronectin Levels in Trabecular Meshwork Cells

    PubMed Central

    Keller, Kate E.; Sun, Ying Ying; Vranka, Janice A.; Hayashi, Lauren; Acott, Ted S.

    2012-01-01

    Hyaluronan (HA) is a major component of the extracellular matrix (ECM) and is synthesized by three HA synthases (HAS). Similarities between the HAS2 knockout mouse and the hdf mutant mouse, which has a mutation in the versican gene, suggest that HA and versican expression may be linked. In this study, the relationship between HA synthesis and levels of versican, fibronectin and several other ECM components in trabecular meshwork cells from the anterior segment of the eye was investigated. HA synthesis was inhibited using 4-methylumbelliferone (4MU), or reduced by RNAi silencing of each individual HAS gene. Quantitative RT-PCR and immunoblotting demonstrated a reduction in mRNA and protein levels of versican and fibronectin. Hyaluronidase treatment also reduced versican and fibronectin levels. These effects could not be reversed by addition of excess glucose or glucosamine or exogenous HA to the culture medium. CD44, tenascin C and fibrillin-1 mRNA levels were reduced by 4MU treatment, but SPARC and CSPG6 mRNA levels were unaffected. Immunostaining of trabecular meshwork tissue after exposure to 4MU showed an altered localization pattern of HA-binding protein, versican and fibronectin. Reduction of versican by RNAi silencing did not affect HA concentration as assessed by ELISA. Together, these data imply that HA concentration affects synthesis of certain ECM components. Since precise regulation of the trabecular meshwork ECM composition and organization is required to maintain the aqueous humor outflow resistance and intraocular pressure homeostasis in the eye, coordinated coupling of HA levels and several of its ECM binding partners should facilitate this process. PMID:23139787

  17. High-molecular weight hyaluronan reduced renal PKC activation in genetically diabetic mice.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; Micali, Antonio; Nastasi, Giancarlo; Squadrito, Francesco; Altavilla, Domenica; Bitto, Alessandra; Polito, Francesca; Rinaldi, Maria Grazia; Calatroni, Alberto; D'Ascola, Angela; Campo, Salvatore

    2010-11-01

    The cluster determinant (CD44) seems to play a key role in tissues injured by diabetes type 2. CD44 stimulation activates the protein kinase C (PKC) family which in turn activates the transcriptional nuclear factor kappa B (NF-κB) responsible for the expression of the inflammation mediators such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-18 (IL-18), inducible nitric oxide synthase (iNOS), and matrix metalloproteinases (MMPs). Regulation of CD44 interaction with its ligands depends greatly upon PKC. We investigated the effect of the treatment with high-molecular weight hyaluronan (HA) on diabetic nephropathy in genetically diabetic mice. BKS.Cg-m+/+Lepr(db) mice had elevated plasma insulin from 15 days of age and high blood sugar levels at 4 weeks. The severe nephropathy that developed was characterized by a marked increased in CD44 receptors, protein kinase C betaI, betaII, and epsilon (PKC(βI), PKC(βII), and PKCε) mRNA expression and the related protein products in kidney tissue. High levels of mRNA and related protein levels were also detected in the damaged kidney for NF-κB, TNF-α, IL-6, IL-18, MMP-7, and iNOS. Chronic daily administration of high-molecular mass HA for 2 weeks significantly reduced CD44, PKC(βI), PKC(βII), and PKCα gene expression and the related protein production in kidney tissue and TNF-α, IL-6, IL-18, MMP-7, and iNOS expression and levels also decreased. Histological analysis confirmed the biochemical data. However, blood parameters of diabetes were unchanged. These results suggest that the CD44 and PKC play an important role in diabetes and interaction of high-molecular weight HA with these proteins may reduce inflammation and secondary pathologies due to this disease.

  18. Inhibition of hyaluronan synthesis alters sulfated glycosaminoglycans deposition during chondrogenic differentiation in ATDC5 cells.

    PubMed

    Yoshioka, Yutaka; Kozawa, Eiji; Urakawa, Hiroshi; Arai, Eisuke; Futamura, Naohisa; Zhuo, Lisheng; Kimata, Koji; Ishiguro, Naoki; Nishida, Yoshihiro

    2015-08-01

    In chondrogenic differentiation, expression and collaboration of specific molecules, such as aggrecan and type II collagen, in extracellular matrix (ECM) are crucial. However, few studies have clarified the roles of hyaluronan (HA) in proteoglycan aggregation during chondrogenic differentiation. We assessed the roles of HA in sulfated glycosaminoglycans deposition during chondrogenic differentiation by means of 4-methylumbelliferone (4-MU), an HA synthase inhibitor, using ATDC5 cells. ATDC5 cells were treated with 0.5 mM 4-MU for 7 or 21 days after induction of chondrogenic differentiation with insulin. Depositions of sulfated glycosaminoglycans were evaluated with Alcian blue staining. mRNA expression of ECM molecules was determined using real-time RT-PCR. The deposition of aggrecan and versican was investigated with immunohistochemical staining using specific antibodies. Effects of 4-MU on HA concentrations were analyzed by HA binding assay. 4-MU suppressed the positivity of Alcian blue staining, although this delay was reversible. Interestingly, stronger positivity of Alcian blue staining was observed at day 21 in cultures with 4-MU discontinuation than in the control. 4-MU significantly increased the mRNA expression of aggrecan, versican, and type II collagen, which was consistent with increased deposition of aggrecan and versican. The HA concentration in ECM and cell-associated region was significantly suppressed with 4-MU treatment. We conclude that the inhibition of HA synthesis slows sulfated glycosaminoglycans deposition during chondrogenic differentiation despite the increased deposition of other ECM molecules. Transient starvation of HA with 4-MU accelerates chondrogenic ECM formation, suggesting its potential to stimulate chondrogenic differentiation with adequate use.

  19. Hyaluronan modulates growth factor induced mammary gland branching in a size dependent manner.

    PubMed

    Tolg, Cornelia; Yuan, Han; Flynn, Sarah M; Basu, Kaustuv; Ma, Jenny; Tse, Kenneth Chor Kin; Kowalska, Beatrice; Vulkanesku, Diana; Cowman, Mary K; McCarthy, James B; Turley, Eva A

    2017-02-21

    Mammary gland morphogenesis begins during fetal development but expansion of the mammary tree occurs postnatally in response to hormones, growth factors and extracellular matrix. Hyaluronan (HA) is an extracellular matrix polysaccharide that has been shown to modulate growth factor-induced branching in culture. Neither the physiological relevance of HA to mammary gland morphogenesis nor the role that HA receptors play in these responses are currently well understood. We show that HA synthase (HAS2) is expressed in both ductal epithelia and stromal cells but HA primarily accumulates in the stroma. HA accumulation and expression of the HA receptors CD44 and RHAMM are highest during gestation when gland remodeling, lateral branch infilling and lobulo-alveoli formation is active. Molecular weight analyses show that approximately 98% of HA at all stages of morphogenesis is >300kDa. Low levels of 7-114kDa HA fragments are also detected and in particular the accumulation of 7-21kDa HA fragments are significantly higher during gestation than other morphogenetic stages (p<0.05). Using these in vivo results as a guide, in culture analyses of mammary epithelial cell lines (EpH4 and NMuMG) were performed to determine the roles of high molecular weight, 7-21kDa (10kDa MWavg) and HA receptors in EGF-induced branching morphogenesis. Results of these assays show that while HA synthesis is required for branching and 10kDa HA fragments strongly stimulate branching, the activity of HA decreases with increasing molecular weight and 500kDa HA strongly inhibits this morphogenetic process. The response to 10kDa HA requires RHAMM function and genetic deletion of RHAMM transiently blunts lateral branching in vivo. Collectively, these results reveal distinct roles for HA polymer size in modulating growth factor induced mammary gland branching and implicates these polymers in both the expansion and sculpting of the mammary tree during gestation.

  20. Hyaluronan Is Crucial for Stem Cell Differentiation into Smooth Muscle Lineage

    PubMed Central

    Simpson, Russell M.L.; Hong, Xuechong; Wong, Mei Mei; Karamariti, Eirini; Bhaloo, Shirin Issa; Warren, Derek; Kong, Wei; Hu, Yanhua

    2016-01-01

    Abstract Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital to further our understanding of the pathogenesis of vascular disease and for development of cell‐based therapies and tissue engineering. Hyaluronan (HA) has emerged as an important component of the stem cell niche, however its role during stem cell differentiation is a complicated and inadequately defined process. This study aimed to investigate the role of HA in embryonic stem cell (ESC) differentiation toward a SMC lineage. ESCs were seeded on collagen‐IV in differentiation medium to generate ESC‐derived SMCs (esSMCs). Differentiation coincided with increased HA synthase (HAS) 2 expression, accumulation of extracellular HA and its assembly into pericellular matrices. Inhibition of HA synthesis by 4‐methylumbelliferone (4MU), removal of the HA coat by hyaluronidase (HYAL) or HAS2 knockdown led to abrogation of SMC gene expression. HA activates ERK1/2 and suppresses EGFR signaling pathways via its principle receptor, CD44. EGFR inactivation coincided with increased binding to CD44, which was further augmented by addition of high molecular weight (HMW)‐HA either exogenously or via HAS2 overexpression through adenoviral gene transfer. HMW‐HA‐stimulated esSMCs displayed a functional role in vascular tissue engineering ex vivo, vasculogenesis in a matrigel plug model and SMC accumulation in neointimal lesions of vein grafts in mice. These findings demonstrate that HAS2‐induced HA synthesis and organization drives ESC‐SMC differentiation. Thus, remodeling of the HA microenvironment is a critical step in directing stem cell differentiation toward a vascular lineage, highlighting HA as a potential target for treatment of vascular diseases. Stem Cells 2016;34:1225–1238 PMID:26867148

  1. Molecular Identification and Epidemiological Tracing of Pasteurella multocida Meningitis in a Baby

    PubMed Central

    Boerlin, Patrick; Siegrist, Hans H.; Burnens, André P.; Kuhnert, Peter; Mendez, Purita; Prétat, Gérard; Lienhard, Reto; Nicolet, Jacques

    2000-01-01

    We report a case of Pasteurella multocida meningitis in a 1-month-old baby exposed to close contact with two dogs and a cat but without any known history of injury by these animals. 16S rRNA gene sequencing of the isolate from the baby allowed identification at the subspecies level and pointed to the cat as a possible source of infection. Molecular typing of Pasteurella isolates from the animals, from the baby, and from unrelated animals clearly confirmed that the cat harbored the same P. multocida subsp. septica strain on its tonsils as the one isolated from the cerebrospinal fluid of the baby. This case stresses the necessity of informing susceptible hosts at risk of contracting zoonotic agents about some basic hygiene rules when keeping pets. In addition, this study illustrates the usefulness of molecular methods for identification and epidemiological tracing of Pasteurella isolates. PMID:10699029

  2. Pasteurella multocida infection in a cirrhotic patient: case report, microbiological aspects and a review of literature.

    PubMed

    Migliore, E; Serraino, C; Brignone, C; Ferrigno, D; Cardellicchio, A; Pomero, F; Castagna, E; Osenda, M; Fenoglio, L

    2009-01-01

    Pasteurellosis is a zoonosis often caused by cat or dog bites or scratches, or by direct exposure to their secretions. Pasteurella multocida is the main pathogen involved in infections through domestic animal bites; generally a local infection characterized by its particular virulence with consequent rapid onset. Serious infection has also been reported in persons affected by comorbidity without domestic animal bite injuries. Here we report the case of a woman with lower limb exudating vesicular skin ulcers affected by liver cirrhosis, bilateral knee arthritis, septicemia with positive blood culture and synovial fluid culture for Pasteurella multocida. The etiology of Pasteurella multocida must be borne in mind in cases of sepsis in immunodeficient individuals, such as the cirrhotic patient, as well as exposure to domestic animals.

  3. An atypical presentation of a Pasteurella multocida infection following a cat bite: a case report.

    PubMed

    Collins, Chris; Flanagan, Brigitte; Henning, J Scott

    2012-06-01

    Pasteurella multocida is a bacterial organism that commonly causes cellulitis after animal bites, especially cat bites. We report an unusual vesiculopustular infection of the hand following a domestic cat bite. Pasteurella multocida and Staphylococcus aureus were cultured from the wound and the patient was treated with amoxicillin-clavulanate potassium. Further history revealed that the patient's cat had nibbled on her hand. Pasteurella usually is resistant to many of the typical empiric antibiotics used to treat skin infections. Amoxicillin-clavulanate potassium (500 mg 3 times daily) is the treatment of choice for patients who have an infected cat or dog bite with no known bacterial cause. A thorough patient history is needed to promptly arrive at a proper diagnosis for an atypical presentation of a common disease.

  4. The Development of a Novel Therapeutic Strategy to Target Hyaluronan in the Extracellular Matrix of Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Kudo, Daisuke; Suto, Akiko; Hakamada, Kenichi

    2017-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases to affect humans, regardless of whether patients receive multimodal therapy (including surgery, radiotherapy, and chemotherapy). This resistance to intervention is currently considered to be caused by the desmoplastic change of the extracellular matrix (ECM) in PDAC tissues, which is characterized by the accumulation of cancer-associated fibroblasts, collagen, proteoglycan, and hyaluronan. Among these ECM components, hyaluronan has attracted interest because various studies have indicated that hyaluronan-rich PDAC is correlated with the progressive properties of cancer cells, both in experimental and clinical settings. Hence, the reduction of hyaluronan in cancer tissue may represent a novel therapeutic approach for PDAC. 4-methylumbelliferone (4-MU) is a derivative of coumarin that was reported to suppress the synthesis of hyaluronan in cultured human skin fibroblasts in 1995. As an additional study, our group firstly reported that 4-MU reduced the hyaluronan synthesis of mouse melanoma cells and exerted anti-cancer activity. Subsequently, we have showed that 4-MU inhibited liver metastasis in mice inoculated with human pancreatic cancer cells. Thereafter, 4-MU has been accepted as an effective agent for hyaluronan research and is expected to have clinical applications. This review provides an overview of the interaction between PDAC and hyaluronan, the properties of 4-MU as a suppressor of the synthesis of hyaluronan, and the perspectives of PDAC treatment targeting hyaluronan. PMID:28282922

  5. Comparison of methods to detect Pasteurella multocida in carrier waterfowl

    USGS Publications Warehouse

    Samuel, M.D.; Shadduck, D.J.; Goldberg, D.R.; Johnson, W.P.

    2003-01-01

    We conducted laboratory challenge trials using mallard ducks (Anas platyrhynchos) to compare methods for detecting carriers of Pasteurella multocida, the bacterium that causes avian cholera, in wild birds. Birds that survived the initial infection were euthanized at 2-4 wk intervals up to 14 wk post challenge. Isolates of P. multocida were obtained at necropsy from 23% of the birds that survived initial infection. We found that swab samples (oral, cloacal, nasal, eye, and leg joint) were most effective for detecting carrier birds up to 14 wk post infection. No detectable differences in isolation were observed for samples stored in either 10% dimethysulfoxide or brain heart infusion broth. The frequency of detecting carriers in our challenge trials appeared to be related to mortality rates observed during the trial, but was not related to a number of other factors including time after challenge, time delays in collecting tissues postmortem, and route of infection. In our trials, there was little association between antibody levels and carrier status. We concluded that swabs samples collected from recently dead birds, stored in liquid nitrogen, and processed using selective broth provide a feasible field method for detecting P. multocida carriers in wild waterfowl.

  6. Transcriptional Response of Pasteurella multocida to Nutrient Limitation

    PubMed Central

    Paustian, Michael L.; May, Barbara J.; Kapur, Vivek

    2002-01-01

    Bacteria often encounter environments where nutrient availability is limited, and they must adapt accordingly. To identify Pasteurella multocida genes that are differentially expressed during nutrient limitation, we utilized whole-genome microarrays to compare levels of gene expression during growth in rich and minimal media. Our analysis showed that the levels of expression of a total of 669 genes, representing approximately one-third of the genome, were detectably altered over the course of the experiment. A large number (n = 439) of genes, including those involved in energy metabolism, transport, protein synthesis, and binding, were expressed at higher levels in rich medium, suggesting that, upon exposure to a rich environment, P. multocida immediately begins to turn on many energy-intensive biosynthetic pathways or, conversely, turns these genes off when it is exposed to a nutrient-deficient environment. Genes with increased expression in minimal medium (n = 230) included those encoding amino acid biosynthesis and transport systems, outer membrane proteins, and heat shock proteins. Importantly, our analysis also identified a large number (n = 164) of genes with unknown functions whose expression was altered during nutrient limitation. Overall, the results of our study show that a wide repertoire of genes, many of which have yet to be functionally classified, undergo transcriptional regulation in P. multocida in response to growth in minimal medium and provide a strong foundation to investigate the transcriptional response of this multispecies pathogen to growth in a nutrient-limited environment. PMID:12057970

  7. Antigenic and virulence properties of Pasteurella haemolytica leukotoxin mutants.

    PubMed Central

    Petras, S F; Chidambaram, M; Illyes, E F; Froshauer, S; Weinstock, G M; Reese, C P

    1995-01-01

    Antigenic properties of two mutants of Pasteurella haemolytica, strains 59B0071 and 59B0072, that do not produce detectable leukotoxin were investigated. Western blot (immunoblot) analysis with a number of polyclonal sera from animals recovering from pasteurellosis revealed that both mutants secreted a variety of antigens that were also present in cultures of several wild-type strains. These antigens ranged from about 100 to 15 kDa. Mutant strain 59B0071 was found to be totally deficient in leukotoxin, as judged not only by Western blotting but also by cytotoxicity assays with bovine lymphoma (BL-3) cells or bovine polymorphonuclear cells as targets. The mutant strain 59B0071 had normal levels of a secreted sialylglycoprotease, however. When strains were tested for virulence in goat and cattle challenge experiments, a reduction in mortality and lung lesions was observed with the mutant 59B0071 in comparison with results obtained with wild-type strains. These results are consistent with an important role for leukotoxin in P. haemolytica virulence and suggest that leukotoxin-negative mutants may be useful tools in the investigation of other virulence properties involved in P. haemolytica infections. PMID:7868224

  8. Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis.

    PubMed Central

    Mosier, D A; Simons, K R; Confer, A W; Panciera, R J; Clinkenbeard, K D

    1989-01-01

    Antigens associated with whole Pasteurella haemolytica biotype A serotype 1, a capsular carbohydrate-protein extract of the organism, and P. haemolytica leukotoxin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antigens of the electrophoresed preparations were detected by Western blotting (immunoblotting) with sera from cattle which were either nonvaccinated or vaccinated with live or killed P. haemolytica vaccines and had variable degrees of resistance to experimental pneumonic pasteurellosis. Distinct, easily recognizable antigens of these preparations were identified, and the antibody responses to these antigens were quantified by densitometry. To determine their importance to disease resistance, we then compared antibody responses with experimental lesion scores. Antibody reactivity to surface antigens which were significantly correlated with resistance and present in two or more of the preparations were detected at 86, 66, 51, 49, 34, 31, and 16 kilodaltons (kDa). Of these, antibody responses to antigens at 86, 49, and 31 kDa appeared most important based on their concentration and significance levels. Antibody reactivity to leukotoxin antigens which were significantly correlated with resistance and common with important surface antigens were detected at 86, 66, and 49 kDa. Antibody responses to unique leukotoxin antigens which were significantly correlated with resistance were present at 92 and 58 kDa. Images PMID:2917783

  9. Outer membrane vesicles of Pasteurella multocida contain virulence factors

    PubMed Central

    Fernández-Rojas, Miguel A; Vaca, Sergio; Reyes-López, Magda; de la Garza, Mireya; Aguilar-Romero, Francisco; Zenteno, Edgar; Soriano-Vargas, Edgardo; Negrete-Abascal, Erasmo

    2014-01-01

    Pasteurella multocida (Pm) is a gram-negative bacterium able to infect different animal species, including human beings. This bacterium causes economic losses to the livestock industry because of its high morbidity and mortality in animals. In this work, we report the characterization of outer membrane vesicles (OMVs) released into the culture medium by different Pm serogroups. Purified OMVs in the range of 50–300 nm were observed by electron microscopy. Serum obtained from chickens infected with Pm recognized several proteins from Pm OMVs. Additionally, rabbit antiserum directed against a secreted protease from Actinobacillus pleuropneumoniae recognized a similar protein in the Pm OVMs, suggesting that OMVs from these bacterial species contain common immunogenic proteins. OmpA, a multifunctional protein, was identified in OMVs from different Pm serogroups, and its concentration was twofold higher in OMVs from Pm serogroups B and D than in OMVs from other serogroups. Three outer membrane proteins were also identified: OmpH, OmpW, and transferrin-binding protein. Three bands of 65, 110, and 250 kDa with proteolytic activity were detected in Pm OMVs of serogroups A and E. Additionally, β-lactamase activity was detected only in OMVs from Pm 12945 Ampr (serogroup A). Pm OMVs may be involved in different aspects of disease pathogenesis. PMID:25065983

  10. The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage.

    PubMed

    Pullinger, Gillian D; Bevir, Thomas; Lax, Alistair J

    2004-01-01

    Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.

  11. Antigenicity of partial fragments of recombinant Pasteurella multocida toxin.

    PubMed

    Lee, Jeongmin; Woo, Hee-Jong

    2010-12-01

    Pasteurella multocida serogroup D strain, which produces P. multocida toxin (PMT), is a widespread and harmful pathogen of respiratory diseases such as pneumonia and progressive atrophic rhinitis (PAR) in swine. Vaccination has been considered the most desirable and effective approach for controlling the diseases caused by toxigenic P. multocida. To investigate the antigenicity and immunogenicity of partial fragments of recombinant PMT, recombinant proteins of the N-terminal (PMT-A), middle (PMT-B), Cterminal (PMT-C), and middle-C-terminal (PMT2.3) regions of PMT were successfully produced in an Escherichia coli expression system. The molecular masses of PMT-A, PMT-B, PMT-C, and PMT2.3 were ca. 53, 55, 35, and 84 kDa, respectively, purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. All the recombinant proteins except for PMT-A showed immune responses to antisera obtained from a swine showing symptoms of PAR. Moreover, high titers of PMT-specific antibodies were raised from mice immunized with each of the recombinant proteins; however, the immunoreactivities of the antibodies to authentic PMT and heat-inactivated whole bacteria were different, respectively. In the protection study, the highest protection against homologous challenge was shown in the case of PMT2.3; relatively poor protections occurred for the other PMT fragments.

  12. Membrane interaction of Pasteurella multocida toxin involves sphingomyelin.

    PubMed

    Brothers, Michael C; Ho, Mengfei; Maharjan, Ram; Clemons, Nathan C; Bannai, Yuka; Waites, Mark A; Faulkner, Melinda J; Kuhlenschmidt, Theresa B; Kuhlenschmidt, Mark S; Blanke, Steven R; Rienstra, Chad M; Wilson, Brenda A

    2011-12-01

    Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC-overlay binding experiments with (125) I-labeled PMT, PMT-N or the C-terminus of PMT; (b) pull-down experiments using reconstituted membrane liposomes with full-length PMT; (c) surface plasmon resonance analysis of PMT-N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including monoasialogangliosides GM(1) , GM(2) or GM(3) , but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co-receptor.

  13. Persistence of Pasteurella multocida in wetlands following avian cholera outbreaks

    USGS Publications Warehouse

    Blanchong, Julie A.; Samuel, M.D.; Goldberg, D.R.; Shadduck, D.J.; Lehr, M.A.

    2006-01-01

    Avian cholera, caused by Pasteurella multocida, affects waterbirds across North America and occurs worldwide among various avian species. Once an epizootic begins, contamination of the wetland environment likely facilitates the transmission of P. multocida to susceptible birds. To evaluate the ability of P. multocida serotype-1, the most common serotype associated with avian cholera in waterfowl in western and central North America, to persist in wetlands and to identify environmental factors associated with its persistence, we collected water and sediment samples from 23 wetlands during winters and springs of 1996a??99. These samples were collected during avian cholera outbreaks and for up to 13 wk following initial sampling. We recovered P. multocida from six wetlands that were sampled following the initial outbreaks, but no P. multocida was isolated later than 7 wk after the initial outbreak sampling. We found no significant relationship between the probability of recovery of P. multocida during resampling and the abundance of the bacterium recovered during initial sampling, the substrate from which isolates were collected, isolate virulence, or water quality conditions previously suggested to be related to the abundance or survival of P. multocida. Our results indicate that wetlands are unlikely to serve as a long-term reservoir for P. multocida because the bacterium does not persist in wetlands for long time periods following avian cholera outbreaks.

  14. Drug conjugation to hyaluronan widens therapeutic indications for ovarian cancer

    PubMed Central

    Montagner, Isabella Monia; Merlo, Anna; Carpanese, Debora; Zuccolotto, Gaia; Renier, Davide; Campisi, Monica; Pasut, Gianfranco; Zanovello, Paola; Rosato, Antonio

    2015-01-01

    Management of ovarian cancer still requires improvements in therapeutic options. A drug delivery strategy was tested that allows specific targeting of tumor cells in combination with a controlled release of a cytotoxic molecule. To this aim, the efficacy of a loco-regional intraperitoneal treatment with a bioconjugate (ONCOFID-S) derived by chemical linking of SN-38, the active metabolite of irinotecan (CPT-11), to hyaluronan was assessed in a mouse model of ovarian carcinomatosis. In vitro, the bioconjugate selectively interacted with ovarian cancer cells through the CD44 receptor, disclosed a dose-dependent tumor growth inhibition efficacy comparable to that of free SN-38 drug, and inhibited Topoisomerase I function leading to apoptosis by a mechanism involving caspase-3 and -7 activation and PARP cleavage. In vivo, the intraperitoneal administration of ONCOFID-S in tumor-bearing mice did not induce inflammation, and evidenced an improved therapeutic efficacy compared with CPT-11. In conclusion, SN-38 conjugation to hyaluronan significantly improved the profile of in vivo tolerability and widened the field of application of irinotecan. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of ovarian cancer. PMID:26097871

  15. Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury

    PubMed Central

    Lazrak, Ahmed; Creighton, Judy; Yu, Zhihong; Komarova, Svetlana; Doran, Stephen F.; Aggarwal, Saurabh; Emala, Charles W.; Stober, Vandy P.; Trempus, Carol S.; Garantziotis, Stavros

    2015-01-01

    Chlorine (Cl2) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl2 reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl2 (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl2 exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl2 exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl2 (100 ppm, 10 min) or incubation with Cl2-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca2+, and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca2+, blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca2+ channels of airway smooth muscle cells, increasing their contractility and thus causing AHR. PMID:25747964

  16. Effect of Carboxymethylation on the Rheological Properties of Hyaluronan

    PubMed Central

    Wendling, Rian J.; Christensen, Amanda M.; Quast, Arthur D.; Atzet, Sarah K.; Mann, Brenda K.

    2016-01-01

    Chemical modifications made to hyaluronan to enable covalent crosslinking to form a hydrogel or to attach other molecules may alter the physical properties as well, which have physiological importance. Here we created carboxymethyl hyaluronan (CMHA) with varied degree of modification and investigated the effect on the viscosity of CMHA solutions. Viscosity decreased initially as modification increased, with a minimum viscosity for about 30–40% modification. This was followed by an increase in viscosity around 45–50% modification. The pH of the solution had a variable effect on viscosity, depending on the degree of carboxymethyl modification and buffer. The presence of phosphates in the buffer led to decreased viscosity. We also compared large-scale production lots of CMHA to lab-scale and found that large-scale required extended reaction times to achieve the same degree of modification. Finally, thiolated CMHA was disulfide crosslinked to create hydrogels with increased viscosity and shear-thinning aspects compared to CMHA solutions. PMID:27611817

  17. Green synthesis of hyaluronan fibers with silver nanoparticles.

    PubMed

    Abdel-Mohsen, A M; Hrdina, Radim; Burgert, Ladislav; Krylová, Gabriela; Abdel-Rahman, Rasha M; Krejčová, Anna; Steinhart, Miloš; Beneš, Ludvík

    2012-06-20

    The application of green chemistry in the nano-science and technology is very important in the area of the preparation of various materials. In this work, an eco-friendly chemical method was successfully used for the preparation of hyaluronan fibers containing silver nanoparticles (AgNPs). Thus, hyaluronic acid (HA) was dissolved in an aqueous solution of sodium hydroxide to prepare a transparent solution, which was used for the preparation of fibers by a wet-spinning technique. Consequently, silver nanoparticles inside the fiber were prepared. Different parameters affecting the preparation of final product, such as concentration of silver nitrate, hyaluronan fiber concentration, time and temperature of the reaction, pH of the reaction mixture, were studied. AgNPs were confirmed by transmission electron microscopy (TEM), X-ray diffraction (XRD), two-dimensional X-ray scattering (2D SWAXS), UV/Vis spectroscopy, inductively coupled plasma optical emission spectrometry (ICP OES) and scan electron microscopy (SEM). Mechanical properties of prepared fibers were also measured.

  18. Characterisation of Pasteurella dagmatis-like isolates recovered from the feline oral cavity.

    PubMed

    Sellyei, Boglárka; Wehmann, Eniko; Makrai, László; Magyar, Tibor

    2010-10-26

    Three Pasteurella dagmatis-like strains recovered from the feline oral cavity were analysed by traditional biochemical tests, the Biolog Microstation™ ID System, and 16S rRNA and sodA gene sequence analysis. The molecular biological methods revealed that these strains differ from P. dagmatis, forming a new genomospecies in the genus Pasteurella sensu stricto. Furthermore, sequence analysis and multiple alignments of 16S rRNA and the sodA gene established that the P. pneumotropica NCTC10827 and the P. dagmatis-like strains described here possess high genetic similarity.

  19. Pasteurella haemolytica bacteriophage: identification, partial characterization, and relationship of temperate bacteriophages from isolates of Pasteurella haemolytica (biotype A, serotype 1).

    PubMed

    Richards, A B; Renshaw, H W; Sneed, L W

    1985-05-01

    Pasteurella haemolytica (biotype A, serotype 1) isolates (n = 15) from the upper respiratory tract of clinically normal cattle, as well as from lung lesions from cases of fatal bovine pasteurellosis, were examined for the presence of bacteriophage after irradiation with UV light. Treatment of all P haemolytica isolates with UV irradiation resulted in lysis of bacteria due to the induction of vegetative development of bacteriophages. The extent of growth inhibition and bacterial lysis in irradiated cultures was UV dose-dependent. Bacterial cultures exposed to UV light for 20 s reached peak culture density between 60 and 70 minutes after irradiation; thereafter, culture density declined rapidly, so that by 120 minutes, it was approximately 60% of the original value. When examined ultrastructurally, lytic cultures from each isolate revealed bacteriophages with an overall length of approximately 200 nm and that appeared to have a head with icosahedral symmetry and a contractile tail. Cell-free filtrate from each noninduced bacterial isolate was inoculated onto the other bacterial isolates in a cross-culture sensitivity assay for the presence of phages lytic for the host bacterial isolates. Zones of lysis (plaques) did not develop when bacterial lawns grown from the different isolates were inoculated with filtrates from the heterologous isolates.

  20. Hyaluronan-modified magnetic nanoclusters for detection of CD44-overexpressing breast cancer by MR imaging.

    PubMed

    Lim, Eun-Kyung; Kim, Hyun-Ouk; Jang, Eunji; Park, Joseph; Lee, Kwangyeol; Suh, Jin-Suck; Huh, Yong-Min; Haam, Seungjoo

    2011-11-01

    We fabricated hyaluronan-modified magnetic nanoclusters (HA-MNCs) for detection of CD44-overexpressing breast cancer using magnetic resonance (MR) imaging. CD44 is closely associated with cancer growth, including proliferation, metastasis, invasion, and angiogenesis. Hence, pyrenyl hyaluronan (Py-HA) conjugates were synthesized as CD44-targetable surfactants with hyaluronan (HA) and 1-pyrenylbutyric acid (Py) to modify hyaluronan on hydrophobic magnetic nanocrystals. Subsequently, HA-MNCs were fabricated using the nano-emulsion method; magnetic nanocrystals were simultaneously self-assembled with Py-HA conjugates, and their physical and magnetic properties depended on the degree of substitution (DS) of Py in Py-HA conjugates. HA-MNCs exhibited superior targeting efficiency with MR sensitivity as well as excellent biocompatibility through in vitro/in vivo studies. This suggests that HA-MNCs can be a potent cancer specific molecular imaging agent via targeted detection of CD44 with MR imaging.

  1. Uptake of hyaluronan in hepatic metastases after blocking of liver endothelial cell receptors.

    PubMed

    Mahteme, H; Graf, W; Larsson, B S; Gustafson, S

    1998-09-01

    To follow the biodistribution of exogenous hyaluronan in tumor-bearing animals, a total of seventeen inbred rats with hepatic metastases from a colonic adenocarcinoma received 125I-labelled hyaluronan by intravenous injections. Group I received only labeled hyaluronan (25 microg), whereas group II received 2.5 mg chondroitin sulphate prior to labeled hyaluronan, to block receptor uptake in normal liver endothelial cells. Animals in group III received intravenous, as well as intraperitoneal chondroitin sulphate (2.5 mg), to see if a better and prolonged blocking could be achieved. Radioactivity was visualized by whole body autoradiography, using phosphorimaging and the average radioactivity determined as phosphoimaging density units of the total area of hepatic metastases, normal liver, and skeletal muscle by computer-based image analysis. At 5 h, tumors in groups II and III showed higher uptake (4.8+/-1.8, P = .01 and 3.6+/-1.1, P = .01, respectively), in comparison to group I (1.8+/-0.6), and the mean normal liver/tumor concentration ratio was reduced from 21.4+/-10.1 in group I to 5.7+/-2.7 in group II and 3.5+/-1.1 in group III (P = .008 and P = .01, respectively). Our study shows that hyaluronan targets liver metastases of a colon adenocarcinoma. Furthermore, chondroitin sulphate pretreatment increases tumor uptake, while uptake at normal receptor sites is significantly reduced. The results also suggest that after blocking of normal hyaluronan/chondroitin sulphate receptors in healthy tissue, hyaluronan may be used to deliver drugs to specific hyaluronan receptor-positive sites of pathology.

  2. A Hyaluronan-Based Scaffold for the in Vitro Construction of Dental Pulp-Like Tissue

    PubMed Central

    Ferroni, Letizia; Gardin, Chiara; Sivolella, Stefano; Brunello, Giulia; Berengo, Mario; Piattelli, Adriano; Bressan, Eriberto; Zavan, Barbara

    2015-01-01

    Dental pulp tissue supports the vitality of the tooth, but it is particularly vulnerable to external insults, such as mechanical trauma, chemical irritation or microbial invasion, which can lead to tissue necrosis. In the present work, we present an endodontic regeneration method based on the use of a tridimensional (3D) hyaluronan scaffold and human dental pulp stem cells (DPSCs) to produce a functional dental pulp-like tissue in vitro. An enriched population of DPSCs was seeded onto hyaluronan-based non-woven meshes in the presence of differentiation factors to induce the commitment of stem cells to neuronal, glial, endothelial and osteogenic phenotypes. In vitro experiments, among which were gene expression profiling and immunofluorescence (IF) staining, proved the commitment of DPSCs to the main components of dental pulp tissue. In particular, the hyaluronan-DPSCs construct showed a dental pulp-like morphology consisting of several specialized cells growing inside the hyaluronan fibers. Furthermore, these constructs were implanted into rat calvarial critical-size defects. Histological analyses and gene expression profiling performed on hyaluronan-DPSCs grafts showed the regeneration of osteodentin-like tissue. Altogether, these data suggest the regenerative potential of the hyaluronan-DPSC engineered tissue. PMID:25739081

  3. [Hyaluronic acid (hyaluronan) levels in pathological human saphenous veins. Effects of procyanidol oligomers].

    PubMed

    Drubaix, I; Maraval, M; Robert, L; Robert, A M

    1997-01-01

    We investigated the hyaluronan content in the pathologic human venous wall using an ELSA assay with hyaluronectin according to the method of Delpech et al. The mean hyaluronan content in the 74 fragments from 12 venous walls studied was 596 +/- 528 ng/mg dry weight. These 12 venous walls could be separated in 3 distinct groups according to their hyaluronan content, low (277 +/- 141 ng/mg dry weight), moderate (552 +/- 361 ng/m dry weight) or high (1299 +/- 568 ng/mg dry weight). The differences between these groups are significant (p < 0.001). The presence of a veino-lymphatic oedema was generally associated with a high hyaluronan level (in 65% of cases). The 3H-glucosamine incorporation in cultured venous wall explants showed a 35% increase (p < 0.002) in varicosis as compared with the non or less modified segments of the vein and a 29% (p < 0.001) increase in presence of a veino-lymphatic oedema. The addition of 1 mg/ml of PCO (Procyanidolic Oligomers) to the culture media induced near to 20% decrease of the 3H-glucosamine incorporation and a 34% decrease of the hyaluronan content. Our results confirm the role of local overproduction of hyaluronan in the establishment of oedema and the potential effect of PCO to counteract it.

  4. Clinical Features and Outcomes of Pasteurella multocida Infection

    PubMed Central

    Giordano, Antonio; Dincman, Toros; Clyburn, Benjamin E.; Steed, Lisa L.; Rockey, Don C.

    2015-01-01

    Abstract Pasteurella multocida, a zoonotic infectious organism, has most often been described in patients after an animal bite. Here, we characterize the clinical features and outcomes of P multocida infection in a large cohort of patients according to the presence or absence of an animal bite. We retrospectively searched MUSC's laboratory information system for all patients with positive P multocida cultures from 2000 to 2014. Extensive data were abstracted, including clinical and outcome data. The Charlson comorbidity index (CCI) was used to assess comorbidities among patients. We identified 44 patients with P multocida infections, including 25 with an animal bite. The average age was 64 years and the majority of patients were women (N = 30). There was no difference in age and sex distribution among those with and without a bite (P = 0.38 and 0.75, respectively). A CCI ≥1 was significantly associated with the absence of a bite (P = 0.006). Patients presenting without a bite were more frequently bacteremic (37% vs 4%, respectively, P = 0.001), and were hospitalized more often (84% vs 44%, respectively, P = 0.012). Of the 8 patients who required intensive care unit (ICU)-based care, 7 were non-bite-related. There were 4 deaths, all occurring in patients not bitten. P multocida infections not associated with an animal bite were often associated with bacteremia, severe comorbidity(ies), immune-incompetent states, the need for ICU management, and were associated with substantial mortality. PMID:26356688

  5. Secretion and expression of the Pasteurella haemolytica Leukotoxin.

    PubMed Central

    Highlander, S K; Engler, M J; Weinstock, G M

    1990-01-01

    The Pasteurella haemolytica leukotoxin gene cluster (lktCABD) is homologous to the Escherichia coli hemolysin locus (hlyCABD). Since the cloned leukotoxin (LktA) is not secreted from E. coli cells, a heteroplasmid complementation system was developed that permits secretion of the leukotoxin from cells expressing the hemolysin transport proteins HlyB and HlyD. We observed that the secreted leukotoxin protein had weak hemolytic activity when activated by either the HlyC or LktC proteins and that LktC expressed in E. coli could confer weak hemolytic activity upon hemolysin. Thus, it appears that the accessory proteins of the leukotoxin and hemolysin gene clusters are functionally similar, although their expression in E. coli is not equivalent. Northern (RNA) blot analysis of the P. haemolytica leukotoxin gene cluster revealed a major 3.5-kilobase transcript that includes the lktC and lktA genes. The start site for this transcript mapped to a cytosine residue 30 nucleotides upstream from the putative start of lktC; a similar initiation site was observed in E. coli, although adjacent cytosine and adenine residues were also utilized. The 3.5-kilobase transcript terminated near the rho-independent terminator structure between lktA and lktB, but transcription may continue, via antitermination or de novo transcription initiation, into the downstream lktB and lktD genes. We propose that the lack of LktB and LktD function in E. coli is a result, at least in part, of poor lktBD transcription and suggest that a P. haemolytica-specific regulator is required for optimal expression of the leukotoxin genes. Images PMID:2185213

  6. The actions of Pasteurella multocida toxin on neuronal cells.

    PubMed

    Surguy, Susan M; Duricki, Denise A; Reilly, Joanne M; Lax, Alistair J; Robbins, Jon

    2014-02-01

    Pasteurella multocida toxin (PMT) activates the G-proteins Gαi(₁₋₃), Gα(q), Gα₁₁, Gα₁₂ and Gα₁₃ by deamidation of specific glutamine residues. A number of these alpha subunits have signalling roles in neurones. Hence we studied the action of this toxin on rat superior cervical ganglion (SCG) neurones and NG108-15 neuronal cells. Both Gα(q) and Gα₁₁ could be identified in SCGs with immunocytochemistry. PMT had no direct action on Kv7 or Cav2 channels in SCGs. However PMT treatment enhanced muscarinic receptor mediated inhibition of M-current (Kv7.2 + 7. 3) as measured by a 19-fold leftward shift in the oxotremorine-M concentration-inhibition curve. Agonists of other receptors, such as bradykinin or angiotensin, that inhibit M-current did not produce this effect. However the amount of PIP₂ hydrolysis could be enhanced by PMT for all three agonists. In a transduction system in SCGs that is unlikely to be affected by PMT, Go mediated inhibition of calcium current, PMT was ineffective whereas the response was blocked by pertussis toxin as expected. M1 muscarinic receptor evoked calcium mobilisation in transformed NG108-15 cells was enhanced by PMT. The calcium rises evoked by uridine triphosphate acting on endogenous P2Y₂ receptors in NG108-15 cells were enhanced by PMT. The time and concentration dependence of the PMT effect was different for the resting calcium compared to the calcium rise produced by activation of P2Y₂ receptors. PMT's action on these neuronal cells would suggest that if it got into the brain, symptoms of a hyperexcitable nature would be seen, such as seizures.

  7. [The effect of local and systemic immunization of suckling pigs on bronchoalveolar clearance and on intrabronchial infections with Pasteurella multocida].

    PubMed

    Müller, G; Heilmann, P

    1990-01-01

    In vivo investigations of bactericidal bronchoalveolar clearance of weaning pigs locally or systemically immunized with Pasteurella antigens exhibited clearly distinctive results, as early as 3 days after the last immunization. They differed from controls by significant increase in the elimination of the homologous Pasteurella multocida A wild strain. That increase in the clearance remains for 21 days and is specific only to the immunized capsular type. There are no relations of this result to the cytological findings of the lavage fluids and to results of the intrabronchial challenge infection. Frequency and severity of pneumonias were reduced by as little as 3 aerogenic immunizations with Pasteurella mutants.

  8. Characterization of TolC Efflux Pump Proteins from Pasteurella multocida▿ †

    PubMed Central

    Hatfaludi, Tamas; Al-Hasani, Keith; Dunstone, Michelle; Boyce, John; Adler, Ben

    2008-01-01

    Two TolC homologs, PM0527 and PM1980, were identified for Pasteurella multocida. A pm0527 mutant displayed increased susceptibility to a range of chemicals, including rifampin (512-fold) and acridine orange (128-fold). A pm1980 mutant showed increased susceptibility to rifampin, ceftazidime, and vancomycin. PMID:18725450

  9. Sialic Acid Uptake Is Necessary for Virulence of Pasteurella multocida in turkeys

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many pathogenic bacteria employ systems to incorporate sialic acid into their membranes as a means of protection against host defense mechanisms. Pasteurella multocida is an opportunistic pathogen which causes diseases of economic importance in a wide range of animal species and sialic acid uptake p...

  10. Draft Genome Sequence of the Rodent Opportunistic Pathogen Pasteurella pneumotropica ATCC 35149T.

    PubMed

    Sasaki, Hiraku; Ishikawa, Hiroki; Asano, Ryoki; Ueshiba, Hidehiro; Matsumoto, Tetsuya; Boot, Ron; Kawamoto, Eiichi

    2014-08-07

    Pasteurella pneumotropica is an opportunistic pathogen in rodents that is commonly isolated from upper respiratory tracts in laboratory rodents. Here, we report the draft genome sequence of the P. pneumotropica type strain ATCC 35149, which was first isolated and characterized as biotype Jawetz.

  11. Proximity-dependent inhibition of growth of mannheimia haemolytica by pasteurella multocida.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. hae...

  12. Pasteurella species peritoneal dialysis-associated peritonitis: Household pets as a risk factor

    PubMed Central

    Poliquin, Philippe Guillaume; Lagacé-Wiens, Philippe; Verrelli, Mauro; Allen, David W; Embil, John M

    2015-01-01

    BACKGROUND: Pasteurella species are Gram-negative coccobacilli that are a part of the normal oropharyngeal flora of numerous domestic animals. They have been recognized as a rare but significant cause of peritonitis in patients undergoing peritoneal dialysis (PD). A consensus about management strategies for PD-associated peritonitis caused by Pasteurella species currently does not exist. METHODS: The microbiological database serving the Manitoba Renal Program was searched from 1997 to 2013 for cases of Pasteurella species PD-associated peritonitis, and charts were reviewed. PubMed was searched for case reports and data were abstracted. RESULTS: Seven new local cases and 30 previously reported cases were analyzed. This infection is clinically similar to other forms of PD peritonitis, with household pet exposure appearing to be the strongest risk factor. Cats are the most commonly implicated pet. Direct contact between the pet and the equipment was commonly reported (25 of 37 patients) but was not necessary for infection to develop. The mean duration of treatment was 15 days. Complication rates were low, with only 11% of patients requiring PD catheter removal. There was no mortality reported. CONCLUSION: Pasteurella species are a rare cause of PD-associated peritonitis that can be successfully treated with a two-week course of intraperitoneal antibiotics with a high likelihood of catheter salvage. PMID:25798157

  13. Hyaluronan as a promising excipient for ocular drug delivery.

    PubMed

    Guter, Michaela; Breunig, Miriam

    2017-04-01

    Hyaluronan (HA) is a naturally occurring polysaccharide and well known for its exceptional properties such as high biocompatibility and biodegradability, along with a low immunogenicity. Besides its use for various biomedical applications it recently came into focus as a favorable excipient for the formulation of various ocular therapeutics. This review article summarizes the ocular distribution of HA and its most heavily investigated binding protein "cluster of differentiation 44" (CD44) which is the rationale for the clinical use of HA, primarily as an additive in ocular applications ranging from eye drops to contact lenses. Moreover, examples will be given for using HA in various pre-clinical approaches to generate entirely new therapeutics, most notably in the field of nanotechnology.

