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Sample records for pasteurella hyaluronan synthase

  1. Role of hyaluronan and hyaluronan synthase in endometrial cancer.

    PubMed

    Yabushita, Hiromitsu; Kishida, Tameko; Fusano, Kanako; Kanyama, Kouhei; Zhuo, Lisheng; Itano, Naoki; Kimata, Koji; Noguchi, Masayoshi

    2005-06-01

    The aim of this study was to determine if the immunohistochemical expression of hyaluronan synthase (HAS) and serum levels of hyaluronan correlate with the clinicopathological manifestations of endometrial carcinoma. Sera were obtained from 59 endometrial cancer patients and 22 post-menopausal healthy women. Concentration of hyaluronan in sera was measured by an inhibitory ELISA using a hyaluronan-binding protein. Tissues obtained from 59 endometrial cancer patients were immunostained by the avidin-biotin-peroxidase complex method using anti-HAS1, anti-HAS2, anti-HAS3 and anti-CD44 antibody. A section was defined as having positive expression when >50% of the tumor cells were intensely stained. The expression of HAS1 was related to the depth of myometrial invasion, histological grade and lymph-vascular space involvement, but the expression of HAS2 and HAS3 was unrelated to these parameters. CD44 expression occurred more frequently in the HAS2- or HAS3-positive groups than in the HAS2- or HAS3-negative groups, and the expression of HAS1 was unrelated to CD44 expression. Serum levels of hyaluronan were higher in the endometrial cancer group than in the healthy control group, and increased with depth of myometrial invasion, histological grade and lymph-vascular space involvement. Serum hyaluronan levels were higher in the HAS1-positive group than in the HAS1-negative group, but the expression of HAS2 and HAS3 was unrelated to serum hyaluronan levels. HAS1 expression and an increase in serum hyaluronan in endometrial cancer may be associated with disease progression through myometrial invasion and lymph-vascular space involvement. PMID:15870928

  2. Hyaluronan synthases and hyaluronidases in nasal polyps.

    PubMed

    Panogeorgou, T; Tserbini, E; Filou, S; Vynios, D H; Naxakis, S S; Papadas, T A; Goumas, P D; Mastronikolis, N S

    2016-07-01

    Nasal polyps (NPs) are benign lesions of nasal and paranasal sinuses mucosa affecting 1-4 % of all adults. Nasal polyposis affects the quality of patient's life as it causes nasal obstruction, postnasal drainage, purulent nasal discharge, hyposmia or anosmia, chronic sinusitis, facial pain and snoring. Without treatment, the disease can alter the craniofacial skeleton in cases of extended growth of polyps. The development of NPs is caused by the hyperplasia of nasal or paranasal sinuses mucosa, and edema of extracellular matrix. This is usually the result of high concentration of high molecular mass hyaluronan (HA) which is either overproduced or accumulated from blood supply. The size of HA presents high diversity and, especially in pathologic conditions, chains of low molecular mass can be observed. In NPs, chains of about 200 kDa have been identified and considered to be responsible for the inflammation. The purpose of the present study was the investigation, in NPs and normal nasal mucosa (NM), of the expression of the wild-type and alternatively spliced forms of hyaluronidases, their immunolocalization, and the expression of HA synthases to examine the isoform(s) responsible for the increased amounts of HA in NPs. Hyaluronidases' presence was examined on mRNA (RT-PCR analysis) and protein (immunohistochemistry) levels. Hyaluronan synthases' presence was examined on mRNA levels. Hyaluronidases were localized in the cytoplasm of epithelial and inflammatory cells, as well as in the matrix. On mRNA level, it was found that hyal-1-wt was decreased in NPs compared to NM and hyal-1-v3, -v4 and -v5 were substantially increased. Moreover, HAS2 and HAS3 were the only hyaluronan synthases detected, the expression of which was almost similar in NPs and NM. Overall, the results of the present study support that hyaluronidases are the main enzymes responsible for the decreased size of hyaluronan observed in NPs; thus they behave as inflammatory agents. Therefore, they

  3. 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.

  4. Regulation of hyaluronan synthases in mouse uterine cervix.

    PubMed

    Uchiyama, Taro; Sakuta, Tomohiro; Kanayama, Toshiji

    2005-02-18

    We examined the expression pattern of hyaluronan synthase (HAS) mRNAs in the uterine cervix of pregnant mice. The expression levels of HAS-1 and -2 mRNAs peaked at delivery, whereas that of HAS-3 mRNA peaked on the 15th day of pregnancy. The regulation of HAS mRNA expression was examined in pregnant mouse uterine cervical fibroblasts. The expression of HAS-1, -2, and -3 mRNAs was significantly augmented by interleukin-1beta (IL-1beta). Progesterone significantly interfered with expression of HAS-1 and -2 mRNAs, but significantly increased the expression of HAS-3 mRNA. Low-molecular-weight hyaluronan significantly enhanced only the expression of HAS-1 mRNA. These results indicate that HAS in the uterine cervix is regulated in a complex manner by IL-1beta, progesterone, and low-molecular-weight hyaluronan, of which changes in the cervical tissue and serum closely participate in uterine cervical ripening and/or inflammation. PMID:15649434

  5. Accumulation of Extracellular Hyaluronan by Hyaluronan Synthase 3 Promotes Tumor Growth and Modulates the Pancreatic Cancer Microenvironment

    PubMed Central

    Zhao, Chunmei; Singha, Netai C.; Osgood, Ryan J.; Symons, Rebecca; Jiang, Ping; Li, Xiaoming; Thompson, Curtis B.; Infante, Jeffrey R.; Jacobetz, Michael A.; Tuveson, David A.; Frost, Gregory I.; Shepard, H. Michael; Huang, Zhongdong

    2014-01-01

    Extensive accumulation of the glycosaminoglycan hyaluronan is found in pancreatic cancer. The role of hyaluronan synthases 2 and 3 (