  14. Hyaluronan- and heparin-reduced silver nanoparticles with antimicrobial properties

    PubMed Central

    Kemp, Melissa M; Kumar, Ashavani; Clement, Dylan; Ajayan, Pulickel; Mousa, Shaker

    2009-01-01

    Aims Silver nanoparticles exhibit unique antibacterial properties that make these ideal candidates for biological and medical applications. We utilized a clean method involving a single synthetic step to prepare silver nanoparticles that exhibit antimicrobial activity. Materials & methods These nanoparticles were prepared by reducing silver nitrate with diaminopyridinylated heparin (DAPHP) and hyaluronan (HA) polysaccharides and tested for their efficacy in inhibiting microbial growth. Results & discussion The resulting silver nanoparticles exhibit potent antimicrobial activity against Staphylococcus aureus and modest activity against Escherichia coli. Silver–HA showed greater antimicrobial activity than silver–DAPHP, while silver–glucose nanoparticles exhibited very weak antimicrobial activity. Neither HA nor DAPHP showed activity against S. aureus or E. coli. Conclusion These results suggest that DAPHP and HA silver nanoparticles have potential in antimicrobial therapeutic applications. PMID:19505245

  15. Potential therapeutic applications of hyaluronan in the lung

    PubMed Central

    Cantor, Jerome O

    2007-01-01

    Hyaluronan (HA), a long-chain polysaccharide, is currently being evaluated as a potential therapeutic agent for a number of inflammatory disorders. The effect of HA on inflammation appears to be related to its molecular size, with larger polysaccharide chains having anti-inflammatory activity and smaller ones having proinflammatory properties. This dichotomous behavior is particularly relevant to the work of our laboratory on an aerosolized preparation of HA to treat pulmonary emphysema. The breakdown of inhaled HA into smaller fragments could possibly induce an inflammatory reaction in the lung that counteracts any beneficial effect. Consequently, the proposed therapeutic use of HA will require development of treatment strategies aimed at minimizing its proinflammatory activity. PMID:18229566

  16. A novel photopolymerizable derivative of hyaluronan for designed hydrogel formation.

    PubMed

    Bobula, Tomáš; Buffa, Radovan; Hermannová, Martina; Kohutová, Lenka; Procházková, Pavlína; Vágnerová, Hana; Čepa, Martin; Wolfová, Lucie; Židek, Ondřej; Velebný, Vladimír

    2017-04-01

    A new photopolymerizable derivative of hyaluronan (methacrylhydrazide-HA, MAHA) was prepared by carbodiimide chemistry. The reaction conditions were optimized for molecular weight (Mw), reaction time and amount of reagents with a degree of methacrylation (DM) ranging from 2% to 58%. Methacrylhydrazide-HA was hydrolytically stable (PBS, 7days, 37°C) in contrast to commonly used methacrylester analoque (23% hydrolyzed). MAHA readily photopolymerized into densely crosslinked hydrogels under physiological conditions. The varied DM, Mw, irradiation time (texp) and macromer concentration in photocrosslinking afforded hydrogels with different physical (swelling ratio, degradation rate) and mechanical properties (stiffness, toughness). Three-dimensional fabrication and surface patterning of MAHA hydrogels were demonstrated by photolithography and light mediated micromolding. A live-dead assay with skin fibroblasts showed convenient biocompatibility of MAHA (16%, 116kDa) for potential scaffolding applications in tissue engineering and regenerative medicine.

  17. Human milk hyaluronan enhances innate defense of the intestinal epithelium.

    PubMed

    Hill, David R; Rho, Hyunjin K; Kessler, Sean P; Amin, Ripal; Homer, Craig R; McDonald, Christine; Cowman, Mary K; de la Motte, Carol A

    2013-10-04

    Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.

  18. Preparation of biologically intact radioiodinated hyaluronan of high specific radioactivity: coupling of /sup 125/I-tyramine-cellobiose to amino groups after partial N-deacetylation

    SciTech Connect

    Dahl, L.B.; Laurent, T.C.; Smedsrod, B.

    1988-12-01

    Hyaluronan was substituted with tyramine-cellobiose on amino residues exposed after hydrazinolytic N-deacetylation of the polysaccharide. Nonsubstituted amino groups were reacetylated, and the carboxylic hydrazides were removed by treatment with HIO/sub 3/. The adduct was labeled with /sup 125/I before or after coupling to hyaluronan. N-deacetylation increased with prolonged pretreatment with hydrazine, which also reduced the chain length of hyaluronan. Hydrazinolysis for 30 min produced hyaluronan with Mr 2.2-2.9 x 10(5). This material was substituted with varying amounts of tyramine-cellobiose (from 1 per 20 to 1 per 130 disaccharides). Hyaluronan labeled in this way was recognized by Streptomyces hyaluronidase, hyaluronan affinity protein of cartilage proteoglycan, and receptors for specific endocytosis of hyaluronan in liver endothelial cells. Since tyramine-cellobiose is nondegradable and therefore is arrested intralysosomally at the site of uptake, turnover studies of hyaluronan can be easily carried out with this ligand.

  19. Neonatal Pasteurella multocida subsp. septica Meningitis Traced to Household Cats: Molecular Linkage Analysis Using Repetitive-Sequence-Based PCR

    PubMed Central

    Freij, Bishara J.; Robinson-Dunn, Barbara; Makin, Jacob; Runge, Jessica K.; Luna, Ruth Ann

    2015-01-01

    Pasteurella multocida is a rare cause of neonatal bacterial meningitis. We describe such a case and verify two household cats as the source of infection using repetitive-element PCR (rep-PCR) molecular fingering. PMID:26491173

  20. Pasteurella haemolytica complicated respiratory infections in sheep and goats.

    PubMed

    Brogden, K A; Lehmkuhl, H D; Cutlip, R C

    1998-01-01

    Respiratory infections which commonly occur in sheep and goats often result from adverse physical and physiological stress combined with viral and bacterial infections. Inevitably, Pasteurella haemolytica pneumonia occurs as a result of these interactions. In this review, we present recent advances in research on the complex etiology of pneumonia involving P. haemolytica. Initially stress, induced by factors such as heat, overcrowding, exposure to inclement weather, poor ventilation, handling and transport is a major predisposing factor. Respiratory viruses including parainfluenza 3 (PI-3) virus, adenovirus type 6 and respiratory syncytial virus (RSV), and to a lesser extent bovine adenovirus type 2, ovine adenovirus types 1 and 5, and reovirus type 1 cause respiratory infections and pneumonia. More importantly these viruses also dramatically increase the susceptibility of sheep and goats to secondary P. haemolytica infection. Primary infection of the lower respiratory tract, with Mycoplasma ovipneumoniae and Bordetella parapertussis can increase the susceptibility of sheep and goats to secondary P. haemolytica infection. It is possible that initial infections with viral or primary bacterial agents break down the antimicrobial barrier consisting of beta defensins and anionic peptides found in epithelial cells, resident and inflammatory cells, and serous and mucous secretions of the respiratory tract. Loss of barrier integrity may release P. haemolytica from its usual commensal status. Once in the lung, P. haemolytica becomes opportunistic. To grow and colonize, P. haemolytica uses extracellular products like O-sialoglycoprotein endopeptidase, neuraminidase and RTX leukotoxin, as well as cell-associated products such as capsular polysaccharide, lipopolysaccharide, outer membrane proteins, proteins involved in iron acquisition and a periplasmic superoxide dismutase. In lambs and kids, pneumonic pasteurellosis can be acute, characterized by fever, listlessness, poor

  1. Perturbation of hyaluronan metabolism predisposes patients with type 1 diabetes mellitus to atherosclerosis

    PubMed Central

    Holleman, F.; de Groot, E.; Vink, H.; Gort, J.; Kontush, A.; Chapman, M. J.; Hutten, B. A.; Brouwer, C. B.; Hoekstra, J. B. L.; Kastelein, J. J. P.; Stroes, E. S. G.

    2007-01-01

    Aims/hypothesis Cardiovascular disease contributes to mortality in type 1 diabetes mellitus, but the specific pathophysiological mechanisms remain to be established. We recently showed that the endothelial glycocalyx, a protective layer of proteoglycans covering the endothelium, is severely perturbed in type 1 diabetes, with concomitantly increased plasma levels of hyaluronan and hyaluronidase. In the present study, we evaluated the relationship between hyaluronan and hyaluronidase with carotid intima-media thickness (cIMT), an established surrogate marker for cardiovascular disease. Subjects and methods Non-smoking type 1 diabetes patients without micro- or macrovascular complications and matched controls were recruited and cIMT of both carotid arteries was measured. To evaluate the relationship between cIMT and hyaluronan and hyaluronidase as well as other parameters, uni- or multivariate regression analyses were performed. Results We included 99 type 1 diabetes patients (age 10–72 years) and 99 age- and sex-matched controls. Mean cIMT, HbA1c, high sensitivity C-reactive protein, hyaluronan and hyaluronidase were significantly increased in type 1 diabetes vs controls. Plasma hyaluronan and hyaluronidase were correlated in type 1 diabetes. In univariate regression analyses, mean IMT was associated with plasma hyaluronan, age and male sex, whereas after multivariate analysis only age and sex remained statistically significant. Conclusions/interpretation We conclude that type 1 diabetes patients show structural changes of the arterial wall associated with increased hyaluronan metabolism. These data may lend further support to altered glycosaminoglycan metabolism in type 1 diabetes as a potential mechanism involved in accelerated atherogenesis. PMID:17415544

  2. Inhibition of Ovarian Cancer Chemoresistance and Metastasis with Antagonists of Hyaluronan-CD44-CD147 Interactions

    DTIC Science & Technology

    2013-07-01

    chitosan nanoparticles (Han et al., 2011), which will be used to determine whether these particles, with and without loading with cisplatin, doxorubicin...carcinoma cells to cisplatin treatment in a mouse xenograft model, as well as in cell culture. Hyaluronan oligosaccharide-decorated nanoparticles ...the regulator of hyaluronan synthesis, CD147, sensitize cisplatin-resistant ovarian carcinoma cells to cisplatin in cell culture. Nanoparticles

  3. A Pasteurella sp associated with respiratory disease in captive desert tortoises.

    PubMed

    Snipes, K P; Biberstein, E L; Fowler, M E

    1980-11-01

    Bacteria isolated from captive healthy desert tortoises were compared with bacteria from captive tortoises with respiratory illness and with bacteria from free-ranging tortoises from the Mojave Desert. Major differences were not observed among these groups when bacteria from the mouth, nares, trachea, lungs, and cloaca were compared. Frequently encountered organisms in all 3 groups included: coagulase-negative, catalase-positive, gram-positive cocci; Corynebacterium sp; members of Enterobacteriaceae, including Proteus spp; and a bacterium apparently belonging to the genus Pasteurella. The Pasteurella sp was consistently found to be associated with respiratory lesions in captive tortoises with signs of respiratory disease but was also found to be part of the gastrointestinal and nasal flora of healthy tortoises. It was hypothesized that respiratory disease in captive desert tortoises involves a commensal bacterium with the potential to be an opportunistic pathogen when the tortoise is stressed by a captive environment.

  4. Exposure of human lung fibroblasts to ozone: cell mortality and hyaluronan metabolism

    SciTech Connect

    Mayer, D.; Branscheid, D. )

    1992-04-01

    Exposure of cultures of human lung fibroblasts to 0.5 ppm ozone for 20 h resulted in a significant increase in cellular mortality by 29%; after exposure to 2.5 ppm ozone for 4 h, the increase amounted to 74%. A marked difference in sensitivity to ozone was observed between fibroblast lines from different individuals. This variability in resistance to ozone was more evident after exposure to 0.5 ppm ozone for 20 h, when compared with 2.5 ppm ozone for 4 h. In one fibroblast line, synthesis of hyaluronan was enhanced by exposure to 0.5 ppm ozone for 20 h. The concentrations of hyaluronan in culture media increased in experiments using different fibroblast cell lines, a phenomenon that was obvious both if cell numbers and combined protein concentrations of cells and media are selected as references for hyaluronan concentrations.

  5. Renal abscess caused by a Providencia stuartii isolate biochemically misidentified as Pasteurella.

    PubMed

    Chamberland, Robin R; McElvania TeKippe, Erin; Burnham, Carey-Ann D; Kennedy, Donald J

    2013-08-01

    Providencia stuartii is associated with urinary tract infection (UTI) in catheterized patients. Here we report an abscess containing P. stuartii in a patient with a history of UTI, renal stones, and stent placement. This organism was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and 16S rRNA gene sequencing following biochemical identification as Pasteurella.

  6. In-silico analysis of Pasteurella multocida to identify common epitopes between fowl, goat and buffalo.

    PubMed

    Ghaffar, Ammarah; Tariq, Aamira

    2016-04-10

    Pasteurella multocida represents a highly diverse group of bacteria infecting various hosts like the fowl, goat and buffalo leading to huge economic loss to the poultry and cattle industry. Previous reports indicated that the outer membrane proteins contribute significantly to the pathogenesis of Pasteurella multocida. The comparative in-silico genome wide analysis of four pathogenic Pasteurella multocida strains (Anand1-poultry, Anand1-goat, PMTB and VTCCBAA264) with their respective hosts was performed. A pipeline was developed to identify the list of non-homologous proteins of Pasteurella multocida strains and their hosts. The list was further analyzed for the identification of the essential outer membrane proteins responsible for the pathogenicity. Outer membrane proteins were further selected from these antigenic proteins on the basis of their pathogenic potential. A common B-cell epitope (TDYRNRDRS, ARRSVTSKEN, and KINDQWRW) determined via sequential and structural approach from the lipopolysaccharide (LPS) assembly outer membrane complex protein was predicted from fowl, goat and buffalo. Furthermore, we identified T-cell epitopes based on the lipopolysaccharide (LPS) assembly outer membrane complex protein via docking studies which were either similar to the B-cell epitopes or were occurring in the same patch except for MHC class II M fowl. We propose that this difference in epitope sequence is due to different interacting MHC class II protein predicted from the fowl. Hence, in the current study we found that a unique epitope based on the common antigenic lipopolysaccharide (LPS) outer membrane complex protein present in fowl, goat and buffalo can be a suitable target for vaccine development against the two economic devastating diseases; fowl cholera (FC) and hemorrhagic septicemia (HS).

  7. Regulation of Hyaluronan Synthesis in Vascular Diseases and Diabetes

    PubMed Central

    Moretto, Paola; Karousou, Evgenia; Viola, Manuela; Caon, Ilaria; Passi, Alberto; Vigetti, Davide

    2015-01-01

    Cell microenvironment has a critical role determining cell fate and modulating cell responses to injuries. Hyaluronan (HA) is a ubiquitous extracellular matrix glycosaminoglycan that can be considered a signaling molecule. In fact, interacting with several cell surface receptors can deeply shape cell behavior. In vascular biology, HA triggers smooth muscle cells (SMCs) dedifferentiation which contributes to vessel wall thickening. Furthermore, HA is able to modulate inflammation by altering the adhesive properties of endothelial cells. In hyperglycemic conditions, HA accumulates in vessels and can contribute to the diabetic complications at micro- and macrovasculature. Due to the pivotal role in favoring atherogenesis and neointima formation after injuries, HA could be a new target for cardiovascular pathologies. This review will focus on the recent findings regarding the regulation of HA synthesis in human vascular SMCs. In particular, the effects of the intracellular HA substrates availability, adenosine monophosphate-activated protein kinase (AMPK), and protein O-GlcNAcylation on the main HA synthetic enzyme (i.e., HAS2) will be discussed. PMID:25834831

  8. Chemico-physical properties of hyaluronan-based sponges.

    PubMed

    Milella, E; Brescia, E; Massaro, C; Ramires, P A

    2000-12-15

    The aim of this study was to obtain information on the chemico-physical and surface properties of the hyaluronan total benzylic ester sponges to evaluate their stability, surface "cleanliness" and handling for the applications in the tissue engineering. The thermal analysis, the characterization of surface chemical composition and the swelling test were performed on these materials. Moreover, the morphological changes, the rheological behavior, and the molecular weight loss in function of the time were monitored when the sponges were incubated in cell culture medium. The results showed that the sponges were thermally stable up to 220 degrees C and the surface composition was different from that of the bulk for C-O contribution. No contaminants were detected. In culture medium, the samples swelled assuming the rheological properties of biopolymer gel. When the sponges were in contact with the culture medium, their molecular weight remained stable for the first day and a loss of 11% and 31% was recorded for samples removed from culture medium after 3 and 7 days, respectively. With the scanning electron microscopy analysis, the spongy structure appeared with open interconnecting pores. The micrographs related to the samples after incubation in culture medium showed that the degradation was evident on the surface after 1 day. The deterioration of the pore walls and the presence of craters increased with time and, after 3 days, the phenomena were present also in the section.

  9. Characterization of Hyaluronan-Protein Microstructures and Polymer Solutions

    NASA Astrophysics Data System (ADS)

    Curtis, J. E.; McLane, L.; Bedoya, M.; Beatty, R.; Kramer, A.; Boehm, H.; Scrimgeour, J.

    2010-03-01

    Evidence is mounting that mechanical and topographical features of biomaterials can be as critical for cellular behavior as chemical properties. A case in point is hyaluronan (HA), a large polysaccharide with unique mechanical and hydrodynamic properties, found in many tissues and bodily fluids. Thanks to a large variety of accessible conformations and aggregation states, this remarkable polymer can impart on its biological environment a diverse range of structural and viscoelastic properties with far-reaching consequences for cell physiology (migration, inflammation, cancer). Supramolecular assembly of HA is typically mediated by HA-binding proteins. These specialized molecules are known to assist the formation of organized structures, such as cross-linked bundles, gels, or the all-important pericellular coat, a polymer network anchored to many cell surfaces. Precisely how the material properties of HA-rich matrices and aggregates are modified by the associated proteins, however, is largely a matter of speculation. We will present new insights concerning the cell coat and HA-protein solutions characterized using passive microrheology, fluorescence recovery after photobleaching (FRAP), and optical force probe microscopy.

  10. In vitro hemocompatibility testing of UV-modified hyaluronan hydrogels.

    PubMed

    Amarnath, Leena Pravina; Srinivas, Arvind; Ramamurthi, Anand

    2006-03-01

    Hydrogels (hylans) based on cross-linked hyaluronan (HA) are potentially good biomaterials for vascular tissue engineering applications because they are highly non-antigenic and -immunogenic. To facilitate surface endothelialization, vital to vascular deployment, we irradiated the gel surface with low wavelength UV light. This process micro-textures the smooth gel surface to provide sites for cell anchorage and causes limited scission of native long-chain HA yielding smaller fragments that elicit an enhanced cell response. In the current in vitro study, we assessed the effects of UV irradiation on the short-term (<45 min) interaction between hylan gels and human blood cells (RBCs, platelets) and coagulation proteins at physiologic temperature. Although the lowered hydrophilicity of irradiated (UV) hylans elicited greater vascular cell response relative to unmodified (U) hylans, platelet deposition was unaffected and much lower compared to collagen-coated glass controls. The adhered platelets were rounded or mildly pseudopodic and did not express p-selectin, an activation marker. Both gel types induced identical, and minimal platelet release as measured using an platelet factor 4 ELISA, and identically deferred the intrinsic and extrinsic coagulation pathways. Both gel types induced elevated levels of contact activation of bound, but not plasma-phase factor XII relative to controls. Hemolysis rates were also identical and within accepted standards. We conclude that UV-treatment of hylans, useful to improve surface endothelialization, does not compromise their short-term hemocompatibility, vital to their use as vascular implant materials.

  11. Determination of Hyaluronan Molecular Mass Distribution in Human Breast Milk

    PubMed Central

    Yuan, Han; Amin, Ripal; Ye, Xin; De La Motte, Carol A.; Cowman, Mary K.

    2015-01-01

    Hyaluronan (HA) in human milk mediates host responses to microbial infection, via TLR4- and CD44-dependent signaling. Signaling by HA is generally size-specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low M component. Here we report the size distribution of HA in human milk samples from twenty unique donors. A new method for HA analysis, employingion exchange (IEX) chromatography to fractionate HA by size, and specific quantification of each size fraction by competitive Enzyme Linked Sorbent Assay (ELSA), was developed. When separated into four fractions, milk HA with M ≤ 20 kDa, M ≈20-60 kDa, and M ≈ 60-110 kDa comprised an average of 1.5%, 1.4% and 2% of the total HA, respectively. The remaining 95% was HA with M≥110 kDa. Electrophoretic analysis of the higher M HA from thirteen samples showed nearly identical M distributions, with an average M of ∼440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low M HA components. PMID:25579786

  12. CD147: regulator of hyaluronan signaling in invasiveness and chemoresistance.

    PubMed

    Grass, G Daniel; Dai, Lu; Qin, Zhiqiang; Parsons, Chris; Toole, Bryan P

    2014-01-01

    Major determinants that influence negative outcome in cancer patients are the abilities of cancer cells to resist current therapies and to invade surrounding host tissue, consequently leading to local and metastatic dissemination. Hyaluronan (HA), a prominent constituent of the tumor microenvironment, not only provides structural support but also interacts with cell surface receptors, especially CD44, that influence cooperative signaling pathways leading to chemoresistance and invasiveness. CD147 (emmprin; basigin) is a member of the Ig superfamily that has also been strongly implicated in chemoresistance and invasiveness. CD147 both regulates HA synthesis and interacts with the HA receptors, CD44, and LYVE-1. Increased CD147 expression induces formation of multiprotein complexes containing CD44 (or LYVE-1) as well as members of the membrane-type matrix metalloproteinase, receptor tyrosine kinase, ABC drug transporter, or monocarboxylate transporter families, which become assembled in specialized lipid raft domains along with CD147 itself. In each case, multivalent HA-receptor interactions are essential for formation or stabilization of the lipid raft complexes and for downstream signaling pathways or transporter activities that are driven by these complexes. We conclude that cooperativity between HA, HA receptors, and CD147 may be a major driver of the interconnected pathways of invasiveness and chemoresistance widely critical to malignancy.

  13. Carcinoma Cell Hyaluronan as a "Portable" Cancerized Prometastatic Microenvironment.

    PubMed

    Turley, Eva A; Wood, David K; McCarthy, James B

    2016-05-01

    Hyaluronan (HA) is a structurally simple polysaccharide, but its ability to act as a template for organizing pericellular matrices and its regulated synthesis and degradation are key to initiating repair responses. Importantly, these HA functions are usurped by tumor cells to facilitate progression and metastasis. Recent advances have identified the functional complexities associated with the synthesis and degradation of HA-rich matrices. Three enzymes synthesize large HA polymers while multiple hyaluronidases or tissue free radicals degrade these into smaller bioactive fragments. A family of extracellular and cell-associated HA-binding proteins/receptors translates the bioinformation encrypted in this complex polymer mixture to activate signaling networks required for cell survival, proliferation, and migration in an actively remodeling microenvironment. Changes in HA metabolism within both the peritumor stroma and parenchyma are linked to tumor initiation, progression, and poor clinical outcome. We review evidence that metastatic tumor cells must acquire the capability to autonomously synthesize, assemble, and process their own "portable" HA-rich microenvironments to survive in the circulation, metastasize to ectopic sites, and escape therapeutic intervention. Strategies to disrupt the HA machinery of primary tumor and circulating tumor cells may enhance the effectiveness of current conventional and targeted therapies. Cancer Res; 76(9); 2507-12. ©2016 AACR.

  14. Oral administration of hyaluronan reduces bone turnover in ovariectomized rats.

    PubMed

    Ma, Jenny; Granton, Patrick V; Holdsworth, David W; Turley, Eva A

    2013-01-16

    The effect of oral hyaluronan (HA) on bone loss in ovariectomized (OVX) 3-month-old rats was measured using serum markers of bone turnover and bone mineral density. OVX rats were administered 1 mg/kg HA (OVX + HA) or phosphate-buffered saline (PBS) (OVX + PBS) by oral gavage (5 days/week for 54 days). Additional controls included sham ovariectomy with PBS gavage (Sham + PBS) and no treatment. Oral administration of HA resulted in approximately 50% (p < 0.05) increases in serum HA. Gel filtration analyses showed this was high molecular weight HA (300-500 kDa). Osteopenia was mild due to the young age of the animals. Thus, ovariectomy resulted in a 30% increase in serum collagen N-terminal telopeptides (p < 0.001), a 20% increase in serum nitrate/nitrite levels (p = 0.05), and a 5-6% decrease in femur bone mineral density/content (p < 0.05). HA gavage blunted the development of osteopenia in this model as determined by preventing the 30% increase in serum collagen N-terminal telopeptide levels (p < 0.001) and by reducing bone mineral content loss from 6 to 4%. These results show that oral supplements of HA (gavage solution, 0.12% solution) significantly reduce bone turnover associated with mild osteopenia in rats.

  15. Evaluation of bacterial cellulose/hyaluronan nanocomposite biomaterials.

    PubMed

    Li, Ying; Qing, Shuang; Zhou, Jianhai; Yang, Guang

    2014-03-15

    Bacterial cellulose (BC) is useful in the biomedical field because of its unique structure and properties. The high nano-porosity of BC allows other materials to be incorporated and form reinforced composites. Here we describe the preparation and characterization of novel BC/hyaluronan (HA) nanocomposites with a 3-D network structure. BC/HA was obtained using a solution impregnation method. Elemental and ATR-FTIR analyses showed that this method is highly effective to form composites with BC. Weight loss analysis showed that BC/HA have a lower water loss than BC at 37 °C. The total surface area and pore volume of BC/HA films gradually decreased with the HA content, as followed by FE-SEM analysis. The elongation at break of BC/HA films gradually increased as the HA content increased. Thermogravimetric analysis showed that the weight loss for the BC/HA composites were lower than for pure BC between 250 and 350 °C. The results of weight loss, elongation at break and thermal stability suggested that these novel BC/HA films could be applied potentially as wound dressing materials.

  16. IL-10 Induction from Implants Delivering Pancreatic Islets and Hyaluronan

    PubMed Central

    Bollyky, Paul L.; Vernon, Robert B.; Falk, Ben A.; Preisinger, Anton; Gooden, Michel D.; Nepom, Gerald T.; Gebe, John A.

    2013-01-01

    Local induction of pro-tolerogenic cytokines, such as IL-10, is an appealing strategy to help facilitate transplantation of islets and other tissues. Here, we describe a pair of implantable devices that capitalize on our recent finding that hyaluronan (HA) promotes IL-10 production by activated T cells. The first device is an injectable hydrogel made of crosslinked HA and heparan sulfate loaded with anti-CD3/anti-CD28 antibodies and IL-2. T cells embedded within this hydrogel prior to polymerization go on to produce IL-10 in vivo. The second device is a bioengineered implant consisting of a polyvinyl alcohol sponge scaffold, supportive collagen hydrogel, and alginate spheres mediating sustained release of HA in fluid form. Pancreatic islets that expressed ovalbumin (OVA) antigen were implanted within this device for 14 days into immunodeficient mice that received OVA-specific DO.11.10 T cells and a subsequent immunization with OVA peptide. Splenocytes harvested from these mice produced IL-10 upon re-challenge with OVA or anti-CD3 antibodies. Both of these devices represent model systems that will be used, in future studies, to further evaluate IL-10 induction by HA, with the objective of improving the survival and function of transplanted islets in the setting of autoimmune (type 1) diabetes. PMID:23971054

  17. Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.

    PubMed

    Nagy, Nadine; Kaber, Gernot; Johnson, Pamela Y; Gebe, John A; Preisinger, Anton; Falk, Ben A; Sunkari, Vivekananda G; Gooden, Michel D; Vernon, Robert B; Bogdani, Marika; Kuipers, Hedwich F; Day, Anthony J; Campbell, Daniel J; Wight, Thomas N; Bollyky, Paul L

    2015-10-01

    We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

  18. IL-10 induction from implants delivering pancreatic islets and hyaluronan.

    PubMed

    Bollyky, Paul L; Vernon, Robert B; Falk, Ben A; Preisinger, Anton; Gooden, Michel D; Nepom, Gerald T; Gebe, John A

    2013-01-01

    Local induction of pro-tolerogenic cytokines, such as IL-10, is an appealing strategy to help facilitate transplantation of islets and other tissues. Here, we describe a pair of implantable devices that capitalize on our recent finding that hyaluronan (HA) promotes IL-10 production by activated T cells. The first device is an injectable hydrogel made of crosslinked HA and heparan sulfate loaded with anti-CD3/anti-CD28 antibodies and IL-2. T cells embedded within this hydrogel prior to polymerization go on to produce IL-10 in vivo. The second device is a bioengineered implant consisting of a polyvinyl alcohol sponge scaffold, supportive collagen hydrogel, and alginate spheres mediating sustained release of HA in fluid form. Pancreatic islets that expressed ovalbumin (OVA) antigen were implanted within this device for 14 days into immunodeficient mice that received OVA-specific DO.11.10 T cells and a subsequent immunization with OVA peptide. Splenocytes harvested from these mice produced IL-10 upon re-challenge with OVA or anti-CD3 antibodies. Both of these devices represent model systems that will be used, in future studies, to further evaluate IL-10 induction by HA, with the objective of improving the survival and function of transplanted islets in the setting of autoimmune (type 1) diabetes.

  19. Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis

    PubMed Central

    Nagy, Nadine; Kaber, Gernot; Johnson, Pamela Y.; Gebe, John A.; Preisinger, Anton; Falk, Ben A.; Sunkari, Vivekananda G.; Gooden, Michel D.; Vernon, Robert B.; Bogdani, Marika; Kuipers, Hedwich F.; Day, Anthony J.; Campbell, Daniel J.; Wight, Thomas N.; Bollyky, Paul L.

    2015-01-01

    We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals. PMID:26368307

  20. The Rise and Fall of Hyaluronan in Respiratory Diseases

    PubMed Central

    Lauer, Mark E.; Dweik, Raed A.; Garantziotis, Stavros; Aronica, Mark A.

    2015-01-01

    In normal airways, hyaluronan (HA) matrices are primarily located within the airway submucosa, pulmonary vasculature walls, and, to a lesser extent, the alveoli. Following pulmonary injury, elevated levels of HA matrices accumulate in these regions, and in respiratory secretions, correlating with the extent of injury. Animal models have provided important insight into the role of HA in the onset of pulmonary injury and repair, generally indicating that the induction of HA synthesis is an early event typically preceding fibrosis. The HA that accumulates in inflamed airways is of a high molecular weight (>1600 kDa) but can be broken down into smaller fragments (<150 kDa) by inflammatory and disease-related mechanisms that have profound effects on HA pathobiology. During inflammation in the airways, HA is often covalently modified with heavy chains from inter-alpha-inhibitor via the enzyme tumor-necrosis-factor-stimulated-gene-6 (TSG-6) and this modification promotes the interaction of leukocytes with HA matrices at sites of inflammation. The clearance of HA and its return to normal levels is essential for the proper resolution of inflammation. These data portray HA matrices as an important component of normal airway physiology and illustrate its integral roles during tissue injury and repair among a variety of respiratory diseases. PMID:26448757

  1. Group B Streptococcus evades host immunity by degrading hyaluronan

    PubMed Central

    Kolar, Stacey L.; Kyme, Pierre; Tseng, Ching Wen; Soliman, Antoine; Kaplan, Amber; Liang, Jiurong; Nizet, Victor; Jiang, Dianhua; Murali, Ramachandran; Arditi, Moshe; Underhill, David M.; Liu, George Y.

    2015-01-01

    SUMMARY In response to tissue injury, hyaluronan (HA) polymers are cleaved by host hyaluronidases generating small fragments that ligate Toll-Like Receptors to elicit inflammatory responses. Pathogenic bacteria such as Group B Streptococci (GBS) express and secrete hyaluronidases as a mechanism for tissue invasion, but it is not known how this activity relates to immune detection of HA. We found that bacterial hyaluronidases secreted by GBS and other Gram-positive pathogens degrade pro-inflammatory HA fragments to their component disaccharides. Additionally, HA disaccharides block TLR2/4 signaling elicited by both host-derived HA fragments and other TLR2/4 ligands, including LPS. Application of GBS hyaluronidase or HA disaccharides reduced pulmonary pathology and pro-inflammatory cytokine levels in an acute lung injury model. We conclude that breakdown of host-generated pro-inflammatory HA fragments to disaccharides allows bacterial pathogens to evade immune detection and could be exploited as a strategy to treat inflammatory diseases. PMID:26651945

  2. Group B Streptococcus Evades Host Immunity by Degrading Hyaluronan.

    PubMed

    Kolar, Stacey L; Kyme, Pierre; Tseng, Ching Wen; Soliman, Antoine; Kaplan, Amber; Liang, Jiurong; Nizet, Victor; Jiang, Dianhua; Murali, Ramachandran; Arditi, Moshe; Underhill, David M; Liu, George Y

    2015-12-09

    In response to tissue injury, hyaluronan (HA) polymers are cleaved by host hyaluronidases, generating small fragments that ligate Toll-like receptors (TLRs) to elicit inflammatory responses. Pathogenic bacteria such as group B Streptococcus (GBS) express and secrete hyaluronidases as a mechanism for tissue invasion, but it is not known how this activity relates to immune detection of HA. We found that bacterial hyaluronidases secreted by GBS and other Gram-positive pathogens degrade pro-inflammatory HA fragments to their component disaccharides. In addition, HA disaccharides block TLR2/4 signaling elicited by both host-derived HA fragments and other TLR2/4 ligands, including lipopolysaccharide. Application of GBS hyaluronidase or HA disaccharides reduced pulmonary pathology and pro-inflammatory cytokine levels in an acute lung injury model. We conclude that breakdown of host-generated pro-inflammatory HA fragments to disaccharides allows bacterial pathogens to evade immune detection and could be exploited as a strategy to treat inflammatory diseases.

  3. A new selective enrichment procedure for isolating Pasteurella multocida from avian and environmental samples

    USGS Publications Warehouse

    Moore, M.K.; Cicnjak-Chubbs, L.; Gates, R.J.

    1994-01-01

    A selective enrichment procedure, using two new selective media, was developed to isolate Pasteurella multocida from wild birds and environmental samples. These media were developed by testing 15 selective agents with six isolates of P. multocida from wild avian origin and seven other bacteria representing genera frequently found in environmental and avian samples. The resulting media—Pasteurella multocida selective enrichment broth and Pasteurella multocida selective agar—consisted of a blood agar medium at pH 10 containing gentamicin, potassium tellurite, and amphotericin B. Media were tested to determine: 1) selectivity when attempting isolation from pond water and avian carcasses, 2) sensitivity for detection of low numbers of P. multocida from pure and mixed cultures, 3) host range specificity of the media, and 4) performance compared with standard blood agar. With the new selective enrichment procedure, P. multocida was isolated from inoculated (60 organisms/ml) pond water 84% of the time, whereas when standard blood agar was used, the recovery rate was 0%.

  4. Link protein hyaluronan-binding motif abrogates CD44-hyaluronan-mediated leukemia-liver cell adhesion.

    PubMed

    Chen, Jing; Li, Na; Li, Gongchu

    2013-05-01

    The liver is a frequent site for the metastasis of cancer cells originating from other sites. Leukemic liver metastasis is associated with poor prognosis. The ligation of CD44 with hyaluronan (HA) has been shown to contribute to the drug resistance of leukemic cells. In this study, a link protein HA-binding motif was genetically fused with enhanced green fluorescence protein (EGFP) to generate an EGFP-L fusion protein. Furthermore, a coculture system was established to investigate the interaction of leukemic cells with liver cells. CD44-positive Kasumi-1, but not CD44-negative HL-60 cells, were observed to adhere to the liver cell line L02. This cell-cell adhesion was significantly blocked by HA, indicating that Kasumi-L02 cell adhesion was mediated by the CD44-HA interaction. Compared to EGFP, EGFP-L fusion protein bound to L02 and BEL7404 liver cells. EGFP-L partially abrogated the Kasumi-L02 adhesion, suggesting that the link protein-binding motif is able to inhibit CD44-HA-mediated leukemia-liver adhesion. These results may help provide insight into novel therapeutic methods for leukemic patients diagnosed with liver metastasis.

  5. Truncated variants of hyaluronan-binding protein 1 bind hyaluronan and induce identical morphological aberrations in COS-1 cells.

    PubMed Central

    Sengupta, Aniruddha; Tyagi, Rakesh K; Datta, Kasturi

    2004-01-01

    Hyaluronan (HA)-binding protein 1 (HABP1) is multifunctional in nature and exists as a trimer through coiled-coil interaction between alpha-helices at its N- and C-termini. To investigate the importance of trimeric assemblage and HA-binding ability of HABP1, we generated and overexpressed variants of HABP1 by truncating the alpha-helices at its termini. Subsequently, these variants were transiently expressed in COS-1 cells to examine the influence of these structural variations on normal cell morphology, as compared with those imparted by HABP1. Substantiating the centrality of coiled-coil interaction for maintaining the trimeric assembly of HABP1, we demonstrate that disruption of trimerization does not alter the affinity of variants towards its ligand HA. Transient expression of HABP1 altered the morphology of COS-1 cells by generating numerous cytoplasmic vacuoles along with disruption of the f-actin network. Interestingly, the truncated variants also imparted identical morphological changes. Characterization of the cytoplasmic vacuoles revealed that most of these vacuoles were autophagic in nature, resembling those generated under stress conditions. The identical morphological changes manifested in COS-1 cells on transient expression of HABP1 or its variants is attributed to their comparable HA-binding ability, which in concert with endogenous HABP1, may deplete the cellular HA pool. Such quenching of HA below a threshold level in the cellular milieu could generate a stress condition, manifested through cytoplasmic vacuoles and a disassembly of the f-actin network. PMID:15005653

  6. Phylogenomic and molecular demarcation of the core members of the polyphyletic pasteurellaceae genera actinobacillus, haemophilus, and pasteurella.

    PubMed

    Naushad, Sohail; Adeolu, Mobolaji; Goel, Nisha; Khadka, Bijendra; Al-Dahwi, Aqeel; Gupta, Radhey S

    2015-01-01

    The genera Actinobacillus, Haemophilus, and Pasteurella exhibit extensive polyphyletic branching in phylogenetic trees and do not represent coherent clusters of species. In this study, we have utilized molecular signatures identified through comparative genomic analyses in conjunction with genome based and multilocus sequence based phylogenetic analyses to clarify the phylogenetic and taxonomic boundary of these genera. We have identified large clusters of Actinobacillus, Haemophilus, and Pasteurella species which represent the "sensu stricto" members of these genera. We have identified 3, 7, and 6 conserved signature indels (CSIs), which are specifically shared by sensu stricto members of Actinobacillus, Haemophilus, and Pasteurella, respectively. We have also identified two different sets of CSIs that are unique characteristics of the pathogen containing genera Aggregatibacter and Mannheimia, respectively. It is now possible to demarcate the genera Actinobacillus sensu stricto, Haemophilus sensu stricto, and Pasteurella sensu stricto on the basis of discrete molecular signatures. The other members of the genera Actinobacillus, Haemophilus, and Pasteurella that do not fall within the "sensu stricto" clades and do not contain these molecular signatures should be reclassified as other genera. The CSIs identified here also provide useful diagnostic targets for the identification of current and novel members of the indicated genera.

  7. Phylogenomic and Molecular Demarcation of the Core Members of the Polyphyletic Pasteurellaceae Genera Actinobacillus, Haemophilus, and Pasteurella

    PubMed Central

    Naushad, Sohail; Adeolu, Mobolaji; Goel, Nisha; Khadka, Bijendra; Al-Dahwi, Aqeel; Gupta, Radhey S.

    2015-01-01

    The genera Actinobacillus, Haemophilus, and Pasteurella exhibit extensive polyphyletic branching in phylogenetic trees and do not represent coherent clusters of species. In this study, we have utilized molecular signatures identified through comparative genomic analyses in conjunction with genome based and multilocus sequence based phylogenetic analyses to clarify the phylogenetic and taxonomic boundary of these genera. We have identified large clusters of Actinobacillus, Haemophilus, and Pasteurella species which represent the “sensu stricto” members of these genera. We have identified 3, 7, and 6 conserved signature indels (CSIs), which are specifically shared by sensu stricto members of Actinobacillus, Haemophilus, and Pasteurella, respectively. We have also identified two different sets of CSIs that are unique characteristics of the pathogen containing genera Aggregatibacter and Mannheimia, respectively. It is now possible to demarcate the genera Actinobacillus sensu stricto, Haemophilus sensu stricto, and Pasteurella sensu stricto on the basis of discrete molecular signatures. The other members of the genera Actinobacillus, Haemophilus, and Pasteurella that do not fall within the “sensu stricto” clades and do not contain these molecular signatures should be reclassified as other genera. The CSIs identified here also provide useful diagnostic targets for the identification of current and novel members of the indicated genera. PMID:25821780

  8. Hyaluronan degrading silica nanoparticles for skin cancer therapy

    NASA Astrophysics Data System (ADS)

    Scodeller, P.; Catalano, P. N.; Salguero, N.; Duran, H.; Wolosiuk, A.; Soler-Illia, G. J. A. A.

    2013-09-01

    We report the first nanoformulation of Hyaluronidase (Hyal) and its enhanced adjuvant effect over the free enzyme. Hyaluronic acid (HA) degrading enzyme Hyal was immobilized on 250 nm silica nanoparticles (SiNP) maintaining specific activity of the enzyme via the layer-by-layer self-assembly technique. This process was characterized by dynamic light scattering (DLS), zeta potential, infrared and UV-Vis spectroscopy, transmission electron microscopy (TEM) and enzymatic activity measurements. The nanoparticles were tested in vivo as adjuvants of carboplatin (CP), peritumorally injected in A375 human melanoma bearing mice and compared with the non-immobilized enzyme, on the basis of equal enzymatic activity. Alcian Blue staining of A375 tumors indicated large overexpression of hyaluronan. At the end of the experiment, tumor volume reduction with SiNP-immobilized Hyal was significantly enhanced compared to non-immobilized Hyal. Field emission scanning electron microscopy (FE-SEM) images together with energy dispersive X-ray spectroscopy (EDS) spectra confirmed the presence of SiNP on the tumor. We mean a proof of concept: this extracellular matrix (ECM) degrading enzyme, immobilized on SiNP, is a more effective local adjuvant of cancer drugs than the non-immobilized enzyme. This could prove useful in future therapies using other or a combination of ECM degrading enzymes.We report the first nanoformulation of Hyaluronidase (Hyal) and its enhanced adjuvant effect over the free enzyme. Hyaluronic acid (HA) degrading enzyme Hyal was immobilized on 250 nm silica nanoparticles (SiNP) maintaining specific activity of the enzyme via the layer-by-layer self-assembly technique. This process was characterized by dynamic light scattering (DLS), zeta potential, infrared and UV-Vis spectroscopy, transmission electron microscopy (TEM) and enzymatic activity measurements. The nanoparticles were tested in vivo as adjuvants of carboplatin (CP), peritumorally injected in A375 human

  9. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  10. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  11. Alginate-hyaluronan composite hydrogels accelerate wound healing process.

    PubMed

    Catanzano, O; D'Esposito, V; Acierno, S; Ambrosio, M R; De Caro, C; Avagliano, C; Russo, P; Russo, R; Miro, A; Ungaro, F; Calignano, A; Formisano, P; Quaglia, F

    2015-10-20

    In this paper we propose polysaccharide hydrogels combining alginate (ALG) and hyaluronan (HA) as biofunctional platform for dermal wound repair. Hydrogels produced by internal gelation were homogeneous and easy to handle. Rheological evaluation of gelation kinetics of ALG/HA mixtures at different ratios allowed understanding the HA effect on ALG cross-linking process. Disk-shaped hydrogels, at different ALG/HA ratio, were characterized for morphology, homogeneity and mechanical properties. Results suggest that, although the presence of HA does significantly slow down gelation kinetics, the concentration of cross-links reached at the end of gelation is scarcely affected. The in vitro activity of ALG/HA dressings was tested on adipose derived multipotent adult stem cells (Ad-MSC) and an immortalized keratinocyte cell line (HaCaT). Hydrogels did not interfere with cell viability in both cells lines, but significantly promoted gap closure in a scratch assay at early (1 day) and late (5 days) stages as compared to hydrogels made of ALG alone (p<0.01 and 0.001 for Ad-MSC and HaCaT, respectively). In vivo wound healing studies, conducted on a rat model of excised wound indicated that after 5 days ALG/HA hydrogels significantly promoted wound closure as compared to ALG ones (p<0.001). Overall results demonstrate that the integration of HA in a physically cross-linked ALG hydrogel can be a versatile strategy to promote wound healing that can be easily translated in a clinical setting.

  12. Hyaluronan: A Simple Polysaccharide with Diverse Biological Functions

    PubMed Central

    Dicker, Kevin T.; Gurski, Lisa A.; Pradhan-Bhatt, Swati; Witt, Robert L.; Farach-Carson, Mary C.; Jia, Xinqiao

    2014-01-01

    Hyaluronan (HA) is a linear polysaccharide with disaccharide repeats of D-glucuronic acid and N-acetyl-D-glucosamine. It is evolutionary conserved and abundantly expressed in the extracellular matrix (ECM), on the cell surface and even inside cells. Being a simple polysaccharide, HA exhibits an astonishing array of biological functions. HA interacts with various proteins or proteoglycans to organize the ECM and to maintain tissue homeostasis. The unique physical and mechanical properties of HA contribute to the maintenance of tissue hydration, the mediation of solute diffusion through the extracellular space and the lubrication of certain tissues. The diverse biological functions of HA are manifested through its complex interactions with matrix components and resident cells. Binding of HA with cell surface receptors activates various signaling pathways that regulate cell function, tissue development, inflammation, wound healing and tumor progression and metastasis. Taking advantage of the inherent biocompatibility and biodegradability of HA, as well as its susceptibility to chemical modification, researchers have developed various HA-based biomaterials and tissue constructs with promising and broad clinical potential. In this article, we illustrate the properties of HA from a matrix biology perspective by first introducing principles underlying the biosynthesis and biodegradation of HA, as well as the interactions of HA with various proteins and proteoglycans. We next highlight the roles of HA in physiological and pathological states, including morphogenesis, wound healing and tumor metastasis. A deeper understanding of the mechanisms underlying the roles of HA in various physiological processes can provide new insights and tools for the engineering of complex tissues and tissue models. PMID:24361428

  13. Hyaluronan within fascia in the etiology of myofascial pain.

    PubMed

    Stecco, Carla; Stern, R; Porzionato, A; Macchi, V; Masiero, S; Stecco, A; De Caro, R

    2011-12-01

    The layers of loose connective tissue within deep fasciae were studied with particular emphasis on the histochemical distribution of hyaluronan (HA). Samples of deep fascia together with the underlying muscles were taken from neck, abdomen and thigh from three fresh non-embalmed cadavers. Samples were stained with hematoxylin-eosin, Azan-Mallory, Alcian blue and a biotinylated HA-binding protein specific for HA. An ultrasound study was also performed on 22 voluntary subjects to analyze the thickness of these deep fasciae and their sublayers. The deep fascia presented a layer of HA between fascia and the muscle and within the loose connective tissue that divided different fibrous sublayers of the deep fascia. A layer of fibroblast-like cells that stained prominently with Alcian blue stain was observed. It was postulated that these are cells specialized for the biosynthesis of the HA-rich matrix. These cells we have termed "fasciacytes", and may represent a new class of cells not previously recognized. The ultrasound study highlighted a mean thickness of 1.88 mm of the fascia lata, 1.68 mm of the rectus sheath, and 1.73 mm of the sternocleidomastoid fascia. The HA within the deep fascia facilitates the free sliding of two adjacent fibrous fascial layers, thus promoting the normal function associated with the deep fascia. If the HA assumes a more packed conformation, or more generally, if the loose connective tissue inside the fascia alters its density, the behavior of the entire deep fascia and the underlying muscle would be compromised. This, we predict, may be the basis of the common phenomenon known as "myofascial pain."

  14. Effect of Fetal Size on Fetal Placental Hyaluronan and Hyaluronoglucosaminidases Throughout Gestation in the Pig

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous results indicated that the trophoblast-endometrial epithelial cell bilayer of porcine placenta undergoes microscopic folding during gestation, and the folded bilayer is embedded in placental stroma. We hypothesized that hyaluronan was a component of placental stroma, and that hyaluronidases...

  15. Local application of hyaluronan gel in conjunction with periodontal surgery: a randomized controlled trial.

    PubMed

    Fawzy El-Sayed, Karim M; Dahaba, Moushira A; Aboul-Ela, Shadw; Darhous, Mona S

    2012-08-01

    Hyaluronic acid application has been proven to be beneficial in a number of medical disciplines. The aim of the current study was to clinically evaluate the effect of local application of hyaluronan gel in conjunction with periodontal surgery. Fourteen patients with chronic periodontitis having four interproximal intrabony defects (≥3 mm) with probing depth values >5 mm were included in this split-mouth study. Following initial nonsurgical periodontal therapy and re-evaluation, defects were randomly assigned to be treated with modified Widman flap (MWF) surgery in conjunction with either 0.8% hyaluronan gel (test) or placebo gel (control) application. Clinical attachment level (CAL), probing depth (PD), gingival recession (GR), plaque index (PI), and bleeding on probing (BOP) values were taken at baseline and 3 and 6 months. Differences between test and control sites were evaluated using a Wilcoxon signed-rank and a McNemar test. A Friedman and a Cochran test were used to test equal ranks over time. Statistically significant differences were noted for CAL and GR (P < 0.05) in favor of the test sites. No significant differences were found regarding PD, BOP, or PI values (P > 0.05). Hyaluronan gel application in conjunction with periodontal surgery appears to result in significant improvement of CAL and in a reduction in GR. Hyaluronan gel application appears to improve the clinical outcome of MWF surgery.

  16. Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding

    SciTech Connect

    Ruffell, Brian; Johnson, Pauline . E-mail: pauline@interchange.ubc.ca

    2005-08-26

    CD44 is a widely expressed cell adhesion molecule that binds hyaluronan, an extracellular matrix glycosaminoglycan, in a tightly regulated manner. This regulated interaction has been implicated in inflammation and tumor metastasis. CD44 exists in the standard form, CD44H, or as higher molecular mass isoforms due to alternative splicing. Here, we identify serine 180 in human CD44H as the site of chondroitin sulfate addition and show that lack of chondroitin sulfate addition at this site enhances hyaluronan binding by CD44. A CD44H-immunoglobulin fusion protein expressed in HEK293 cells, and CD44H expressed in murine L fibroblast cells were modified by chondroitin sulfate, as determined by reduced sulfate incorporation after chondroitinase ABC treatment. Mutation of serine 180 or glycine 181 in CD44H reduced chondroitin sulfate addition and increased hyaluronan binding, indicating that serine 180 is the site for chondroitin sulfate addition in CD44H and that this negatively regulates hyaluronan binding.

  17. A practical synthesis of capped 4-methylumbelliferyl hyaluronan disaccharides and tetrasaccharides as potential hyaluronidase substrates.

    PubMed

    Gold, Henrik; Munneke, Stefan; Dinkelaar, Jasper; Overkleeft, Herman S; Aerts, Johannes M F G; Codée, Jeroen D C; van der Marel, Gijs A

    2011-09-06

    The synthesis of hyaluronan dimers and tetramers equipped with a 4-methylumbelliferyl group at the reducing end to potentially allow monitoring of hyaluronidase activities is described. The 4-OH at the non-reducing glucuronate in the presented series is either removed or methylated to prohibit transglycosylase reactions, leading to a total of four probes.

  18. Development and characterization of chitosan/hyaluronan film for transdermal delivery of thiocolchicoside.

    PubMed

    Bigucci, Federica; Abruzzo, Angela; Saladini, Bruno; Gallucci, Maria Caterina; Cerchiara, Teresa; Luppi, Barbara

    2015-10-05

    The objective of this study was the development of chitosan/hyaluronan transdermal films to improve bioavailability of thiocolchicoside. This approach offers the possibility to elude the first-pass metabolism and at the same time it is able to provide a predictable and extended duration of activity. Films were prepared by casting and drying of aqueous solutions containing different weight ratios of chitosan and hyaluronan and characterized for their physico-chemical and functional properties. In accordance with polymeric composition of films and, therefore, with the amount of the net charge after the complexation, films containing the same weight ratio of chitosan and hyaluronan showed lower water uptake ability with respect to films containing only one polymeric species or an excess of chitosan or hyaluronan. Moreover, the lower the hydration of the polymeric network, the lower is the drug diffusion through the films and its permeation through the skin. This study clearly confirmed that the selection of a suitable polymeric weight ratio and appropriate preparative conditions allows the modulation of film functional properties, suggesting that these formulations could be used as a novel technological platform for transdermal drug delivery.

  19. The high and low molecular weight forms of hyaluronan have distinct effects on CD44 clustering.

    PubMed

    Yang, Cuixia; Cao, Manlin; Liu, Hua; He, Yiqing; Xu, Jing; Du, Yan; Liu, Yiwen; Wang, Wenjuan; Cui, Lian; Hu, Jiajie; Gao, Feng

    2012-12-14

    CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA.

  20. Pyrene-conjugated hyaluronan facilitated exfoliation and stabilisation of low dimensional nanomaterials in water.

    PubMed

    Zhang, Fei; Chen, Xianjue; Boulos, Ramiz A; Yasin, Faizah Md; Lu, Haibo; Raston, Colin; Zhang, Hongbin

    2013-05-25

    Pyrene-conjugated hyaluronan (Py-HA) facilitates the exfoliation of low-dimensional nanomaterials including graphite, hexagonal boron nitride (h-BN) and molybdenum disulfide (MoS2), and the dispersion of carbon nanotubes (CNTs) and carbon nano-onions (CNOs) in water (and PBS solutions), with the assistance of sonication.

  1. Hyaluronan accumulation and arrested oligodendrocyte progenitor maturation in vanishing white matter disease.

    PubMed

    Bugiani, Marianna; Postma, Nienke; Polder, Emiel; Dieleman, Nikki; Scheffer, Peter G; Sim, Fraser J; van der Knaap, Marjo S; Boor, Ilja

    2013-01-01

    Vanishing white matter disease is a genetic leukoencephalopathy caused by mutations in eukaryotic translation initiation factor 2B. Patients experience a slowly progressive neurological deterioration with episodes of rapid clinical worsening triggered by stress. The disease may occur at any age and leads to early death. Characteristic neuropathological findings include cystic degeneration of the white matter with feeble, if any, reactive gliosis, dysmorphic astrocytes and paucity of myelin despite an increase in oligodendrocytic density. These features have been linked to a maturation defect of astrocytes and oligodendrocytes. However, the nature of the link between glial immaturity and the observed neuropathological features is unclear. We hypothesized that the defects in maturation and function of astrocytes and oligodendrocytes are related. Brain tissue of seven patients with genetically proven vanishing white matter disease was investigated using immunohistochemistry, western blotting, quantitative polymerase chain reaction and size exclusion chromatography. The results were compared with those obtained from normal brain tissue of age-matched controls, from chronic demyelinated multiple sclerosis lesions and from other genetic and acquired white matter disorders. We found that the white matter of patients with vanishing white matter disease is enriched in CD44-expressing astrocyte precursor cells and accumulates the glycosaminoglycan hyaluronan. Hyaluronan is a major component of the extracellular matrix, and CD44 is a hyaluronan receptor. We found that a high molecular weight form of hyaluronan is overabundant, especially in the most severely affected areas. Comparison between the more severely affected frontal white matter and the relatively spared cerebellum confirms that high molecular weight hyaluronan accumulation is more pronounced in the frontal white matter than in the cerebellum. High molecular weight hyaluronan is known to inhibit astrocyte and

  2. Molecular mass dependence of hyaluronan detection by sandwich ELISA-like assay and membrane blotting using biotinylated hyaluronan binding protein

    PubMed Central

    Yuan, Han; Tank, Mihir; Alsofyani, Abeer; Shah, Naman; Talati, Nishant; LoBello, Jaclyn C; Kim, Jin Ryoun; Oonuki, Yoji; de la Motte, Carol A; Cowman, Mary K

    2013-01-01

    Hyaluronan (HA) is widely detected in biological samples and its concentration is most commonly determined by the use of a labeled specific HA binding protein (aggrecan G1-IGD-G2, HABP), employing membrane blotting and sandwich enzyme-linked immunosorbent assay (ELISA)-like methods. However, the detected signal intensity or the quantified value obtained by using these surface-based methods is related to the molecular mass (M) of HA, especially for HA in the low M range below ∼150 kDa. At the same mass or mass concentration, higher M HA gives a higher signal than lower M HA. We have experimentally determined the quantitative relationship between the M of HA (in the range 20–150 kDa) and the relative signal intensity in comparison with a standard HA, in a sandwich ELISA-like assay. An M-dependent signal correction factor (SCF) was calculated and used to correct the signal intensity, so that the corrected concentration value would more accurately reflect the true HA concentration in solution. The SCF for polydisperse low M HA was also calculated and compared with experimental results. When the molecular mass distribution of an HA sample is determined by a method such as gel electrophoresis, then its appropriately averaged SCF can be calculated and used to correct the signal in sandwich ELISA to obtain a more accurate concentration estimation. The correction method works for HA with M between ∼150 and 20 kDa, but lower M HA is too poorly detected for useful analysis. The physical basis of the M-dependent detection is proposed to be the increase in detector-accessible fraction of each surface-bound molecule as M increases. PMID:23964097

  3. Stabilin-1 and -2 constitute a novel family of fasciclin-like hyaluronan receptor homologues.

    PubMed Central

    Politz, Oliver; Gratchev, Alexei; McCourt, Peter A G; Schledzewski, Kai; Guillot, Pierre; Johansson, Sophie; Svineng, Gunbjorg; Franke, Peter; Kannicht, Christoph; Kzhyshkowska, Julia; Longati, Paola; Velten, Florian W; Johansson, Staffan; Goerdt, Sergij

    2002-01-01

    MS-1, a high-molecular-mass protein expressed by non-continuous and angiogenic endothelial cells and by alternatively activated macrophages (Mphi2), and the hepatic sinusoidal endothelial hyaluronan clearance receptor are similar with respect to tissue distribution and biochemical characteristics. In the present study we purified these proteins by immuno- and hyaluronan-affinity chromatography respectively, sequenced tryptic peptides and generated full-length cDNA sequences in both mouse and human. The novel genes, i.e. stabilin-1 and stabilin-2, code for homologous transmembrane proteins featuring seven fasciclin-like adhesion domains, 18-20 epidermal-growth-factor domains, one X-link domain and three to six B-(X(7))-B hyaluronan-binding motifs. Northern-blotting experiments revealed the presence of both stabilins in organs with predominant endothelial sinuses such as liver, spleen and lymph node: stabilin-1 mRNA was also detected in organs with predominant Mphi2 cells, such as placenta, and in interleukin-4/glucocorticoid-stimulated Mphi2 cells in vitro. A polyclonal antibody made against human recombinant stabilin-1 confirmed the expression of stabilin-1 protein in splenic sinus endothelial cells in vivo and in Mphi2 in vitro. On the basis of high similarity at the protein level and the unique domain composition, which differs from that of all other known fasciclin-like proteins and hyaluronan receptors, stabilin-1 and stabilin-2 define a novel family of fasciclin-like hyaluronan receptor homologues that might play a role in cell-cell and cell-matrix interactions in vascular function and inflammatory processes. PMID:11829752

  4. Heparin prevents intracellular hyaluronan synthesis and autophagy responses in hyperglycemic dividing mesangial cells and activates synthesis of an extensive extracellular monocyte-adhesive hyaluronan matrix after completing cell division.

    PubMed

    Wang, Aimin; Ren, Juan; Wang, Christina P; Hascall, Vincent C

    2014-03-28

    Growth-arrested rat mesangial cells (RMCs) at a G0/G1 interphase stimulated to divide in hyperglycemic medium initiate intracellular hyaluronan synthesis that induces autophagy/cyclin D3-induced formation of a monocyte-adhesive extracellular hyaluronan matrix after completing cell division. This study shows that heparin inhibits the intracellular hyaluronan synthesis and autophagy responses, but at the end of cell division it induces synthesis of a much larger extracellular monocyte-adhesive hyaluronan matrix. Heparin bound to RMC surfaces by 1 h, internalizes into the Golgi/endoplasmic reticulum region by 2 h, and was nearly gone by 4 h. Treatment by heparin for only the first 4 h was sufficient for its function. Streptozotocin diabetic rats treated daily with heparin showed similar results. Glomeruli in sections of diabetic kidneys showed extensive accumulation of autophagic RMCs, increased hyaluronan matrix, and influx of macrophages over 6 weeks. Hyaluronan staining in the glomeruli of heparin-treated diabetic rats was very high at week 1 and decreased to near control level by 6 weeks without any RMC autophagy. However, the influx of macrophages by 6 weeks was as pronounced as in diabetic glomeruli. The results are as follows: 1) heparin blocks synthesis of hyaluronan in intracellular compartments, which prevents the autophagy and cyclin D3 responses thereby allowing RMCs to complete cell division and sustain function; 2) interaction of heparin with RMCs in early G1 phase is sufficient to induce signaling pathway(s) for its functions; and 3) influxed macrophages effectively remove the hyaluronan matrix without inducing pro-fibrotic responses that lead to nephropathy and proteinurea in diabetic kidneys.

  5. Putative chitin synthases from Branchiostoma floridae show extracellular matrix-related domains and mosaic structures.

    PubMed

    Guerriero, Gea

    2012-08-01

    The transition from unicellular to multicellular life forms requires the development of a specialized structural component, the extracellular matrix (ECM). In Metazoans, there are two main supportive systems, which are based on chitin and collagen/hyaluronan, respectively. Chitin is the major constituent of fungal cell walls and arthropod exoskeleton. However, presence of chitin/chitooligosaccharides has been reported in lower chordates and during specific stages of vertebrate development. In this study, the occurrence of chitin synthases (CHSs) was investigated with a bioinformatics approach in the cephalochordate Branchiostoma floridae, in which the presence of chitin was initially reported in the skeletal rods of the pharyngeal gill basket. Twelve genes coding for proteins containing conserved amino acid residues of processive glycosyltransferases from GT2 family were found and 10 of them display mosaic structures with novel domains never reported previously in a chitin synthase. In particular, the presence of a discoidin (DS) and a sterile alpha motif (SAM) domain was found in nine identified proteins. Sequence analyses and homology modelling suggest that these domains might interact with the extracellular matrix and mediate protein-protein interaction. The multi-domain putative chitin synthases from B. floridae constitute an emblematic example of the explosion of domain innovation and shuffling which predate Metazoans.

  6. Improving hydrophilicity of silicone elastomer by IPN formation with hyaluronan

    NASA Astrophysics Data System (ADS)

    Koch, Richard L.

    Soft contact lenses have been available to consumers for the past several decades. By far, the most popular form on the market today is the silicone hydrogel, with nearly 70% of the market share. However, many contact lens wearers still have issues which cause them to discontinue lens use. It is estimated that between 25-35% of people discontinue use permanently. This can be traced back to two main issues with modern hydrogel lenses: a lack of adequate oxygen permeability across the lens; and lens-induced dehydration of the cornea. The corneal epithelium lining the lens of the eye is an avascular environment. As such, the cells must get their oxygen by diffusion through the tear film, or any material covering the lens. The silicone hydrogel SCLs have reduced oxygen gas permeability compared to traditional silicone elastomers. Additionally, when the hydrogel lenses lose water to evaporation, they pull water from the wearer's eye, contributing to dryness. Beyond simple discomfort, these issues can lead to pathologies such as hyperemia and even corneal cell death in severe cases. It was determined that a solution to these issues would be a new ocular lens material which had superior oxygen gas permeability and was hydrophilic without containing water in its bulk. The aim of this research was to create an interpenetrating polymer network (IPN) materials of poly(dimethyl siloxane) (PDMS) and hyaluronan (HA) with such properties. The results in this work indicate the successful synthesis of these HA-PDMS IPN materials. These elastomeric materials had improved hydrophilicity compared to untreated PDMS. Additionally, new chemical species (ATR/FTIR and XPS spectroscopy) and surface morphologies (SEM imaging) indicated the introduction of HA into the PDMS. Furthermore, analysis of the oxygen gas permeability showed no significant change for the treated samples as compared to the PDMS base material. As silicone materials have use in many biomedical fields, the material was

  7. Hyaluronidase and Hyaluronan Oligosaccharides Promote Neurological Recovery after Intraventricular Hemorrhage

    PubMed Central

    Vinukonda, Govindaiah; Dohare, Preeti; Arshad, Arslan; Zia, Muhammad T.; Panda, Sanjeet; Korumilli, Ritesh; Kayton, Robert; Hascall, Vincent C.; Lauer, Mark E.

    2016-01-01

    Intraventricular hemorrhage (IVH) in premature infants results in inflammation, arrested oligodendrocyte progenitor cell (OPC) maturation, and reduced myelination of the white matter. Hyaluronan (HA) inhibits OPC maturation and complexes with the heavy chain (HC) of glycoprotein inter-α-inhibitor to form pathological HA (HC–HA complex), which exacerbates inflammation. Therefore, we hypothesized that IVH would result in accumulation of HA, and that either degradation of HA by hyaluronidase treatment or elimination of HCs from pathological HA by HA oligosaccharide administration would restore OPC maturation, myelination, and neurological function in survivors with IVH. To test these hypotheses, we used the preterm rabbit model of glycerol-induced IVH and analyzed autopsy samples from premature infants. We found that total HA levels were comparable in both preterm rabbit pups and human infants with and without IVH, but HA receptors—CD44, TLR2, TLR4—were elevated in the forebrain of both humans and rabbits with IVH. Hyaluronidase treatment of rabbits with IVH reduced CD44 and TLR4 expression, proinflammatory cytokine levels, and microglia infiltration. It also promoted OPC maturation, myelination, and neurological recovery. HC–HA and tumor necrosis factor-stimulated gene-6 were elevated in newborns with IVH; and depletion of HC–HA levels by HA oligosaccharide treatment reduced inflammation and enhanced myelination and neurological recovery in rabbits with IVH. Hence, hyaluronidase or HA oligosaccharide treatment represses inflammation, promotes OPC maturation, and restores myelination and neurological function in rabbits with IVH. These therapeutic strategies might improve the neurological outcome of premature infants with IVH. SIGNIFICANCE STATEMENT Approximately 12,000 premature infants develop IVH every year in the United States, and a large number of survivors with IVH develop cerebral palsy and cognitive deficits. The onset of IVH induces inflammation

  8. Hybrid polyketide synthases

    SciTech Connect

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  9. Effects of intratumoral administration of a hyaluronan-cisplatin nanoconjugate to five dogs with soft tissue sarcomas

    PubMed Central

    Venable, Rachel O.; Worley, Deanna R.; Gustafson, Daniel L.; Hansen, Ryan J.; Ehrhart, E. J.; Cai, Shuang; Cohen, Mark S.; Forrest, M. Laird

    2013-01-01

    Objective To determine the effects of intratumoral injection of a hyaluronan-cisplatin nanoconjugate on local and systemic platinum concentrations and systemic toxicosis. Animals 5 dogs with spontaneous soft tissue sarcomas (STSs). Procedures For each dog, approximately 1.5 mL of hyaluronan nanocarrier conjugated with 20 mg of cisplatin was injected into an external STS. Blood samples were collected immediately before (0 hours) and at 0.5, 1, 2, 3, 4, 24, and 96 hours after hyaluronan-cisplatin injection for pharmacokinetic analyses. Urine samples were obtained at 0 and at 96 hours after hyaluronan-cisplatin injection for urinalysis. Each treated STS and its sentinel lymph nodes were surgically removed 96 hours after the hyaluronan-cisplatin injection. Inductively coupled plasma mass spectrometry was used to measure platinum concentrations in blood samples, tumors, and lymph nodes. Results No tissue reactions were detected 96 hours after hyaluronan-cisplatin injection. Mean ± SD area under the curve, peak concentration, and terminal half-life for unbound (plasma) and total (serum) platinum were 774.6 ± 221.1 ng·h/mL and 3,562.1 ± 2,031.1 ng·h/mL, 56.5 ± 20.9 ng/mL and 81.6 ± 40.4 ng/mL, and 33.6 ± 16.1 hours and 51.2 ± 29.1 hours, respectively. Platinum concentrations ranged from 3,325 to 8,229 ng/g in STSs and 130 to 6,066 ng/g in STS-associated lymph nodes. Conclusions and Clinical Relevance Intratumoral injection of the hyaluronan-cisplatin nanoconjugate was well tolerated in treated dogs. Following intratumoral hyaluronan-cisplatin injection, platinum concentration was 1,000-fold and 100-fold greater within treated tumors and tumor-draining lymphatics, respectively, compared with that in plasma. PMID:23176425

  10. Characterization of Pasteurella species isolated from lungs of calves with pneumonia.

    PubMed Central

    Madsen, E B; Bisgaard, M; Mutters, R; Pedersen, K B

    1985-01-01

    During routine bacteriological examination of pneumonic calf lungs it was experienced that many Pasteurella multocida-like isolates had a fermentation pattern different from what is generally accepted for P. multocida sensu stricto. Forty-one out of 50 strains selected for further investigation were phenotypically related and formed a group of indole-, mannitol-and sorbitol-negative P. multocida-like strains, which was tentatively designated taxon 13. Deoxyribonucleic acid/deoxyribonucleic acid hybridizations including both ornithine positive and ornithine negative strains of taxon 13 allowed the classification of the former as P. multocida biovar 6 and the latter as V factor independent strains of Haemophilus avium. PMID:3986681

  11. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.

    PubMed Central

    Yang, B; Yang, B L; Savani, R C; Turley, E A

    1994-01-01

    We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R431, spaced seven amino acids apart, are critical for HA binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA--Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X7)B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, R431 and K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7)B motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses

  12. NLRP3/Cryopyrin Is Necessary for Interleukin-1β (IL-1β) Release in Response to Hyaluronan, an Endogenous Trigger of Inflammation in Response to Injury*S⃞

    PubMed Central

    Yamasaki, Kenshi; Muto, Jun; Taylor, Kristen R.; Cogen, Anna L.; Audish, David; Bertin, John; Grant, Ethan P.; Coyle, Anthony J.; Misaghi, Amirhossein; Hoffman, Hal M.; Gallo, Richard L.

    2009-01-01

    Inflammation under sterile conditions is a key event in autoimmunity and following trauma. Hyaluronan, a glycosaminoglycan released from the extracellular matrix after injury, acts as an endogenous signal of trauma and can trigger chemokine release in injured tissue. Here, we investigated whether NLRP3/cryopyrin, a component of the inflammasome, participates in the inflammatory response to injury or the cytokine response to hyaluronan. Mice with a targeted deletion in cryopyrin showed a normal increase in Cxcl2 in response to sterile injuries but had decreased inflammation and release of interleukin-1β (IL-1β). Similarly, the addition of hyaluronan to macrophages derived from cryopyrin-deficient mice increased release of Cxcl2 but did not increase IL-1β release. To define the mechanism of hyaluronan-mediated activation of cryopyrin, elements of the hyaluronan recognition process were studied in detail. IL-1β release was inhibited in peritoneal macrophages derived from CD44-deficient mice, in an MH-S macrophage cell line treated with antibodies to CD44, or by inhibitors of lysosome function. The requirement for CD44 binding and hyaluronan internalization could be bypassed by intracellular administration of hyaluronan oligosaccharides (10–18-mer) in lipopolysaccharide-primed macrophages. Therefore, the action of CD44 and subsequent hyaluronan catabolism trigger the intracellular cryopyrin → IL-1β pathway. These findings support the hypothesis that hyaluronan works through IL-1β and the cryopyrin system to signal sterile inflammation. PMID:19258328

  13. Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb.

    PubMed

    Matsumoto, Kazu; Li, Yingcui; Jakuba, Caroline; Sugiyama, Yoshinori; Sayo, Tetsuya; Okuno, Misako; Dealy, Caroline N; Toole, Bryan P; Takeda, Junji; Yamaguchi, Yu; Kosher, Robert A

    2009-08-01

    The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb.

  14. Pasteurella multocida septic shock after a cat scratch in an elderly otherwise healthy woman: a case report.

    PubMed

    Fernández-Valencia, Jenaro A; García, Sebastian; Prat, Salvio

    2008-03-01

    Pasteurella multocida, a gram-negative coccobacillus, is a commensal in the nasopharynx of many animals. P. multocida infections most commonly involve the skin, soft tissues, and respiratory tract, particularly in immunosuppressed patients. The present case illustrates a severe articular infection caused by this bacterium, leading to septic shock, in an elderly, otherwise healthy woman, after a simple scratch of a cat.

  15. Experimental study of pathogenicity of Pasteurella multocida serogroup F in rabbits.

    PubMed

    Jaglic, Zoran; Jeklova, Edita; Leva, Lenka; Kummer, Vladimir; Kucerova, Zdenka; Faldyna, Martin; Maskova, Jarmila; Nedbalcova, Katerina; Alexa, Pavel

    2008-01-01

    The role of Pasteurella multocida serogroup F in inducing disease in rabbits was investigated in this study. Three groups of 12 Pasteurella-free rabbits each were intranasally (i.n.), subcutaneously (s.c.), and perorally (p.o.) challenged, respectively. Six rabbits of each group were immunosuppressed using dexamethasone. Eight rabbits (four of them immunosuppressed) inoculated i.n. showed symptoms of respiratory distress resulting in respiratory failure and died or were euthanized in the terminal stage of the disease 3-6 days post-infection (p.i.). The main pathological findings were fibrinopurulent pleuropneumonia (immunocompetent rabbits) or diffuse haemorrhagic pneumonia (immunosuppressed rabbits). Septicemic syndrome ending with shock occurred in 11 rabbits (6 of them immunosuppressed) inoculated s.c., which died or were euthanized in the terminal stage of the disease 2-3 days p.i. The most significant pathological findings were extensive cutaneous and subcutaneous lesions. All of the p.o. inoculated rabbits survived the challenge showing no clinical signs of the disease and no macroscopic lesions. The observations in this study indicate that in addition to serogroups A and D of P. multocida, serogroup F also can be highly pathogenic for rabbits and therefore might be a cause of considerable economic loss in commercial rabbit production.

  16. SELECTIVE INHIBITION BY TRYPTOPHAN ANALOGUES OF MURINE TOXIN SYNTHESIS IN PASTEURELLA PESTIS

    PubMed Central

    Montie, Thomas C.; Ajl, Samuel J.

    1964-01-01

    Montie, Thomas C. (Albert Einstein Medical Center, Philadelphia, Pa.), and Samuel J. Ajl. Selective inhibition by tryptophan analogues of murine toxin synthesis in Pasteurella pestis. J. Bacteriol. 88:1467–1475. 1964.—Washed-cell suspensions of Pasteurella pestis, avirulent strain “Tjiwidej,” exhibited a preferential inhibition of toxin synthesis relative to total protein formation, when grown in the presence of various tryptophan analogues. Growth was partially inhibited in the presence of methyl analogues. High concentrations of 5-fluorotryptophan induced slight growth-inhibitory effects. However, toxin production was more sensitive to these levels of the analogue. Growth inhibition appeared not to relate to toxin inhibition. Inhibition of toxin synthesis by analogues was reversed by l-tryptophan and indole. Shikimic acid but not anthranilic acid antagonized the action of 4-methyltryptophan on selective toxin synthesis. The formation of tryptophanless protein accounted for continued protein synthesis in tryptophan-depleted cells. Protein resolved by acrylamide gel electrophoresis from crude cell extracts exhibited two toxic protein bands. The synthesis of one toxin-protein band, the less-mobile of the two, appeared to be associated with the membrane fraction of the cell, and was selectively blocked in cells grown in the presence of tryptophan analogues. Cellular tryptophan levels may determine the quantity and quality of proteins made. Images PMID:14234807

  17. Pasteurella multocida-associated sinusitis in khaki Campbell ducks (Anas platyrhynchos).

    PubMed

    Songserm, Thaweesak; Viriyarampa, A Srisamai; Sae-Heng, Namdee; Chamsingh, Wilairat; Bootdee, Orawan; Pathanasophon, Pornpen

    2003-01-01

    Pasteurella multocida group B, serotype 3, was isolated from sinusitis-affected khaki Campbell ducks. To study the role of P. multocida in sinusitis, commercial khaki Campbell ducks were experimentally infected with P. multocida alone or combined with Escherichia coli. In Expt. 1, experimental ducks were infected with P. multocida intranasally or ocularly. A comparison was done by intranasal inoculation with pooled nasal discharge from the affected ducks or phosphate-buffered saline. The ducks intranasally inoculated with the nasal discharge or P. multocida showed sinusitis. In Expt. 2, E. coli alone or a combination of P. multocida and E. coli was intranasally inoculated into experimental ducks. The ducks intranasally inoculated with the combination of P. multocida and E. coli had sinusitis, the same as found in the field but less severe than that of the field cases. Pasteurella multocida was already present in litter/floor of duck farms. We concluded that P. multocida played a role in induction of sinusitis. However, the sinusitis in ducks may be initiated by poor management, especially in the brooding period of ducks.

  18. Using amplified fragment length polymorphism analysis to differentiate isolates of Pasteurella multocida serotype 1

    USGS Publications Warehouse

    Blehert, D.S.; Jefferson, K.L.; Heisey, D.M.; Samuel, M.D.; Berlowski, B.M.; Shadduck, D.J.

    2008-01-01

    Avian cholera, an infectious disease caused by the bacterium Pasteurella multocida, kills thousands of North American wild waterfowl annually. Pasteurella multocida serotype 1 isolates cultured during a laboratory challenge study of Mallards (Anas platyrhynchos) and collected from wild birds and environmental samples during avian cholera outbreaks were characterized using amplified fragment length polymorphism (AFLP) analysis, a whole-genome DNA fingerprinting technique. Comparison of the AFLP profiles of 53 isolates from the laboratory challenge demonstrated that P. multocida underwent genetic changes during a 3-mo period. Analysis of 120 P. multocida serotype 1 isolates collected from wild birds and environmental samples revealed that isolates were distinguishable from one another based on regional and temporal genetic characteristics. Thus, AFLP analysis had the ability to distinguish P. multocida isolates of the same serotype by detecting spatiotemporal genetic changes and provides a tool to advance the study of avian cholera epidemiology. Further application of AFLP technology to the examination of wild bird avian cholera outbreaks may facilitate more effective management of this disease by providing the potential to investigate correlations between virulence and P. multocida genotypes, to identify affiliations between bird species and bacterial genotypes, and to elucidate the role of specific bird species in disease transmission. ?? Wildlife Disease Association 2008.

  19. Mammalian ceramide synthases.

    PubMed

    Levy, Michal; Futerman, Anthony H

    2010-05-01

    In mammals, ceramide, a key intermediate in sphingolipid metabolism and an important signaling molecule, is synthesized by a family of six ceramide synthases (CerS), each of which synthesizes ceramides with distinct acyl chain lengths. There are a number of common biochemical features between the CerS, such as their catalytic mechanism, and their structure and intracellular localization. Different CerS also display remarkable differences in their biological properties, with each of them playing distinct roles in processes as diverse as cancer and tumor suppression, in the response to chemotherapeutic drugs, in apoptosis, and in neurodegenerative diseases.

  20. Monoterpene synthases from common sage (Salvia officinalis)

    DOEpatents

    Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  1. Characterization of polyelectrolyte behavior of the polysaccharides chitosan, heparin, and hyaluronan, by light scattering and viscometry.

    NASA Astrophysics Data System (ADS)

    Boddohi, Soheil; Yonemura, Susan; Kipper, Matt

    2008-03-01

    This study on the polyelectrolyte behavior of polysaccharides in solution is motivated by our recent work in development of nanostructured polysaccharide-based surface coatings. Chitosan behaves as a weak polycation, and hyaluronan behaves as a weak polyanion, while heparin behaves as a strong polyanion. The ability to control the conformation of these polysaccharides in solution, by changing the solution ionic strength and pH may offer the opportunity to further tune the nanoscale features of polysaccharide-based surface coatings assembled from solution. In the work reported here, the solution conformation of these polymers is determined from gel permeation chromatography coupled to differential refractive index, light scattering, and viscometry detection. These results are related to the nanostructure of chitosan-heparin and chitosan-hyaluronan surface coatings based on polyelectrolyte multilayers.

  2. X-ray ablation of hyaluronan hydrogels: Fabrication of three-dimensional microchannel networks

    NASA Astrophysics Data System (ADS)

    Weon, B. M.; Chang, S.; Yeom, J.; Hahn, S. K.; Je, J. H.; Hwu, Y.; Margaritondo, G.

    2009-09-01

    We present a simple and highly versatile protocol for polymer ablation: hard x-ray irradiation makes it possible to rapidly depolymerize hyaluronan hydrogels and fabricate three-dimensional network of microchannels. Photodynamic and photochemical analyses show that x-ray irradiation directly cleaves the polymer backbone and the total dose controls the degradation kinetics. This nonthermal ablation protocol may offer opportunities for processing organic polymers and biological materials.

  3. Gold-Nanoclustered Hyaluronan Nano-Assemblies for Photothermally Maneuvered Photodynamic Tumor Ablation.

    PubMed

    Han, Hwa Seung; Choi, Ki Young; Lee, Hansang; Lee, Minchang; An, Jae Yoon; Shin, Sol; Kwon, Seunglee; Lee, Doo Sung; Park, Jae Hyung

    2016-12-27

    Optically active nanomaterials have shown great promise as a nanomedicine platform for photothermal or photodynamic cancer therapies. Herein, we report a gold-nanoclustered hyaluronan nanoassembly (GNc-HyNA) for photothermally boosted photodynamic tumor ablation. Unlike other supramolecular gold constructs based on gold nanoparticle building blocks, this system utilizes the nanoassembly of amphiphilic hyaluronan conjugates as a drug carrier for a hydrophobic photodynamic therapy agent verteporfin, a polymeric reducing agent, and an organic nanoscaffold upon which gold can grow. Gold nanoclusters were selectively installed on the outer shell of the hyaluronan nanoassembly, forming a gold shell. Given the dual protection effect by the hyaluronan self-assembly as well as by the inorganic gold shell, verteporfin-encapsulated GNc-HyNA (Vp-GNc-HyNA) exhibited outstanding stability in the bloodstream. Interestingly, the fluorescence and photodynamic properties of Vp-GNc-HyNA were considerably quenched due to the gold nanoclusters covering the surface of the nanoassemblies; however, photothermal activation by 808 nm laser irradiation induced a significant increase in temperature, which empowered the PDT effect of Vp-GNc-HyNA. Furthermore, fluorescence and photodynamic effects were recovered far more rapidly in cancer cells due to certain intracellular enzymes, particularly hyaluronidases and glutathione. Vp-GNc-HyNA exerted a great potential to treat tumors both in vitro and in vivo. Tumors were completely ablated with a 100% survival rate and complete skin regeneration over the 50 days following Vp-GNc-HyNA treatment in an orthotopic breast tumor model. Our results suggest that photothermally boosted photodynamic therapy using Vp-GNc-HyNA can offer a potent therapeutic means to eradicate tumors.

  4. Freeze-dried eudragit-hyaluronan multicompartment liposomes to improve the intestinal bioavailability of curcumin.

    PubMed

    Catalan-Latorre, Ana; Ravaghi, Maryam; Manca, Maria Letizia; Caddeo, Carla; Marongiu, Francesca; Ennas, Guido; Escribano-Ferrer, Elvira; Peris, José Esteban; Diez-Sales, Octavio; Fadda, Anna Maria; Manconi, Maria

    2016-10-01

    This work aimed at finding an innovative vesicle-type formulation able to improve the bioavailability of curcumin upon oral administration. To this purpose, phospholipid, Eudragit® S100 and hyaluronan sodium salt were combined to obtain eudragit-hyaluronan immobilized vesicles using an easy and environmentally-friendly method. For the first time, the two polymers were combined in a system intended for oral delivery, to enhance curcumin stability when facing the harsh environment of the gastrointestinal tract. Four different formulations were prepared, keeping constant the amount of the phospholipid and varying the eudragit-hyaluronan ratio. The freeze-drying of the samples, performed to increase their stability, led to a reduction of vesicle size and a good homogeneity of the systems, after simple rehydration with water. X-ray diffraction study demonstrated that after the freeze-drying process, curcumin remained successfully incorporated within the vesicles. All the vesicles displayed similar features: size ranging from 220 to 287nm, spherical or oval shape, multilamellar or large unilamellar morphology with a peculiar multicompartment organization involving 1-4 smaller vesicles inside. In vitro studies demonstrated the ability of the combined polymers to protect the vesicles from the harsh conditions of the gastro-intestinal tract (i.e., ionic strength and pH variation), which was confirmed in vivo by the greater deposition of curcumin in the intestinal region, as compared to the free drug in dispersion. This enhanced accumulation of curcumin provided by the eudragit-hyaluronan immobilized vesicles, together with an increase in Caco-2 cell viability exposed to hydrogen peroxide, indicated that vesicles can ensure a local protection against oxidative stress and an increase in its intestinal absorption.

  5. Hyaluronan tetrasaccharide in the cerebrospinal fluid is associated with self-repair of rats after chronic spinal cord compression.

    PubMed

    Wang, J; Rong, W; Hu, X; Liu, X; Jiang, L; Ma, Y; Dang, G; Liu, Z; Wei, F

    2012-05-17

    The objective of this study was to explore changes in hyaluronan levels in the cerebrospinal fluid (CSF) in a spinal cord compression model, to investigate whether hyaluronan tetrasaccharide was involved in this process, and to test the effects of hyaluronan tetrasaccharide on neuron and oligodendrocyte repair. We developed a chronic spinal cord compression model with various sizes of polymer sheets (1.5×0.7×0.3 mm(3); 5×1.5×0.7 mm(3)) that were implanted microsurgically underneath the C(5-6) laminae. The rats were divided into three groups: a sham group, a mildly compressed (MC) group, and a widely compressed (WC) group. Locomotor functional evaluations revealed that the behavioral function of the MC and WC groups dropped to their lowest level from the fourth to fifth week and gradually recovered thereafter. The hyaluronan levels in the CSF gradually increased after spinal cord compression. Furthermore, hyaluronan tetrasaccharide was involved in the hyaluronan change. In addition, we found that nuclear factor kappa B (NF-κB) and cellular inhibitor-of-apoptosis protein 2 (c-IAP(2)) were co-expressed in neurons and oligodendrocytes, and caspase-3 expression gradually decreased in the compression model. The brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) expression was upregulated in astrocytes at the fourth week post-compression. Hyaluronan tetrasaccharide (HA(4)) induced NF-κB and c-IAP(2) to suppress the H(2)O(2)-induced apoptosis in primary neuronal cultures and increased BDNF and VEGF expression in astrocytic cultures in vitro. These findings suggest that HA(4) in the CSF may associate with behavioral recovery by increasing the levels of NF-κB, c-IAP(2), and neurotrophic factors after chronic spinal cord compression.

  6. Influence of synthetic polyelectrolytes on the growth and properties of hyaluronan-chitosan multilayers.

    PubMed

    Salomäki, Mikko; Kankare, Jouko

    2009-02-09

    Both hyaluronan (HA) and chitosan (CHI) are biocompatible polysaccharide electrolytes. The multilayers formed by these polyelectrolytes alone are known to be rather soft and strongly viscoelastic. In this work we study multilayers formed by incorporating synthetic nonsaccharide polyelectrolytes such as polyallylamine (PAH) and poly(acrylic acid) (PAA) in various proportions into the HA/CHI layers. The buildup was followed on a quartz crystal resonator. Surface acoustic impedance recorded in these measurements, in suitable conditions, gives a spiral when plotted in the complex plane. The shape of this spiral depends on the viscoelasticity of the layer material and regularity of the growth process. We found that poly(acrylic acid) destroys the soft diffuse matrix formed by hyaluronan. It forms diffusion barriers when deposited sparsely between the layers. If its proportion is higher, the film growth adopts a linear buildup in the layer-by-layer process. The linear buildup of CHI/PAA reveals that the buildup regime of a multilayer film does not determine the viscoelastic properties of the film. Linearly and exponentially growing films may have very similar mechanical properties. Polyacrylic acid forms a kind of scaffold inside the film giving the natively soft hyaluronan/chitosan film more mechanical strength. The optimal combination gave more than 100-fold increase in the shear modulus.

  7. Basic FGF and PDGF-BB synergistically stimulate hyaluronan and IL-6 production by orbital fibroblasts.

    PubMed

    Virakul, Sita; Heutz, Judith W; Dalm, Virgil A S H; Peeters, Robin P; Paridaens, Dion; van den Bosch, Willem A; Hirankarn, Nattiya; van Hagen, P Martin; Dik, Willem A

    2016-09-15

    Orbital fibroblast activation is a central pathologic feature of Graves' Ophthalmopathy (GO). Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have been proposed to contribute to GO, but their effects on orbital fibroblasts are largely unknown. We found that bFGF stimulated proliferation and hyaluronan production, but not IL-6 production by orbital fibroblasts, while VEGF hardly affected orbital fibroblast activity. Remarkably, co-stimulation of orbital fibroblasts with bFGF and PDGF-BB synergistically enhanced IL-6 and hyaluronan production and displayed an additive effect on proliferation compared to either bFGF or PDGF-BB stimulation. Nintedanib, a FGF- and PDGF-receptor targeting drug, more efficiently blocked bFGF + PDGF-BB-induced IL-6 and hyaluronan production than dasatinib that only targets PDGF-receptor. In conclusion, bFGF may contribute to orbital inflammation and tissue remodeling in GO, especially through synergistic interaction with PDGF-BB. Multi-target therapy directed at the bFGF and PDGF pathways may potentially be of interest for the treatment of GO.

  8. Free radical depolymerization of hyaluronan by Maillard reaction products: role in liquefaction of aging vitreous.

    PubMed

    Deguine, V; Menasche, M; Ferrari, P; Fraisse, L; Pouliquen, Y; Robert, L

    1998-02-01

    The degradation of hyaluronan was followed by viscosimetry and by HPLC in order to study the possible role of Maillard products (lysine-glucose) on the alteration of the vitreous gel in aging and diabetes. Lysine-glucose generated Maillard products produced a decrease of viscosity and of the number average molecular weight (Mn) of hyaluronan during a 1 h incubation at 37 degrees C. This effect was comparable to that produced by 1 U/ml of testicular hyaluronidase but was weaker than the effect of a Fenton-type reagent (Udenfriend's reagent). The polydispersity of hyaluronan incubated with Maillard products appeared higher than with hyaluronidase suggesting a more random reaction. Antioxydant enzymes (SOD, catalase), the iron chelators (desferrioxamine, transferrin) and the free radical scavengers (uric acid, carnosine) inhibited the degradation by Maillard products confirming its free radical nature and the intervention of trace metals. Maillard products have been detected in diabetic vitreous and may play a role in its accelerated modifications (liquefaction) in diabetes as compared to normal aging.

  9. Metastatin: a hyaluronan-binding complex from cartilage that inhibits tumor growth.

    PubMed

    Liu, N; Lapcevich, R K; Underhill, C B; Han, Z; Gao, F; Swartz, G; Plum, S M; Zhang, L; Green, S J

    2001-02-01

    In this study, a hyaluronan-binding complex, which we termed Metastatin, was isolated from bovine cartilage by affinity chromatography and found to have both antitumorigenic and antiangiogenic properties. Metastatin was able to block the formation of tumor nodules in the lungs of mice inoculated with B16BL6 melanoma or Lewis lung carcinoma cells. Single i.v. administration of Metastatin into chicken embryos inhibited the growth of both B16BL6 mouse melanoma and TSU human prostate cancer cells growing on the chorioallantoic membrane. The in vivo biological effect may be attributed to the antiangiogenic activity because Metastatin is able to inhibit the migration and proliferation of cultured endothelial cells as well as vascular endothelial growth factor-induced angiogenesis on the chorioallantoic membrane. In each case, the effect could be blocked by either heat denaturing the Metastatin or premixing it with hyaluronan, suggesting that its activity critically depends on its ability to bind hyaluronan on the target cells. Collectively, these results suggest that Metastatin is an effective antitumor agent that exhibits antiangiogenic activity.

  10. A Case of Lower Respiratory Tract Infection with Canine-associated Pasteurella canis in a Patient with Chronic Obstructive Pulmonary Disease

    PubMed Central

    Acharya, Preetam R.; Biranthabail, Dhanashree; Rangnekar, Aseem; Shiragavi, Sachin

    2015-01-01

    This is the report of lower respiratory tract infection with Pasteurella canis in a chronic obstructive pulmonary disease (COPD) patient with history of casual exposure to cats. Pasteurella species are part of the oral and gastrointestinal flora in the canine animals. These organisms are usually implicated in wound infection following animal bites, but can also be associated with a variety of infections including respiratory tract infections. PMID:26435948

  11. Self-reinforcement and protein sustained delivery of hyaluronan hydrogel by tailoring a dually cross-linked network.

    PubMed

    Luo, Chunhong; Xu, Guoguang; Wang, Xinghui; Tu, Mei; Zeng, Rong; Rong, Jianhua; Zhao, Jianhao

    2015-01-01

    A series of self-reinforcing hyaluronan hydrogels were developed to improve mechanical properties and protein sustained delivery thanks to a dually cross-linked network. Hyaluronan gel particles (HGPs, 1-5 μm in diameter) with different cross-linking densities, i.e. HGPs-1.5, HGPs-3 and HGPs-15, were prepared in an inverse emulsion system and used as the reinforcing phase after glycidyl methacrylation, while glycidyl methacrylated hyaluronan with a substitution degree of 45.2% was synthesized as the matrix phase. These two phases were cross-linked under ultraviolet irradiation to form self-reinforcing hyaluronan hydrogels (srHAs) that showed typical cross-linked structure of HGPs connecting the matrix phase by cross-section observation. In comparison to hyaluronan bulk gels and their blends with HGPs, srHAs distinctly enhanced the mechanical properties and BSA long-term sustained delivery, especially srHA-1.5 showed the highest compressive modulus of 220±15 kPa and the slowest BSA delivery (67% release at 14 d). The 3T3 fibroblast cell culture showed that all the srHAs had no cytotoxicity.

  12. Mechanisms of acetohydroxyacid synthases.

    PubMed

    Chipman, David M; Duggleby, Ronald G; Tittmann, Kai

    2005-10-01

    Acetohydroxyacid synthases are thiamin diphosphate- (ThDP-) dependent biosynthetic enzymes found in all autotrophic organisms. Over the past 4-5 years, their mechanisms have been clarified and illuminated by protein crystallography, engineered mutagenesis and detailed single-step kinetic analysis. Pairs of catalytic subunits form an intimate dimer containing two active sites, each of which lies across a dimer interface and involves both monomers. The ThDP adducts of pyruvate, acetaldehyde and the product acetohydroxyacids can be detected quantitatively after rapid quenching. Determination of the distribution of intermediates by NMR then makes it possible to calculate individual forward unimolecular rate constants. The enzyme is the target of several herbicides and structures of inhibitor-enzyme complexes explain the herbicide-enzyme interaction.

  13. Simple visual assay for determination of Pasteurella haemolytica cytotoxin neutralizing antibody titers in cattle sera.

    PubMed Central

    Gentry, M J; Confer, A W; Kreps, J A

    1985-01-01

    A simple visual assay is described for determining the capacity of bovine serum to neutralize the cytotoxin produced by Pasteurella haemolytica serotype 1. The test was reproducible from day to day with different target cell populations and cytotoxin preparations. Cytotoxin neutralization titers obtained by the visual assay were comparable to those determined by the trypan blue exclusion and 51Cr-release methods. The visual assay was used to measure neutralization titers of bovine sera obtained from vaccination experiments and fractions of purified serum obtained by gel filtration. The major advantages of the visual assay over other assays are that it is rapid, inexpensive, and does not use radioisotopes. It also does not require specialized equipment, making it adaptable to most laboratories. Images PMID:3905853

  14. Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor.

    PubMed Central

    Ogunnariwo, J A; Schryvers, A B

    1990-01-01

    Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure. Images PMID:2365453

  15. Deadly case of Pasteurella multocida aortitis and mycotic aneurysm following a cat bite

    PubMed Central

    Cho, Dennis Dane; Berliner, Yaniv; Carr, David

    2016-01-01

    Animal bites are frequently encountered in the emergency department (ED). Aortitis leading to mycotic abdominal aortic aneurysm is a rare and potentially deadly complication of Pasteurella multocida (P. multocida) following an animal bite. We present the case of a 68-year-old male who presented to the ED after falling at home. He complained of weakness and abdominal pain. He was in septic shock and was treated empirically with broad-spectrum antibiotics and intravenous fluids. He reported previous antibiotic treatment of a cellulitis secondary to a cat bite injury to his right thumb four weeks prior. Abdominal ultrasound and subsequent computed tomography scan revealed a leaking mycotic abdominal aneurysm that was surgically repaired. Blood cultures and aortic wall tissue cultures grew P. multocida. Given how common animal bite presentations are in the ED, this case highlights the need to consider aortitis and mycotic abdominal aortic aneurysm in an unwell patient with an animal bite. PMID:27326399

  16. Udder orf infection and its role in ovine clinical mastitis caused by Pasteurella haemolytica.

    PubMed

    Burriel, A R

    1997-04-01

    During an experimental study of ovine subclinical mastitis caused by coagulase-negative staphylococci, an outbreak of contagious ecthyma occurred among ewes unvaccinated against parapox virus. The same group of ewes developed a high rate (43.7%) of clinical mastitis caused by Pasteurella haemolytica. The rate of clinical mastitis among ewes vaccinated against parapox virus was very low (3.7%) suggesting that the presence of orf in the unvaccinated ewes was contributing to the high rate of clinical mastitis. An examination of the iron, sodium, potassium and albumin concentration of milk collected from 16 unvaccinated and nine randomly selected vaccinated ewes before experimental infection with coagulase-negative staphylococci or their uninfected control mammary glands indicated significant differences in the iron (p < 0.0001) and sodium (p = 0.01) concentration. Increased iron concentration in the milk may have assisted in the development of udder infection caused by P. haemolytica as iron is easily utilised by this bacterium.

  17. Efficacy of recombinant leukotoxin in protection against pneumonic challenge with live Pasteurella haemolytica A1.

    PubMed Central

    Conlon, J A; Shewen, P E; Lo, R Y

    1991-01-01

    The recombinant leukotoxin (rLKT) of the bacterium Pasteurella haemolytica A1 was examined for its ability to protect cattle from experimental challenge with logarithmic-phase P. haemolytica. Six different vaccines were utilized in the experiment: P. haemolytica culture supernatant, P. haemolytica culture supernatant enriched with rLKT, rLKT alone, P. haemolytica culture supernatant enriched with Escherichia coli supernatant not containing LKT, E. coli supernatant alone, and phosphate-buffered saline. rLKT alone showed no protective capacity against development of clinical signs of respiratory disease or against development of postmortem lung lesions after experimental challenge. It was, however, shown to enhance the efficacy of the culture supernatant vaccine and decrease clinical signs and pneumonic lesions. The complexity of protective immunity in this disease is emphasized in this study, and, although LKT is an important virulence factor of the organism, an immune response to LKT alone does not protect animals against disease. PMID:1987075

  18. Pasteurella multocida septicemia caused by close contact with a domestic cat: case report and literature review.

    PubMed

    Kimura, Ryosuke; Hayashi, Yoshinari; Takeuchi, Toyo; Shimizu, Manabu; Iwata, Masaru; Tanahashi, Junji; Ito, Makoto

    2004-08-01

    We report here a case of Pasteurella multocida infection caused by cat exposure presenting with septic shock, sinusitis, and pneumonia. The patient was a febrile 20-year-old woman who had been experiencing disturbed consciousness progressively. She had close contact with a domestic cat and had received some scratches on both arms. A magnetic resonance imaging (MRI) scan of the head showed a high intensity in the paranasal cavity, and a computed tomographic (CT) scan of the chest showed bilateral lung consolidations. The pathogen was identified as P. multocida by the cultures from blood and nasal discharge. She was given intensive antibiotic therapy with ceftriaxone and piperacillin, continuous hemodiafiltration (CHDF) therapy, and anticoagulation therapy. Owing to these therapeutic regimens, the septic shock was successfully treated without complications. We also review the literature on P. multocida septicemia.

  19. Deadly case of Pasteurella multocida aortitis and mycotic aneurysm following a cat bite.

    PubMed

    Cho, Dennis Dane; Berliner, Yaniv; Carr, David

    2016-06-16

    Animal bites are frequently encountered in the emergency department (ED). Aortitis leading to mycotic abdominal aortic aneurysm is a rare and potentially deadly complication of Pasteurella multocida (P. multocida) following an animal bite. We present the case of a 68-year-old male who presented to the ED after falling at home. He complained of weakness and abdominal pain. He was in septic shock and was treated empirically with broad-spectrum antibiotics and intravenous fluids. He reported previous antibiotic treatment of a cellulitis secondary to a cat bite injury to his right thumb four weeks prior. Abdominal ultrasound and subsequent computed tomography scan revealed a leaking mycotic abdominal aneurysm that was surgically repaired. Blood cultures and aortic wall tissue cultures grew P. multocida. Given how common animal bite presentations are in the ED, this case highlights the need to consider aortitis and mycotic abdominal aortic aneurysm in an unwell patient with an animal bite.

  20. Tracheal versus pulmonary deposition and clearance of inhaled Pasteurella haemolytica or Staphylococcus aureus in mice.

    PubMed Central

    Rodríguez, L M; López, A; Merino-Moncada, M; Martínez-Burnes, J; Mondragón, I

    1985-01-01

    The aim of this investigation was to do a comparative study on the deposition and clearance of inhaled bacteria between the lungs and tracheae of mice exposed to aerosols of bacteria. Two hundred and eighty-eight mice were divided into four groups (n = 72) and exposed to aerosols of Pasteurella haemolytica or Staphylococcus aureus in four replicates. The numbers of bacteria were determined in the trachea and lungs of mice sacrificed 0, 2, 4, 8, 12, 24, 48 and 72 hours postexposure. Results indicated that bacterial deposition was greater in lungs than in tracheae. No significant (p greater than 0.05) difference was observed between P. haemolytica and S. aureus clearance rates. Although bacteria were rapidly eliminated from the whole respiratory tract, bacterial clearance was significantly (p less than 0.002) faster in tracheae than lungs. A significant (p less than 0.05) replicate effect was also observed. PMID:4041977

  1. Immunological roles of Pasteurella multocida toxin (PMT) using a PMT mutant strain.

    PubMed

    Kim, Tae Jung; Toan, Nguyen Tat; Jang, Eun Jin; Jung, Bock Gie; Lee, Jae Il; Lee, Bong Joo

    2007-08-01

    The immunological role of the Pasteurella multocida toxin (PMT) in mice was examined using a PMT mutant strain. After a nasal inoculation, the mutant strain failed to induce interstitial pneumonia. Moreover, PMT had no significant effect on the populations of CD4+, CD8+, CD3+, and CD19+ immunocytes in blood or on the populations of CD4+ and CD8+ splenocytes (P<0.01). However, there was a significant increase in the total number of cells in the BAL samples obtained from the wild-type P. multocida-inoculated mice. On the other hand, the level of IL-1 expression decreased when the macrophages from the bronchio-alveolar lavage were stimulated with PMT. Overall, PMT appears to play some role (stimulating and/or inhibiting) in the immunological responses but further studies will be required to confirm this.

  2. Persistence of Pasteurella multocida in Nebraska (USA) wetlands under epizootic conditions

    USGS Publications Warehouse

    Price, J.I.; Brand, C.J.

    1984-01-01

    Gleason Basin, a marsh located in the western part of the Rainwater Basin in Nebraska, was selected during the 1980 spring waterfowl migration as a study site to determine the presence and persistence of virulent Pasteurella multocida. Avian cholera mortality in migratory waterfowl using the Basin increased during a 2-wk period of a die-off beginning the first week of March when 2,409 carcasses were collected from the marsh. Study sites within the marsh were established for sampling water associated with and not associated with intact and scavenged carcasses. Isolations of virulent P. multocida were made from five of six study sites associated with either intact or scavenged carcasses for 3 days and from three of five non-carcass-associated study sites for 2 days. Recovery of these bacteria from this environment suggested a possible source of infection for susceptible waterfowl using the contaminated site.

  3. The pulmonary clearance of Pasteurella haemolytica in calves infected with bovine virus diarrhea or Mycoplasma bovis.

    PubMed Central

    Lopez, A; Maxie, M G; Savan, M; Ruhnke, H L; Thomson, R G; Barnum, D A; Geissinger, H D

    1982-01-01

    Based on current literature which commonly associates bovine virus diarrhea virus and Mycoplasma bovis with "pneumonic pasteurellosis," an investigation was conducted into the effect of these two pathogens on the capacity of bovine lung to clear inhaled Pasteurella haemolytica. There was no significant effect (p less than 0.05) of either bovine virus diarrhea virus or M. bovis on the mean clearance rate of P. haemolytica, nor did the time interval of three, five or seven days between the first inoculation and exposure to P. haemolytica and adversely affect the lung clearance rates. However, it was found that the left lungs and a higher bacterial retention (p less than 0.05) than the right lungs. PMID:7127194

  4. Interaction between Ascaris suum and Pasteurella multocida in the lungs of mice.

    PubMed

    Tjørnehøj, K; Eriksen, L; Aalbaek, B; Nansen, P

    1992-01-01

    In an experiment including 8 groups of 15 mice, the effect of migrating Ascaris suum larvae in the lungs on the establishment and pathogenicity of aerosol exposure to Pasteurella multocida was investigated. Following aerosol exposure to P. multocida, mice with migrating A. suum in their lungs developed more severe pneumonia and septicaemia than did parasite-free mice. The parasite-induced effect on bacterial pathogenicity was more marked for a non-toxin-producing P. multocida as compared with a toxin-producing strain of P. multocida, possibly due to the higher spontaneous pathogenicity of the non-toxigenic strain of P. multocida. The present results should encourage controlled experiments on possible interactions between A. suum and various airborne microbial infections in pigs.

  5. Toll-like receptor 4-positive macrophages protect mice from Pasteurella pneumotropica-induced pneumonia

    NASA Technical Reports Server (NTRS)

    Hart, Marcia L.; Mosier, Derek A.; Chapes, Stephen K.

    2003-01-01

    This study investigates Toll-like receptor 4 (TLR4)-positive macrophages in early recognition and clearance of pulmonary bacteria. TLR4 is a trans-membrane receptor that is the primary recognition molecule for lipopolysaccharide of gram-negative bacteria. The TLR4(Lps-del) mouse strains C57BL10/ScN (B10) and STOCK Abb(tm1) TLR4(Lps-del) Slc11a1(s)(B10 x C2D) are susceptible to pulmonary infections and develop pneumonia when naturally or experimentally infected by the opportunistic bacterium Pasteurella pneumotropica. Since these mice have the TLR4(Lps-del) genotype, we hypothesized that reconstitution of mice with TLR4-positive macrophages would provide resistance to this bacterium. A cultured macrophage cell line (C2D macrophages) and bone marrow cells from C2D mice were adoptively transferred to B10 and B10 x C2D mice by intraperitoneal injection. C2D macrophages increased B10 and B10 x C2D mouse resistance to P. pneumotropica. In C2D-recipient mice there was earlier transcription of tumor necrosis factor alpha and chemokines JE and macrophage inflammatory protein 2 (MIP-2) in the lungs of B10 and B10 x C2D mice, and there was earlier transcription of KC and MIP-1alpha in B10 x C2D mice. In addition, the course of inflammation following experimental Pasteurella challenge was altered in C2D recipients. C2D macrophages also protected B10 x C2D mice, which lack CD4(+) T cells. These data indicate that macrophages are critical for pulmonary immunity and can provide host resistance to P. pneumotropica. This study indicates that TLR4-positive macrophages are important for early recognition and clearance of pulmonary bacterial infections.

  6. Cloning of a serotype-specific antigen from Pasteurella haemolytica A1.

    PubMed Central

    Gonzalez-Rayos, C; Lo, R Y; Shewen, P E; Beveridge, T J

    1986-01-01

    Recombinant plasmids coding for a soluble (or surface) antigen of Pasteurella haemolytica A1 were identified. Two plasmids, both containing the same 5.4 kilobase pairs of insert DNA, were recovered independently by screening a clone band of P. haemolytica A1 genomic DNA in Escherichia coli for the expression of P. haemolytica A1 soluble antigens (R. Y. C. Lo and L.A. Cameron, Can. J. Biochem. Cell Biol. 64:73-76, 1986). E. coli cells carrying the plasmids were found to be agglutinated by an antiserum raised against the P. haemolytica A1 soluble antigens. Analysis of the E. coli clones by electron microscopy revealed patches of amorphous material on the surface of the cells which were not present on the controls. Further characterization with protein A-colloidal gold labeled both these patches and the outer membranes of these cloned cells pretreated with the specific antiserum. These results indicated that the cloned antigen was expressed on the surface of the E. coli cells. The cloned antigen was found to be specific for serotype 1 when tested by slide agglutination against a collection of P. haemolytica typing antisera. Southern blot hybridization, using the cloned DNA as a probe, labeled the genomic DNA from P. haemolytica serotype 1 as well as the cross-agglutinating serotypes 2 and 7, but not DNA from the non-cross-agglutinating serotypes 3 and 4 and Pasteurella multocida. These results demonstrated that serotype specificity could be attributed to the particular antigenic determinants in the genome of the organism. Images PMID:3527985

  7. Ovine pulmonary surfactant induces killing of Pasteurella haemolytica, Escherichia coli, and Klebsiella pneumoniae by normal serum.

    PubMed Central

    Brogden, K A

    1992-01-01

    Pulmonary surfactant has been shown to play an increasingly important role in bacterial clearance at the alveolar surface in the lung. This study describes a bactericidal mechanism in which ovine pulmonary surfactant induces killing of Pasteurella haemolytica by normal serum. To demonstrate killing, six bacterial species were incubated first with pulmonary surfactant for 60 min at 37 degrees C and then with serum for an additional 60 min at 37 degrees C. P. haemolytica type A1 strains 82-25 and L101, a P. haemolytica type 2 strain, Escherichia coli, and Klebsiella pneumoniae were susceptible and Pasteurella multocida, Serratia marcescens, and Pseudomonas aeruginosa were not susceptible to killing by ovine pulmonary surfactant and normal serum. No bacteria incubated with bovine pulmonary surfactant were killed by normal serum. Although the species origin of pulmonary surfactant was selective, the species origin of serum was not. P. haemolytica incubated with ovine pulmonary surfactant was killed by fetal calf serum, gnotobiotic calf serum, pooled normal sheep serum, pooled normal rabbit serum, and pooled guinea pig serum. Ultrastructurally, killed P. haemolytica suspensions contained dead cells and cells distorted with vacuoles between the cytoplasmic membrane and the cytoplasm. The mechanism of killing did not correlate with concentrations of complement or lysozyme or titers of residual antibody in either the pulmonary surfactant or the serum, and killing was reduced by preincubation of surfactant with P. haemolytica lipopolysaccharide. Preliminary characterization of both surfactant and serum implicate a low-molecular-weight proteinaceous component in the surfactant and serum albumin in the serum. This mechanism may help clear certain gram-negative bacteria from the lungs of sheep as a part of the pulmonary innate defense system. Images PMID:1452351

  8. Superovulation of beef cattle with a split-single intramuscular administration of Folltropin-V in two concentrations of hyaluronan.

    PubMed

    Tríbulo, Andrés; Rogan, Dragan; Tríbulo, Humberto; Tríbulo, Ricardo; Mapletoft, Reuben J; Bó, Gabriel A

    2012-05-01

    Three experiments were designed to evaluate the superovulatory response of beef cows following two intramuscular (IM) administrations 48 h apart of Folltropin-V diluted in reduced concentrations of hyaluronan (Split-single IM administrations; Experiment 1-300 mg Folltropin-V on the first day and 100 mg 48 h later; Experiment 2-200 mg Folltropin-V on the first day and 100 mg 48 h later). In Experiments 1 and 2, superovulatory response and ova embryo/embryo production did not differ between donors receiving twice daily IM of Folltropin-V over 4 days and those given a Split-single IM administration of Folltropin-V diluted in 10 mg/mL hyaluronan solution. Experiment 3 compared Split-single IM administration of Folltropin-V diluted in two hyaluronan concentrations (5 or 10 mg/mL) with Folltropin-V diluted in saline and administered twice-daily over 4 days. Beef cows (17 Angus and 12 Simmental) were randomly assigned to one of three treatment groups to be superstimulated three times in a cross-over design, so that all cows received all treatments. A total dose of 300 mg Folltropin-V was divided into twice-daily IM over 4 days, or in two IM treatment 48 h apart (200 mg on first day and 100 mg 48 h later) in the hyaluronan groups. Mean (± SEM) numbers of transferable embryos did not differ among treatment groups (Control: 4.0 ± 0.8; 10 mg/mL hylauronan: 5.0 ± 0.9; 5 mg/mL hyaluronan: 6.1 ± 1.3). We concluded that the Split-single IM administration of Folltropin-V diluted in either concentration of hyaluronan resulted in a comparable superovulatory response to the traditional twice-daily protocol.

  9. Identification of a chondroitin synthase from an unexpected source, the green sulfur bacterium Chlorobium phaeobacteroides.

    PubMed

    Green, Dixy E; DeAngelis, Paul L

    2017-01-19

    Glycosaminoglycans [GAGs] are known to be present in all animals as well as some pathogenic microbes. Chondroitin sulfate is the most abundant GAG in mammals where it has various structural and adhesion roles. The Gram-negative bacteria Pasteurella multocida Type F and Escherichia coli K4 produce extracellular capsules composed of unsulfated chondroitin or a fructosylated chondroitin, respectively. Such polysaccharides that are structurally related to host molecules do not generally provoke a strong antibody response thus are thought to be employed as molecular camouflage during infection. We observed a sequence from the photosynthetic green sulfur bacteria, Chlorobium phaeobacteroides DSM 266, which was very similar (~62% identical) to the open reading frames of the known bi-functional chondroitin synthases (PmCS and KfoC); some segments are strikingly conserved amongst the three proteins. Recombinant E. coli-derived Chlorobium enzyme preparations were found to possess bona fide chondroitin synthase activity in vitro This new catalyst, CpCS, however, has a more promiscuous acceptor usage than the prototypical PmCS, which may be of utility in novel chimeric GAG syntheses. The finding of such a similar chondroitin synthase enzyme in C. phaeobacteroides is unexpected for several reasons including: (a) a free-living non-pathogenic organism should not "need" an animal self molecule for protection, (b) the Proteobacteria and the green sulfur bacterial lineages diverged ~2.5-3 billion years ago, and (c) the ecological niches of these bacteria are not thought to overlap substantially to facilitate horizontal gene transfer. CpCS provides insight into the structure/function relationship of this class of enzymes.

  10. Phosphanilic Acid Inhibits Dihydropteroate Synthase

    DTIC Science & Technology

    1989-11-01

    dihydropteroate synthases of P. aeruginosa and E . coli were about equally susceptible to inhibition by PA. These results suggest that cells of P. aeruginosa...are more permeable to PA than cells of E . coli . Although a weak inhibitor, PA acted on dihydropteroate synthase in the same manner as the sulfonamides...with which PA is structurally related. Inhibition of E . coli by PA in a basal salts-glucose medium was prevented by p-aminobenzoic acid (pABA). However

  11. Bacterial nitric oxide synthases.

    PubMed

    Crane, Brian R; Sudhamsu, Jawahar; Patel, Bhumit A

    2010-01-01

    Nitric oxide synthases (NOSs) are multidomain metalloproteins first identified in mammals as being responsible for the synthesis of the wide-spread signaling and protective agent nitric oxide (NO). Over the past 10 years, prokaryotic proteins that are homologous to animal NOSs have been identified and characterized, both in terms of enzymology and biological function. Despite some interesting differences in cofactor utilization and redox partners, the bacterial enzymes are in many ways similar to their mammalian NOS (mNOS) counterparts and, as such, have provided insight into the structural and catalytic properties of the NOS family. In particular, spectroscopic studies of thermostable bacterial NOSs have revealed key oxyheme intermediates involved in the oxidation of substrate L-arginine (Arg) to product NO. The biological functions of some bacterial NOSs have only more recently come to light. These studies disclose new roles for NO in biology, such as taking part in toxin biosynthesis, protection against oxidative stress, and regulation of recovery from radiation damage.

  12. Identification, Design and Synthesis of Tubulin-Derived Peptides as Novel Hyaluronan Mimetic Ligands for the Receptor for Hyaluronan-Mediated Motility (RHAMM/HMMR)

    PubMed Central

    Esguerra, Kenneth V. N.; Tolg, Cornelia; Akentieva, Natalia; Price, Matthew; Cho, Choi-Fong; Lewis, John D.; McCarthy, James B.; Turley, Eva A.; Luyt, Leonard G.

    2016-01-01

    Fragments of the extracellular matrix component hyaluronan (HA) promote tissue inflammation, fibrosis and tumor progression. HA fragments act through HA receptors including CD44, LYVE1, TLR2,4 and the receptor for hyaluronan mediated motility (RHAMM/HMMR). RHAMM is a multifunctional protein with both intracellular and extracellular roles in cell motility and proliferation. Extracellular RHAMM binds directly to HA fragments while intracellular RHAMM binds directly to ERK1 and tubulin. Both HA and regions of tubulin (s-tubulin) are anionic and bind to basic amino acid-rich regions in partner proteins, such as in HA and tubulin binding regions of RHAMM. We used this as a rationale for developing bioinformatics and SPR (surface plasmon resonance) based screening to identify high affinity anionic RHAMM peptide ligands. A library of 12-mer peptides was prepared based on the carboxyl terminal tail sequence of s-tubulin isoforms and assayed for their ability to bind to the HA/tubulin binding region of recombinant RHAMM using SPR. This approach resulted in the isolation of three 12-mer peptides with nanomolar affinity for RHAMM. These peptides bound selectively to RHAMM but not to CD44 or TLR2,4 and blocked RHAMM:HA interactions. Furthermore, fluorescein-peptide uptake by PC3MLN4 prostate cancer cells was blocked by RHAMM mAb but not by CD44 mAb. These peptides also reduced the ability of prostate cancer cells to degrade collagen type I. The selectivity of these novel HA peptide mimics for RHAMM suggest their potential for development as HA mimetic imaging and therapeutic agents for HA-promoted disease. PMID:26456171

  13. Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HA-dependent primary adhesion.

    PubMed Central

    Mohamadzadeh, M; DeGrendele, H; Arizpe, H; Estess, P; Siegelman, M

    1998-01-01

    The localization of circulating leukocytes within inflamed tissues occurs as the result of interactions with and migration across vascular endothelium, and is governed, in part, by the expression of adhesion molecules on both cell types. Recently, we have described a novel primary adhesion interaction between the structurally activated form of the adhesion molecule CD44 on lymphocytes and its major ligand hyaluronan on endothelial cells under physiologic laminar flow conditions, and have proposed that this interaction functions in an extravasation pathway for lymphocytes in vascular beds at sites of inflammation. While the regulation of activated CD44 on leukocytes has been characterized in depth, regulation of hyaluronate (HA) on endothelial cells has not been extensively studied. Here we demonstrate that the expression of HA on cultured endothelial cell lines and primary endothelial cultures is inducible by the proinflammatory cytokines TNFalpha and IL-1beta, as well as bacterial lipopolysaccharide. In addition, this inducibility appears strikingly restricted to endothelial cells derived from microvascular, but not large vessel, sources. The elevated HA levels thus induced result in increased CD44-dependent adhesive interactions in both nonstatic shear and laminar flow adhesion assays. Changes in mRNA levels for the described HA synthetic and degradative enzymes were not found, suggesting other more complex mechanisms of regulation. Together, these data add to the selectin and immunoglobulin gene families a new inducible endothelial adhesive molecule, hyaluronan, and help to further our understanding of the potential physiologic roles of the CD44/HA interaction; i.e., local cytokine production within inflamed vascular beds may enhance surface hyaluronan expression on endothelial cells, thereby creating local sites receptive to the CD44/HA interaction and thus extravasation of inflammatory cells. PMID:9421471

  14. Extracellular matrix hyaluronan signals via its CD44 receptor in the increased responsiveness to mechanical stimulation.

    PubMed

    Ferrari, L F; Araldi, D; Bogen, O; Levine, J D

    2016-06-02

    We propose that the extracellular matrix (ECM) signals CD44, a hyaluronan receptor, to increase the responsiveness to mechanical stimulation in the rat hind paw. We report that intradermal injection of hyaluronidase induces mechanical hyperalgesia, that is inhibited by co-administration of a CD44 receptor antagonist, A5G27. The intradermal injection of low (LMWH) but not high (HMWH) molecular weight hyaluronan also induces mechanical hyperalgesia, an effect that was attenuated by pretreatment with HMWH or A5G27. Pretreatment with HMWH also attenuated the hyperalgesia induced by hyaluronidase. Similarly, intradermal injection of A6, a CD44 receptor agonist, produced hyperalgesia that was inhibited by HMWH and A5G27. Inhibitors of protein kinase A (PKA) and Src, but not protein kinase C (PKC), significantly attenuated the hyperalgesia induced by both A6 and LMWH. Finally, to determine if CD44 receptor signaling is involved in a preclinical model of inflammatory pain, we evaluated the effect of A5G27 and HMWH on the mechanical hyperalgesia associated with the inflammation induced by carrageenan. Both A5G27 and HMWH attenuated carrageenan-induced mechanical hyperalgesia. Thus, while LMWH acts at its cognate receptor, CD44, to induce mechanical hyperalgesia, HMWH acts at the same receptor as an antagonist. That the local administration of HMWH or A5G27 inhibits carrageenan-induced hyperalgesia supports the suggestion that carrageenan produces changes in the ECM that contributes to inflammatory pain. These studies define a clinically relevant role for signaling by the hyaluronan receptor, CD44, in increased responsiveness to mechanical stimulation.

  15. Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling

    PubMed Central

    Konopka, Anna; Zeug, Andre; Skupien, Anna; Kaza, Beata; Mueller, Franziska; Chwedorowicz, Agnieszka; Ponimaskin, Evgeni; Wilczynski, Grzegorz M.; Dzwonek, Joanna

    2016-01-01

    Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a “stellate”-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of

  16. A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides.

    PubMed

    Lauder, R M; Huckerby, T N; Nieduszynski, I A

    2000-04-01

    A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology,4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to approximately 0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sbeta(1-3)GalNAc6S structure.

  17. A role for the extracellular matrix component hyaluronan in kidney dysfunction during ACE-inhibitor fetopathy.

    PubMed

    Hansell, P; Palm, F

    2015-04-01

    Despite data showing that inhibitors of the renin-angiotensin system increase the risks of fetal morbidity and dysfunctionality later in life, their use during pregnancy has increased. The fetopathy induced by angiotensin converting enzyme (ACE) inhibitors is characterized by anuria, hypotension and growth restriction, but can also be associated with pulmonary hypoplasia. In the kidney, this fetopathy includes atrophy of the medulla, reduced number of glomeruli, developmental lesions of tubules and vessels, tubulointerstitial inflammation and extracellular matrix accumulation. Although angiotensin II (Ang II) inhibition during nephrogenesis interferes with normal growth and development, this review will focus on effects of the heavily accumulated matrix component hyaluronan (HA). An important mechanism of HA accumulation during nephrogenesis is disruption of its normal reduction as a consequence of lack of Ang II activation of hyaluronidase. Hyaluronan has very large water-attracting properties and is pro-inflammatory when fragmented. The ensuing inflammation and interstitial oedema affect kidney function. Hyaluronan is colocalized with CD44 overexpression and infiltrating immune cells. These properties make HA a plausible contributor to the observed structural and functional kidney defects associated with the fetopathy. Available data support an involvement of HA in kidney dysfunction of the foetus and during adulthood due to the physico-chemical characteristics of HA. No clinical treatment for HA accumulation exists. Treatment with the HA-degrading enzyme hyaluronidase and an HA synthesis inhibitor has been tested successfully in experimental models in the kidney, heart and pancreas. Reduced HA accumulation to reduce interstitial oedema and inflammation may improve organ function, but this concept needs to be tested in a controlled study before causal relationships can be established.

  18. Hyaluronan tetrasaccharides stimulate ceramide production through upregulated mRNA expression of ceramide synthesis-associated enzymes.

    PubMed

    Kage, Madoka; Tokudome, Yoshihiro

    2016-03-01

    It has been reported that hyaluronan has different physiological functions as suggested by variation in molecular weight. In addition, it has also been reported that CD44, the major hyaluronan receptor, was demonstrated to induce keratinocyte differentiation and lipid synthesis of cholesterol. We focus attention on the hyaluronan tetrasaccharides (HA4) which is the smallest unit of hyaluronan. We previously reported that HA4 induced keratinocyte differentiation and that CD44 may be involved. For the purpose of clarifying the influence of HA4 on ceramide synthesis, we evaluated both of these factors in keratinocytes in vitro and in vivo. The mRNA expression of ceramide synthesis-associated enzymes and intracellular ceramide content were evaluated after HA4 treatment in normal human epidermal keratinocytes. In addition, the ceramide increasing effect of HA4 on skin in UVA-irradiated hairless mice was assessed by water content of stratum corneum (SC) and transepidermal water loss (TEWL) methods. The mRNA expression of ceramide synthesis-associated enzymes and intracellular ceramide content after HA4 treatment were increased compared with the control. Furthermore, HA4 treatment increased water content of SC and decreased TEWL. These findings suggest that HA4 affected ceramide synthesis and is involved in the improvement of UV-induced skin damage.

  19. Peritoneal Tumor Carcinomatosis: Pharmacological Targeting with Hyaluronan-Based Bioconjugates Overcomes Therapeutic Indications of Current Drugs

    PubMed Central

    Montagner, Isabella Monia; Merlo, Anna; Zuccolotto, Gaia; Renier, Davide; Campisi, Monica; Pasut, Gianfranco; Zanovello, Paola; Rosato, Antonio

    2014-01-01

    Peritoneal carcinomatosis still lacks reliable therapeutic options. We aimed at testing a drug delivery strategy allowing a controlled release of cytotoxic molecules and selective targeting of tumor cells. We comparatively assessed the efficacy of a loco-regional intraperitoneal treatment in immunocompromised mice with bioconjugates formed by chemical linking of paclitaxel or SN-38 to hyaluronan, against three models of peritoneal carcinomatosis derived from human colorectal, gastric and esophageal tumor cell xenografts. In vitro, bioconjugates were selectively internalized through mechanisms largely dependent on interaction with the CD44 receptor and caveolin-mediated endocytosis, which led to accumulation of compounds into lysosomes of tumor cells. Moreover, they inhibited tumor growth comparably to free drugs. In vivo, efficacy of bioconjugates or free drugs against luciferase-transduced tumor cells was assessed by bioluminescence optical imaging, and by recording mice survival. The intraperitoneal administration of bioconjugates in tumor-bearing mice exerted overlapping or improved therapeutic efficacy compared with unconjugated drugs. Overall, drug conjugation to hyaluronan significantly improved the profiles of in vivo tolerability and widened the field of application of existing drugs, over their formal approval or current use. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of peritoneal carcinomatosis. PMID:25383653

  20. Improving the distribution of Doxil® in the tumor matrix by depletion of tumor hyaluronan

    PubMed Central

    Kohli, Aditya G.; Kivimäe, Saul; Tiffany, Matthew R.; Szoka, Francis C.

    2014-01-01

    Liposomes improve the pharmacokinetics and safety of rapidly cleared drugs, but have not yet improved the clinical efficacy compared to the non-encapsulated drug. This inability to improve efficacy may be partially due to the non-uniform distribution of liposomes in solid tumors. The tumor extra-cellular matrix is a barrier to distribution and includes the high molecular weight glycosaminoglycan, hyaluronan (HA). Strategies to remove HA or block its synthesis may improve drug delivery into solid tumors. Orally administered methylumbelliferone (MU) is an inhibitor of HA synthesis, but it is limited by low potency and limited solubility. In this study, we encapsulate a water-soluble phosphorylated prodrug of MU (MU-P) in a liposome (L-MU-P). We demonstrate that L-MU-P is a more potent inhibitor of HA synthesis than oral MU in the 4T1 murine mammary carcinoma model using both a quantitative ELISA and histochemistry. We show that HA depletion improves the tumor distribution of liposomes computed using Mander’s colocalization analysis of liposomes with the tumor vasculature. Hyaluronan depletion also increases the fraction of the tumor area positive for liposomes. This improved distribution extends the overall survival of mice treated with Doxil®. PMID:24852095

  1. Improving the distribution of Doxil® in the tumor matrix by depletion of tumor hyaluronan.

    PubMed

    Kohli, Aditya G; Kivimäe, Saul; Tiffany, Matthew R; Szoka, Francis C

    2014-10-10

    Liposomes improve the pharmacokinetics and safety of rapidly cleared drugs, but have not yet improved the clinical efficacy compared to the non-encapsulated drug. This inability to improve efficacy may be partially due to the non-uniform distribution of liposomes in solid tumors. The tumor extra-cellular matrix is a barrier to distribution and includes the high molecular weight glycosaminoglycan, hyaluronan (HA). Strategies to remove HA or block its synthesis may improve drug delivery into solid tumors. Orally administered methylumbelliferone (MU) is an inhibitor of HA synthesis, but it is limited by low potency and limited solubility. In this study, we encapsulate a water-soluble phosphorylated prodrug of MU (MU-P) in a liposome (L-MU-P). We demonstrate that L-MU-P is a more potent inhibitor of HA synthesis than oral MU in the 4T1 murine mammary carcinoma model using both a quantitative ELISA and histochemistry. We show that HA depletion improves the tumor distribution of liposomes computed using Mander's colocalization analysis of liposomes with the tumor vasculature. Hyaluronan depletion also increases the fraction of the tumor area positive for liposomes. This improved distribution extends the overall survival of mice treated with Doxil®.

  2. Migration of bovine aortic smooth muscle cells after wounding injury. The role of hyaluronan and RHAMM.

    PubMed Central

    Savani, R C; Wang, C; Yang, B; Zhang, S; Kinsella, M G; Wight, T N; Stern, R; Nance, D M; Turley, E A

    1995-01-01

    The migration of smooth muscle cells is a critical event in the pathogenesis of vascular diseases. We have investigated the role of hyaluronan (HA) and the hyaluronan receptor RHAMM in the migration of adult bovine aortic smooth muscle cells (BASMC). Cultured BASMC migrated from the leading edge of a single scratch wound with increased velocity between 1 and 24 h. Polyclonal anti-RHAMM antisera that block HA binding with this receptor abolished smooth muscle cell migration following injury. HA stimulated the random locomotion of BASMC and its association with the cell monolayer increased following wounding injury. Immunoblot analysis of wounded monolayers demonstrated a novel RHAMM protein isoform that appeared within one hour after injury. At the time of increased cell motility after wounding, FACS analysis demonstrated an increase in the membrane localization in approximately 25% of the cell population. Confocal microscopy of injured monolayers confirmed that membrane expression of this receptor was limited to cells at the wound edge. Collectively, these data demonstrate that RHAMM is necessary for the migration of smooth muscle cells and that expression and distribution of this receptor is tightly regulated following wounding of BASMC monolayers. Images PMID:7533785

  3. Hyaluronan hydration generates three-dimensional meso-scale structure in engineered collagen tissues.

    PubMed

    Anandagoda, Nelomi; Ezra, Daniel G; Cheema, Umber; Bailly, Maryse; Brown, Robert A

    2012-10-07

    Here, we show that the local incorporation of osmotically active hyaluronan into previously compressed collagen constructs results in further rapid dehydration/compression of collagen layers, channel formation and generation of new interfaces; these novel structures, at the nano-micro (i.e. meso-scale) were formed within native collagen gels, in a highly predictable spatial manner and offer important new methods of fabricating scaffolds (e.g. tubes and open-spirals) with potential for use in tissue regeneration such as in peripheral nerves and small vessels. This paper tests the possibility that the local fluid content of a dense collagen network can be controlled by incorporation of an osmotically active (native) macromolecule--hyluronan. This is an exemplar physiological, osmotic swelling agent. Hyaluronan is commonly secreted by cells deep in connective tissues, so is a good candidate for this role in a cell-driven system balancing mechanical compaction of bulk tissue collagen. These constructs may have potential as functional in vitro models representing developmental and pathological processes.

  4. Modified High-Molecular-Weight Hyaluronan Promotes Allergen-Specific Immune Tolerance.

    PubMed

    Gebe, John A; Yadava, Koshika; Ruppert, Shannon M; Marshall, Payton; Hill, Paul; Falk, Ben A; Sweere, Johanna M; Han, Hongwei; Kaber, Gernot; Medina, Carlos; Mikecz, Katalin; Ziegler, Steven F; Balaji, Swathi; Keswani, Sundeep G; Perez, Vinicio A de Jesus; Butte, Manish J; Nadeau, Kari; Altemeier, William A; Fanger, Neil; Bollyky, Paul L

    2017-01-01

    The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.

  5. Wound dressing based on chitosan/hyaluronan/nonwoven fabrics: Preparation, characterization and medical applications.

    PubMed

    Abdel-Rahman, Rasha M; Abdel-Mohsen, A M; Hrdina, R; Burgert, L; Fohlerova, Z; Pavliňák, D; Sayed, O N; Jancar, J

    2016-08-01

    Thin layers of chitosan (positively charged)/sodium hyaluronate (negatively charged)/nonwoven fabrics were constructed by polyelectrolyte multilayer pad-dry-cure technique. Pure chitosan (CS) was isolated from shrimp shell and immobilized onto nonwoven fabrics (NWFs) using citric acid (CTA) as cross linker and solvent agents through a pad-dry-cure method. The prepared thin layer of chitosan citrate/nonwoven fabrics (CSCTA/NWFs) were consequently impregnated with hyaluronan (CSCTA/HA/NWFs) in the second path through a pad-dry-cure method. Chitosan/hyaluronan/nonwoven fabrics wound dressing was characterized by different techniques such as FTIR-ATR, TGA and SEM. The antibacterial activity and the cytotoxicity of the dressing sheets were evaluated against Escherichia coli (E. coli) and Streptococcus aureus (S. aureus), mouse fibroblast (NIH-3T3) and keratinocytes (HaCaT) cell lines, respectively. The cell-fabrics interaction was also investigated using fluorescence microscope, based on live/dead staining assay of 3T3 cells. The healing properties of the new wound dressing were evaluated and compared with the control sample.

  6. Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry.

    PubMed

    Demange, Elise; Kassim, Yusra; Petit, Cyrille; Buquet, Catherine; Dulong, Virginie; Cerf, Didier Le; Buchonnet, Gérard; Vannier, Jean-Pierre

    2013-11-01

    Haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) grow in a specified niche in close association with the microenvironment, the so-called 'haematopoietic niche'. Scaffolds have been introduced to overcome the liquid culture limitations, mimicking the presence of the extracellular matrix (ECM). In the present study the hyaluronic acid scaffold, already developed in the laboratory, has been used for the first time to maintain long-term cultures of CD34⁺ haematopoietic cells obtained from human cord blood. One parameter investigated was the impact on ex vivo survival of CD34⁺ cord blood cells (CBCs) on the hyaluronic acid surface, immobilized with peptides containing the RGD motif. This peptide was conjugated by coating the hyaluronan hydrogel and cultured in serum-free liquid phase complemented with stem cell factor (SCF), a commonly indispensable cytokine for haematopoiesis. Our work demonstrated that these hyaluronan hydrogels were superior to traditional liquid cultures by maintaining and expanding the HPCs without the need for additional cytokines, and a colonization of 280-fold increment in the hydrogel compared with liquid culture after 28 days of ex vivo expansion.

  7. Pulmonary delivery of cisplatin-hyaluronan conjugates via endotracheal instillation for the treatment of lung cancer

    PubMed Central

    Xie, Yumei; Aillon, Kristin L.; Cai, Shuang; Christian, Jason M.; Davies, Neal M.; Berkland, Cory J.; Forrest, M. Laird

    2010-01-01

    Cisplatin (CDDP) intravenous treatments suffer several dose-limiting toxicity issues. Hyaluronan (HA), a naturally occurring biopolymer in the interstitium, is primarily cleared by the lymphatic system. An alteration in input rate and administration route through pulmonary delivery of hyaluronan-cisplatin conjugate (HA-Pt) may increase local lung CDDP concentrations and decrease systemic toxicity. Sprague-Dawley rats were split into four groups: i.v. CDDP (3.5 mg/kg), i.v. HA-Pt conjugate (3.5 mg/kg equivalent CDDP), lung instillation CDDP and lung instillation HA-Pt conjugate. Total platinum level in the lungs of the HA-Pt lung instillation group was 5.7-fold and 1.2-fold higher than the CDDP intravenous group at 24 h and 96 h, respectively. A 1.1-fold increase of Pt accumulation in lung draining nodes for the HA-Pt lung instillation group was achieved at 24 h relative to the CDDP i.v. group. In the brain and kidneys, the CDDP i.v. group had higher tissue/plasma ratios compared to the HA-Pt lung instillation group. Augmented tissue distribution from CDDP i.v. could translate into enhanced tissue toxicity compared to the altered input rate and distribution of the intrapulmonary nanoformulation. In conclusion, a local pulmonary CDDP delivery system was developed with increased platinum concentration in the lungs and draining nodes compared to i.v. therapy. PMID:20363303

  8. Immunohistochemical localization and expression of the hyaluronan receptor CD44 in the epithelium of the pig oviduct during oestrus.

    PubMed

    Tienthai, P; Yokoo, M; Kimura, N; Heldin, P; Sato, E; Rodriguez-Martinez, H

    2003-01-01

    Hyaluronan is related to essential reproductive processes in pigs. Hyaluronan produced by cumulus cells builds, via specific cell surface receptors, an extracellular matrix responsible for cumulus cell cloud expansion during final oocyte maturation, a preparatory event for ovulation and fertilization. In addition, hyaluronan that has been localized in the pig oviduct both in the intraluminal fluid and on the surface of the lining epithelium of the preovulatory sperm reservoir, has proven beneficial during in vitro fertilization and embryo culture, thus indicating that it has a role in vivo. This study monitored the immunolocalization, protein determination and gene expression of the major cell surface hyaluronan receptor CD44 in the epithelial lining of the pig oviduct during selected stages of standing oestrus, in relation to spontaneous ovulation. The CD44 immunostaining in the lining epithelium was localized to the surface membrane and the supranuclear domain of mainly the secretory cells, particularly in the sperm reservoir of both treatment (inseminated) and control (non-inseminated) specimens. Up to four hyaluronan-binding protein (HABP) bands (60, 90, 100 and 200 kDa) were detected in the tubal epithelium, and the 200 kDa band was determined as CD44 by immunoblotting. The expression of CD44 mRNA was higher before than after ovulation (P < 0.05), most conspicuously in the uterotubal junction (UTJ). In addition, CD44 expression in the preovulatory UTJ and the ampullary-isthmic junction (AIJ) of control animals was higher than in those that were inseminated (P < 0.05 and P < 0.01 for UTJ and AIJ, respectively). The results demonstrate for the first time that the specific hyaluronan receptor CD44 is expressed by the oviduct epithelial cells during spontaneous oestrus, and is particularly abundant in the sperm reservoir before ovulation. Presence of spermatozoa in this segment seemed to downregulate the receptor. The variation in the expression of CD44 in

  9. Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

    SciTech Connect

    Zhao, Ningbo Wang, Xin Qin, Lei Guo, Zhengze Li, Dehua

    2015-09-25

    Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.

  10. Loading mitomycin C inside long circulating hyaluronan targeted nano-liposomes increases its antitumor activity in three mice tumor models.

    PubMed

    Peer, Dan; Margalit, Rimona

    2004-02-20

    The frequent overexpression of the hyaluronan receptors CD44 and RHAMM in cancer cells opens the door for targeting by the naturally-occurring high-M(r) hyaluronan. This is the first time effective in vivo tumor targeting is reported for mitomycin C (MMC) loaded inside nano-sized hyaluronan-liposomes (denoted tHA-LIP). The severe adverse effects of free MMC made it a rational candidate for an effective targeted carrier. In vitro, loading MMC inside tHA-LIP increased drug potency 100-fold, in cells overexpressing, but not in cells underexpressing, hyaluronan receptors. Both types of liposomes were non-toxic and reduced MMC-related toxicity in healthy C57BL/6 mice. In 3 tumor models, BALB/c bearing C-26 solid tumors; C57BL/6 bearing B16F10.9 or (separately) D122 lung metastasis, tHA-LIP were long-circulating, 7-fold and 70-fold longer than nt-LIP and free MMC, respectively. tHA-LIP-mediated MMC accumulation in tumor-bearing lungs was 20% of injected dose, compared to 0.6% and 4% with free drug and nt-LIP, respectively. Tumor-free lungs showed low accumulation, irrespective of drug formulation. Key indicators of therapeutic responses, tumor progression, metastatic burden and survival, were superior (p < 0.001) in animals receiving MMC-loaded tHA-LIP, no treatment, MMC-loaded nt-LIP and free drug. In conclusion, tHA-LIP perform as tumor-targeted carriers, with promising prospects for treatment of tumors overexpressing hyaluronan receptors.

  11. Development of OMP based indirect ELISA to gauge the antibody titers in bovines against Pasteurella multocida

    PubMed Central

    Dogra, V; Verma, S; Singh, G; Wani, A. H; Chahota, R; Dhar, P; Verma, L; Sharma, M

    2015-01-01

    Pasteurella multocida (P. multocida) is an important pathogen of various domestic animals. The outer membrane proteins (OMPs) play a major role in pathogenesis and immunogenicity of P. multocida. The aim of the study was to develop indirect enzyme linked immuno sorbant assay (ELISA) based on OMPs to ascertain the antibody titers in animals post-infection or to gauge the potency of vaccine. The OMPs were extracted and purified from P. multocida P:52 (vaccine strain) and P. multocida B:2 isolated from natural outbreak of Haemorrhagic septicaemia (HS) and analyzed on SDS PAGE and through western blot. The OMPs profile of the vaccine strain and the isolate from the natural outbreak of HS were found to be similar. Optimization of various components viz. coating antigens, anti-species conjugate, etc. were carried out against both anti-P. multocida hyper immune and pre immune serum. Validation of OMP based indirect ELISA assay to measure immune response against P. multocida in bovine revealed 91% diagnostic sensitivity (DSN) and about 100% diagnostic specificity (DSP) at 25% cut off. OMP based indirect ELISA was found to be more specific, but less sensitive as compared to WCL based assay. PMID:27175202

  12. Efficacy of a novel Pasteurella multocida vaccine against progressive atrophic rhinitis of swine

    USGS Publications Warehouse

    Hsuan, Shih-Ling; Liao, Chih-Ming; Huang, Chienjin; Winton, James R.; Chen, Zeng-Weng; Lee, Wei-Cheng; Liao, Jiunn-Wang; Chen, Ter-Hsin; Chiou, Chwei-Jang; Yeh, Kuang-Sheng; Chien, Maw-Sheng

    2009-01-01

    The efficacy of a novel vaccine composed of three short recombinant subunit Pasteurella multocida toxin (PMT) proteins in combination with a bi-valent P. multocida whole-cell bacterin (rsPMT–PM) was evaluated in field studies for prevention and control of progressive atrophic rhinitis (PAR) of swine at 15 conventional farrow-to-finish farms. Experimental piglets that were immunized twice with the rsPMT–PM vaccine developed detectable titers of neutralizing antibodies (greater than 1:8) that prevented the growth retardation and pathological lesions typically observed following challenge with authentic PMT. A total of 542 sows were vaccinated once or twice prior to parturition and serum neutralizing antibody titers were evaluated. Both single and double vaccination protocols induced neutralizing antibody titers of 1:16 or higher in 62% and 74% of sows, respectively. Notably, neither sows nor piglets at a farm experiencing a severe outbreak of PAR at the time of the vaccination trial had detectable antibody titers, but antibody titers increased significantly to 1:16 or higher in 40% of sows following double vaccination. During the year after vaccination, clinical signs of PAR decreased in fattening pigs and growth performance improved sufficiently to reduce the rearing period until marketing by 2 weeks. Collectively, these results indicate that the rsPMT–PM vaccine could be used to provide protective immunity for controlling the prevalence and severity of PAR among farm-raised swine.

  13. Effect of simulated stress on susceptibility of bighorn sheep neutrophils to Pasteurella haemolytica leukotoxin.

    PubMed

    Kraabel, B J; Miller, M W

    1997-07-01

    We examined the effects of simulated stress on susceptibility of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) neutrophils to Pasteurella haemolytica leukotoxin in a blocked, crossover experiment. Ten captive-raised bighorn sheep were sampled 10 hr after separate administrations of long-acting adrenocorticotrophic hormone (ACTH) gel and normal saline (control). We then compared in vitro leukotoxin-dependent neutrophil death rates after exposure to culture supernatants from four unique P. haemolytica isolates (one from domestic and three from bighorn sheep). Simulated stress effects were evidenced by elevated (P = 0.002) mean plasma cortisol concentrations, more neutrophils (P = 0.037), and fewer lymphocytes and eosinophils (P < or = 0.043) in ACTH-treated bighorn sheep. Maximum leukotoxin-dependent neutrophil death rates were > or = 61% for three of four P. haemolytica isolates tested. For all three cytotoxic isolates, neutrophil death rates at 150 micrograms/50 microliters supernatant were about 1.13 times higher (P = 0.0001) after bighorns received ACTH; for two of these, overall neutrophil death rates were higher (P < or = 0.001) in ACTH-treated bighorn sheep. Although variable leukotoxin production among P. haemolytica strains appeared principally responsible for differences in leukotoxin-dependent neutrophil death rates, susceptibility of bighorn sheep neutrophils to leukotoxin was increased by prior exposure to elevated plasma cortisol concentrations. It follows that if similar processes occur in neutrophils and alveolar macrophages in vivo, they could contribute to greater susceptibility of stressed bighorn sheep to pneumonic pasteurellosis.

  14. Susceptibility of phagocytes from elk, deer, bighorn sheep, and domestic sheep to Pasteurella haemolytica cytotoxins.

    PubMed

    Silflow, R M; Foreyt, W J

    1994-10-01

    Alveolar macrophages and peripheral blood neutrophils from elk (Cervus elaphus), bighorn sheep (Ovis canadensis canadensis), and domestic sheep were exposed to culture supernatants from Pasteurella haemolytica isolated from bighorn sheep and domestic sheep. In a second experiment, peripheral blood neutrophils from mule deer (Odocoileus hemionus), elk, and bighorn sheep were exposed to culture supernatants from P. haemolytica isolated from elk, bighorn sheep and domestic sheep. Alveolar macrophages from elk, bighorn sheep and domestic sheep were resistant to killing by P. haemolytica supernatants from bighorn sheep and domestic sheep; susceptibility of neutrophils to cell death, as measured by release of lactate dehydrogenase, differed significantly (P < 0.05) between the four species tested. Bighorn sheep and domestic sheep neutrophils were susceptible to cytotoxin damage by the P. haemolytica isolates used; bighorn sheep neutrophils were four- to eight-fold more susceptible to cytotoxin damage than domestic sheep neutrophils. Neutrophils from deer and elk were resistant to killing by P. haemolytica cytotoxins from any species tested.

  15. Evaluation of transport media for Pasteurella multocida isolates from rabbit nasal specimens.

    PubMed

    Kawamoto, E; Sawada, T; Maruyama, T

    1997-08-01

    A suitable medium for the transport of Pasteurella multocida in nasal specimens from rabbits was investigated by using pure cultures of the organism and nasal swabs from infected rabbits. First, the ability of eight transport media to preserve the viabilities of P. multocida strains isolated from rabbits was studied. Cary-Blair medium and Leibovitz medium no. 15 (L-15) were found to be superior to the other six media tested, enabling survival of the organism for more than 14 days at room temperature. Second, the survival of P. multocida in nasal specimens was evaluated on both Cary-Blair medium and L-15. The recovery rate of the organism from these two media was more than 80 to 90% during 4 days of storage and decreased gradually with increasing preservation time. There were no significant differences (P > 0.05) in recovery rates of the organism between Cary-Blair medium and L-15. On the basis of these results, we recommend the use of Cary-Blair medium for the transport of P. multocida in rabbit nasal specimens because of the ease of transport of nasal swabs by mail.

  16. Evaluation of transport media for Pasteurella multocida isolates from rabbit nasal specimens.

    PubMed Central

    Kawamoto, E; Sawada, T; Maruyama, T

    1997-01-01

    A suitable medium for the transport of Pasteurella multocida in nasal specimens from rabbits was investigated by using pure cultures of the organism and nasal swabs from infected rabbits. First, the ability of eight transport media to preserve the viabilities of P. multocida strains isolated from rabbits was studied. Cary-Blair medium and Leibovitz medium no. 15 (L-15) were found to be superior to the other six media tested, enabling survival of the organism for more than 14 days at room temperature. Second, the survival of P. multocida in nasal specimens was evaluated on both Cary-Blair medium and L-15. The recovery rate of the organism from these two media was more than 80 to 90% during 4 days of storage and decreased gradually with increasing preservation time. There were no significant differences (P > 0.05) in recovery rates of the organism between Cary-Blair medium and L-15. On the basis of these results, we recommend the use of Cary-Blair medium for the transport of P. multocida in rabbit nasal specimens because of the ease of transport of nasal swabs by mail. PMID:9230361

  17. Differentiation of toxigenic from nontoxigenic isolates of Pasteurella multocida by PCR.

    PubMed Central

    Nagai, S; Someno, S; Yagihashi, T

    1994-01-01

    A PCR assay was developed for the differentiation of toxigenic Pasteurella multocida subsp. multocida strains, the major etiologic agent for progressive atrophic rhinitis in pigs, from nontoxigenic strains. The PCR targeted a toxA gene encoding a 143-kDa dermonecrotic toxin that is considered to be the central etiologic factor in progressive atrophic rhinitis. toxA fragments were amplified from toxigenic P. multocida isolates but not from nontoxigenic isolates or other bacteria isolated from pigs. The sensitivity of the reaction was as low as 10 pg of chromosomal DNA from a toxigenic strain. The results obtained by PCR of the DNAs of 187 field isolates of P. multocida were consistent with those obtained by the guinea pig skin test and Western blot (immunoblot) analysis. Restriction fragment analysis of the PCR-amplified fragments from 67 field isolates and comparison of the DNA sequences of fragments from capsular serotype A and D strains suggest that the PCR-amplified region, which is considered to encode the major immunologic determinants of the toxin, would be the same among P. multocida strains. The PCR that we describe should be useful for the diagnosis and the etiologic survey of progressive atrophic rhinitis. Images PMID:8027302

  18. Inhibition of Pasteurella multocida Adhesion to Rabbit Respiratory Epithelium Using Lectins

    PubMed Central

    Carrillo, Magda Patricia; Martinez, Nhora María; Patiño, María del Pilar; Iregui, Carlos Arturo

    2015-01-01

    This study aimed to evaluate the ability of a panel of lectins to inhibit the ability of Pasteurella multocida to adhere to and affect the rabbit respiratory epithelium. Nasal septa from rabbit fetuses were cultured with various lectins before the addition of P. multocida. The percentage of bacteria adhering to the epithelium was evaluated semiquantitatively by indirect immunoperoxidase (IIP) staining. The goblet cells (GCs) were counted in semithin sections stained with toluidine blue and served as the main morphological criterion to evaluate the inhibitory effect of the lectins. The lectins PNA, WGA, RCA120, and DBA significantly inhibited the adhesion of P. multocida to the ciliated epithelium (P < 0.05) and prevented the pathogen-induced increase in the number of GCs (P < 0.05) compared with those of positive control tissues. In addition, VVA, SJA, UEA I, DSL, SBA, and ECL significantly inhibited the increase in GCs compared with that of the control tissues. The results suggest that less aggressive therapeutic strategies, such as treatment with lectins, may represent alternative approaches to control bacterial respiratory infections. PMID:25810949

  19. THE SURVIVAL OF PASTEURELLA PESTIS IN MATERIALS PRESERVED BY SOLID CARBON DIOXIDE (DRY-ICE).

    PubMed

    GOLDENBERG, M I; QUAN, S F; PRINCE, F M

    1964-01-01

    In the investigation of outbreaks of plague it is frequently more satisfactory to send specimens of suspected material collected in the field to a central laboratory, where usually better facilities for their more detailed examination are available. In the present study the authors have investigated the suitability of solid CO(2) (dry-ice) for the preservation of such material during transit. Three types of preparation were tested: broth suspensions of Pasteurella pestis, the livers and spleens of guinea-pigs dying after being infected with two different strains of P. pestis, and whole carcasses of mice and ground-squirrels infected with the organism. An additional test to ascertain the rate at which animal specimens became frozen and thawed was also carried out.These studies showed that (1) organisms in the various tissues frozen in dry-ice were not adversely affected by such treatment; (2) the survival of P. pestis cells did not depend on the number of organisms present in broth cultures or tissue suspensions, small numbers surviving equally well as large; and (3) plague bacilli contained in whole carcasses, even when present in small numbers, were also successfully preserved. It is concluded from these results, and also from the authors' practical use of the method over several years, that for the transport of plague-suspect materials from the field to the laboratory freezing with dry-ice can be confidently recommended.

  20. In vivo antimicrobial activity of marbofloxacin against Pasteurella multocida in a tissue cage model in calves

    PubMed Central

    Cao, Changfu; Qu, Ying; Sun, Meizhen; Qiu, Zhenzhen; Huang, Xianhui; Huai, Binbin; Lu, Yan; Zeng, Zhenling

    2015-01-01

    Marbofloxacin is a fluoroquinolone specially developed for use in veterinary medicine with broad-spectrum antibacterial activity. The objective of our study was to re-evaluate in vivo antimicrobial activity of marbofloxacin against Pasteurella multocida using subcutaneously implanted tissue cages in calves. Calves were infected by direct injection into tissue cages with P. multocida(type B, serotype 2), then intramuscularly received a range of marbofloxacin doses 24 h after inoculation. The ratio of 24 h area under the concentration-time curve divided by the minimum inhibitory concentration or the mutant prevention concentration (AUC24 h/MIC or AUC24 h/MPC) was the pharmacokinetic-pharmacodynamic (PK/PD) index that best described the effectiveness of marbofloxacin against P. multocida (R2 = 0.8514) by non-linear regression analysis. Marbofloxacin exhibited a good antimicrobial activity in vivo. The levels of AUC24 h/MIC and AUC24 h/MPC that produced 50% (1.5log10 CFU/mL reduction) and 90% (3log10 CFU/mL reduction) of maximum response were 18.60 and 50.65 h, 4.67 and 12.89 h by using sigmoid Emax model WINNONLIN software, respectively. The in vivo PK/PD integrated methods by tissue cage model display the advantage of the evaluation of antimicrobial activity and the optimization of the dosage regimen for antibiotics in the presence of the host defenses, especially in target animal of veterinary interest. PMID:26257726

  1. Studies on the presence and persistence of Pasteurella multocida serovars and genotypes in fowl cholera outbreaks.

    PubMed

    Singh, Reema; Blackall, Patrick J; Remington, Bruce; Turni, Conny

    2013-12-01

    Pasteurella multocida was isolated from poultry on six farms (one free-range duck farm, one free-range turkey farm, one conventional enclosed turkey farm, and three free-range layer farms) suffering fowl cholera outbreaks. In addition, historical isolates from previous outbreaks were available for the conventional turkey farm and the three free-range layer farms. The isolates were serotyped using the Heddleston scheme and genotyped using multi-locus sequencing typing. In the current outbreaks, two of the farms had two different sequence types (STs) of P. multocida in the investigated outbreak (the free-range turkey farm and one of the free-range layer farms). The remaining four farms had one ST within the investigated outbreak. In looking at the historical isolates, two of the four farms had multiple genotypes involved. On the four farms with historical isolates from previous outbreaks, at least one new genotype was present in the investigated outbreak as compared with the historical isolates. On one layer farm, one genotype persisted over a 10-year period. Serotyping revealed the presence of multiple serovars in the current and historical outbreaks, with serovars sometimes changing over time. This study has shown that several STs of P. multocida can be present during some outbreaks of fowl cholera, although other outbreaks involve a single ST. Also, the STs present on a property suffering repeated fowl cholera can both persist and change over time.

  2. Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida.

    PubMed

    Subaaharan, S; Blackall, L L; Blackall, P J

    2010-03-24

    A total of 63 isolates of Pasteurella multocida from Australian poultry, all associated with fowl cholera outbreaks, and three international reference strains, representing the three subspecies within P. multocida were used to develop a multi-locus sequence typing scheme. Primers were designed for conserved regions of seven house-keeping enzymes -adk, est, gdh, mdh, pgi, pmi and zwf - and internal fragments of 570-784 bp were sequenced for all isolates and strains. The number of alleles at the different loci ranged from 11 to 20 and a total of 29 allelic profiles or sequence types were recognised amongst the 66 strains. There was a strong concordance between the MLST data and the existing multi-locus enzyme electrophoresis and ribotyping data. When used to study a sub-set of isolates with a known detailed epidemiological history, the MLST data matched the results given by restriction endonuclease analysis, pulsed-field gel electrophoresis, ribotyping and REP-PCR. The MLST scheme provides a high level of resolution and is an excellent tool for studying the population structure and epidemiology of P. multocida.

  3. Identification and antimicrobial susceptibility patterns of Pasteurella multocida isolated from chickens and japanese quails in Brazil.

    PubMed

    Rigobelo, Everlon Cid; Blackall, Patrick Joseph; Maluta, Renato Pariz; de Ávila, Fernando Antonio

    2013-01-01

    A study was performed to verify the presence of Pasteurella multocida in eight different poultry groups of 90 birds each. Groups I to IV were chickens (I being > 6 weeks of age with a history of respiratory illness, II > 6 weeks of age and free of respiratory illness, III < 6 weeks of age with respiratory illness and IV being < 6 weeks of age and with no respiratory illness. Groups V to VIII had the matching characteristics of Groups I to V but consisted of Japanese Quails. The P. multocida isolation rate from the groups was as follows; Group I 56/90 (62.3%) Group II 18/90 (20.0%), Group III 12/90 (13.3%), Group IV 3/90 (3.33%), Group V 8/90 (8.88%), Group VI 2/90 (2.22%) Group VII 2/90 (2.22%) and Group VIII 1/90 (1.11%). These isolation rates were not significantly different within the groups of a bird type but the overall chicken isolation rate was significantly higher than the quail isolation rate (p < 0.01). All isolates were examined for their sensitivity to four antimicrobial agents. The results showed only low levels of resistance to the agents tested. The highest level of resistance detected was to cephalothin (5.1% of isolates) followed by amikacin (3.4%).

  4. A Quantitative Flurometric Assay for the Measurement of Antibody to Pasteurella haemolytica in Cattle

    PubMed Central

    Confer, A.W.; Fox, J.C.; Newman, P.R.; Lawson, G.W.; Corstvet, R.E.

    1983-01-01

    A rapid, simple fluorometric method is described for measuring antibody to Pasteurella haemolytica in sera of cattle. Various antigen preparations were compared for the test including live, formalin-killed and phenol-killed P. haemolytica. A preparation composed of formalin-killed organisms from a 22 hour culture gave consistent results and was used in the studies. The test was reproduciable with percent coefficients of variation for fluorescent signal unit values on ten or more replicate samples ranging from 5.7 to 28.0. Sera from calves vaccinated by aerosol exposure to live P. haemolytica had up to a five-fold increase in antibody titer as measured by the flurometric method test during a 21 day period. Fluorometric method titers were comparable to those obtained by the indirect bacterial agglutination test. There was no seroconversion to P. haemolytica in calves vaccinated by aerosol exposure of P. multocida. The major advantages of the fluorometric method test over conventional methods are that the assay does not require serial dilutions of serum samples and thus limits time and effort to determine antibody titers. PMID:6339016

  5. Comparative analysis of Pasteurella multocida strains isolated from bovine respiratory infections.

    PubMed

    Sellyei, Boglárka; Rónai, Zsuzsanna; Jánosi, Szilárd; Makrai, László

    2015-12-01

    Bovine respiratory disease (BRD) is the leading cause of significant economic losses in the intensive beef industry worldwide. Beside numerous risk factors Pasteurella multocida, which is regarded as a secondary pathogen, may play a role in the development of the disease. Previous studies of strains from swine pneumonia revealed that there are a few clones associated with clinical disease, suggesting that some strains may be more virulent than others. This linkage may be true in the BRD, however composition of P. multocida populations in the herds are slightly characterized. Thus, we decided to perform phenotypic and genotypic characterisation of strains isolated from calves with respiratory infection at 31 different herds in Hungary. The results demonstrated the presence of two dominant strain types. At the identical taxonomic background (P. multocida subsp. multocida) with slight phenotypic variability they could be separated by trehalose fermentation capacity, α-glucosidase activity and molecular fingerprint patterns of ERIC- and M13-PCR. Independent prevalence and geographical origin of the strain types may refer to their significance in the illness, but their comparison with strains isolated from healthy individuals is taken into consideration.

  6. Identification and antimicrobial susceptibility patterns of Pasteurella multocida isolated from chickens and japanese quails in Brazil

    PubMed Central

    Rigobelo, Everlon Cid; Blackall, Patrick Joseph; Maluta, Renato Pariz; de Ávila, Fernando Antonio

    2013-01-01

    A study was performed to verify the presence of Pasteurella multocida in eight different poultry groups of 90 birds each. Groups I to IV were chickens (I being > 6 weeks of age with a history of respiratory illness, II > 6 weeks of age and free of respiratory illness, III < 6 weeks of age with respiratory illness and IV being < 6 weeks of age and with no respiratory illness. Groups V to VIII had the matching characteristics of Groups I to V but consisted of Japanese Quails. The P. multocida isolation rate from the groups was as follows; Group I 56/90 (62.3%) Group II 18/90 (20.0%), Group III 12/90 (13.3%), Group IV 3/90 (3.33%), Group V 8/90 (8.88%), Group VI 2/90 (2.22%) Group VII 2/90 (2.22%) and Group VIII 1/90 (1.11%). These isolation rates were not significantly different within the groups of a bird type but the overall chicken isolation rate was significantly higher than the quail isolation rate (p < 0.01). All isolates were examined for their sensitivity to four antimicrobial agents. The results showed only low levels of resistance to the agents tested. The highest level of resistance detected was to cephalothin (5.1% of isolates) followed by amikacin (3.4%). PMID:24159299

  7. Pasteurella haemolytica serotype 2 contains the gene for a noncapsular serotype 1-specific antigen.

    PubMed Central

    Gonzalez, C T; Maheswaran, S K; Murtaugh, M P

    1995-01-01

    An ssa1-homologous genomic fragment cloned from Pasteurella haemolytica serotype 2 (ST2) enabled transformation of Escherichia coli DH5 alpha to a serotype 1 (ST1) phenotype through expression of the ST1-specific antigen (Ssa1). The Ssa1 protein expressed by ssa1-transformed E. coli was susceptible to heat and protease treatment and was distinct from P. haemolytica ST1-specific capsular polysaccharide. Electrophoretic analysis of in vitro-translated proteins, as well as the predicted amino acid sequence, demonstrated that Ssa1 proteins encoded from either ST1- or ST2-derived ssa1 genes were essentially identical. A comparison of the nucleotide sequences of ssa1 genes derived from P. haemolytica ST1 and ST2 revealed greater than 99% homology. Amino acid sequence homology of the predicted products of ST1- and ST2-derived ssa1 genes was greater than 98%. Northern (RNA) blot studies revealed that the presence of an increased level of ssa1 transcript in P. haemolytica ST1 grown as surface-adherent cultures on solid medium was correlated with a serologically detectable Ssa1 protein. Expression of the ssa1 transcript in ST1 was similarly upregulated by a high iron concentration in the growth medium. PMID:7890392

  8. Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica A1 glycoprotease gene.

    PubMed Central

    Abdullah, K M; Lo, R Y; Mellors, A

    1991-01-01

    Pasteurella haemolytica serotype A1 secretes a glycoprotease which is specific for O-sialoglycoproteins such as glycophorin A. The gene encoding the glycoprotease enzyme has been cloned in the recombinant plasmid pH1, and its nucleotide sequence has been determined. The gene (designated gcp) codes for a protein of 35.2 kDa, and an active enzyme protein of this molecular mass can be observed in Escherichia coli clones carrying pPH1. In vivo labeling of plasmid-encoded proteins in E. coli maxicells demonstrated the expression of a 35-kDa protein from pPH1. The amino-terminal sequence of the heterologously expressed protein corresponds to that predicted from the nucleotide sequence. The glycoprotease is a neutral metalloprotease, and the predicted amino acid sequence of the glycoprotease contains a putative zinc-binding site. The gene shows no significant homology with the genes for other proteases of procaryotic or eucaryotic origin. However, there is substantial homology between gcp and an E. coli gene, orfX, whose product is believed to function in the regulation of macromolecule biosynthesis. Images PMID:1885539

  9. Sialoglycoprotease of Pasteurella haemolytica A1: detection of antisialoglycoprotease antibodies in sera of calves.

    PubMed Central

    Lee, C W; Shewen, P E; Cladman, W M; Conlon, J A; Mellors, A; Lo, R Y

    1994-01-01

    Log phase culture supernate from Pasteurella haemolytica biotype A, serotype 1 contains a proteolytic enzyme specific for O-sialoglycoproteins. Using two methods, Western immunoblotting and enzyme neutralization assay, it was demonstrated that certain bovine sera from two previous P. haemolytica A1 vaccination and challenge trials contained antibodies (Ab) (isotypes IgG1 and IgG2 on Western immunoblot) to the sialoglycoprotease (Gcp). In these trials, selected calves were vaccinated twice with either the commercial culture supernate vaccine Presponse or given phosphate-buffered saline (PBS). One trial was conducted during spring, P. haem XIX, and the other during the winter, P. haem XXI. Although there was no clear evidence for induction of anti-Gcp in response to vaccination, several calves seroconverted following intrapulmonary challenge with live P. haemolytica A1. This is the first report of anti-Gcp Ab in bovine sera. The results indicated that the Gcp is immunogenic and that the bacterium produces the enzyme in vivo. Further, animals with an anti-Gcp response had less pneumonia at necropsy, suggesting the Gcp may induce protective immunity. Images Fig. 1. Fig. 2. PMID:8004547

  10. Molecular studies of Ssa1, a serotype-specific antigen of Pasteurella haemolytica A1.

    PubMed Central

    Lo, R Y; Strathdee, C A; Shewen, P E; Cooney, B J

    1991-01-01

    A serotype-specific antigen of Pasteurella haemolytica A1 encoded on the recombinant plasmid pSSA1 is characterized. Nucleotide sequence analysis of the insert DNA in pSSA1 identified the gene ssaI, which codes for a protein of approximately 100 kDa. In vivo labeling of pSSA1-encoded protein in Escherichia coli maxicells showed the expression of a 100-kDa protein from the insert DNA on the recombinant plasmid. Northern blot and primer extension analyses were used to identify the mRNA transcript in P. haemolytica A1 and the putative promoter of ssaI. The antigen (designated Ssa1) could be localized to the outer membrane of P. haemolytica A1 and E. coli clones carrying pSSA1. A rabbit serum against Ssa1 was produced by using whole cells of E. coli expressing Ssa1 on the surface as the immunogen, demonstrating that Ssa1 is immunogenic in rabbits. The results from colony immunoblot analysis with calf serum from animals that were resistant to P. haemolytica A1-induced pneumonia suggest indirectly that Ssa1 is also immunogenic in the animals. Images PMID:1840576

  11. Comparative Genomics Analysis of Two Different Virulent Bovine Pasteurella multocida Isolates

    PubMed Central

    Pan, Tingting; Li, Tian; Wu, Rui

    2016-01-01

    The Pasteurella multocida capsular type A isolates can cause pneumonia and bovine respiratory disease (BRD). In this study, comparative genomics analysis was carried out to identify the virulence genes in two different virulent P. multocida capsular type A isolates (high virulent PmCQ2 and low virulent PmCQ6). The draft genome sequence of PmCQ2 is 2.32 Mbp and contains 2,002 protein-coding genes, 9 insertion sequence (IS) elements, and 1 prophage region. The draft genome sequence of PmCQ6 is 2.29 Mbp and contains 1,970 protein-coding genes, 2 IS elements, and 3 prophage regions. The genome alignment analysis revealed that the genome similarity between PmCQ2 and PmCQ6 is 99% with high colinearity. To identify the candidate genes responsible for virulence, the PmCQ2 and PmCQ6 were compared together with that of the published genomes of high virulent Pm36950 and PmHN06 and avirulent Pm3480 and Pm70 (capsular type F). Five genes and two insertion sequences are identified in high virulent strains but not in low virulent or avirulent strains. These results indicated that these genes or insertion sequences might be responsible for the virulence of P. multocida, providing prospective candidates for further studies on the pathogenesis and the host-pathogen interactions of P. multocida. PMID:28070502

  12. Antibodies against Pasteurella multocida in snow geese in the western arctic

    USGS Publications Warehouse

    Samuel, M.D.; Shadduck, D.J.; Goldberg, D.R.; Baranyuk, V.; Sileo, L.; Price, J.I.

    1999-01-01

    To determine if lesser snow geese (Chen caerulescens caerulescens) are a potential reservoir for the Pasteurella multocida bacterium that causes avian cholera, serum samples and/or pharyngeal swabs were collected from > 3,400 adult geese breeding on Wrangel Island (Russia) and Banks Island (Canada) during 1993-1996. Pharyngeal swab sampling rarely (> 0.1%) detected birds that were exposed to P. multocida in these populations. Geese with serum antibody levels indicating recent infection with P. multocida were found at both breeding colonies. Prevalence of seropositive birds was 3.5% at Wrangel Island, an area that has no recorded history of avian cholera epizootics. Prevalence of seropositive birds was 2.8% at Banks Island in 1994, but increased to 8.2% during 1995 and 1996 when an estimated 40,000-60,000 snow geese were infected. Approximately 50% of the infected birds died during the epizootic and a portion of the surviving birds may have become carriers of the disease. This pattern of prevalence indicated that enzootic levels of infection with P. multocida occurred at both breeding colonies. When no avian cholera epizootics occurred (Wrangel Island, Banks Island in 1994), female snow geese (4.7%) had higher antibody prevalence than males (2.0%).

  13. Identification of avian strains of Pasteurella multocida in India by conventional and PCR assays.

    PubMed

    Shivachandra, S B; Kumar, A A; Gautam, R; Singh, Vijendra P; Saxena, M K; Srivastava, S K

    2006-11-01

    The prevalence of capsular and somatic serotypes were studied among 123 Pasteurella multocida strains isolated from chickens (n=94), ducks (22), quails (4), turkeys (2) and geese (1) from different geographical regions of India. All strains exhibited similar cultural and morphological characteristics. Ninety-two of the isolates belonged to serotype A:1, the most prevalent serotype, with serotypes A:3, A:1,3, D:3 and F:3 having two isolates each. Only one isolate was positive for serotypes A:4 and D:1. Twenty isolates were untyped. A multiplex capsular PCR assay generated amplicons of sizes approximately 460, approximately 1044, approximately 657 and approximately 854 bp in 106 isolates identified as capsular serotype-A, 15 in serotype D and two in serotype F. Capsular types B and E were not detected in any of the avian isolates studied. The present findings suggest that a multiplex capsular PCR assay may be suitable for the rapid initial identification serotypes P. multocida during epidemiological studies of fowl cholera.

  14. Pulmonary lesions induced by Pasteurella haemolytica in neutrophil sufficient and neutrophil deficient calves.

    PubMed Central

    Breider, M A; Walker, R D; Hopkins, F M; Schultz, T W; Bowersock, T L

    1988-01-01

    The role of neutrophils in the development of peracute lung lesions of bovine pneumonic pasteurellosis was investigated. Eight calves were divided into two groups of four calves each. Group I was treated with intravenous phosphate-buffered saline and served as the neutrophil sufficient calves. Group II was treated with intravenous hydroxyurea which produced a state of neutropenia. When peripheral blood neutrophil numbers dropped below 300 cells/microL in group II, all calves were challenged with an intrabronchial bolus of Pasteurella haemolytica in the log phase of growth. An acute inflammatory process occurred in both groups of calves indicated by a rise in body temperature. While pulmonary lesions occurred in both groups by six hours postinoculation, they varied in pathological characteristics. Pulmonary lesions in the neutrophil sufficient calves consisted of fibrinopurulent alveolitis-bronchiolitis with associated alveolar septal necrosis, interlobular edema, and intravascular thrombi. The neutrophil deficient calves had extensive intra-alveolar edema, interlobular edema, intraalveolar hemorrhage, atelectasis, and focal areas of alveolar septal necrosis. These results show that P. haemolytica can induce severe pulmonary tissue damage through both neutrophil dependent and neutrophil independent mechanisms. Images Fig. 1. Fig. 2. PMID:3370555

  15. Serotypes and DNA fingerprint profiles of Pasteurella multocida isolated from raptors

    USGS Publications Warehouse

    Wilson, M.A.; Duncan, R.M.; Nordholm, G.E.; Berlowski, B.M.

    1995-01-01

    Pasteurella multocida isolates from 21 raptors were examined by DNA fingerprint profile and serotyping methods. Isolates were obtained from noncaptive birds of prey found in 11 states from November 28, 1979, through February 10, 1993. Nine isolates were from bald eagles, and the remaining isolates were from hawks, falcons, and owls. Seven isolates were members of capsule group A, and 14 were nonencapsulated. One isolate was identified as somatic type 3, and another was type 3,4,7; both had unique HhaI DNA fingerprint profiles. Nineteen isolates expressed somatic type 1 antigen; HhaI profiles of all type 1 isolates were identical to each other and to the HhaI profile of the reference somatic type 1, strain X-73. The 19 type 1 isolates were differentiated by sequential digestion of DNA with HpaII; four HpaII fingerprint profiles were obtained. The HpaII profile of one isolate was identical to the HpaII profile of strain X-73. Incidence of P. multocida somatic type 1 in raptors suggests that this type may be prevalent in other wildlife or wildlife environments.

  16. Application of ozone disinfection to remove Enterococcus seriolicida, Pasteurella piscicida, and Vibrio anguillarum from seawater.

    PubMed Central

    Sugita, H; Asai, T; Hayashi, K; Mitsuya, T; Amanuma, K; Maruyama, C; Deguchi, Y

    1992-01-01

    Survival of bacterial fish pathogens, including Enterococcus seriolicida, Vibrio anguillarum, and Pasteurella piscicida, in ozonated seawater was determined in a batch system. Bacterial counts of all fish pathogens decreased at more than 0.040 to 0.060 mg of total residual oxidants (TROs) per liter, whereas no decrease in viable counts was observed at less than 0.018 to 0.028 mg of TROs per liter. The 99% inactivation point was achieved at concentrations of 0.111 mg/liter for E. seriolicida, 0.063 mg/liter for P. piscicida, and 0.064 mg/liter for V. anguillarum within 1 min. Moreover, the mean 99 and 99.9% killing concentration-contact time (C.t) products were 0.123 and 0.186 mg.min/liter for E. seriolicida, 0.056 and 0.084 mg.min/liter for P. piscicida, and 0.081 and 0.123 mg.min/liter for V. anguillarum, respectively. However, the mean 99 and 99.9% C.t products for the mixed population in coastal seawater were 0.200 and 0.621 mg.min/liter. These results strongly suggest that ozone treatment at more than 1.0 mg of TROs per liter for several minutes is able to disinfect seawater for mariculture efficiently. PMID:1476447

  17. Plasmid and restriction endonuclease patterns in Pasteurella multocida isolated from a swine pyramid.

    PubMed

    Rúbies, Xavier; Casal, Jordi; Pijoan, Carlos

    2002-01-03

    Restriction endonuclease analysis (REA) and plasmid profile were used to study the epidemiology of Pasteurella multocida in a swine pyramid structure. The studied pyramid was comprised of a group of 12 swine farrow-to-finish farms related by unidirectional animal movement. P. multocida isolates were obtained from the lungs of 275 slaughtered pigs. Serotyping was performed by hyaluronidase sensitivity test and toxicity was investigated by the ELISA test. HpaII was used to cleave the P. multocida extracted DNA. REA patterns relationships were studied using the Sokal-Michener coefficients, and the dendrogram was built using the UPGMA system. The 218 P. multocida isolates obtained were distributed in 17 REA patterns. In 9 of the 12 farms studied only 2-3 REA patterns were detected, with one clearly predominant pattern. The 81 strains with plasmids were assigned to six plasmid profiles. REA and plasmid profiles proved to be good epidemiological tools for identifying different strains of P. multocida with the same phenotype.

  18. Ultrastructural Comparison of the Nasal Epithelia of Healthy and Naturally Affected Rabbits with Pasteurella multocida A

    PubMed Central

    Esquinas, Paula; Botero, Lucía; Patiño, María del Pilar; Iregui, Carlos

    2013-01-01

    An ultrastructural comparison between the nasal cavities of healthy rabbits and those suffering from two forms of spontaneous infection with Pasteurella multocida was undertaken. Twelve commercially produced rabbits of different ages and respiratory health status were divided into four groups: healthy from 0 to 21 days (G1, n = 2); healthy from 23 to 49 days (G2, n = 2); healthy from 51 to 69 days (G3, n = 2); diseased rabbits with septicemia and the rhinitic form of P. multocida infection (G4, n = 3). The main ultrastructural changes observed were a widening of the interepithelial spaces, increased activity and number of goblet cells, the formation of two types of vacuoles in epithelial cells, the degranulation and migration of heterophils between the epithelial cells, and the association of this migration with some of the other changes. No bacteria were observed adhering to the epithelium, and very few were observed free in the mucus. Scant inter-epithelial spaces were found in healthy rabbits, but they were not as large and numerous as those found in diseased animals. We discuss the origin and meaning of these changes but, we focus on the significance of the inter-epithelial spaces and goblet cells for the defense of the upper respiratory airways against the bacterium and its lipopolysaccharide. PMID:23577280

  19. A 5-year retrospective report of Gallibacterium anatis and Pasteurella multocida isolates from chickens in Mississippi.

    PubMed

    Jones, K H; Thornton, J K; Zhang, Y; Mauel, M J

    2013-12-01

    A 5-yr retrospective study (November 2006-December 2011) was conducted to determine the isolation frequency of Pasteurella multocida and Gallibacterium anatis and their antibiograms from chickens submitted to the Mississippi Poultry Research and Diagnostic Laboratory. The number of isolations of G. anatis increased over the last 5 yr in broiler and broiler breeder type chickens. For P. multocida, the number of isolations increased from 2006 to 2010, but decreased through 2011 with all isolations being from boiler breeder type chickens. Gallibacterium anatis demonstrated almost complete resistance to novobiocin, tylosin, lincosamide, and tetracycline antimicrobials with moderate to high sensitivity to sulfonamides, fluoroquinolones, and florfenicol. There was intermediate sensitivity for spectinomycin and erythromycin and variable resistance to β-lactam and aminoglycoside antimicrobials. In sharp contrast, P. multocida showed moderate to high sensitivity to β-lactam, novobiocin, and tetracycline antimicrobials, but had antibiograms similar to G. anatis for the other antimicrobials. Sensitivities were determined using minimum inhibitory concentration. This study examines the trends over a 5-yr period of the number of isolates of P. multocida and G. anatis and their sensitivities. These 2 pathogens produce very similar clinical signs and lesions (fowl cholera-like) in breeders despite having extremely antagonistic sensitivity patterns. This study shows the necessity for producers to attempt culture and sensitivity in suspect fowl cholera-like flocks before initiating antimicrobial treatment commonly used with P. multocida for fear that the culprit may actually be the more antimicrobial-resistant G. anatis.

  20. Characterization of Pasteurella multocida isolates from wetland ecosystems during 1996 to 1999

    USGS Publications Warehouse

    Samuel, M.D.; Shadduck, D.J.; Goldberg, D.R.; Wilson, M.A.; Joly, D.O.; Lehr, M.A.

    2003-01-01

    We cultured 126 Pasteurella multocida isolates, 92 from water and 34 from sediment samples collected from wetlands in the Pacific and Central flyways of the United States between 1996 and 1999. Most (121) of the isolates were P. multocida serotype 1, but serotypes 3, 3/4, 10, and 11 were also found. Many (82) of the isolates were further characterized by DNA fingerprinting procedures and tested in Pekin ducks for virulence. Almost all the serotype 1 isolates we tested caused mortality in Pekin ducks. Serotype 1 isolates varied in virulence, but the most consistent pattern was higher mortality in male ducks than in females. We found no evidence that isolates found in sediment vs. water, between Pacific and Central flyways, or during El Nino years had consistently different virulence. We also found a number of non-serotype 1 isolates that were avirulent in Pekin ducks. Isolates had DNA fingerprint profiles similar to those found in birds that died during avian cholera outbreaks.

  1. Surveillance of antimicrobial resistance in clinical isolates of Pasteurella multocida and Streptococcus suis from Ontario swine.

    PubMed

    Glass-Kaastra, Shiona K; Pearl, David L; Reid-Smith, Richard J; McEwen, Beverly; Slavic, Durda; Fairles, Jim; McEwen, Scott A

    2014-10-01

    Susceptibility results for Pasteurella multocida and Streptococcus suis isolated from swine clinical samples were obtained from January 1998 to October 2010 from the Animal Health Laboratory at the University of Guelph, Guelph, Ontario, and used to describe variation in antimicrobial resistance (AMR) to 4 drugs of importance in the Ontario swine industry: ampicillin, tetracycline, tiamulin, and trimethoprim-sulfamethoxazole. Four temporal data-analysis options were used: visualization of trends in 12-month rolling averages, logistic-regression modeling, temporal-scan statistics, and a scan with the "What's strange about recent events?" (WSARE) algorithm. The AMR trends varied among the antimicrobial drugs for a single pathogen and between pathogens for a single antimicrobial, suggesting that pathogen-specific AMR surveillance may be preferable to indicator data. The 4 methods provided complementary and, at times, redundant results. The most appropriate combination of analysis methods for surveillance using these data included temporal-scan statistics with a visualization method (rolling-average or predicted-probability plots following logistic-regression models). The WSARE algorithm provided interesting results for quality control and has the potential to detect new resistance patterns; however, missing data created problems for displaying the results in a way that would be meaningful to all surveillance stakeholders.

  2. Pasteurella multocida toxin as a transporter of non-cell-permeating proteins.

    PubMed

    Bergmann, Stefan; Jehle, Doris; Schwan, Carsten; Orth, Joachim H C; Aktories, Klaus

    2013-07-01

    The protein toxin Pasteurella multocida toxin (PMT) is the causative agent of atrophic rhinitis in pigs, leading to atrophy of the nasal turbinate bones by affecting osteoblasts and osteoclasts. The mechanism of PMT-induced intoxication is a deamidation of α-subunits of heterotrimeric G proteins, including Gαq, Gα13, and Gαi, thereby causing persistent activation of the G proteins. Here we utilized PMT as a transporter of the non-cell-permeating A domain of diphtheria toxin (DTa). Fusion proteins of PMT and DTa ADP-ribosylated elongation factor 2, the natural target of diphtheria toxin, leading to cell toxicity. PMT-DTa effects were competed by PMT, indicating binding to the same cell surface receptor. Fluorescently labeled PMT-DTa and PMT colocalized with specific markers of early and late endosomes. Bafilomycin A, which inhibits vacuolar H(+)-ATPase, blocked PMT-DTa-induced intoxication of HEK-293 cells. By constructing various PMT-DTa chimeras, we identified a minimal region of PMT necessary for uptake of DTa. The data suggest that PMT is able to transport cargo proteins into eukaryotic cells by utilizing the PMT-specific uptake route.

  3. A systemic Pasteurella multocida toxin aggravates cardiac hypertrophy and fibrosis in mice.

    PubMed

    Weise, Markus; Vettel, Christiane; Spiger, Katharina; Gilsbach, Ralf; Hein, Lutz; Lorenz, Kristina; Wieland, Thomas; Aktories, Klaus; Orth, Joachim H C

    2015-09-01

    Pasteurella multocida toxin (PMT) persistently activates heterotrimeric G proteins of the Gαq/11 , Gα12/13 and Gαi family without interaction with G protein-coupled receptors (GPCRs). We show that PMT acts on heart tissue in vivo and on cardiomyocytes and cardiac fibroblasts in vitro by deamidation of heterotrimeric G proteins. Increased normalized ventricle weights and fibrosis were detected after intraperitoneal administration of PMT in combination with the GPCR agonist phenylephrine. In neonatal rat cardiomyocytes, PMT stimulated the mitogen-activated protein kinase pathway, which is crucial for the development of cellular hypertrophy. The toxin induced phosphorylation of the canonical phosphorylation sites of the extracellular-regulated kinase 1/2 and, additionally, caused phosphorylation of the recently recognized autophosphorylation site, which appears to be important for the development of cellular hypertrophy. Moreover, PMT stimulated the small GTPases Rac1 and RhoA. Both switch proteins are involved in cardiomyocyte hypertrophy. In addition, PMT stimulated RhoA and Rac1 in neonatal rat cardiac fibroblasts. RhoA and Rac1 have been implicated in the regulation of connective tissue growth factor (CTGF) secretion and expression. Accordingly, we show that PMT treatment increased secretion and expression of CTGF in cardiac fibroblasts. Altogether, the data indicate that PMT is an inducer of pathological remodelling of cardiac cells and identifies the toxin as a promising tool for studying heterotrimeric G protein-dependent signalling in cardiac cells.

  4. Pasteurella multocida toxin: Targeting mast cell secretory granules during kiss-and-run secretion.

    PubMed

    Danielsen, Elisabeth M; Christiansen, Nina; Danielsen, E Michael

    2016-02-01

    Pasteurella multocida toxin (PMT), a virulence factor of the pathogenic Gram-negative bacterium P. multocida, is a 146 kDa protein belonging to the A-B class of toxins. Once inside a target cell, the A domain deamidates the α-subunit of heterotrimeric G-proteins, thereby activating downstream signaling cascades. However, little is known about how PMT selects and enters its cellular targets. We therefore studied PMT binding and uptake in porcine cultured intestinal mucosal explants to identify susceptible cells in the epithelium and underlying lamina propria. In comparison with Vibrio cholera B-subunit, a well-known enterotoxin taken up by receptor-mediated endocytosis, PMT binding to the epithelial brush border was scarce, and no uptake into enterocytes was detected by 2h, implying that none of the glycolipids in the brush border are a functional receptor for PMT. However, in the lamina propria, PMT distinctly accumulated in the secretory granules of mast cells. This also occurred at 4 °C, ruling out endocytosis, but suggestive of uptake via pores that connect the granules to the cell surface. Mast cell granules are known to secrete their contents by a "kiss-and-run" mechanism, and we propose that PMT may exploit this secretory mechanism to gain entry into this particular cell type.

  5. Selective Membrane Redistribution and Depletion of Gαq-Protein by Pasteurella multocida Toxin.

    PubMed

    Clemons, Nathan C; Luo, Shuhong; Ho, Mengfei; Wilson, Brenda A

    2016-08-01

    Pasteurella multocida toxin (PMT), the major virulence factor responsible for zoonotic atrophic rhinitis, is a protein deamidase that activates the alpha subunit of heterotrimeric G proteins. Initial activation of G alpha-q-coupled phospholipase C-beta-1 signaling by PMT is followed by uncoupling of G alpha-q-dependent signaling, causing downregulation of downstream calcium and mitogenic signaling pathways. Here, we show that PMT decreases endogenous and exogenously expressed G alpha-q protein content in host cell plasma membranes and in detergent resistant membrane (DRM) fractions. This membrane depletion of G alpha-q protein was dependent upon the catalytic activity of PMT. Results indicate that PMT-modified G alpha-q redistributes within the host cell membrane from the DRM fraction into the soluble membrane and cytosolic fractions. In contrast, PMT had no affect on G alpha-s or G beta protein levels, which are not substrate targets of PMT. PMT also had no affect on G alpha-11 levels, even though G alpha-11 can serve as a substrate for deamidation by PMT, suggesting that membrane depletion of PMT-modified G-alpha-q has specificity.

  6. Associations between water quality, Pasteurella multocida, and avian cholera at Sacramento National Wildlife Refuge

    USGS Publications Warehouse

    Lehr, M.A.; Botzler, R.G.; Samuel, M.D.; Shadduck, D.J.

    2005-01-01

    We studied patterns in avian cholera mortality, the presence of Pasteurella multocida in the water or sediment, and water chemistry characteristics in 10 wetlands at the Sacramento National Wildlife Refuge Complex (California, USA), an area of recurrent avian cholera epizootics, during the winters of 1997 and 1998. Avian cholera outbreaks (a?Y50 dead birds) occurred on two wetlands during the winter of 1997, but no P. multocida were recovered from 390 water and 390 sediment samples from any of the 10 wetlands. No mortality events were observed on study wetlands during the winter of 1998; however, P. multocida was recovered from water and sediment samples in six of the 10 study wetlands. The pH levels were higher for wetlands experiencing outbreaks during the winter of 1997 than for nonoutbreak wetlands, and aluminum concentrations were higher in wetlands from which P. multocida were recovered during the winter of 1998. Water chemistry parameters (calcium, magnesium, sodium, and dissolved protein) previously linked with P. multocida and avian cholera mortality were not associated with the occurrence of avian cholera outbreaks or the presence of P. multocida in our study wetlands. Overall, we found no evidence to support the hypothesis that wetland characteristics facilitate the presence of P. multocida and, thereby, allow some wetlands to serve as long-term sources (reservoirs) for P. multocida.

  7. A Pasteurella multocida strain affecting nulliparous heifers and calves in different ways.

    PubMed

    Turni, Conny; Dayao, Denise; Aduriz, Gorka; Cortabarria, Nekane; Tejero, Carolina; Ibabe, Jose C; Singh, Reema; Blackall, Pat

    2016-11-15

    Pasteurella multocida isolates from dairy cattle on a farm in Spain were associated with pneumonia of calves (six isolates) and mastitis of heifers (five isolates). The objective was to determine if the P. multocida isolates retrieved from both disease scenarios were the same strain or whether more than one strain was present. The isolates were identified by a species-specific polymerase chain (PCR) assay, serotyped by the Heddleston scheme and then typed by a number of molecular genotyping assays including multi-locus sequence typing (MLST). The 11 isolates were confirmed as P. multocida but failed to react with any of the 16 Heddleston antisera. The PCR targeting the genes associated with the lipopolysaccharide outer core biosynthesis locus assigned all the isolates to L3-the type that contains Heddleston serovars 3 and 4. The MLST analysis showed all isolates belonging to ST 79 within the clonal complex of ST13. Only one of the isolates showed a slight different profile by the repetitive extragenic palindromic PCR. The conclusion was that the same strain was associated with pneumonia in calves and mastitis in heifers.

  8. Isolation and Sequencing of a Temperate Transducing Phage for Pasteurella multocida

    PubMed Central

    Campoy, Susana; Aranda, Jesús; Àlvarez, Gerard; Barbé, Jordi; Llagostera, Montserrat

    2006-01-01

    A temperate bacteriophage (F108) has been isolated through mitomycin C induction of a Pasteurella multocida serogroup A strain. F108 has a typical morphology of the family Myoviridae, presenting a hexagonal head and a long contractile tail. F108 is able to infect all P. multocida serogroup A strains tested but not those belonging to other serotypes. Bacteriophage F108, the first P. multocida phage sequenced so far, presents a 30,505-bp double-stranded DNA genome with cohesive ends (CTTCCTCCCC cos site). The F108 genome shows the highest homology with those of Haemophilus influenzae HP1 and HP2 phages. Furthermore, an F108 prophage attachment site in the P. multocida chromosome has been established to be inside a gene encoding tRNALeu. By using several chromosomal markers that are spread along the P. multocida chromosome, it has been demonstrated that F108 is able to perform generalized transduction. This fact, together with the absence of pathogenic genes in the F108 genome, makes this bacteriophage a valuable tool for P. multocida genetic manipulation. PMID:16672452

  9. Cellular and Molecular Action of the Mitogenic Protein-Deamidating Toxin from Pasteurella multocida

    PubMed Central

    Wilson, Brenda A.; Ho, Mengfei

    2011-01-01

    Summary The mitogenic toxin from Pasteurella multocida (PMT) is a member of the dermonecrotic toxin family, which includes toxins from Bordetella, E. coli and Yersinia. Members of the dermonecrotic toxin family modulate G-protein targets in host cells through selective deamidation and/or transglutamination of a critical active site glutamine residue in the G-protein target, which results in activation of the intrinsic GTPase activity. Structural and biochemical data point to the uniqueness of PMT among these toxins in its structure and action. Whereas the other dermonecrotic toxins act on small Rho GTPases, PMT acts on the α subunits of heterotrimeric Gq, Gi and G12/13 protein families. To date, experimental evidence support a model whereby PMT potently stimulates various mitogenic and survival pathways through activation of Gq and G12/13 signaling, ultimately leading to cellular proliferation, while strongly inhibiting pathways involved in cellular differentiation through activation of Gi signaling. The resulting cellular outcomes account for the global physiological effects observed during infection with toxinogenic P. multocida, as well as hint at potential long-term sequelae that may result from PMT exposure. PMID:21569202

  10. Pneumonia in Calves Produced with Aerosols of Bovine Herpesvirus 1 and Pasteurella haemolytica

    PubMed Central

    Jericho, K. W. F.; Langford, E. V.

    1978-01-01

    In each of 11 experiments, four calves were exposed first to an aerosol of bovine herpesvirus 1 (BHV1, virus of infectious bovine rhinotracheitis) and second to an aerosol of Pasteurella haemolytica. The interval between aerosols was three to five days. In two other experiments, calves were exposed only to a bacterial aerosol. Climate was controlled for all experiments from the day of viral exposure and for eight of the experiments it was also controlled for four to six days before the first aerosol. The concentration of infectious doses of virus in the aerosols and the number of bacteria in the aerosols of each calf were determined. Macroscopically recognizable rhinitis, tonsillitis, laryngitis, tracheitis and pneumonia of lobar distribution in 42 lobes from 11 calves were seen in five experiments in which bacterial aerosol followed the viral aerosol by at least four days. One calf died with marked respiratory disease in each of four experiments within four days of exposure to the bacterial aerosol. Production of pneumonia was dependent on an interval between aerosols of at least four days but not on the condition of controlled climate on the environmental chamber either before or after the viral aerosol nor on the period of habituation allowed calves of some experiments. ImagesFig. 1.Fig. 2.Fig. 3. PMID:210912

  11. Virulence Genes and Antimicrobial Resistance Profiles of Pasteurella multocida Strains Isolated from Rabbits in Brazil

    PubMed Central

    Ferreira, Thais Sebastiana Porfida; Felizardo, Maria Roberta; Sena de Gobbi, Débora Dirani; Gomes, Cleise Ribeiro; Nogueira Filsner, Pedro Henrique de Lima; Moreno, Marina; Paixão, Renata; Pereira, Jucélia de Jesus; Micke Moreno, Andrea

    2012-01-01

    Pasteurella multocida is responsible for a wide range of diseases in domestic animals. In rabbits, the agent is related to nasal discharge, pneumonia, otitis media, pyometra, orchitis, abscess, and septicemia. One hundred and forty rabbits with respiratory diseases from four rabbitries in São Paulo State, Brazil were evaluated for the detection of P. multocida in their nasal cavities. A total of twenty-nine animals were positive to P. multocida isolation, and 46 strains were selected and characterized by means of biochemical tests and PCR. P. multocida strains were tested for capsular type, virulence genes, and resistance profile. A total of 45.6% (21/46) of isolates belonged to capsular type A, and 54.34% (25/46) of the isolates were untypeable. None of the strains harboured toxA or pfhA genes. The frequency of the other twenty genes tested was variable, and the data generated was used to build a dendrogram, showing the relatedness of strains, which were clustered according to origin. Resistance revealed to be more common against sulfonamides and cotrimoxazole, followed by erythromycin, penicillin, and amoxicillin. PMID:22919347

  12. Prevalence of Pasteurella multocida and other respiratory pathogens in the nasal tract of Scottish calves.

    PubMed

    Hotchkiss, E J; Dagleish, M P; Willoughby, K; McKendrick, I J; Finlayson, J; Zadoks, R N; Newsome, E; Brulisauer, F; Gunn, G J; Hodgson, J C

    2010-10-09

    The prevalence of Pasteurella multocida, a cause of bovine respiratory disease, was studied in a random sample of beef suckler and dairy farms throughout Scotland, by means of a cross-sectional survey. A total of 637 calves from 68 farms from six geographical regions of Scotland were sampled between February and June 2008. Deep nasal swabs were taken, and samples that were culture-positive for P multocida were confirmed by PCR. Prevalence of P multocida was 17 per cent (105 of 616 calves); 47 per cent of farms had at least one positive animal. A higher prevalence was detected in dairy calves than beef calves (P=0.04). It was found that P multocida was associated with Mycoplasma-like organisms (P=0.06) and bovine parainfluenza type 3 virus (BPI-3) (P=0.04), detected by culture and quantitative PCR of nasal swabs, respectively. Detection of P multocida was not associated with bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1 (BoHV-1) or bovine viral diarrhoea virus (BVDV). Mycoplasma-like organisms, BPI-3, BRSV, BoHV-1 and BVDV were detected in 58, 17, four, 0 and eight calves, on 25, five, two, 0 and five of the 68 farms, respectively.

  13. Antibodies against Pasteurella multocida in snow geese in the western Arctic.

    PubMed

    Samuel, M D; Shadduck, D J; Goldberg, D R; Baranyuk, V; Sileo, L; Price, J I

    1999-07-01

    To determine if lesser snow geese (Chen caerulescens caerulescens) are a potential reservoir for the Pasteurella multocida bacterium that causes avian cholera, serum samples and/or pharyngeal swabs were collected from > 3,400 adult geese breeding on Wrangel Island (Russia) and Banks Island (Canada) during 1993-1996. Pharyngeal swab sampling rarely (> 0.1%) detected birds that were exposed to P. multocida in these populations. Geese with serum antibody levels indicating recent infection with P. multocida were found at both breeding colonies. Prevalence of seropositive birds was 3.5% at Wrangel Island, an area that has no recorded history of avian cholera epizootics. Prevalence of seropositive birds was 2.8% at Banks Island in 1994, but increased to 8.2% during 1995 and 1996 when an estimated 40,000-60,000 snow geese were infected. Approximately 50% of the infected birds died during the epizootic and a portion of the surviving birds may have become carriers of the disease. This pattern of prevalence indicated that enzootic levels of infection with P. multocida occurred at both breeding colonies. When no avian cholera epizootics occurred (Wrangel Island, Banks Island in 1994), female snow geese (4.7%) had higher antibody prevalence than males (2.0%).

  14. Hyaluronan Upregulates Mitochondrial Biogenesis and Reduces Adenoside Triphosphate Production for Efficient Mitochondrial Function in Slow-Proliferating Human Mesenchymal Stem Cells.

    PubMed

    Solis, Mairim Alexandra; Wei, Yau-Huei; Chang, Chiung-Hsin; Yu, Chen-Hsiang; Kuo, Pao-Lin; Huang, Lynn L H

    2016-10-01

    Hyaluronan-coated surfaces preserve the proliferation and differentiation potential of mesenchymal stem cells by prolonging their G1-phase transit, which maintains cells in a slow-proliferative mode. Mitochondria are known to play a crucial role in stem cell self-renewal and differentiation. In this study, for the first time, the metabolic mechanism underlying the hyaluronan-regulated slow-proliferative maintenance of stem cells was investigated by evaluating mitochondrial functions. Human placenta-derived mesenchymal stem cells (PDMSCs) cultured on hyaluronan-coated surfaces at 0.5, 3.0, 5.0, and 30 µg/cm(2) were found to have an average 58% higher mitochondrial mass and an increase in mitochondrial DNA copy number compared to noncoated tissue culture surfaces (control), as well as a threefold increase in the gene expression of the mitochondrial biogenesis-related gene PGC-1α. Increase in mitochondrial biogenesis led to a hyaluronan dose-dependent increase in mitochondrial membrane potential, ATP content, and oxygen consumption rate, with reactive oxygen species levels shown to be at least three times lower compared to the control. Although hyaluronan seemed to favor mitochondrial function, cell entry into a hyaluronan-regulated slow-proliferative mode led to a fivefold reduction in ATP production and coupling efficiency levels. Together, these results suggest that hyaluronan-coated surfaces influence the metabolic proliferative state of stem cells by upregulating mitochondrial biogenesis and function with controlled ATP production. This more efficiently meets the energy requirements of slow-proliferating PDMSCs. A clear understanding of the metabolic mechanism induced by hyaluronan in stem cells will allow future applications that may overcome the current limitations faced in stem cell culture. Stem Cells 2016;34:2512-2524.

  15. Sucrose Synthase: Expanding Protein Function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. Th...

  16. Is molecular size a discriminating factor in hyaluronan interaction with human cells?

    PubMed

    D'Agostino, Antonella; Stellavato, Antonietta; Corsuto, Luisana; Diana, Paola; Filosa, Rosanna; La Gatta, Annalisa; De Rosa, Mario; Schiraldi, Chiara

    2017-02-10

    Nowadays there is a great interest in investigating the effect of particular hyaluronan fragments in the biomedical field and in cosmeceutical applications. Literature has reported that very low molecular weight HA (Mw<5kDa) has an inflammatory effect, whilst HA ranging from 15 to 250 has shown controversial effects. This work aims to give better elucidation on the correlation between the different sized HA fragments and their biological functions. In this respect, a simple and effective degradation strategy is used to obtain several HA fragments. Also, an hydrodynamic and structural characterization was performed in order to obtain samples suitable to evaluate cellular response. In particular an in vitro scratch test in time lapse experiments was used to study the effect of HA fragments, ranging from 1800 to 6kDa on wound dermal reparation based on human keratinocytes. All high and low Mw HA used in this study allowed for faster wound closure compared to the un-treated cells, except for 6kDa that, on the contrary, prevented repair. In addition, TGF-β 1, TNFα and IL-6, representative biomarkers of the inflammation phase occurring in wound healing process, were quantified by RT-PCR. A general up-regulation trend of these biomarkers was found with the HA molecular weight reduction. LHA6kDa was the only treatment that induced a major inflammatory response (over 30 fold increase respect to control) confirming the recent literature outcomes. IL-6 protein level evaluated through ELISA assay corroborated the previous results. Furthermore, activation of key HA receptors, such as CD44, RHAMM, TLR4, with respect to hyaluronan size, was evaluated, at transcriptional level showing selective recognition by HA 1800, 1400, 500 for CD44, whilst the lower Mw fragments activated TLR-4 moderately at 50 and 15kDa. An increase to "alarm" level was found for 6kDa fragments. Immunofluorescence staining confirmed this data. The present research work demonstrated that the diverse pharma

  17. Synthesis and characterization of collagen/hyaluronan/chitosan composite sponges for potential biomedical applications.

    PubMed

    Lin, Yen-Chih; Tan, Fa-Jui; Marra, Kacey G; Jan, Shyh-Shyan; Liu, Deng-Cheng

    2009-09-01

    Cells, scaffolds and growth factors are three main components of a tissue-engineered construct. Collagen type I, a major protein of the extracellular matrix (ECM) in mammals, is a suitable scaffold material for regeneration. Another important constituent of the ECM, hyaluronic acid (hyaluronan, HA), has been used for medical purposes due to its hydrogel properties and biodegradability. Chitosan is a linear polysaccharide comprised of beta1- to beta4-linked d-glucosamine residues, and its potential as a biomaterial is based on its cationic nature and high charge density in solution. This study was conducted to evaluate the characteristics of scaffolds composed of different ratios of type I comb collagen and chitosan with added HA in order to obtain the optimum conditions for the manufacture of collagen-hyaluronan-chitosan (Col-HA-Ch; comprising collagen, HA and chitosan mixed in different ratios: 10:1:0, Col10HACh0; 9:1:1, Col9HACh1; 8:1:2, Col8HACh2; 7:1:3, Col7HACh3; 6:1:4, Col6HACh4; and 5:1:5, Col5HACh5) composite porous scaffolds. Microstructural observation of the composite scaffolds was performed using scanning electron microscopy. The mean pore diameters ranged from 120 to 182microm and decreased as the chitosan composition increased. All scaffolds showed high pore interconnectivity. Swelling ratio measurements showed that all specimens could bind 35- to 40-fold of physiological fluid and still maintain their form and stability. For tensile strength, the optimal ratio of collagen and chitosan was 9:1. Thermal stability was investigated using a differential scanning calorimeter and showed that Col5HACh5 and Col6HACh4 were significantly more stable than the other groups. In enzymatic sensitivity, a steady increase in the biostability of the scaffolds was achieved as the chitosan concentration was increased. In biocompatibility testing, the proliferation of the fibroblasts cultured in Co-HA-Ch tri-copolymer scaffolds was high. Overall, we observed the 9

  18. Paclitaxel-loaded hyaluronan solid nanoemulsions for enhanced treatment efficacy in ovarian cancer

    PubMed Central

    Kim, Joo-Eun; Park, Young-Joon

    2017-01-01

    Paclitaxel-loaded hyaluronan solid nanoemulsions (PTX-HSNs) were successfully fabricated for the delivery of PTX to improve ovarian cancer treatment via active tumor targeting. PTX-HSNs were fabricated using high-pressure homogenization with a microfluidizer and were lyophilized with d-mannitol. Hyaluronan was coated on the outside of the PTX-HSN sphere. The mean size of the PTX-HSNs was maintained less than 100 nm, with a relatively narrow size distribution. The PTX loading content was 3 mg/mL, and encapsulation efficiency (EE) was close to 100%. In vitro cell affinity studies using SK-OV-3 (cluster of differentiation 44 [CD44+]) and OVCAR-3 (CD44−) cells showed that PTX-HSN had a targeting capability hundredfold higher than that of PTX-loaded solid nanoemulsions (PTX-SNs) without hyaluronan. Further, the in vitro PTX release by PTX-SNs and PTX-HSNs lasted more than 6 days without showing a release burst, which was more sustained than that of Taxol®, suggesting a more constant effect on cancer cells at the tumor site than was observed for Taxol. The in vivo toxicity, in vivo antitumor effects, and pharmacokinetics of PTX-HSNs and Taxol were evaluated in nude mice and rats. The maximum tolerated dose (MTD) for PTX-HSNs, PTX-SNs, and Taxol was determined by measuring changes in clinical symptoms after administering 20–50 mg/kg PTX via the caudal vein. The MTD of PTX-HSNs had a dosing capacity greater than 50 mg PTX/kg, which was 2.5-fold higher than that of Taxol when administered as a PTX injection. In vivo, PTX-HSN treatment effectively inhibited tumor growth and showed less toxicity in tumor-transplanted mice compared to that observed for Taxol treatments. The pharmacokinetic parameters of PTX-HSNs were more desirable than those of Taxol. After PTX-HSN treatment, the circulation time of PTX was prolonged and retention of PTX in ovarian tumor tissues increased. Therefore, PTX-HSN is a highly effective nanosystem with a high MTD for delivering PTX to ovarian

  19. Differentiation among closely related organisms of the Actinobacillus-Haemophilus-Pasteurella group by means of lysozyme and EDTA.

    PubMed Central

    Olsen, I; Brondz, I

    1985-01-01

    Bacteriolysis in Tris-maleate buffer (0.005 M, pH 7.2) supplemented with EDTA (0.01 M) and hen egg white lysozyme (HEWL, 1.0 microgram/ml) was set up to assist differentiation between the taxonomically closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. A. actinomycetemcomitans was more sensitive to lysis in this system than H. aphrophilus. The standard method for bacteriolysis separated the 10 tested strains of A. actinomycetemcomitans into two groups (I and II) based on their lysis patterns, whereas the 7 strains of H. aphrophilus examined were homogeneous. In group I of A. actinomycetemcomitans, EDTA displayed a considerable lytic effect, which was not increased by supplementation with HEWL. In group II, the lytic effect of EDTA was much less, but HEWL had a considerable supplementary lytic effect. When the turbidity of A. actinomycetemcomitans (ATCC 29522) or H. aphrophilus (ATCC 33389) suspended in Tris buffer was monitored at close pH intervals (0.2) from pH 5.2 to 9.2, maximal lysis of ATCC 29522 occurred with EDTA at pH 8.0 and with EDTA-HEWL at pH 7.6, while ATCC 33389 lysed with EDTA at pH 9.0 and with EDTA-HEWL at pH 9.2. When other members of the family Pasteurellaceae (Haemophilus influenzae type b, Haemophilus paraphrophilus, Pasteurella multocida, Pasteurella haemolytica, and Pasteurella ureae) were included for comparison, the group I strains of A. actinomycetemcomitans were the most rapidly lysed by EDTA. H. paraphrophilus was the least sensitive of the gram-negative strains tested, but not as resistant as Micrococcus luteus (control). M. luteus was the organism most sensitive to lysozyme, followed by P. ureae and the group II strains of A. actinomycetemcomitans, while the group I strains of A. actinomycetemcomitans, H. paraphrophilus, and P. haemolytica were the least sensitive organisms. Images PMID:3935663

  20. Hyaluronan and Hyaluronan-Binding Proteins Accumulate in Both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate With Inflammatory Cells in Insulitis

    PubMed Central

    Bogdani, Marika; Johnson, Pamela Y.; Potter-Perigo, Susan; Nagy, Nadine; Day, Anthony J.; Bollyky, Paul L.

    2014-01-01

    Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes. PMID:24677718

  1. A complete set of hyaluronan fragments obtained from hydrolysis catalyzed by hyaluronidase: Application to studies of hyaluronan mass distribution by simple HPLC devices.

    PubMed

    Tranchepain, Frédéric; Deschrevel, Brigitte; Courel, Marie-Noëlle; Levasseur, Nicolas; Le Cerf, Didier; Loutelier-Bourhis, Corinne; Vincent, Jean-Claude

    2006-01-15

    Hyaluronan (HA) has different biological functions according to its molar mass; short HA fragments are involved in inflammation processes and angiogenesis, whereas native HA is not. Physicochemically, studies of native HA hydrolysis catalyzed by bovine testicular hyaluronidase (HAase) have suggested that kinetic parameters depend on HA chain length. To study the influence of HA chain length in more detail, and to try to correlate the physicochemical and biological properties of HA, HA hydrolysis catalyzed by HAase was used in a new procedure to obtain HA fragments of different molar masses. HA fragments (10-mg scale) with a molar mass from 800 to 300,000 g mol(-1) were prepared, purified using low-pressure size exclusion chromatography (SEC), lyophilized, and characterized in molar mass by either mass spectrometry or HPLC-SEC-multiangle laser light scattering. The polydispersity index of the purified fractions was less than 1.25. The complete set of HA standards obtained was used to calibrate our routine HPLC-SEC device using only a refractive index (RI) detector. We showed that the N-acetyl-d-glucosamine reducing end assay and the calibrated HPLC-SEC-RI gave equivalent kinetic data. In addition, the HPLC-SEC-RI furnished the mass distribution of the polysaccharide during its hydrolysis.

  2. Unusual Aetiology of Pasteurella canis Biovar 2 Causing Dacryocystitis in HIV Patient: A Case Report and Review of Literature

    PubMed Central

    Negi, Sanjay Singh; Gade, Neeta

    2017-01-01

    Pasteurella species are zoonotic bacterial pathogens implicated very infrequently in various human infections following animal bites or licks usually of dogs and cats. This case report described a rare clinical presentation of dacryocystitis caused by P.canis in a Human Immunodeficiency Virus (HIV) positive young male patient involved in caring of cattle. It advocates the utmost need of recognizing the wide clinical manifestation spectrum of P.canis even without prior penetrating injury. P.canis associated clinical infection is more extensive than had been thought previously especially in immunocompromised patient. Early accurate identification and evidence based anti-microbial therapy may prove crucial in preventing further potential complications. PMID:28384864

  3. [A case of cat-scratch-induced Pasteurella multocida infection presenting with disseminated intravascular coagulation and acute renal failure].

    PubMed

    Fukuchi, Takahiko; Morisawa, Yuji

    2009-09-01

    Domestic animals are the main reservoirs of Pasteurella species for human zoonosis due to bites and scratches. Pasterurella multocida may cause serious soft-tissue infection and, less commonly, sepsis or septic shock, particularly in insufficient initial therapy and an immunocompromised host. We report a case of cat-scratch-induced P. multocida infection, presenting with disseminated intravascular coagulation and acute renal failure. A febrile 83-year-old woman with consciousness disturbance and a subcutaneous left-foot abscess due to a scratch from a pet cat. She was successfully treated with antibiotic piperacillin and clindamycin therapy and aggressive wound drainage.

  4. Murine homologue of the human KIAA1199 is implicated in hyaluronan binding and depolymerization.

    PubMed

    Yoshida, Hiroyuki; Nagaoka, Aya; Nakamura, Sachiko; Sugiyama, Yoshinori; Okada, Yasunori; Inoue, Shintaro

    2013-01-01

    Recently, we have disclosed that human KIAA1199 (hKIAA1199) is a hyaluronan (HA) binding protein implicated in HA depolymerization. Although a murine homologue (mKiaa1199) was previously cloned, no information about the function of the molecule was available. Here, we show that cells transfected with mKiaa1199 cDNA selectively catabolized HA via the clathrin-coated pit pathway. A glycosaminoglycan-binding assay demonstrated the specific binding of mKiaa1199 to HA. These results were similar to our observations with hKIAA1199, although slight differences were found in the peak sizes of the minimum degradates of HA. We conclude that like hKIAA1199, mKiaa1199 is a hyaladherin, leading to HA depolymerization.

  5. The Potency of Hyaluronan of Different Molecular Weights in the Stimulation of Blood Phagocytes

    PubMed Central

    Safrankova, Barbora; Gajdova, Silvie; Kubala, Lukas

    2010-01-01

    The regulatory functions of glycosaminoglycan hyaluronan (HA) are suggested to be dependent on its molecular weight (MW). Proinflammatory and stimulatory effects are proposed mainly for the low MW HA. However, the complex response of blood phagocytes to HA of different MW is unclear. Herein, the effects of highly purified HA of precisely defined MW (52, 250, and 970 kDa) on human blood phagocytes were tested. All MW HA activated blood phagocytes, including the spontaneous production of ROS, degranulation, and the production of tumor necrosis factor alpha, with low MW HA 52 kDa having the highest potency and high MW HA 970 kDa having the lowest potency. Interestingly, HA inhibited ROS production stimulated by opsonized zymosan particles and, in contrast, potentiated starch-activated ROS production, mostly independent of MW. Data showed a significant effect of HA of different MW on blood phagocytes, including high MW HA. PMID:21403830

  6. Enhanced multiparametric hyaluronan degradation for production of molar-mass-defined fragments.

    PubMed

    Holubova, Lucie; Korecka, Lucie; Podzimek, Stepan; Moravcova, Veronika; Rotkova, Jana; Ehlova, Tereza; Velebny, Vladimir; Bilkova, Zuzana

    2014-11-04

    Hyaluronic acid (HA) is known to serve as a dynamic mediator intervening in many physiological functions. Its specific effect has been repeatedly confirmed to be strongly influenced by the molecular size of hyaluronan fragments. However common technological approaches of HA fragments production have their limitations. In many cases, the final products do not meet the strict pharmaceutical requirements, specifically due to size polydispersity and reaction contaminants. We present novel methodology based on combination of unique incidental ability of the plant-derived protease papain to split the glycosidic bonds and an indispensable advantages of biocompatible macroporous material with incorporated ferrous ions serving as carrier for covalent papain fixation. This atypical and yet unpublished highly efficient multiparametric approach allows enhanced HA fragmentation for easily and safely producing molar-mass-defined HA fragments with narrow size distribution. Native polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography/multi-angle light scattering (SEC-MALS) confirmed the effectiveness of our multiparametric approach.

  7. Studies on the formation and characterization of macroporous electron-beam generated hyaluronan cryogels

    NASA Astrophysics Data System (ADS)

    Reichelt, Senta; Naumov, Sergej; Knolle, Wolfgang; Prager, Andrea; Decker, Ulrich; Becher, Jana; Weisser, Jürgen; Schnabelrauch, Matthias

    2014-12-01

    Macroporous cryogels from methacrylated hyaluronan were synthesized at sub-zero temperatures using a novel electron-beam based approach with and without the use of tetra(ethylene glycol) diacrylate as additional cross-linker. Mechanically stable materials possessing a spongy morphology with pore diameters ranging from 50 μm to 70 μm were obtained and characterized by scanning electron microscopy. The excellent cytocompatibility was proven by seeding and cultivation of 3T3 mouse cells on top of the gels. The primary reaction and the chain growth radicals were investigated by quantum chemical calculation and electron paramagnetic resonance spectroscopy and experimentally verified by X-ray photoelectron spectroscopy. The additional cross-linker enhances the reaction efficiency of the cryogel formation.

  8. 4-Methylumbelliferone Treatment and Hyaluronan Inhibition as a Therapeutic Strategy in Inflammation, Autoimmunity, and Cancer

    PubMed Central

    Nagy, Nadine; Kuipers, Hedwich F.; Frymoyer, Adam R.; Ishak, Heather D.; Bollyky, Jennifer B.; Wight, Thomas N.; Bollyky, Paul L.

    2015-01-01

    Hyaluronan (HA) is a prominent component of the extracellular matrix at many sites of chronic inflammation, including type 1 diabetes (T1D), multiple sclerosis, and numerous malignancies. Recent publications have demonstrated that when HA synthesis is inhibited using 4-methylumbelliferone (4-MU), beneficial effects are observed in several animal models of these diseases. Notably, 4-MU is an already approved drug in Europe and Asia called “hymecromone” where it is used to treat biliary spasm. However, there is uncertainty regarding how 4-MU treatment provides benefit in these animal models and the potential long-term consequences of HA inhibition. Here, we review what is known about how HA contributes to immune dysregulation and tumor progression. Then, we review what is known about 4-MU and hymecromone in terms of mechanism of action, pharmacokinetics, and safety. Finally, we review recent studies detailing the use of 4-MU to treat animal models of cancer and autoimmunity. PMID:25852691

  9. Effects of inhaled high-molecular weight hyaluronan in inflammatory airway disease.

    PubMed

    Lamas, Adelaida; Marshburn, Jamie; Stober, Vandy P; Donaldson, Scott H; Garantziotis, Stavros

    2016-10-03

    Cystic fibrosis (CF) is a chronic inflammatory disease that is affecting thousands of patients worldwide. Adjuvant anti-inflammatory treatment is an important component of cystic fibrosis treatment, and has shown promise in preserving lung function and prolonging life expectancy. Inhaled high molecular weight hyaluronan (HMW-HA) is reported to improve tolerability of hypertonic saline and thus increase compliance, and has been approved in some European countries for use as an adjunct to hypertonic saline treatment in cystic fibrosis. However, there are theoretical concerns that HMW-HA breakdown products may be pro-inflammatory. In this clinical pilot study we show that sputum cytokines in CF patients receiving HMW-HA are not increased, and therefore HMW-HA does not appear to adversely affect inflammatory status in CF airways.

  10. Hyaluronan and RHAMM in wound repair and the "cancerization" of stromal tissues.

    PubMed

    Tolg, Cornelia; McCarthy, James B; Yazdani, Arjang; Turley, Eva A

    2014-01-01

    Tumors and wounds share many similarities including loss of tissue architecture, cell polarity and cell differentiation, aberrant extracellular matrix (ECM) remodeling (Ballard et al., 2006) increased inflammation, angiogenesis, and elevated cell migration and proliferation. Whereas these changes are transient in repairing wounds, tumors do not regain tissue architecture but rather their continued progression is fueled in part by loss of normal tissue structure. As a result tumors are often described as wounds that do not heal. The ECM component hyaluronan (HA) and its receptor RHAMM have both been implicated in wound repair and tumor progression. This review highlights the similarities and differences in their roles during these processes and proposes that RHAMM-regulated wound repair functions may contribute to "cancerization" of the tumor microenvironment.

  11. Hyaluronan and RHAMM in Wound Repair and the “Cancerization” of Stromal Tissues

    PubMed Central

    Tolg, Cornelia; McCarthy, James B.; Yazdani, Arjang; Turley, Eva A.

    2014-01-01

    Tumors and wounds share many similarities including loss of tissue architecture, cell polarity and cell differentiation, aberrant extracellular matrix (ECM) remodeling (Ballard et al., 2006) increased inflammation, angiogenesis, and elevated cell migration and proliferation. Whereas these changes are transient in repairing wounds, tumors do not regain tissue architecture but rather their continued progression is fueled in part by loss of normal tissue structure. As a result tumors are often described as wounds that do not heal. The ECM component hyaluronan (HA) and its receptor RHAMM have both been implicated in wound repair and tumor progression. This review highlights the similarities and differences in their roles during these processes and proposes that RHAMM-regulated wound repair functions may contribute to “cancerization” of the tumor microenvironment. PMID:25157350

  12. Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B1 expression

    PubMed Central

    1996-01-01

    The hyaluronan (HA) receptor RHAMM is an important regulator of cell growth. Overexpression of RHAMM is transforming and is required for H- ras transformation. The molecular mechanism underlying growth control by RHAMM and other extracellular matrix receptors remains largely unknown. We report that soluble RHAMM induces G2/M arrest by suppressing the expression of Cdc2/Cyclin B1, a protein kinase complex essential for mitosis. Down-regulation of RHAMM by use of dominant negative mutants or antisense of mRNA also decreases Cdc2 protein levels. Suppression of Cdc2 occurs as a result of an increased rate of cdc2 mRNA degradation. Moreover, tumor cells treated with soluble RHAMM are unable to form lung metastases. Thus, we show that mitosis is directly linked to RHAMM through control of Cdc2 and Cyclin B1 expression. Failure to sustain levels of Cdc2 and Cyclin B1 proteins leads to cell cycle arrest. PMID:8666924

  13. Chitosan-hyaluronan/nano chondroitin sulfate ternary composite sponges for medical use.

    PubMed

    Anisha, B S; Sankar, Deepthi; Mohandas, Annapoorna; Chennazhi, K P; Nair, Shantikumar V; Jayakumar, R

    2013-02-15

    In this work chitosan-hyaluronan composite sponge incorporated with chondroitin sulfate nanoparticle (nCS) was developed. The fabrication of hydrogel was based on simple ionic cross-linking using EDC, followed by lyophilization to obtain the composite sponge. nCS suspension was characterized using DLS and SEM and showed a size range of 100-150 nm. The composite sponges were characterized using SEM, FT-IR and TG-DTA. Porosity, swelling, biodegradation, blood clotting and platelet activation of the prepared sponges were also evaluated. Nanocomposites showed a porosity of 67% and showed enhanced swelling and blood clotting ability. Cytocompatibility and cell adhesion studies of the sponges were done using human dermal fibroblast (HDF) cells and the nanocomposite sponges showed more than 90% viability. Nanocomposite sponges also showed enhanced proliferation of HDF cells within two days of study. These results indicated that this nanocomposite sponges would be a potential candidate for wound dressing.

  14. Poly(ethylene glycol)-or silicone-modified hyaluronan for contact lens wetting agent applications.

    PubMed

    Paterson, Stefan M; Liu, Lina; Brook, Michael A; Sheardown, Heather

    2015-08-01

    Hyaluronan (HA) is a hydrophilic biopolymer that has been explored as a wetting agent in contact lens applications. In this study, HA was modified with siloxy or polyethylene glycol moieties using click chemistry to make it more soluble in monomer solutions used to synthesize model contact lens materials; unmodified HA was not soluble in the same monomer solutions. The water contents of the silicone hydrogels were not increased by the presence of modified HA, nor was there a decrease in the surface contact angle. However, modified HA did lead to a reduction in lysozyme adsorption in some cases. The leaching rate of HA modified with polyethylene glycol from a 78:22 DMA:TRIS(OH) hydrogel was significantly slower than for unmodified HA.

  15. Nanofilms of hyaluronan/chitosan assembled layer-by-layer: An antibacterial surface for Xylella fastidiosa.

    PubMed

    Hernández-Montelongo, Jacobo; Nascimento, Vicente F; Murillo, Duber; Taketa, Thiago B; Sahoo, Prasana; de Souza, Alessandra A; Beppu, Marisa M; Cotta, Monica A

    2016-01-20

    In this work, nanofilms of hyaluronan/chitosan (HA/CHI) assembled layer by layer were synthesized; their application as a potential antimicrobial material was demonstrated for the phytopathogen Xylella fastidiosa, a gram-negative bacterium, here used as a model. For the synthesis, the influence of pH and ionic strength of these natural polymer stem-solutions on final characteristics of the HA/CHI nanofilms was studied in detail. The antibacterial effect was evaluated using widefield fluorescence microscopy. These results were correlated with the chemical properties of the nanofilms, studied by FTIR and Raman spectroscopy, as well as with their morphology and surface properties characterized using SEM and AFM. The present findings can be extended to design and optimize HA/CHI nanofilms with enhanced antimicrobial behavior for other type of phytopathogenic gram-negative bacteria species, such as Xanthomonas citri, Xanthomas campestri and Ralstonia solanacearum.

  16. Intra-articular hyaluronans: the treatment of knee pain in osteoarthritis

    PubMed Central

    Goldberg, Victor M; Goldberg, Laura

    2010-01-01

    The etiology of pain in osteoarthritis is multifactoral, and includes mechanical and inflammatory processes. Intra-articular injections of hyaluronans (HAs) are indicated when non-pharmacological and simple analgesics have failed to relieve symptoms. The HAs appear to reduce pain by restoring both mechanical and biomechanical homeostasis in the joint. There are five FDA-approved injectable preparations of HAs: Hyalgan®, Synvisc®, Supartz®, Orthovisc® and Euflexxa®. They all appear to relieve pain from 4 to 14 weeks after injection and may have disease-modification properties. Although several randomized controlled trials have established the efficacy of this treatment modality, additional high quality randomized control studies with appropriate comparison are still required to clearly define the role of intra-articular HA injections in the treatment of osteoarthritis. PMID:21197309

  17. Intra-articular hyaluronans: the treatment of knee pain in osteoarthritis.

    PubMed

    Goldberg, Victor M; Goldberg, Laura

    2010-05-10

    The etiology of pain in osteoarthritis is multifactoral, and includes mechanical and inflammatory processes. Intra-articular injections of hyaluronans (HAs) are indicated when non-pharmacological and simple analgesics have failed to relieve symptoms. The HAs appear to reduce pain by restoring both mechanical and biomechanical homeostasis in the joint. There are five FDA-approved injectable preparations of HAs: Hyalgan(®), Synvisc(®), Supartz(®), Orthovisc(®) and Euflexxa(®). They all appear to relieve pain from 4 to 14 weeks after injection and may have disease-modification properties. Although several randomized controlled trials have established the efficacy of this treatment modality, additional high quality randomized control studies with appropriate comparison are still required to clearly define the role of intra-articular HA injections in the treatment of osteoarthritis.

  18. Cross-Linked Hyaluronan Gel Reduces the Acute Rectal Toxicity of Radiotherapy for Prostate Cancer

    SciTech Connect

    Wilder, Richard B.; Barme, Greg A.; Gilbert, Ronald F.; Holevas, Richard E.; Kobashi, Luis I.; Reed, Richard R.; Solomon, Ronald S.; Walter, Nancy L.; Chittenden, Lucy; Mesa, Albert V.; Agustin, Jeffrey; Lizarde, Jessica; Macedo, Jorge; Ravera, John; Tokita, Kenneth M.

    2010-07-01

    Purpose: To prospectively analyze whether cross-linked hyaluronan gel reduces the mean rectal dose and acute rectal toxicity of radiotherapy for prostate cancer. Methods and Materials: Between September 2008 and March 2009, we transperitoneally injected 9mL of cross-linked hyaluronan gel (Hylaform; Genzyme Corporation, Cambridge, MA) into the anterior perirectal fat of 10 early-stage prostate cancer patients to increase the separation between the prostate and rectum by 8 to 18mm at the start of radiotherapy. Patients then underwent high-dose rate brachytherapy to 2,200cGy followed by intensity-modulated radiation therapy to 5,040cGy. We assessed acute rectal toxicity using the National Cancer Institute Common Terminology Criteria for Adverse Events v3.0 grading scheme. Results: Median follow-up was 3 months. The anteroposterior dimensions of Hylaform at the start and end of radiotherapy were 13 {+-} 3mm (mean {+-} SD) and 10 {+-} 4mm, respectively. At the start of intensity-modulated radiation therapy, daily mean rectal doses were 73 {+-} 13cGy with Hylaform vs. 106 {+-} 20cGy without Hylaform (p = 0.005). There was a 0% incidence of National Cancer Institute Common Terminology Criteria for Adverse Events v3.0 Grade 1, 2, or 3 acute diarrhea in 10 patients who received Hylaform vs. a 29.7% incidence (n = 71) in 239 historical controls who did not receive Hylaform (p = 0.04). Conclusions: By increasing the separation between the prostate and rectum, Hylaform decreased the mean rectal dose. This led to a significant reduction in the acute rectal toxicity of radiotherapy for prostate cancer.

  19. A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

    USGS Publications Warehouse

    Rocke, Tonie E.; Smith, Susan R.; Miyamoto, Amy; Shadduck, Daniel J.

    2002-01-01

    A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 × 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

  20. Pasteurella multocida infections. Report of 34 cases and review of the literature.

    PubMed

    Weber, D J; Wolfson, J S; Swartz, M N; Hooper, D C

    1984-05-01

    Pasteurella multocida, a small, gram-negative coccobacillus , is part of the normal oral flora of many animals, including the dog and cat. P. multocida is the etiologic agent in a variety of infectious disease syndromes. We have reported 34 cases of infection caused by P. multocida and have reviewed the English literature. P. multocida infections may be divided into three broad groups: 1. Infections resulting from animal bites and scratches : The most common infections caused by P. multocida are local wound infections following animal bites or scratches . Cats are the source of infection in 60 to 80% of cases and dogs in the great majority of the remainder. Local infections are characterized by the rapid appearance of erythema, warmth, tenderness, and frequently purulent drainage. The most common local complications are abscess formation and tenosynovitis. Serious local complications include septic arthritis proximal to bites or scratches , osteomyelitis resulting from direct inoculation or extension of cellulitis, and the combination of septic arthritis and osteomyelitis, most commonly involving a finger or hand after a cat bite. 2. Isolation of P. multocida from the respiratory tract: The isolation of P. multocida from the respiratory tract must be interpreted differently than its isolation from other systemic sites. Most commonly P. multocida found in the respiratory tract is a commensal organism in patients with underlying pulmonary disease, but serious respiratory tract infections including pneumonia, empyema, and lung abscesses may develop. Most patients with respiratory tract colonization or infection have a history of animal exposure. 3. Other systemic infections: P. multocida is recognized as a pathogen in a variety of systemic infections including bacteremia, meningitis, brain abscess, spontaneous bacterial peritonitis, and intra-abdominal abscess. P. multocida often acts as an opportunistic pathogen with a predilection for causing bacteremia in patients

  1. Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish.

    PubMed Central

    Magariños, B; Romalde, J L; Bandín, I; Fouz, B; Toranzo, A E

    1992-01-01

    We compared Pasteurella piscicida strains isolated from different fish species in several European countries with strains isolated in Japan and the United States. The taxonomic analysis revealed that, regardless of the geographic origin and source of isolation, all the strains exhibited the same biochemical and physiological characteristics. Serological assays with different rabbit antisera demonstrated a high level of antigenic similarity among strains, with cross-agglutination titers of 20,480 to 40,960. This serological homogeneity was supported by the lipopolysaccharide (LPS) and membrane protein profiles. All the P. piscicida strains had the same electrophoretic LPS pattern, showing O side chains with a ladder-like structure, and shared at least four major outer membrane proteins, of 20, 30, 42, and 53 kDa. Western blot (immunoblot) analysis with LPS and protein indicated that all the P. piscicida strains are immunologically related. In addition, the chromosomal DNA fingerprint patterns obtained for the European strains with the enzymes EcoRI and BamHI were practically identical to those of the Japanese and U.S. strains. Although some differences were found in the plasmid profiles of P. piscicida, a large number of strains possessed in common plasmid bands of 20 and 7 MDa. In addition, a plasmid of 50 MDa was present in the majority of the European strains. Restriction endonuclease analysis demonstrated the genetic homology of the plasmid bands shared by most of the European strains. All the P. piscicida strains had the same drug resistance patterns, indicating that a correlation between plasmid carriage and resistance to a specific antimicrobial agent cannot be established. The high levels of phenotypic, serological, and genetic homogeneity found among the P. piscicida strains should facilitate the development of DNA probes with diagnostic purposes as well as the design of effective vaccines. Images PMID:1444366

  2. Hematological changes in calves exposed to a mixture of lipopolysaccharide and crude leukotoxin of Pasteurella haemolytica.

    PubMed Central

    Bowersock, T L; Walker, R D; Maddux, J M; Fenner, D; Moore, R N

    1990-01-01

    The purpose of this investigation was to determine if culture supernatants of Pasteurella haemolytica containing crude leukotoxin and lipopolysaccharide (CLCL) causes disseminated intravascular coagulopathy (DIC) when injected into calves. The effect of intraduodenal (ID) exposure followed by a subsequent subcutaneous (SC) inoculation of either heat-treated or untreated CLCL was evaluated. The relative contribution of the crude leukotoxin and lipopolysaccharide (LPS) to the virulence of P. haemolytica was evaluated. One group of calves received an ID inoculation of CLCL followed two weeks later by a SC inoculation of CLCL; one group received an ID inoculation of tissue culture medium followed two weeks later by a SC inoculation of CLCL; and a third group received an ID inoculation of CLCL followed two weeks later by a SC inoculation of heat-treated CLCL. Hematological parameters used to evaluate DIC included white cell count, platelet count, neutrophil number, fibrinogen, fibrin degradation products, one stage prothrombin time (OSPT), activated partial thromboplastin time, body temperature and clinical signs. Each parameter was measured in calves at 0, 2, 4, 6, 12 and 24 h following the SC inoculation of CLCL. Each group had significant changes over time in all parameters except body temperature. Calves that received a SC inoculation of heat-treated CLCL had smaller changes in all parameters except OSPT compared to the other groups. Results suggest that the LPS and leukotoxin of P. haemolytica exert additive effects on the coagulation cascade and number of peripheral leukocytes, and that the ID inoculation of CLCL does not affect the response of calves to a SC inoculation of toxin. PMID:2249175

  3. Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

    PubMed Central

    Wilson, Brenda A.; Ponferrada, Virgilio G.; Vallance, Jefferson E.; Ho, Mengfei

    1999-01-01

    We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells. PMID:9864199

  4. Inhibition of growth and alteration of host cell interactions of Pasteurella multocida with natural byproducts.

    PubMed

    Salaheen, S; Almario, J A; Biswas, D

    2014-06-01

    Pasteurella multocida is a leading cause of fowl cholera in both free-range pasture and conventional/commercially raised poultry. Its infection is a serious threat to poultry health and overall flock viability. Organic poultry is comparatively more vulnerable to this pathogen. It is a significant cause of production loss and price increase of poultry products, specifically organic poultry products. Some plant products are well documented as sources of natural antimicrobials such as polyphenols found in different berry pomaces and citrus oil. Pomace, a byproduct (primarily of seeds and skins) of fruits used for juice and wine production, and citrus oil, the byproduct of citrus juice production, show promising antimicrobial activity against various pathogens. Here, we showed for the first time that blackberry and blueberry pomace extracts and citrus oil inhibited P. multocida growth. Minimum bactericidal concentrations were determined as 0.3 and 0.4 mg/mL gallic acid equivalent for blackberry and blueberry pomace extracts, respectively. Similarly, only 0.05% citrus oil (vol/vol) completely inhibited P. multocida growth. Under shaking conditions, the antimicrobial activity of both pomace extracts and citrus oil was more intensive. Even citrus oil vapor also significantly reduced the growth of P. multocida. In addition, cell surface hydrophobicity of P. multocida was increased by 2- to 3-fold and its adherence to chicken fibroblast (DF1) and bovine mammary gland (MacT) cells was reduced significantly in the presence of pomace extracts only. This study indicates that these natural products might be good alternatives to conventional antimicrobial agents, and hence, may be used as feed or water supplements to control fowl cholera and reduce production loss caused by P. multocida.

  5. Florfenicol As a Modulator Enhancing Antimicrobial Activity: Example Using Combination with Thiamphenicol against Pasteurella multocida

    PubMed Central

    Wei, Chia-Fong; Shien, Jui-Hung; Chang, Shao-Kuang; Chou, Chi-Chung

    2016-01-01

    Synergistic effects between the same class of antibiotics are rarely reported. Our previous study found synergistic-like interaction between florfenicol (FFC) and thiamphenicol (TAP) against Staphylococcus aureus. Here, the enhanced antimicrobial activity was evaluated in 97 clinical isolates of both Gram-negative and Gram-positive bacteria. Susceptible strains were initially identified by checkerboard microdilution assay (fractional inhibitory concentration index [FICI] ≤ 0.625), followed by confirmation of synergism using the time-kill methodology (≥2 log10 CFU/ml reduction). In all, 43% of Pasteurella multocida tested were susceptible to the enhanced bactericidal effect. In chicken fowl cholera models, FFC and TAP combination at much lower dosage that is correspondent to their MIC deduction provided maximum protection in vivo. Furthermore, synergistic combination of FFC with oxytetracycline (OTC) against Pseudomonas aeruginosa in vitro was also demonstrated. Based on the enhanced uptake of TAP and OTC, FFC presumably elicits enhanced antimicrobial activity in an orderly manner through alteration of bacterial membrane permeability or efflux systems and subsequent increase of intracellular concentration of the antibiotics used in combination. Results of ethidium bromide accumulation assay and RNA-seq showed little evidence for the involvement of efflux pumps in the synergy but further investigation is required. This study suggests the potentiality of a novel combination regimen involving FFC as an initiating modulator effective against both Gram-positive and Gram-negative bacteria depending on the antibiotics that are combined. The observed improvement of bacteriostatic effect to bactericidal, and the extended effectiveness against FFC-resistant bacterial strains warrant further studies. PMID:27065961

  6. Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish.

    PubMed

    Magariños, B; Romalde, J L; Bandín, I; Fouz, B; Toranzo, A E

    1992-10-01

    We compared Pasteurella piscicida strains isolated from different fish species in several European countries with strains isolated in Japan and the United States. The taxonomic analysis revealed that, regardless of the geographic origin and source of isolation, all the strains exhibited the same biochemical and physiological characteristics. Serological assays with different rabbit antisera demonstrated a high level of antigenic similarity among strains, with cross-agglutination titers of 20,480 to 40,960. This serological homogeneity was supported by the lipopolysaccharide (LPS) and membrane protein profiles. All the P. piscicida strains had the same electrophoretic LPS pattern, showing O side chains with a ladder-like structure, and shared at least four major outer membrane proteins, of 20, 30, 42, and 53 kDa. Western blot (immunoblot) analysis with LPS and protein indicated that all the P. piscicida strains are immunologically related. In addition, the chromosomal DNA fingerprint patterns obtained for the European strains with the enzymes EcoRI and BamHI were practically identical to those of the Japanese and U.S. strains. Although some differences were found in the plasmid profiles of P. piscicida, a large number of strains possessed in common plasmid bands of 20 and 7 MDa. In addition, a plasmid of 50 MDa was present in the majority of the European strains. Restriction endonuclease analysis demonstrated the genetic homology of the plasmid bands shared by most of the European strains. All the P. piscicida strains had the same drug resistance patterns, indicating that a correlation between plasmid carriage and resistance to a specific antimicrobial agent cannot be established. The high levels of phenotypic, serological, and genetic homogeneity found among the P. piscicida strains should facilitate the development of DNA probes with diagnostic purposes as well as the design of effective vaccines.

  7. Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin.

    PubMed

    Kamitani, Shigeki; Kitadokoro, Kengo; Miyazawa, Masayuki; Toshima, Hirono; Fukui, Aya; Abe, Hiroyuki; Miyake, Masami; Horiguchi, Yasuhiko

    2010-08-13

    Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G(q)- and G(12/13)-dependent pathways through the deamidation of a glutamine residue in the alpha-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575-1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590-670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT DeltaC1(4H)) neither localized to the plasma membrane nor stimulated the G(q/12/13)-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT DeltaC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.

  8. Comparative genome analysis of an avirulent and two virulent strains of avian Pasteurella multocida reveals candidate genes involved in fitness and pathogenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fowl cholera is a highly contagious systemic disease affecting wild and domestic birds, frequently resulting in high morbidity and mortality. The causative agent is Pasteurella multocida (P. multocida). The completed genome of P. multocida strain Pm70 has been available for over eleven years and has...

  9. SIRT3 Deacetylates Ceramide Synthases

    PubMed Central

    Novgorodov, Sergei A.; Riley, Christopher L.; Keffler, Jarryd A.; Yu, Jin; Kindy, Mark S.; Macklin, Wendy B.; Lombard, David B.; Gudz, Tatyana I.

    2016-01-01

    Experimental evidence supports the role of mitochondrial ceramide accumulation as a cause of mitochondrial dysfunction and brain injury after stroke. Herein, we report that SIRT3 regulates mitochondrial ceramide biosynthesis via deacetylation of ceramide synthase (CerS) 1, 2, and 6. Reciprocal immunoprecipitation experiments revealed that CerS1, CerS2, and CerS6, but not CerS4, are associated with SIRT3 in cerebral mitochondria. Furthermore, CerS1, -2, and -6 are hyperacetylated in the mitochondria of SIRT3-null mice, and SIRT3 directly deacetylates the ceramide synthases in a NAD+-dependent manner that increases enzyme activity. Investigation of the SIRT3 role in mitochondrial response to brain ischemia/reperfusion (IR) showed that SIRT3-mediated deacetylation of ceramide synthases increased enzyme activity and ceramide accumulation after IR. Functional studies demonstrated that absence of SIRT3 rescued the IR-induced blockade of the electron transport chain at the level of complex III, attenuated mitochondrial outer membrane permeabilization, and decreased reactive oxygen species generation and protein carbonyls in mitochondria. Importantly, Sirt3 gene ablation reduced the brain injury after IR. These data support the hypothesis that IR triggers SIRT3-dependent deacetylation of ceramide synthases and the elevation of ceramide, which could inhibit complex III, leading to increased reactive oxygen species generation and brain injury. The results of these studies highlight a novel mechanism of SIRT3 involvement in modulating mitochondrial ceramide biosynthesis and suggest an important role of SIRT3 in mitochondrial dysfunction and brain injury after experimental stroke. PMID:26620563

  10. Glucose regulated proteins 78 and 75 bind to the receptor for hyaluronan mediated motility in interphase microtubules

    SciTech Connect

    Kuwabara, Hiroko . E-mail: pa2020@art.osaka-med.ac.jp; Yoneda, Masahiko; Hayasaki, Hana; Nakamura, Toshiya; Mori, Hiroshi

    2006-01-20

    The receptor for hyaluronan mediated motility (RHAMM), which is a hyaluronan-binding protein, is a centrosomal and microtubal protein. Here, we have identified two RHAMM-binding proteins, glucose regulated protein (GRP) 78 and GRP75, using co-immunoprecipitation analysis. These two proteins directly bound to glutathione-S-transferase-RHAMM fusion proteins. By double immunostaining, GRP78 and GRP75 colocalized with RHAMM in interphase microtubules, but were separated in mitotic spindles. Prevention of microtubule polymerization by TN-16 and vincristine sulfate induced RHAMM overexpression without a significant change in GRP78/75. Taken together, GRP78/75 and RHAMM complexes may stabilize microtubules in the interphase, associated with a downregulation of RHAMM. These results reveal a new biochemical activity of RHAMM.

  11. Evaluation of immunopathologic effects of aqueous extract of Echinacea purpurea in mice after experimental challenge with Pasteurella multocida serotype A

    PubMed Central

    Rezaie, A; Gharibi, D; Ghorbanpoor, M; Anbari, S; Pourmahdi Broojeni, M

    2014-01-01

    In order to assess the immunopathological effects of aqueous Echinacea purpurea extract (EPE) on mice experimentally challenged with Pasteurella multocida serotype A, forty female BALB/c mice were randomly divided into four groups. The groups included a control group (received sterile distilled water 2 times/week for 2 weeks, intraperitoneally and then 100 µl sterile saline intranasally), a PMA group (received sterile distilled water as the control group and after 2 weeks, 5.6 × 103 CFU/ml of P. multocida serotype A, intranasally), an EPE+PMA group (received E. purpurea extract intraperitoneally 2 times/week for 2 weeks and then challenged as the PMA group) and an EPE group (received E. purpurea extract as EPE+PMA group and then 100 µl sterile saline intranasally). After 24 and 48 h post challenge, half of the animals in each group were sacrificed and analyzed for bacterial counts in their lungs and livers, TNFα serum levels and histapathological changes. The results showed significant differences in lung bacterial counts between PMA and EPE+PMA groups. TNFα serum level was significantly higher in the PMA group. Histopathological examination revealed infiltration of neutrophils in alveolar septa and hyperemia in the PMA group. In addition, the criteria of bronchopneumonia were partially recovered in the EPE+PMA compared to the PMA group. According to the results, it seems that E. purpurea extract has an immunomodulatory effect and can be used to prevent or control of pneumonia caused by Pasteurella. PMID:27175135

  12. Serum haptoglobin concentration in growing swine after intranasal challenge with Bordetella bronchiseptica and toxigenic Pasteurella multocida type D.

    PubMed Central

    Francisco, C J; Shryock, T R; Bane, D P; Unverzagt, L

    1996-01-01

    The acute phase reaction, in association with progressive atrophic rhinitis (AR), was monitored for 3 wk using serum haptoglobin (HPT) quantification in thirty-six, 15 kg swine after intranasal challenge with varying doses of Pasteurella multocida type D (toxigenic strain) and Bordetella bronchiseptica. The challenge doses were administered alone or in combination with pigs divided into 9 isolated treatment groups. Increasing doses of B. bronchiseptica were associated with lower serum HPT (P < 0.05), whereas increasing doses of P. multocida tended to increase serum HPT (0.05 < P < 0.10). Significant and positive correlation of mean HPT and AR score was found in these pigs; increased AR scores were associated with elevated mean HPT concentration (r = 0.41, P < 0.01). A significant interaction between P. multocida and B. bronchiseptica dose indicated that increasing the dose of B. bronchiseptica, for a fixed P. multocida dose, was associated with less AR (P < 0.05). The AR scores were greater in pigs given P. multocida, than B. bronchiseptica alone. These results indicate that a complex interaction between Pasteurella multocida and Bordetella bronchiseptica causes progressive atrophic rhinitis and alters serum HPT concentration in swine. Images Figure I. Figure II. Figure III. Figure IV. PMID:8809387

  13. Inhibition of Ovarian Cancer Chemoresistance and Metastasis with Antagonists of Hyaluronan-CD44-CD147 Interactions

    DTIC Science & Technology

    2015-09-01

    CD147 interactions in cancer stem cell properties, especially drug resistance, to improvement of therapy for malignant ovarian carcinoma. In this grant...we have shown that: a) drug -resistant human ovarian carcinoma cell lines contain CD133-positive/ CD147-positive/ CD44-positive cancer stem-like...small hyaluronan oligosaccharides sensitize drug -resistant human ovarian carcinoma cells to various chemotherapeutic agents in culture and in vivo; c

  14. Binding of Hyaluronan to the Native Lymphatic Vessel Endothelial Receptor LYVE-1 Is Critically Dependent on Receptor Clustering and Hyaluronan Organization*

    PubMed Central

    Lawrance, William; Banerji, Suneale; Day, Anthony J.; Bhattacharjee, Shaumick; Jackson, David G.

    2016-01-01

    The lymphatic endothelial receptor LYVE-1 has been implicated in both uptake of hyaluronan (HA) from tissue matrix and in facilitating transit of leukocytes and tumor cells through lymphatic vessels based largely on in vitro studies with recombinant receptor in transfected fibroblasts. Curiously, however, LYVE-1 in lymphatic endothelium displays little if any binding to HA in vitro, and this has led to the conclusion that the native receptor is functionally silenced, a feature that is difficult to reconcile with its proposed in vivo functions. Nonetheless, as we reported recently, LYVE-1 can function as a receptor for HA-encapsulated Group A streptococci and mediate lymphatic dissemination in mice. Here we resolve these paradoxical findings and show that the capacity of LYVE-1 to bind HA is strictly dependent on avidity, demanding appropriate receptor self-association and/or HA multimerization. In particular, we demonstrate the prerequisite of a critical LYVE-1 threshold density and show that HA binding may be elicited in lymphatic endothelium by surface clustering with divalent LYVE-1 mAbs. In addition, we show that cross-linking of biotinylated HA in streptavidin multimers or supramolecular complexes with the inflammation-induced protein TSG-6 enables binding even in the absence of LYVE-1 cross-linking. Finally, we show that endogenous HA on the surface of macrophages can engage LYVE-1, facilitating their adhesion and transit across lymphatic endothelium. These results reveal LYVE-1 as a low affinity receptor tuned to discriminate between different HA configurations through avidity and establish a new mechanistic basis for the functions ascribed to LYVE-1 in matrix HA binding and leukocyte trafficking in vivo. PMID:26823460

  15. Binding of Hyaluronan to the Native Lymphatic Vessel Endothelial Receptor LYVE-1 Is Critically Dependent on Receptor Clustering and Hyaluronan Organization.

    PubMed

    Lawrance, William; Banerji, Suneale; Day, Anthony J; Bhattacharjee, Shaumick; Jackson, David G

    2016-04-08

    The lymphatic endothelial receptor LYVE-1 has been implicated in both uptake of hyaluronan (HA) from tissue matrix and in facilitating transit of leukocytes and tumor cells through lymphatic vessels based largely onin vitrostudies with recombinant receptor in transfected fibroblasts. Curiously, however, LYVE-1 in lymphatic endothelium displays little if any binding to HAin vitro, and this has led to the conclusion that the native receptor is functionally silenced, a feature that is difficult to reconcile with its proposedin vivofunctions. Nonetheless, as we reported recently, LYVE-1 can function as a receptor for HA-encapsulated Group A streptococci and mediate lymphatic dissemination in mice. Here we resolve these paradoxical findings and show that the capacity of LYVE-1 to bind HA is strictly dependent on avidity, demanding appropriate receptor self-association and/or HA multimerization. In particular, we demonstrate the prerequisite of a critical LYVE-1 threshold density and show that HA binding may be elicited in lymphatic endothelium by surface clustering with divalent LYVE-1 mAbs. In addition, we show that cross-linking of biotinylated HA in streptavidin multimers or supramolecular complexes with the inflammation-induced protein TSG-6 enables binding even in the absence of LYVE-1 cross-linking. Finally, we show that endogenous HA on the surface of macrophages can engage LYVE-1, facilitating their adhesion and transit across lymphatic endothelium. These results reveal LYVE-1 as a low affinity receptor tuned to discriminate between different HA configurations through avidity and establish a new mechanistic basis for the functions ascribed to LYVE-1 in matrix HA binding and leukocyte traffickingin vivo.

  16. Influence of fetuin and hyaluronan on the post-thaw quality and fertilizing ability of Holstein bull semen.

    PubMed

    Sarıözkan, Serpil; Bucak, Mustafa Numan; Tuncer, Pürhan Barbaros; Büyükleblebici, Serhat; Eken, Ayşe; Akay, Cemal

    2015-08-01

    It was determined that fetuin and hyaluronan supplementation did not provide any significant effect on the post-thaw subjective and CASA motility percentages and sperm motion characteristics, in comparison to the controls (P>0.05). Sperm acrosome and total abnormalities were similar in all groups (P>0.05). Groups M (hyaluronan+fetuin) and H (hyaluronan) displayed a higher rate of sperm membrane integrity, compared to that of Group C (control) (P<0.01). According to the results of the comet assay, the lowest percentage of sperm with damaged DNA was achieved in Group H, when compared to all of the experimental groups (P<0.01). Furthermore, all of the additives resulted in a lower rate of sperm with damaged DNA than that of the controls, and thus, reduced DNA damage (P<0.01). For pregnancy rates, there were no significant differences between the extender groups (P>0.05). MDA formation was found to be lower in Groups M and F (P<0.01). In Group M, SOD activity was determined to have significantly increased (23.61±5.62 U/ml) compared to the other groups (P<0.01). All experimental groups had a GSH-Px activity higher than that of the control group (P<0.01).

  17. Single Molecule Microscopy Reveals an Increased Hyaluronan Diffusion Rate in Synovial Fluid from Knees Affected by Osteoarthritis

    PubMed Central

    Kohlhof, Hendrik; Gravius, Sascha; Kohl, Sandro; Ahmad, Sufian S.; Randau, Thomas; Schmolders, Jan; Rommelspacher, Yorck; Friedrich, Max; Kaminski, Tim P.

    2016-01-01

    Osteoarthritis is a common and progressive joint disorder. Despite its widespread, in clinical practice only late phases of osteoarthritis that are characterized by severe joint damage are routinely detected. Since osteoarthritis cannot be cured but relatively well managed, an early diagnosis and thereby early onset of disease management would lower the burden of osteoarthritis. Here we evaluated if biophysical parameters of small synovial fluid samples extracted by single molecule microscopy can be linked to joint damage. In healthy synovial fluid (ICRS-score < 1) hyaluronan showed a slower diffusion (2.2 μm2/s, N = 5) than in samples from patients with joint damage (ICRS-score > 2) (4.5 μm2/s, N = 16). More strikingly, the diffusion coefficient of hyaluronan in healthy synovial fluid was on average 30% slower than expected by sample viscosity. This effect was diminished or missing in samples from patients with joint damage. Since single molecule microscopy needs only microliters of synovial fluid to extract the viscosity and the specific diffusion coefficient of hyaluronan this method could be of use as diagnostic tool for osteoarthritis. PMID:26868769

  18. Single Molecule Microscopy Reveals an Increased Hyaluronan Diffusion Rate in Synovial Fluid from Knees Affected by Osteoarthritis.

    PubMed

    Kohlhof, Hendrik; Gravius, Sascha; Kohl, Sandro; Ahmad, Sufian S; Randau, Thomas; Schmolders, Jan; Rommelspacher, Yorck; Friedrich, Max; Kaminski, Tim P

    2016-02-12

    Osteoarthritis is a common and progressive joint disorder. Despite its widespread, in clinical practice only late phases of osteoarthritis that are characterized by severe joint damage are routinely detected. Since osteoarthritis cannot be cured but relatively well managed, an early diagnosis and thereby early onset of disease management would lower the burden of osteoarthritis. Here we evaluated if biophysical parameters of small synovial fluid samples extracted by single molecule microscopy can be linked to joint damage. In healthy synovial fluid (ICRS-score < 1) hyaluronan showed a slower diffusion (2.2 μm(2)/s, N = 5) than in samples from patients with joint damage (ICRS-score > 2) (4.5 μm(2)/s, N = 16). More strikingly, the diffusion coefficient of hyaluronan in healthy synovial fluid was on average 30% slower than expected by sample viscosity. This effect was diminished or missing in samples from patients with joint damage. Since single molecule microscopy needs only microliters of synovial fluid to extract the viscosity and the specific diffusion coefficient of hyaluronan this method could be of use as diagnostic tool for osteoarthritis.

  19. Acetohydroxyacid synthases: evolution, structure, and function.

    PubMed

    Liu, Yadi; Li, Yanyan; Wang, Xiaoyuan

    2016-10-01

    Acetohydroxyacid synthase, a thiamine diphosphate-dependent enzyme, can condense either two pyruvate molecules to form acetolactate for synthesizing L-valine and L-leucine or pyruvate with 2-ketobutyrate to form acetohydroxybutyrate for synthesizing L-isoleucine. Because the key reaction catalyzed by acetohydroxyacid synthase in the biosynthetic pathways of branched-chain amino acids exists in plants, fungi, archaea, and bacteria, but not in animals, acetohydroxyacid synthase becomes a potential target for developing novel herbicides and antimicrobial compounds. In this article, the evolution, structure, and catalytic mechanism of acetohydroxyacid synthase are summarized.

  20. Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin

    PubMed Central

    Sager, Martin; Benten, W. Peter M.; Engelhardt, Eva; Gougoula, Christina; Benga, Laurentiu

    2015-01-01

    [Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of β-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of β-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms

  1. Purification and characterization of protein H, the major porin of Pasteurella multocida.

    PubMed Central

    Chevalier, G; Duclohier, H; Thomas, D; Shechter, E; Wróblewski, H

    1993-01-01

    Protein H (B. Lugtenberg, R. van Boxtel, D. Evenberg, M. de Jong, P. Storm, and J. Frik, Infect. Immun. 52:175-182, 1986) is the major polypeptide of the outer membrane of Pasteurella multocida, a bacterium pathogenic for humans and animals. We have purified this protein to homogeneity by size exclusion chromatography after selective extraction with surfactants and demonstrated its pore-forming ability after reincorporation into planar lipid bilayers. In these experiments, the current through the pores was a linear function of the applied voltage in the range of -50 to +50 mV. Voltages beyond +/- 50 mV tended to partially close the channels, giving rise to apparent negative resistances. These observations suggest that protein H channels are probably not voltage regulated in vivo. With the patch clamp technique, single-channel conductance fluctuations of 0.33 nS were recorded in 1 M KCl. Electrophoretic and circular dichroism analyses showed that protein H forms homotrimers stable in sodium dodecyl sulfate at room temperature, with a high content of beta-sheet secondary structure. Upon boiling, the trimers were fully dissociated into monomers with an increase of alpha helix and irregular structure, at the expense of beta sheets. The apparent molecular mass of fully denatured monomers ranged between 37 and 41.8 kDa, depending on the electrophoretic system used for analysis. The trimeric arrangement of protein H was confirmed by image analysis of negatively stained, two-dimensional crystal arrays. This morphological study revealed, in agreement with electrophoretical data, a trimeric structure with an overall diameter of 7.7 nm. Each monomer appeared to contain a pore with an average diameter of 1 nm. Quantitative comparisons revealed that the amino acid composition (hydropathy index of -0.40) and the N-terminal sequence (determined over 36 residues) of protein H are similar to those of bacterial general porins, notably porin P2 of Haemophilus influenzae. We conclude

  2. Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin.

    PubMed

    Sager, Martin; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Benga, Laurentiu

    2015-01-01

    [Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of β-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of β-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms

  3. Comparative clinicopathological changes in buffalo and cattle following infection by Pasteurella multocida B:2.

    PubMed

    Annas, S; Zamri-Saad, M; Jesse, F F A; Zunita, Z

    2015-11-01

    Haemorrhagic septicaemia (HS) is an acute, septicaemic disease of cattle and buffalo of Asia and Africa caused by Pasteurella multocida B:2 or E:2. Buffaloes are believed to be more susceptible than cattle. In this study, 9 buffaloes of 8 months old were divided equally into 3 groups (Groups 1, 3, 5). Similarly, 9 cattle of 8 months old were equally divided into 3 groups (Groups 2, 4, 6). Animals of Groups 1 and 2 were inoculated with PBS while Groups 3 and 4 were inoculated subcutaneously with 10(5) cfu/ml of P. multocida B:2. Animals of Groups 5 and 6 were inoculated intranasally with the same inoculum. Both buffaloes and cattle that were inoculated subcutaneously succumbed to the infection at 16 h and 18 h, respectively. Two buffaloes that were inoculated intranasally (Group 5) succumbed at 68 h while the remaining cattle and buffaloes survived the 72-h study period. Endotoxin was detected in the blood of infected cattle (Group 4) and buffaloes (Groups 3 and 5) prior to the detection of P. multocida B:2 in the blood. The endotoxin was detected in the blood of buffaloes of Group 3 and cattle of Group 4 at 0.5 h post-inoculation while buffaloes of Group 5 and cattle of Group 6 at 1.5 h. On the other hand, bacteraemia was detected at 2.5 h in buffaloes of Group 3 and cattle of Group 4 and at 12 h in buffaloes of Group 5 and cattle of Group 6. Affected cattle and buffaloes showed lesions typical of haemorrhagic septicaemia. These included congestion and haemorrhages in the organs of respiratory, gastrointestinal and urinary tracts with evidence of acute inflammatory reactions. The severity of gross and histopathology lesions in cattle and buffalo calves that succumbed to the infection showed insignificant (p > 0.05) difference. However, inoculated buffalo and cattle that survived the infection showed significantly (p < 0.05) less severe gross and histopathological changes than those that succumbed. In general, cattle are more resistant to intranasal infection by P

  4. Gait patterns after intraarticular treatment of patients with osteoarthritis of the Knee - Hyaluronan versus triamcinolone: a prospective, randomized, doubleblind, monocentric study

    PubMed Central

    2009-01-01

    Objective Evaluation of gait performance and muscle activity patterns as well as clinical efficacy and safety after single intraarticular injection with hyaluronan compared with triamcinolone in patients with knee osteoarthritis. Materials and Methods This trial evaluated the influence of a single injection of hyaluronan or triamcinolone on gait pattern and muscle activity. For clinical evaluation a visual analogue scale for pain, Lequesne index, and Knee Society Score were used. Quality of life was assessed with the SF-36. Results The complete analysis was performed in 50 of 60 patients. 26 patients were treated with triamcinolone and 24 with hyaluronan. Hyaluronan treatment led to significant improvement of range of motion at hip and knee. Significant improvement could be either demonstrated for the pain scale, Lequesne and Knee Society score in both groups. Quality of life showed greater improvement in the triamcinolone group. Conclusion Single application of high-viscosity hyaluronan shows superior range of motion and pain reduction as well as improvement in clinical results. Even if there was a lack of significant differences compared to triamcinolone, this therapy classified as safe and effective in the short follow up. PMID:19380288

  5. Producing biofuels using polyketide synthases

    DOEpatents

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  6. Interactive effect of chondroitin sulphate C and hyaluronan on fluid movement across rabbit synovium

    PubMed Central

    Sabaratnam, S; Coleman, P J; Badrick, E; Mason, R M; Levick, J R

    2002-01-01

    The polysaccharide hyaluronan (HA) conserves synovial fluid by keeping outflow low and almost constant over a wide pressure range (‘buffering’), but only at concentrations associated with polymer domain overlap. We therefore tested whether polymer interactions can cause buffering, using HA-chondroitin sulphate C (CSC) mixtures. Also, since it has been found that capillary filtration is insensitive to the Starling force interstitial osmotic pressure in frog mesenteries, this was assessed in synovium. Hyaluronan at non-buffering concentrations (0.50–0.75 mg ml−1) and/or 25 mg ml−1 CSC (osmotic pressure 68 cmH2O) was infused into knees of anaesthetised rabbits in vivo. Viscometry and chromatography confirmed that HA interacts with CSC. Pressure (Pj) versus trans-synovial flow (Q̇s) relations were measured. Q̇s was outwards for HA alone (1.2 ± 0.9 μl min−1 at 3 cmH2O, mean ± s.e.m.; n = 6). CSC diffused into synovium and changed Q̇s to filtration at low Pj (−4.1 μl min−1, 3 cmH2O, n = 5, P < 0.02, t test). Filtration ceased upon circulatory arrest (n = 3). At higher Pj, 0.75 mg ml−1 HA plus CSC buffered Q̇s to ∼3 μl min−1 over a wide range of Pj, with an outflow increase of only 0.04 ± 0.02 μl min−1 cmH2O−1 (n = 4). With HA or CSC alone, buffering was absent (slopes 0.57 ± 0.04 μl min−1 cmH2O−1 (n = 4) and 0.86 ± 0.05 μl min−1 cmH2O−1 (n = 5), respectively). Therefore, polymer interactions can cause outflow buffering in joints. Also, interstitial osmotic pressure promoted filtration in fenestrated synovial capillaries, so the results for frog mesentery capillaries cannot be generalised. The difference is attributed to differences in pore ultrastructure. PMID:11927686

  7. Localized RNAi therapeutics of chemoresistant grade IV glioma using hyaluronan-grafted lipid-based nanoparticles.

    PubMed

    Cohen, Zvi R; Ramishetti, Srinivas; Peshes-Yaloz, Naama; Goldsmith, Meir; Wohl, Anton; Zibly, Zion; Peer, Dan

    2015-02-24

    Glioblastoma multiforme (GBM) is one of the most infiltrating, aggressive, and poorly treated brain tumors. Progress in genomics and proteomics has paved the way for identifying potential therapeutic targets for treating GBM, yet the vast majority of these leading drug candidates for the treatment of GBM are ineffective, mainly due to restricted passages across the blood-brain barrier. Nanoparticles have been emerged as a promising platform to treat different types of tumors due to their ability to transport drugs to target sites while minimizing adverse effects. Herein, we devised a localized strategy to deliver RNA interference (RNAi) directly to the GBM site using hyaluronan (HA)-grafted lipid-based nanoparticles (LNPs). These LNPs having an ionized lipid were previously shown to be highly effective in delivering small interfering RNAs (siRNAs) into various cell types. LNP's surface was functionalized with hyaluronan (HA), a naturally occurring glycosaminoglycan that specifically binds the CD44 receptor expressed on GBM cells. We found that HA-LNPs can successfully bind to GBM cell lines and primary neurosphers of GBM patients. HA-LNPs loaded with Polo-Like Kinase 1 (PLK1) siRNAs (siPLK1) dramatically reduced the expression of PLK1 mRNA and cumulated in cell death even under shear flow that simulate the flow of the cerebrospinal fluid compared with control groups. Next, a human GBM U87MG orthotopic xenograft model was established by intracranial injection of U87MG cells into nude mice. Convection of Cy3-siRNA entrapped in HA-LNPs was performed, and specific Cy3 uptake was observed in U87MG cells. Moreover, convection of siPLK1 entrapped in HA-LNPs reduced mRNA levels by more than 80% and significantly prolonged survival of treated mice in the orthotopic model. Taken together, our results suggest that RNAi therapeutics could effectively be delivered in a localized manner with HA-coated LNPs and ultimately may become a therapeutic modality for GBM.

  8. Green synthesis, characterization, and anticancer activity of hyaluronan/zinc oxide nanocomposite

    PubMed Central

    Namvar, Farideh; Azizi, Susan; Rahman, Heshu Sulaiman; Mohamad, Rosfarizan; Rasedee, Abdullah; Soltani, Mozhgan; Rahim, Raha Abdul

    2016-01-01

    The study describes an in situ green biosynthesis of zinc oxide nanocomposite using the seaweed Sargassum muticum water extract and hyaluronan biopolymer. The morphology and optical properties of the hyaluronan/zinc oxide (HA/ZnO) nanocomposite were determined by Fourier transform infrared spectroscopy, X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy, and ultraviolet–vis analysis. Electron microscopy and X-ray diffraction analysis showed that the zinc oxide nanoparticles were polydispersed with a mean size of 10.2±1.5 nm. The nanoparticles were mostly hexagonal in crystalline form. The HA/ZnO nanocomposite showed the absorption properties in the ultraviolet zone that is ascribed to the band gap of zinc oxide nanocomposite. In the cytotoxicity study, cancer cells, pancreatic adenocarcinoma (PANC-1), ovarian adenocarcinoma (CaOV-3), colonic adenocarcinoma (COLO205), and acute promyelocytic leukemia (HL-60) cells were treated with HA/ZnO nanocomposite. At 72 hours of treatment, the half maximal inhibitory concentration (IC50) value via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was 10.8±0.3 μg/mL, 15.4±1.2 μg/mL, 12.1±0.9 μg/mL, and 6.25±0.5 μg/mL for the PANC-1, CaOV-3, COLO-205, and HL-60 cells, respectively, showing that the composite is most toxic to the HL-60 cells. On the other hand, HA/ZnO nanocomposite treatment for 72 hours did not cause toxicity to the normal human lung fibroblast (MRC-5) cell line. Using fluorescent dyes and flow cytometry analysis, HA/ZnO nanocomposite caused G2/M cell cycle arrest and stimulated apoptosis-related increase in caspase-3 and -7 activities of the HL-60 cells. Thus, the study shows that the HA/ZnO nanocomposite produced through green synthesis has great potential to be developed into an efficacious therapeutic agent for cancers. PMID:27555781

  9. Loss of the Hyaluronan Receptor RHAMM Prevents Constrictive Artery Wall Remodeling

    PubMed Central

    Ma, Xue; Pearce, Jeffrey D.; Wilson, David B.; English, William P.; Edwards, Matthew S.; Geary, Randolph L.

    2013-01-01

    Objective Constrictive extracellular matrix (ECM) remodeling contributes significantly to restenosis after arterial reconstruction but its molecular regulation is poorly defined. Hyaluronan (HA) accumulates within ECM at sites of injury where it is thought to facilitate smooth muscle cell (SMC) trafficking and collagen remodeling analogous to its role in cutaneous wound healing. SMC receptors for HA include receptor for hyaluronan-mediated motility (RHAMM), which mediates HA-induced migration. We hypothesized RHAMM would also mediate SMC-matrix interactions to alter extent of constrictive remodeling. Methods We studied the role of RHAMM in SMC attachment to collagen, migration, and contraction of collagen gels using blocking antibodies and SMC from RHAMM−/− knockout mice (rKO). We then determined the role of RHAMM in constrictive artery wall remodeling by comparing changes in wall geometry in rKO versus wild-type +/+ (WT) controls 1 month after carotid ligation. Results HA increased SMC attachment to collagen-coated plates but blocking RHAMM reduced adhesion (p=0.025). rKO SMC also demonstrated reduced adhesion (% adherent: 36.1±2.2 vs. 76.3±1.9, p< 0.05). SMC contraction of collagen gels was enhanced by HA and further increased by RHAMM blockade (p< 0.01) or knockout (gel diameter, mm: rKO, 6.7±0.1 vs. WT, 9.8±0.1, p=0.015). RHAMM promoted constrictive remodeling in vivo as carotid artery size was significantly larger in rKO mice 1 month after ligation. Neointimal thickening however was not affected in rKO (p=NS vs WT) but lumen size was significantly larger (lumen area, μm2: 52.4±1.4 × 103 vs. 10.4±1.8 × 103, p=0.01) because artery size constricted less (EEL area, μm2: rKO, 92.4±4.7×103 vs. WT, 51.3±5.9 × 103, p=0.015). Adventitial thickening and collagen deposition were also more extensive in ligated rKO carotids (adventitial thickness, μm: 218±12.2 vs. 109±7.9, p=0.01). Conclusion HA activation of RHAMM significantly impacts SMC

  10. Hyaluronan Based Heparin Free Coated Open and Closed Extracorporeal Circuits for High Risk Coronary Revascularization

    PubMed Central

    Gunaydin, Serdar; Ucar, Halil Ibrahim; Serter, Tanzer; McCusker, Kevin; Ozcelik, Gokhan; Salman, Nevriye; Yorgancioglu, Ali Cem

    2010-01-01

    Abstract: This prospective randomized study compares the inflammatory response and fibrinolytic activation of fully coated/uncoated and open/closed extracorporeal circuits (ECC) in high risk patients. Over a 2-month period, 48 patients with EuroSCOREs 6 or greater undergoing coronary revascularization were pro spectively randomized to one of the four perfusion protocols: Group 1: Closed and totally hyaluronan based heparin free coated (Vision HFO-GBS-HF™, Gish Biomedical, Rancho Santa Margarita, CA) ECC with a soft-shell coated venous reservoir (SVR11S2-HFC™, Gish Biomedical) and a hard-shell cardiotomy (CAPVRF44, Gish Biomedical) (n = 12); Group 2: Closed and totally uncoated identical ECC with soft-shell uncoated venous reservoir and a hard-shell cardiotomy (n = 12); Group 3: Open, totally hyaluronan based heparin free coated ECC (n = 12); and Group 4: Control-open, uncoated ECC (n = 12). Blood samples were collected at T1: Baseline; T2: 15 minutes after cardiopulmonary bypass (CPB) initiation; T3: before cessation of CPB; T4: 15 minutes after protamine reversal, and T5: in the intensive care unit. Serum IL-6 levels were significantly lower at T2 in all study groups, at T3 for coated groups, and T4 for closed+coated group (p < .05 versus control). Creatine kinase M-band (MB) levels in coronary sinus blood demonstrated well preserved myocardium after CPB in both coated groups versus Control (p < .05). Neutrophil CD11b/CD18 levels were significantly lower for all study groups versus control at T2, for both coated groups at T3 and only for closed+coated group at T4 (p < .05). Postoperative hemorrhage (mL) was 510 ± 40 in closed+coated and 536 ± 40 in open+coated groups (control: 784 ± 48, p ≤ .05). No significant differences in thrombin-antithrombin complex and free plasma hemoglobin were observed. Desorbed protein amount on ECC (mg/dL) was 1.7 ± .01 in closed+coated, 2.01 ± .01 in open+coated, and 3.3 ± .015 in control groups (p ≤ .05). Use of a

  11. Hyaluronan based heparin free coated open and closed extracorporeal circuits for high risk coronary revascularization.

    PubMed

    Gunaydin, Serdar; Ucar, Halil Ibrahim; Serter, Tanzer; McCusker, Kevin; Ozcelik, Gokhan; Salman, Nevriye; Yorgancioglu, Ali Cem

    2010-12-01

    This prospective randomized study compares the inflammatory response and fibrinolytic activation of fully coated/uncoated and open/closed extracorporeal circuits (ECC) in high risk patients. Over a 2-month period, 48 patients with EuroSCOREs 6 or greater undergoing coronary revascularization were prospectively randomized to one of the four perfusion protocols: Group 1: Closed and totally hyaluronan based heparin free coated (Vision HFO-GBS-HF, Gish Biomedical, Rancho Santa Margarita, CA) ECC with a soft-shell coated venous reservoir (SVR11S2-HFC, Gish Biomedical) and a hard-shell cardiotomy (CAPVRF44, Gish Biomedical) (n = 12); Group 2: Closed and totally uncoated identical ECC with soft-shell uncoated venous reservoir and a hard-shell cardiotomy (n = 12); Group 3: Open, totally hyaluronan based heparin free coated ECC (n = 12); and Group 4: Control-open, uncoated ECC (n = 12). Blood samples were collected at T1: Baseline; T2: 15 minutes after cardiopulmonary bypass (CPB) initiation; T3: before cessation of CPB; T4: 15 minutes after protamine reversal, and T5: in the intensive care unit. Serum IL-6 levels were significantly lower at T2 in all study groups, at T3 for coated groups, and T4 for closed+coated group (p < .05 versus control). Creatine kinase M-band (MB) levels in coronary sinus blood demonstrated well preserved myocardium after CPB in both coated groups versus Control (p < .05). Neutrophil CD11b/CD18 levels were significantly lower for all study groups versus control at T2, for both coated groups at T3 and only for closed + coated group at T4 (p < .05). Postoperative hemorrhage (mL) was 510 +/- 40 in closed + coated and 536 +/- 40 in open + coated groups (control: 784 +/- 48, p < .05). No significant differences in thrombin-antithrombin complex and free plasma hemoglobin were observed. Desorbed protein amount on ECC (mg/dL) was 1.7 +/- .01 in closed+coated, 2.01 +/- .01 in open+coated, and 3.3 +/- .015 in control groups (p < or = .05). Use of a closed

  12. Polyester synthases: natural catalysts for plastics.

    PubMed Central

    Rehm, Bernd H A

    2003-01-01

    Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with

  13. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    PubMed

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

  14. Cirrhosis, cellulitis and cats: a 'purrfect' combination for life-threatening spontaneous bacterial peritonitis from Pasteurella multocida.

    PubMed

    Hey, Penelope; Gow, Paul; Torresi, Joseph; Testro, Adam

    2012-11-11

    Pasteurella multocida is a Gram-negative coccobacillus that colonises the upper airways of many animals, in particular, dogs and cats. It acts as an opportunistic infection in humans following an animal bite or scratch and is associated with soft tissue infections, septicaemia and pneumonia, particularly in patients with a compromised immune response, such as patients with liver failure. Spontaneous bacterial peritonitis (SBP) is a serious complication of cirrhosis with a death rate of 10-15%. We report a case of a 47-year-old man with cirrhosis who presented with life-threatening P multocida SBP and bacteraemia secondary to a lick from a cat to a cellulitic leg wound. This case highlights the potential severity of an infection from domestic animals and an otherwise innocuous organism in an immunocompromised host.

  15. Enhanced recovery of airborne T3 coliphage and Pasteurella pestis bacteriophage by means of a presampling humidification technique.

    PubMed

    Hatch, M T; Warren, J C

    1969-05-01

    This paper reports a series of experiments in which two methods of collecting airborne bacteriophage particles were compared. A standard aerosol sampler, the AGI-30, was evaluated for its competence in measuring the content of bacteriophage aerosols. It was used alone or with a prewetting or humidification device (humidifier bulb) to recover T(3) coliphage and Pasteurella pestis bacteriophage particles from aerosols maintained at 21 C and varied relative humidity. Collection of bacteriophage particles via the humidifier bulb altered both the initial recovery level and the apparent biological decay. Sampling airborne bacteriophage particles by the AGI-30 alone yielded data that apparently underestimated the maximal number of potentially viable particles within the aerosol, sometimes by as much as 3 logs.

  16. Restriction endonuclease analysis and ribotyping differentiate Pasteurella haemolytica serotype A1 isolates from cattle within a feedlot.

    PubMed Central

    Murphy, G L; Robinson, L C; Burrows, G E

    1993-01-01

    Pasteurella haemolytica serotype A1 isolates were collected from cattle within a feedlot during an outbreak of bovine respiratory disease. Genetic heterogeneity among the isolates was examined by restriction endonuclease analysis (REA), ribotyping, and analysis of plasmid content. The susceptibilities of isolates to several antibiotics were also examined. Five different REA patterns and three different ribotypes were observed among the isolates. Fifty percent of the isolates had an identical REA type, ribotype, and plasmid profile. Examination of the plasmid content of the isolates revealed that most (73%) carry a single plasmid which encodes beta-lactamase, 13.5% carry two plasmids, and 13.5% carry no plasmid. The data reveal the presence of genetic differences among isolates of P. haemolytica A1, associated with shipping fever pneumonia within a closed feedlot, and suggest that a combination of REA, ribotyping, plasmid analysis, and antibiotic susceptibility determination will be useful in analyzing the molecular epidemiology of this disease. Images PMID:7691872

  17. BMP-2 induces versican and hyaluronan that contribute to post-EMT AV cushion cell migration.

    PubMed

    Inai, Kei; Burnside, Jessica L; Hoffman, Stanley; Toole, Bryan P; Sugi, Yukiko

    2013-01-01

    Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.

  18. Hyaluronan-modified superparamagnetic iron oxide nanoparticles for bimodal breast cancer imaging and photothermal therapy

    PubMed Central

    Yang, Rui-Meng; Fu, Chao-Ping; Fang, Jin-Zhi; Xu, Xiang-Dong; Wei, Xin-Hua; Tang, Wen-Jie; Jiang, Xin-Qing; Zhang, Li-Ming

    2017-01-01

    Theranostic nanoparticles with both imaging and therapeutic abilities are highly promising in successful diagnosis and treatment of the most devastating cancers. In this study, the dual-modal imaging and photothermal effect of hyaluronan (HA)-modified superparamagnetic iron oxide nanoparticles (HA-SPIONs), which was developed in a previous study, were investigated for CD44 HA receptor-overexpressing breast cancer in both in vitro and in vivo experiments. Heat is found to be rapidly generated by near-infrared laser range irradiation of HA-SPIONs. When incubated with CD44 HA receptor-overexpressing MDA-MB-231 cells in vitro, HA-SPIONs exhibited significant specific cellular uptake and specific accumulation confirmed by Prussian blue staining. The in vitro and in vivo results of magnetic resonance imaging and photothermal ablation demonstrated that HA-SPIONs exhibited significant negative contrast enhancement on T2-weighted magnetic resonance imaging and photothermal effect targeted CD44 HA receptor-overexpressing breast cancer. All these results indicated that HA-SPIONs have great potential for effective diagnosis and treatment of cancer. PMID:28096667

  19. CD44 Transmembrane Receptor and Hyaluronan Regulate Adult Hippocampal Neural Stem Cell Quiescence and Differentiation.

    PubMed

    Su, Weiping; Foster, Scott C; Xing, Rubing; Feistel, Kerstin; Olsen, Reid H J; Acevedo, Summer F; Raber, Jacob; Sherman, Larry S

    2017-03-17

    Adult neurogenesis in the hippocampal subgranular zone (SGZ) is involved in learning and memory throughout life but declines with aging. Mice lacking the CD44 transmembrane receptor for the glycosaminoglycan hyaluronan (HA) demonstrate a number of neurological disturbances including hippocampal memory deficits, implicating CD44 in the processes underlying hippocampal memory encoding, storage, or retrieval. Here, we found that HA and CD44 play important roles in regulating adult neurogenesis, and we provide evidence that HA contributes to age-related reductions in neural stem cell (NSC) expansion and differentiation in the hippocampus. CD44-expressing NSCs isolated from the mouse SGZ are self-renewing and capable of differentiating into neurons, astrocytes, and oligodendrocytes. Mice lacking CD44 demonstrate increases in NSC proliferation in the SGZ. This increased proliferation is also observed in NSCs grown in vitro, suggesting that CD44 functions to regulate NSC proliferation in a cell-autonomous manner. HA is synthesized by NSCs and increases in the SGZ with aging. Treating wild type but not CD44-null NSCs with HA inhibits NSC proliferation. HA digestion in wild type NSC cultures or in the SGZ induces increased NSC proliferation, and CD44-null as well as HA-disrupted wild type NSCs demonstrate delayed neuronal differentiation. HA therefore signals through CD44 to regulate NSC quiescence and differentiation, and HA accumulation in the SGZ may contribute to reductions in neurogenesis that are linked to age-related decline in spatial memory.

  20. Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells

    PubMed Central

    Vogel, Sarah; Arnoldini, Simon; Möller, Stephanie; Schnabelrauch, Matthias; Hempel, Ute

    2016-01-01

    Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC. PMID:27808176

  1. Synthesis and characterization of a Hyaluronan-polyethylene copolymer for biomedical applications.

    PubMed

    Oldinski, Rachael A; Cranson, Cody N; James, Susan P

    2010-08-01

    Hyaluronan (HA)-based biomaterials are of interest for bone and cartilage tissue engineering because HA plays an important role in orthopedic tissue development, function, and repair. The goal of this project was to develop a biomaterial that incorporated the constituents of both a hydrogel and a hydrophobic polymer for biomedical applications. A series of amphiphilic graft copolymers consisting of HA, a glycosaminoglycan, and high-density polyethylene (HDPE), that is, HA-co-HDPE, were fabricated. The chemical characteristics, physical and viscoelastic properties, and cytocompatibility of novel HA-co-HDPE materials were characterized via Fourier Transform infrared (FTIR) spectroscopy, solid state nuclear magnetic resonance (ssNMR) spectroscopy, differential scanning calorimetry (DSC), dynamic shear testing, and an in vitro human osteoblast cell study. The esterification reaction between HA and functionalized HDPE resulted in semicrystalline, insoluble powder. The dynamic shear properties of HA-co-HDPE concentrated solutions were more like natural proteoglycans than the HA control. HA-co-HDPE was successfully compression molded into disks that swelled upon hydration. Osteoblasts were viable and expressed the osteoblast phenotype after 7 days of culture on HA-co-HDPE materials. These HA-co-HDPE materials may have several biomaterial applications in saline suspension or molded form, including orthopedic tissue repair.

  2. Mechanically strong triple network hydrogels based on hyaluronan and poly(N,N-dimethylacrylamide).

    PubMed

    Tavsanli, Burak; Can, Volkan; Okay, Oguz

    2015-11-21

    Hyaluronan (HA) is a natural polyelectrolyte with distinctive biological functions. Cross-linking of HA to generate less degradable hydrogels for use in biomedical applications has attracted interest over many years. One limitation of HA hydrogels is that they are very brittle and/or easily dissolve in physiological environments, which limit their use in load-bearing applications. Herein, we describe the preparation of triple-network (TN) hydrogels based on HA and poly(N,N-dimethylacrylamide) (PDMA) of high mechanical strength by sequential gelation reactions. TN hydrogels containing 81-91% water sustain compressive stresses above 20 MPa and exhibit Young's moduli of up to 1 MPa. HA of various degrees of methacrylation was used as a multifunctional macromer for the synthesis of the brittle first-network component, while loosely cross-linked PDMA was used as the ductile, second and third network components of TN hydrogels. By tuning the methacrylation degree of HA, double-network hydrogels with a fracture stress above 10 MPa and a fracture strain of 96% were obtained. Increasing the ratio of ductile-to-brittle components via the TN approach further increases the fracture stress above 20 MPa. Cyclic mechanical tests show that, although TN hydrogels internally fracture even under small strain, the ductile components hinder macroscopic crack propagation by keeping the macroscopic gel samples together.

  3. Effect of microwave irradiation on the molecular and structural properties of hyaluronan.

    PubMed

    Bezáková, Zuzana; Hermannová, Martina; Dřímalová, Eugenie; Malovíková, Anna; Ebringerová, Anna; Velebný, Vladimír

    2008-09-05

    Hyaluronan (Na(+) salt of hyaluronic acid, HA) was extensively depolymerised by HCl-catalyzed hydrolysis at pH 3 for up to 500min under temperature-controlled microwave irradiation. The effects of microwave heating on the hydrodynamic properties of the polysaccharide were determined by SEC-MALLS and viscometry. The weight-average molecular mass (Mw) of HA decreased from 1.44×10(6) to ∼5000, reaching the region of higher oligosaccharides. The scission of HA chains was found to proceed randomly during the whole degradation process. Treatment of the Mw and intrinsic viscosity data according to the Mark-Houwink equation, [η]=k×Mw(α) suggested three relationships with α1=0.46 for Mw>500,000, α2=0.84 for Mw between 500,000 and 50,000, and α3=1.13 for Mw<50,000. The results revealed that HA with Mw>10,000 adopts a stiffish coil conformation in solution. As monitored by FT-IR and NMR spectroscopic techniques, the primary structure of the HA chains was maintained during the microwave-assisted hydrolysis at pH 3 at 105°C. At reaction times larger than 240min, uv spectroscopy suggested the depolymerisation of HA was accompanied by formation of by-products produced by side reaction.

  4. Novel synthetic method for the preparation of amphiphilic hyaluronan by means of aliphatic aromatic anhydrides.

    PubMed

    Huerta-Angeles, Gloria; Bobek, Martin; Příkopová, Eva; Šmejkalová, Daniela; Velebný, Vladimír

    2014-10-13

    The present work describes a novel and efficient method of synthesis of amphiphilic hyaluronan (HA) by esterification with alkyl fatty acids. These derivatives were synthesized under mild aqueous and well controlled conditions using mixed aliphatic aromatic anhydrides. These anhydrides characterized by the general formula RCOOCOC6H2Cl3 can be easily prepared by the reaction of the corresponding fatty acid (R) with 2,4,6-trichlorobenzoyl chloride (TCBC) in the presence of triethylamine. The aliphatic aromatic anhydrides RCOOCOC6H2Cl3 then react with the polysaccharide and enable the synthesis of aliphatic acid esters of HA in good yields. No hydrolytic degradation of hyaluronic acid could be observed. Parameters controlling the degree of esterification were systematically studied. Fatty acids with different chain lengths can be introduced applying this methodology. The degree of substitution was decreasing with increasing length of hydrophobic chain. The reaction products were fully characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), SEC-MALLS and chromatographic analyses. Although the esterified HA products exhibited aggregation in solution as demonstrated by NMR, microscopy and rheology, they were still water-soluble.

  5. Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization

    PubMed Central

    Kuipers, Hedwich F.; Rieck, Mary; Gurevich, Irina; Nagy, Nadine; Negrin, Robert S.; Wight, Thomas N.; Steinman, Lawrence; Bollyky, Paul L.

    2016-01-01

    The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3+ regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS. PMID:26787861

  6. In vivo carotid artery closure by laser activation of hyaluronan-embedded gold nanorods

    NASA Astrophysics Data System (ADS)

    Matteini, Paolo; Ratto, Fulvio; Rossi, Francesca; Rossi, Giacomo; Esposito, Giuseppe; Puca, Alfredo; Albanese, Alessio; Maira, Giulio; Pini, Roberto

    2010-07-01

    We prove the first application of near-infrared-absorbing gold nanorods (GNRs) for in vivo laser closure of a rabbit carotid artery. GNRs are first functionalized with a biopolymeric shell and then embedded in hyaluronan, which gives a stabilized and handy laser-activable formulation. Four rabbits undergo closure of a 3-mm longitudinal incision performed on the carotid artery by means of a 810-nm diode laser in conjunction with the topical application of the GNRs composite. An effective surgery is obtained by using a 40-W/cm2 laser power density. The histological and electron microscopy evaluation after a 30-day follow-up demonstrates complete healing of the treated arteries with full re-endothelization at the site of GNRs application. The absence of microgranuloma formation and/or dystrophic calcification is evidence that no host reaction to nanoparticles interspersed through the vascular tissue occurred. The observation of a reshaping and associated blue shift of the NIR absorption band of GNRs after laser treatment supports the occurrence of a self-terminating process, and thus of additional safety of the minimally invasive laser procedure. This study underlines the feasibility of using GNRs for in vivo laser soldering applications, which represents a step forward toward the introduction of nanotechnology-based therapies in minimally invasive clinical practices.

  7. Oleyl-hyaluronan micelles loaded with upconverting nanoparticles for bio-imaging

    NASA Astrophysics Data System (ADS)

    Pospisilova, Martina; Mrazek, Jiri; Matuska, Vit; Kettou, Sofiane; Dusikova, Monika; Svozil, Vit; Nesporova, Kristina; Huerta-Angeles, Gloria; Vagnerova, Hana; Velebny, Vladimir

    2015-09-01

    Hyaluronan (HA) represents an interesting polymer for nanoparticle coating due to its biocompatibility and enhanced cell interaction via CD44 receptor. Here, we describe incorporation of oleate-capped β-NaYF4:Yb3+, Er3+ nanoparticles (UCNP-OA) into amphiphilic HA by microemulsion method. Resulting structures have a spherical, micelle-like appearance with a hydrodynamic diameter of 180 nm. UCNP-OA-loaded HA micelles show a good stability in PBS buffer and cell culture media. The intensity of green emission of UCNP-OA-loaded HA micelles in water is about five times higher than that of ligand-free UCNP, indicating that amphiphilic HA effectively protects UCNP luminescence from quenching by water molecules. We found that UCNP-OA-loaded HA micelles in concentrations up to 50 μg mL-1 increase cell viability of normal human dermal fibroblasts (NHDF), while viability of human breast adenocarcinoma cells MDA-MB-231 is reduced at these concentrations. The utility of UCNP-OA-loaded HA micelles as a bio-imaging probe was demonstrated in vitro by successful labelling of NHDF and MDA-MB-231 cells overexpressing the CD44 receptor.

  8. Expression of hyaluronan (hyaluronic acid) in the developing laminar architecture of the human fetal brain.

    PubMed

    Shibata, Shunichi; Cho, Kwang Ho; Kim, Ji Hyun; Abe, Hiroshi; Murakami, Gen; Cho, Baik Hwan

    2013-10-01

    Hyaluronan (also called hyaluronic acid or HA) plays a key role in the morphogenesis of the brain, but little is known about its expression in the human fetal neocortex. Using immunohistochemical methods, we assayed the expression of HA, glial fibrillary acidic protein, vimentin, nestin, and proliferating cell nuclear antigen in paraffin-embedded histologic sections of 8 mid-term fetuses (estimated gestational age, 12-16 weeks; crown-rump length, 75-120mm). At 12-13 weeks, HA was expressed strongly along the membranes of many cells in the cortical plate and the layer 1 or marginal zone, but showed weak, spotty expression in a fiber-rich layer adjacent to the cortical plate, called the cortical stratified transitional field-1 (STF-1 or a primitive form of the subplate). At 15-16 weeks, HA was expressed in the layer 1 and in the early subplate or presubplate, but less strongly in cells of the possible STF-5 near the subventricular zone. However, the positive observation in STF-5 was probably a result of individual difference in development. The developing cortical plate seemed to produce HA in the presubplate to harbor axonal plexus of various afferent systems, while Cajal-Retzius cells were likely to accumulate HA in the layer 1. The HA-rich zones, those sandwiched the cortical plate, might avoid further migration of cortical cells.

  9. Layilin, a cell surface hyaluronan receptor, interacts with merlin and radixin

    SciTech Connect

    Bono, Petri; Cordero, Etchell; Johnson, Kristen; Borowsky, Mark; Ramesh, Vijaya; Jacks, Tyler; Hynes, Richard O. . E-mail: rohynes@mit.edu

    2005-08-01

    Layilin is a widely expressed integral membrane hyaluronan receptor, originally identified as a binding partner of talin located in membrane ruffles. We have identified merlin, the neurofibromatosis type 2 tumor suppressor protein and radixin, as other interactors with the carboxy-terminal domain of layilin. We show that the carboxy-terminal domain of layilin is capable of binding to the amino-terminal domain of radixin. An interdomain interaction between the amino- and the carboxy-terminal domains of radixin inhibits its ability to bind to layilin. In the presence of acidic phospholipids, the interdomain interaction of radixin is inhibited and layilin can bind to full-length radixin. In contrast, layilin binds both full-length and amino-terminal merlin-GST fusion proteins without a requirement for phospholipids. Furthermore, layilin antibody can immunoprecipitate merlin, confirming association in vivo between these two proteins, which also display similar subcellular localizations in ruffling membranes. No interaction was observed between layilin and ezrin or layilin and moesin. These findings expand the known binding partners of layilin to include other members of the talin/band 4.1/ERM (ezrin, radixin, and moesin) family of cytoskeletal-membrane linker molecules. This in turn suggests that layilin may mediate signals from extracellular matrix to the cell cytoskeleton via interaction with different intracellular binding partners and thereby be involved in the modulation of cortical structures in the cell.

  10. Elasticity, biodegradability and cell adhesive properties of chitosan/hyaluronan multilayer films

    NASA Astrophysics Data System (ADS)

    Schneider, Aurore; Richert, Ludovic; Francius, Gregory; Voegel, Jean-Claude; Picart, Catherine

    2007-03-01

    In the bioengineering field, a recent and promising approach to modifying biomaterial surfaces is the layer-by-layer (LbL) technique used to build thin polyelectrolyte multilayer films. In this work, we focused on polyelectrolyte multilayer films made of two polysaccharides, chitosan (CHI) and hyaluronan (HA), and on the control of their physico-chemical and cell adhesive properties by chemical cross-linking. CHI/HA films were cross-linked using a water soluble carbodiimide and observed by confocal laser scanning microscopy (CLSM) with a fluorescently labeled CHI. Film thicknesses were similar for native and cross-linked films. The film nanometer roughness was measured by atomic force microscopy and was found to be higher for cross-linked films. Cross-linking the films also leads to a drastic change in film stiffness. The elastic modulus of the films (Young's modulus) as measured by AFM nano-indentation was about tenfold increased for cross-linked films as compared to native ones. From a biological point of view, cross-liked films are more resistant to enzymatic degradation by hyaluronidase. Furthermore, the increase in film stiffness has a favorable effect on the adhesion and spreading of chondrosarcoma cells. Thus, the CHI/HA cross-linked films could be used for various applications due to their adhesive properties and to their mechanical properties (including stability in enzymatic media).

  11. Hyaluronan-phosphatidylethanolamine polymers form pericellular coats on keratinocytes and promote basal keratinocyte proliferation.

    PubMed

    Symonette, Caitlin J; Kaur Mann, Aman; Tan, Xiao Cherie; Tolg, Cornelia; Ma, Jenny; Perera, Francisco; Yazdani, Arjang; Turley, Eva A

    2014-01-01

    Aged keratinocytes have diminished proliferative capacity and hyaluronan (HA) cell coats, which are losses that contribute to atrophic skin characterized by reduced barrier and repair functions. We formulated HA-phospholipid (phosphatidylethanolamine, HA-PE) polymers that form pericellular coats around cultured dermal fibroblasts independently of CD44 or RHAMM display. We investigated the ability of these HA-PE polymers to penetrate into aged mouse skin and restore epidermal function in vivo. Topically applied Alexa(647)-HA-PE penetrated into the epidermis and dermis, where it associated with both keratinocytes and fibroblasts. In contrast, Alexa(647)-HA was largely retained in the outer cornified layer of the epidermis and quantification of fluorescence confirmed that significantly more Alexa(647)-HA-PE penetrated into and was retained within the epidermis than Alexa(647)-HA. Multiple topical applications of HA-PE to shaved mouse skin significantly stimulated basal keratinocyte proliferation and epidermal thickness compared to HA or vehicle cream alone. HA-PE had no detectable effect on keratinocyte differentiation and did not promote local or systemic inflammation. These effects of HA-PE polymers are similar to those reported for endogenous epidermal HA in youthful skin and show that topical application of HA-PE polymers can restore some of the impaired functions of aged epidermis.

  12. Signal pathways regulating hyaluronan secretion into static and cycled synovial joints of rabbits.

    PubMed

    Ingram, K R; Wann, A K T; Wingate, R M; Coleman, P J; McHale, N; Levick, J R

    2009-09-01

    Joint lubrication, synovial fluid conservation and many pathophysiological processes depend on hyaluronan (HA). Intra-articular HA injection and exercise, which stimulates articular HA production, ameliorate osteoarthritis. We therefore investigated the pathways regulating movement-stimulated articular HA secretion rate ( ) in vivo. Endogenous HA was removed from the knee joint cavity of anaesthetised rabbits by washout. Joints were then cycled passively or remained static for 5 h, with/without intra-articular agonist/inhibitor, after which newly secreted HA was harvested for analysis. Movement almost doubled . Similar or larger increases were elicited in static joints by the intra-articular Ca(2+) ionophore ionomycin, prostaglandin E(2), cAMP-raising agents, serine/threonine phosphatase inhibitor and activation of protein kinase C (PKC). PKC-stimulated secretion was inhibited by the PKC inhibitor bisindolylmaleimide I and inhibitors of the downstream kinases MEK-ERK (U0126, PD98059). These agents inhibited movement-stimulated secretion of HA (MSHA) only when the parallel p38 kinase path was simultaneously inhibited by SB203580 (ineffective alone). The phospholipase C inhibitor U73122 almost fully blocked MSHA (P = 0.001, n = 10), without affecting static . The ENaC channel blocker amiloride inhibited MSHA, whereas other inhibitors of stretch-activated channels (Gd(3+), ruthenium red, SKF96365) did not. It is proposed that MSHA may be mediated by PLC activation, leading to activation of parallel PKC-MEK-ERK and p38 kinase pathways.

  13. Cyclic movement stimulates hyaluronan secretion into the synovial cavity of rabbit joints.

    PubMed

    Ingram, K R; Wann, A K T; Angel, C K; Coleman, P J; Levick, J R

    2008-03-15

    The novel hypothesis that the secretion of the joint lubricant hyaluronan (HA) is coupled to movement has implications for normal function and osteoarthritis, and was tested in the knee joints of anaesthetized rabbits. After washing out the endogenous synovial fluid HA (miscibility coefficient 0.4), secretion into the joint cavity was measured over 5 h in static joints and in passively cycled joints. The net static secretion rate (11.2 +/- 0.7 microg h(-1), mean +/- s.e.m., n = 90) correlated with the variable endogenous HA mass (mean 367 +/- 8 microg), with a normalized value of 3.4 +/- 0.2 microg h(-1) (100 microg)(-1) . Cyclic joint movement approximately doubled the net HA secretion rate to 22.6 +/- 1.2 microg h(-1) (n = 77) and raised the normalized percentage to 5.9 +/- 0.3 microg h(-1) (100 microg)(-1). Secretion was inhibited by 2-deoxyglucose and iodoacetate, confirming active secretion. The net accumulation rate underestimated true secretion rate due to some trans-synovial loss. HA turnover time (endogenous mass/secretion rate) was 17-30 h (static) to 8-15 h (moved) The results demonstrate for the first time that the active secretion of HA is coupled to joint usage. Movement-secretion coupling may protect joints against the damaging effects of repetitive joint use, replace HA lost during periods of immobility (overnight), and contribute to the clinical benefit of exercise therapy in moderate osteoarthritis.

  14. Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization.

    PubMed

    Kuipers, Hedwich F; Rieck, Mary; Gurevich, Irina; Nagy, Nadine; Butte, Manish J; Negrin, Robert S; Wight, Thomas N; Steinman, Lawrence; Bollyky, Paul L

    2016-02-02

    The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3(+) regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

  15. Hyaluronan nanoparticles bearing γ-secretase inhibitor: in vivo therapeutic effects on rheumatoid arthritis.

    PubMed

    Heo, Roun; Park, Jong-Sung; Jang, Hye Jin; Kim, Seol-Hee; Shin, Jung Min; Suh, Yung Doug; Jeong, Ji Hoon; Jo, Dong-Gyu; Park, Jae Hyung

    2014-10-28

    γ-Secretase inhibitors which prevent Notch activation are emerging as potent therapeutics for various inflammatory diseases, including ischemic stroke and rheumatoid arthritis. However, their indiscriminate distribution in the body causes serious side effects after systemic administration, since Notch proteins are ubiquitous receptors that play an important role in cellular functions such as differentiation, proliferation, and apoptosis. In this study, hyaluronan nanoparticles (HA-NPs) bearing a γ-secretase inhibitor (DAPT) were prepared as potential therapeutics for rheumatoid arthritis. In vivo biodistribution of the DAPT-loaded HA-NPs (DNPs), labeled with near-infrared dye, were observed using a non-invasive optical imaging system after systemic administration to a collagen-induced arthritis (CIA) mouse model. The results demonstrated that DNPs were effectively accumulated at the inflamed joint of the CIA mice. From the in vivo therapeutic efficacy tests, DNPs (1mg DAPT/kg) significantly attenuated the severity of RA induction compared to DAPT alone (2mg/kg), which was judged from clinical scores, tissue damage, and neutrophil infiltration. In addition, DNPs dramatically reduced the production of pro-inflammatory cytokines (TNF-α, IFN-γ, MCP-1, and IL-6, -12, -17) and collagen-specific auto-antibodies (IgG1 and IgG2a) in the serum of the CIA mice. These results suggest that DNPs have potential as therapeutics for rheumatoid arthritis.

  16. Computational Study of Nanosized Drug Delivery from Cyclodextrins, Crown Ethers and Hyaluronan in Pharmaceutical Formulations.

    PubMed

    Torrens, Francisco; Castellano, Gloria

    2015-01-01

    The problem in this work is the computational characterization of cyclodextrins, crown ethers and hyaluronan (HA) as hosts of inclusion complexes for nanosized drug delivery vehicles in pharmaceutical formulations. The difficulty is addressed through a computational study of some thermodynamic, geometric and topological properties of the hosts. The calculated properties of oligosaccharides of D-glucopyranoses allow these to act as co-solvents of polyanions in water. In crown ethers, the central channel is computed. Mucoadhesive polymer HA in formulations releases drugs in mucosas. Geometric, topological and fractal analyses are carried out with code TOPO. Reference calculations are performed with code GEPOL. From HA to HA·3Ca and hydrate, the hydrophilic solvent-accessible surface varies with the count of H-bonds. The fractal dimension rises. The dimension of external atoms rises resulting 1.725 for HA. It rises going to HA·3Ca and hydrate. Nonburied minus molecular dimension rises and decays. Hydrate globularity is lower than O(water), Ca(2+) and O(HA). Ca(2+) rugosity is smaller than for hydrate, O(HA) and O(water). Ca(2+) and O(water) accessibilities are greater than hydrate. Conclusions are drawn on: (1) the relative stability of linear/cyclic and shorter/larger polymers; (2) the atomic analysis of properties allows determining the atoms with maximum reactivity.

  17. Fibronectin functional domains coupled to hyaluronan stimulate adult human dermal fibroblast responses critical for wound healing.

    PubMed

    Ghosh, Kaustabh; Ren, Xiang-Dong; Shu, Xiao Zheng; Prestwich, Glenn D; Clark, Richard A F

    2006-03-01

    Fibronectin (FN) facilitates dermal fibroblast migration during normal wound healing. Proteolytic degradation of FN in chronic wounds hampers healing. Previously, three FN functional domains (FNfd) have been shown to be sufficient for optimal adult human dermal fibroblast migration. Here we report the development of an acellular hydrogel matrix comprised of the FNfds coupled to a hyaluronan (HA) backbone to stimulate wound repair. Employing Michael-type addition, the cysteine- tagged FNfds were first coupled to a homobifunctional PEG derivative. Thereafter, these PEG derivative FNfd solutions, containing bifunctional PEG-derivative crosslinker were coupled to thiol-modified HA (HA-DTPH) to obtain a crosslinked hydrogel matrix. When evaluated in vitro, these acellular hydrogels were completely cytocompatible. While spreading and proliferation of adult human dermal fibroblasts plateaued at higher FNfd bulk densities, their rapid and robust migration followed a typical bell-shaped response. When implanted in porcine cutaneous wounds, these acellular matrices, besides being completely biocompatible, induced rapid and en masse recruitment of stromal fibroblasts that was not observed with RGD-tethered or unmodified hydrogels. Such constructs might be of great benefit in clinical settings where rapid formation of new tissue is needed.

  18. Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro.

    PubMed

    Zhao, Ningbo; Wang, Xin; Qin, Lei; Guo, Zhengze; Li, Dehua

    2015-09-25

    Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation.

  19. Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells

    NASA Astrophysics Data System (ADS)

    Vogel, Sarah; Arnoldini, Simon; Möller, Stephanie; Schnabelrauch, Matthias; Hempel, Ute

    2016-11-01

    Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

  20. Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

    PubMed Central

    Secundino, Ismael; Lizcano, Anel; Roupé, K. Markus; Wang, Xiaoxia; Cole, Jason N.; Olson, Joshua; Ali, S. Raza; Dahesh, Samira; Amayreh, Lenah K.; Henningham, Anna; Varki, Ajit; Nizet, Victor

    2015-01-01

    Inhibitory CD33-related Siglec receptors regulate immune cell activation upon engaging ubiquitous sialic acids (Sias) on host cell surface glycans. Through molecular mimicry, Sia-expressing pathogen group B Streptococcus binds inhibitory human Siglec-9 (hSiglec-9) to blunt neutrophil activation and promote bacterial survival. We unexpectedly discovered that hSiglec-9 also specifically binds high molecular weight hyaluronan (HMW-HA), another ubiquitous host glycan, through a region of its terminal Ig-like V-set domain distinct from the Sia-binding site. HMW-HA recognition by hSiglec-9 limited neutrophil extracellular trap (NET) formation, oxidative burst, and apoptosis, defining HMW-HA as a regulator of neutrophil activation. However, the pathogen group A Streptococcus (GAS) expresses a HMW-HA capsule that engages hSiglec-9, blocking NET formation and oxidative burst, thereby promoting bacterial survival. Thus, a single inhibitory lectin receptor detects two distinct glycan “self-associated molecular patterns” to maintain neutrophil homeostasis, and two leading human bacterial pathogens have independently evolved molecular mimicry to exploit this immunoregulatory mechanism. PMID:26411873

  1. Hyaluronan's Role in Fibrosis: A Pathogenic Factor or a Passive Player?

    PubMed Central

    Albeiroti, Sami; Soroosh, Artin; de la Motte, Carol A.

    2015-01-01

    Fibrosis is a debilitating condition that can lead to impairment of the affected organ's function. Excessive deposition of extracellular matrix (ECM) molecules is characteristic of most fibrotic tissues. Fibroblasts activated by cytokines or growth factors differentiate into myofibroblasts that drive fibrosis by depositing ECM molecules, such as collagen, fibronectin, and connective tissue growth factor. Transforming growth factor-β (TGF-β) is one of the major profibrotic cytokines which promotes fibrosis by signaling abnormal ECM regulation. Hyaluronan (HA) is a major ECM glycosaminoglycan that is regulated by TGF-β and whose role in fibrosis is emerging. Aside from its role as a hydrating, space filling polymer, HA regulates different cellular functions and is known to have a role in wound healing and inflammation. Importantly, HA deposition is increased in multiple fibrotic diseases. In this review we highlight studies that link HA to fibrosis and discuss what is known about the role of HA, its receptors, and its anabolic and catabolic enzymes in different fibrotic diseases. PMID:26583132

  2. Hyaluronan-Phosphatidylethanolamine Polymers Form Pericellular Coats on Keratinocytes and Promote Basal Keratinocyte Proliferation

    PubMed Central

    Symonette, Caitlin J.; Tan, Xiao Cherie; Tolg, Cornelia; Ma, Jenny; Perera, Francisco; Turley, Eva A.

    2014-01-01

    Aged keratinocytes have diminished proliferative capacity and hyaluronan (HA) cell coats, which are losses that contribute to atrophic skin characterized by reduced barrier and repair functions. We formulated HA-phospholipid (phosphatidylethanolamine, HA-PE) polymers that form pericellular coats around cultured dermal fibroblasts independently of CD44 or RHAMM display. We investigated the ability of these HA-PE polymers to penetrate into aged mouse skin and restore epidermal function in vivo. Topically applied Alexa647-HA-PE penetrated into the epidermis and dermis, where it associated with both keratinocytes and fibroblasts. In contrast, Alexa647-HA was largely retained in the outer cornified layer of the epidermis and quantification of fluorescence confirmed that significantly more Alexa647-HA-PE penetrated into and was retained within the epidermis than Alexa647-HA. Multiple topical applications of HA-PE to shaved mouse skin significantly stimulated basal keratinocyte proliferation and epidermal thickness compared to HA or vehicle cream alone. HA-PE had no detectable effect on keratinocyte differentiation and did not promote local or systemic inflammation. These effects of HA-PE polymers are similar to those reported for endogenous epidermal HA in youthful skin and show that topical application of HA-PE polymers can restore some of the impaired functions of aged epidermis. PMID:25276814

  3. Hyaluronan in Peritumoral Stroma and Malignant Cells Associates with Breast Cancer Spreading and Predicts Survival

    PubMed Central

    Auvinen, Päivi; Tammi, Raija; Parkkinen, Jyrki; Tammi, Markku; Ågren, Ulla; Johansson, Risto; Hirvikoski, Pasi; Eskelinen, Matti; Kosma, Veli-Matti

    2000-01-01

    Hyaluronan (HA) is an extracellular matrix polysaccharide that promotes cell migration through its cell surface receptors and by effecting changes in the physical environment. HA expression is frequently increased in malignant tumors, whereas its association with the invasive potential and patient outcome in breast cancer has not been reported. The localization and signal intensity of HA was analyzed in 143 paraffin-embedded tumor samples of human breast carcinoma using a biotinylated HA-specific probe. In the immediate peritumoral stroma, HA signal was moderately or strongly increased in 39% and 56% of the cases, respectively. Normal ductal epithelium showed no HA, whereas in 57% of the tumors at least some of the carcinoma cells were HA positive. The intensity of the stromal HA signal and the presence of cell-associated HA were both significantly related to poor differentiation of the tumors, axillary lymph node positivity, and short overall survival of the patients. In Cox’s multivariate analysis, both the intensity of stromal HA signal alone and that combined with the HA positivity in tumor cells were independent prognostic factors for overall survival. These results suggest that HA is directly involved in the spreading of breast cancer and may offer a potential target for new therapies. PMID:10666382

  4. Hyaluronan – A Functional and Structural Sweet Spot in the Tissue Microenvironment

    PubMed Central

    Monslow, James; Govindaraju, Priya; Puré, Ellen

    2015-01-01

    Transition from homeostatic to reactive matrix remodeling is a fundamental adaptive tissue response to injury, inflammatory disease, fibrosis, and cancer. Alterations in architecture, physical properties, and matrix composition result in changes in biomechanical and biochemical cellular signaling. The dynamics of pericellular and extracellular matrices, including matrix protein, proteoglycan, and glycosaminoglycan modification are continually emerging as essential regulatory mechanisms underlying cellular and tissue function. Nevertheless, the impact of matrix organization on inflammation and immunity in particular and the consequent effects on tissue healing and disease outcome are arguably under-studied aspects of adaptive stress responses. Herein, we review how the predominant glycosaminoglycan hyaluronan (HA) contributes to the structure and function of the tissue microenvironment. Specifically, we examine the evidence of HA degradation and the generation of biologically active smaller HA fragments in pathological settings in vivo. We discuss how HA fragments versus nascent HA via alternate receptor-mediated signaling influence inflammatory cell recruitment and differentiation, resident cell activation, as well as tumor growth, survival, and metastasis. Finally, we discuss how HA fragmentation impacts restoration of normal tissue function and pathological outcomes in disease. PMID:26029216

  5. Neocartilage formation from mesenchymal stem cells grown in type II collagen-hyaluronan composite scaffolds.

    PubMed

    Yeh, Hsi-Yi; Lin, Ting-Yu; Lin, Chen-Huan; Yen, B Linju; Tsai, Ching-Lin; Hsu, Shan-Hui

    2013-01-01

    Three-dimensional (3D) collagen type II-hyaluronan (HA) composite scaffolds (CII-HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII-HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII-HA films and SIS sheets upon TGF-β3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII-HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII-HA constructs into NOD-SCID mice confirmed the formation of tissue-engineered cartilage in vivo.

  6. Mechanical properties of tyramine substituted-hyaluronan enriched fascia extracellular matrix.

    PubMed

    Chin, Likang; Calabro, Anthony; Walker, Esteban; Derwin, Kathleen A

    2012-03-01

    Naturally occurring biomaterial scaffolds derived from extracellular matrix (ECM) have been the topic of recent investigation in the context of rotator cuff tendon repair. We previously reported a method to treat fascia ECM with high molecular weight tyramine substituted-hyaluronan (TS-HA) for use as a tendon augmentation scaffold. The presence of crosslinked TS-HA in fascia was associated with an increased macrophage and giant cell response compared to water-treated controls after implantation in a rat abdominal wall model. The objective of this study was to determine the extent to which TS-HA treatment was associated with mechanical property changes of fascia after implantation in the rat model. Fascia samples in all groups demonstrated time-dependent decreases in mechanical properties. TS-HA-treated fascia with crosslinking exhibited a lower toe modulus, a trend toward lower toe stiffness, and a higher transition strain than water-treated controls not only after implantation, but also at time zero. TS-HA treatment, with or without crosslinking, had no significant effect on time-zero or post-implantation load relaxation ratio, load relaxation rate, linear-region stiffness, or linear-region modulus. Our findings demonstrated that the particular TS-HA treatment employed in this study decreased the low-load elastic mechanical properties of fascia ECM, in keeping with the heightened macrophage and giant cell host response seen previously. This work provides a starting point and guidance for investigating alternative HA treatment strategies.

  7. Circular Halbach array for fast magnetic separation of hyaluronan-expressing tissue progenitors.

    PubMed

    Joshi, Powrnima; Williams, P Stephen; Moore, Lee R; Caralla, Tonya; Boehm, Cynthia; Muschler, George; Zborowski, Maciej

    2015-10-06

    Connective tissue progenitors (CTPs) are a promising therapeutic agent for bone repair. Hyaluronan, a high molecular mass glycosaminoglycan, has been shown by us to be a suitable biomarker for magnetic separation of CTPs from bone marrow aspirates in a canine model. For the therapy to be applicable in humans, the magnetic separation process requires scale-up without compromising the viability of the cells. The scaled-up device presented here utilizes a circular Halbach array of diametrically magnetized, cylindrical permanent magnets. This allows precise control of the magnetic field gradient driving the separation, with theoretical analysis favoring a hexapole field. The separation vessel has the external diameter of a 50 mL conical centrifuge tube and has an internal rod that excludes cells from around the central axis. The magnet and separation vessel (collectively dubbed the hexapole magnet separator or HMS) was tested on four human and four canine bone marrow aspirates. Each CTP-enriched cell product was tested using cell culture bioassays as surrogates for in vivo engraftment quality. The magnetically enriched cell fractions showed statistically significant, superior performance compared to the unenriched and depleted cell fractions for all parameters tested, including CTP prevalence (CTPs per 10(6) nucleated cells), proliferation by colony forming unit (CFU) counts, and differentiation by staining for the presence of osteogenic and chondrogenic cells. The simplicity and speed of the HMS operation could allow both CTP isolation and engraftment during a single surgical procedure, minimizing trauma to patients and lowering cost to health care providers.

  8. Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan.

    PubMed

    Cowman, Mary K; Chen, Cherry C; Pandya, Monika; Yuan, Han; Ramkishun, Dianne; LoBello, Jaclyn; Bhilocha, Shardul; Russell-Puleri, Sparkle; Skendaj, Eraldi; Mijovic, Jovan; Jing, Wei

    2011-10-01

    The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agarose gel electrophoresis method for analysis of high molecular mass HA is presented and validated. HA mobility in 0.5% agarose minigels was found to be linearly related to the logarithm of molecular mass in the range from approximately 200 to 6000 kDa. A sample load of 2.5 μg for polydisperse HA samples was employed. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in the sample as well as calculation of weight-average and number-average values. The method was validated for a polydisperse HA sample with a weight-average molecular mass of approximately 2000 kDa. Excellent agreement was found between the weight-average molecular mass determined by electrophoresis and that determined by rheological measurement of the solution viscosity. The revised method was then used to show that heating solutions of HA at 100°C, followed by various cooling procedures, had no effect on the HA molecular mass distribution.

  9. Hyaluronan/Tween 80-assisted synthesis of silver nanoparticles for biological application

    NASA Astrophysics Data System (ADS)

    Li, Hui-Jun; Zhang, An-Qi; Sui, Li; Qian, Dong-Jin; Chen, Meng

    2015-02-01

    Water-soluble and well-stabilized silver nanoparticles (NPs) of small size have been synthesized using hyaluronan (HA) and Tween 80 as reducing and stabilizing agents. The effect of reaction conditions on the formation process of silver NPs was studied, and an aggregative growth mechanism of the silver NPs dominated in HA/Tween 80 system at pH 12 has been proposed. The obtained Ag NPs were characterized by UV-Vis spectroscopy, transmission electron microscopy, X-ray powder diffraction, and X-ray photoelectron spectroscopy. Moreover, the stability of the HA-Tween 80-silver NPs in normal saline was also studied, and a flexible blend membrane containing chitosan, gelatin, and the HA-Tween 80-silver NPs was prepared for further biological applications. Due to the high specific surface area and improved stability of silver NPs, the chitosan-gelatin-silver membrane has shown high antibacterial activity for strains of Escherichia coli. The cell viability tests indicate that the polymer membrane is non-cytotoxic to HepG2 cells, which might be attributed to its good biocompatibility.

  10. Novel enzymatically cross-linked hyaluronan hydrogels support the formation of 3D neuronal networks.

    PubMed

    Broguiere, Nicolas; Isenmann, Luca; Zenobi-Wong, Marcy

    2016-08-01

    Hyaluronan (HA) is an essential component of the central nervous system's extracellular matrix and its high molecular weight (MW) form has anti-inflammatory and anti-fibrotic properties relevant for regenerative medicine. Here, we introduce a new hydrogel based on high MW HA which is cross-linked using the transglutaminase (TG) activity of the activated blood coagulation factor XIII (FXIIIa). These HA-TG gels have significant advantages for neural tissue engineering compared to previous HA gels. Due to their chemical inertness in the absence of FXIIIa, the material can be stored long-term, is stable in solution, and shows no cytotoxicity. The gelation is completely cell-friendly due to the specificity of the enzyme and the gelation rate can be tuned from seconds to hours at physiological pH and independently of stiffness. The gels are injectable, and attach covalently to fibrinogen and fibrin, two common bioactive components in in vitro tissue engineering, as well as proteins present in vivo, allowing the gels to covalently bind to brain or spinal cord defects. These optimal chemical and bioactive properties of HA-TG gels enabled the formation of 3D neuronal cultures of unprecedented performance, showing fast neurite outgrowth, axonal and dendritic speciation, strong synaptic connectivity in 3D networks, and rapidly-occurring and long-lasting coordinated electrical activity.

  11. Hyaluronan modulates TRPV1 channel opening, reducing peripheral nociceptor activity and pain

    PubMed Central

    Caires, Rebeca; Luis, Enoch; Taberner, Francisco J.; Fernandez-Ballester, Gregorio; Ferrer-Montiel, Antonio; Balazs, Endre A.; Gomis, Ana; Belmonte, Carlos; de la Peña, Elvira

    2015-01-01

    Hyaluronan (HA) is present in the extracellular matrix of all body tissues, including synovial fluid in joints, in which it behaves as a filter that buffers transmission of mechanical forces to nociceptor nerve endings thereby reducing pain. Using recombinant systems, mouse-cultured dorsal root ganglia (DRG) neurons and in vivo experiments, we found that HA also modulates polymodal transient receptor potential vanilloid subtype 1 (TRPV1) channels. HA diminishes heat, pH and capsaicin (CAP) responses, thus reducing the opening probability of the channel by stabilizing its closed state. Accordingly, in DRG neurons, HA decreases TRPV1-mediated impulse firing and channel sensitization by bradykinin. Moreover, subcutaneous HA injection in mice reduces heat and capsaicin nocifensive responses, whereas the intra-articular injection of HA in rats decreases capsaicin joint nociceptor fibres discharge. Collectively, these results indicate that extracellular HA reduces the excitability of the ubiquitous TRPV1 channel, thereby lowering impulse activity in the peripheral nociceptor endings underlying pain. PMID:26311398

  12. A semi-multifunctional sialyltransferase from Bibersteinia trehalosi and its comparison to the Pasteurella multocida ST1 mutants.

    PubMed

    Talafová, Klaudia; Hrabárová, Eva; Nahálka, Jozef

    2015-12-20

    Sialic acids are well known for their crucial roles in many physiological and pathological processes. Improvement in the efficacy of protein drugs, an increase in the anti-inflammatory activity of intravenous immunoglobulin, preparation of infant milk and the diagnosis of diseases are examples of why there is a need for efficient in vitro sialylation. Sialyltransferases are crucial enzymes for the synthesis of sialo-oligosaccharides. Here, we introduce a new α2,3-sialyltransferase from bacteria Bibersteinia trehalosi (BtST1), which is homological to sialyltransferase from Pasteurella multocida (PmST1), Pasteurella dagmatis (PdST1) and Haemophilus ducreyi (Hd0053). BtST1 is active in a wide pH range and shows considerable acceptor flexibility. Very good specific activities have been detected with lactose and LacNAc as acceptors, and these activities were comparable to those of efficient multifunctional PmST1 and higher than PdST1, Hd0053 and also PmST1 M144D which was constructed to decrease the high sialidase activity of PmST1. Testing of PmST1 mutant forms revealed that mutations that included S143 caused only the restriction of sialyltransferase activity, whereas mutations including G142 resulted in the loss of activity with lactose. BtST1 possesses only low sialidase and trans-sialidase activities that are comparable to mutant PmST1 M144D, which are detected only in the presence of CMP. The combination of large acceptor flexibility, high activity for lactose and LacNAc and naturally low sialidase activity make BtST1 an attractive enzyme for biotechnological applications.

  13. Experimental pathogenicity and complete genome characterization of a pig origin Pasteurella multocida serogroup F isolate HN07.

    PubMed

    Peng, Zhong; Liang, Wan; Wang, Yuanguo; Liu, Wenjing; Zhang, Hongfeng; Yu, Teng; Zhang, Anding; Chen, Huanchun; Wu, Bin

    2017-01-01

    Pasteurella multocida serotype F isolates are predominately prevalent in avian hosts, but rarely seen in pigs. However, we isolated several strains of P. multocida serotype F from clinical samples of pigs in China. To understand the pathogenicity of these strains, one of the serotype F isolates designated HN07, was used to challenge experimental chickens, as P. multocida of this serotype is predominately prevalent in avian hosts. However, strain HN07 could not resulted in significant clinical signs in experimental chickens even at an infective dose of ∼10(9) CFU, suggesting the isolate was avirulent to chickens and therefore raising the possibility that the porcine serotype F isolate is not transmitted by chickens. We then used HN07 to challenge experimental pigs, as this strain was isolated from pigs. As expected, the strain led to the clinical signs and the pathological lesions in experimental pigs that are similar to the pasteurellosis disease. We then determined the complete genome sequence of the pig origin serogroup F isolate HN07 for the first time. Genome comparison between HN07 and the avian serotype F P. multocida Pm70 identified a novel integrative conjugative element (ICE) ICEpmcn07 which was likely to harbor a series of genes responsible for a putative type IV secretion system (T4SS) in HN07. This is the first time that we determined an ICE carrying a T4SS in P. multocida. Besides, comparative analysis also defined a number of virulence-associated genes in HN07 but absent in Pm70 which may have a contribution to the pathogenicity of the strain. This is the first report of the pathogenicity and genome characterization of a pig origin Pasteurella multocida serogroup F isolate. The pathogenic and genomic definition of the pig origin P. multocida serogroup F in our study would have significance on the pathogenesis and genetic diversity and virulence variability of P. multocida.

  14. The hyaluronan receptors CD44 and RHAMM (CD168) form complexeswith ERK1,2, which sustain high basal motility in breast cancercells

    SciTech Connect

    Hamilton, Sara R.; Fard, Shireen F.; Paiwand, Frouz F.; Tolg,Cornelia; Veiseh, Mandana; Wang, Chao; McCarthy, James B.; Bissell, MinaJ.; Koropatnick, James; Turley, Eva A.

    2007-03-28

    CD44 is an integral hyaluronan receptor that can promote or inhibit motogenic signaling in tumor cells. Rhamm is a non-integral cell surface hyaluronan receptor (CD168) and intracellular protein that promotes cell motility in culture and its expression is strongly upregulated in diseases like arthritis and aggressive cancers. Here we describe an autocrine mechanism utilizing cell surface Rhamm/CD44 interactions to sustain rapid basal motility in invasive breast cancer cell lines. This mechanism requires endogenous hyaluronan synthesis and the formation of Rhamm/CD44/ERK1, 2 complexes. Motile/ invasive MDA-MB-231 and Ras-MCF10A cells produce more endogenous hyaluronan, cell surface CD44 and Rhamm, an oncogenic Rhamm isoform, and exhibit elevated basal activation of ERK1, 2 than less invasive MCF7 and MCF10A breast cancer cells. Furthermore, CD44, Rhamm and ERK1, 2 uniquely co-immunoprecipitate and co-localize in MDA-MB-231 and Ras-MCF10A cells. Rapid motility of the invasive cell lines requires interaction of hyaluronan with cells, activation of ERK1, 2 and the participation of both cell surface CD44 and Rhamm. Combinations of anti-CD44, anti-Rhamm antibodies and a MEK1 inhibitor (PD098059) have less-than-additive blocking effects, suggesting action of all three proteins on a common motogenic signaling pathway. Collectively, these results show that cell surface Rhamm and CD44 act together in a hyaluronan-dependent, autocrine mechanism to coordinate sustained signaling through ERK1, 2 leading to high basal motility of invasive breast cancer cells. Since CD44/Rhamm complexes are not evident in less motile cells, an effect of CD44 on tumor cell motility may depend in part on its ability to partner with additional proteins, in this case cell surface Rhamm.

  15. THE HYALURONAN RECEPTORS CD44 AND RHAMM (CD168) FORM COMPLEXES WITH ERK1,2, WHICH SUSTAIN HIGH BASAL MOTILITY IN BREAST CANCER CELLS

    PubMed Central

    Hamilton, Sara R.; Fard, Shireen F.; Paiwand, Frouz F.; Tolg, Cornelia; Veiseh, Mandana; Wang, Chao; McCarthy, James B.; Bissell, Mina J.; Koropatnick, James; Turley, Eva A.

    2010-01-01

    CD44 is an integral hyaluronan receptor that can promote or inhibit motogenic signaling in tumor cells. Rhamm is a non-integral cell surface hyaluronan receptor (CD168) and intracellular protein that promotes cell motility in culture and its expression is strongly upregulated in diseases like arthritis and aggressive cancers. Here we describe an autocrine mechanism utilizing cell surface Rhamm/CD44 interactions to sustain rapid basal motility in invasive breast cancer cell lines. This mechanism requires endogenous hyaluronan synthesis and the formation of Rhamm/CD44/ERK1,2 complexes. Motile/ invasive MDA-MB-231 and Ras-MCF10A cells produce more endogenous hyaluronan, cell surface CD44 and Rhamm, an oncogenic Rhamm isoform, and exhibit elevated basal activation of ERK1,2 than less invasive MCF7 and MCF10A breast cancer cells. Furthermore, CD44, Rhamm and ERK1,2 uniquely co-immunoprecipitate and co-localize in MDA-MB-231 and Ras-MCF10A cells. Rapid motility of the invasive cell lines requires interaction of hyaluronan with cells, activation of ERK1,2 and the participation of both cell surface CD44 and Rhamm. Combinations of anti-CD44, anti-Rhamm antibodies and a MEK1 inhibitor (PD098059) have less-than-additive blocking effects, suggesting action of all three proteins on a common motogenic signaling pathway. Collectively, these results show that cell surface Rhamm and CD44 act together in a hyaluronan-dependent, autocrine mechanism to coordinate sustained signaling through ERK1,2 leading to high basal motility of invasive breast cancer cells. Since CD44/Rhamm complexes are not evident in less motile cells, an effect of CD44 on tumor cell motility may depend in part on its ability to partner with additional proteins, in this case cell surface Rhamm. PMID:17392272

  16. Crystal structure of riboflavin synthase

    SciTech Connect

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B.

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  17. Physico-chemical properties and degradability of non-woven hyaluronan benzylic esters as tissue engineering scaffolds.

    PubMed

    Milella, E; Brescia, E; Massaro, C; Ramires, P A; Miglietta, M R; Fiori, V; Aversa, P

    2002-02-01

    The development of biocompatible materials which can be processed into three-dimensional scaffolds and the design of appropriate configurations in order to enable the cellular infiltration and proliferation is a major issue in the tissue engineering. The hyaluronan total benzyl ester (Hyaff 11) has been found to be suitable substrate to grow a variety of cell types. Since structural, physical, chemical and biological data can help for tailoring appropriate scaffold for tissue engineering, information on chemicophysical properties on degradability of hyaluronan total benzyl ester non-woven has been obtained. The thermal analysis, the evaluation of the surface chemical composition, the morphology, the mechanical behaviour and the swelling tests were carried out on these materials. The hyaluronan total benzyl ester non-woven showed a thermal stability up to 220 degrees C and the surface composition differed from that of the bulk for C-O and C-C contribution. No contaminant were detected. The non-woven swelled in culture medium. Moreover the mechanical tests showed that when submitted to a press treatment, the samples have best mechanical properties. The pressed Hyaff 11 non-woven undergoes degradation when exposed to DMEM. The frying and breaking of the fibres, a decrease of the mechanical properties and a molecular weight loss have been observed. First, the ester bond of the Hyaff 11 non-woven is hydrolysed and the benzylic alcohol is released and the low molecular weight values indicate that a cleavage of the polymer is promoted by the components of the culture medium. After 11 days, some fragments, constituted by hyaluronic acid with a molecular weight of 23,000 Da became soluble in the medium. No oligomer was detected.

  18. Effects of modified Cary and Blair medium on recovery of nonhemolytic Pasteurella haemolytica from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) pharyngeal swabs.

    PubMed

    Wild, M A; Miller, M W

    1994-01-01

    Modified Cary and Blair transport medium (MCB) was evaluated for recovery of Pasteurella spp. from pharyngeal swabs of healthy Rocky Mountain bighorn sheep (Ovis canadensis canadensis). In experiment one, three pharyngeal swabs were collected from each of 25 bighorns. Pasteurella haemolytica was recovered from 21 of 25 swabs tested almost immediately and from 16 of 25 swabs held in MCB medium at about 22 C for 24 hr before testing (P > 0.10). Recovery of P. haemolytica decreased (P < 0.005) to 1 of 25 when swabs were held in MCB medium at about 22 C for 48 hr before testing. In experiment two, four pharyngeal swabs were collected from each of ten bighorns and held in MCB medium at about 5 C for < or = 5, 24, 48, or 72 hr prior to testing. Recovery was unaffected by storage at 5 C; P. haemolytica was isolated from all 40 of these samples. All Pasteurella spp. isolates were nonhemolytic P. haemolytica. In experiment one, most isolates were serotype 4; in experiment two, serotype 3 was most common. We propose that MCB medium is effective for transporting bighorn sheep pharyngeal swabs for P. haemolytica screening because it imposes minimal or no effect on recovery when held < or = 24 hr at 22 C or < or = 72 hr at 5 C.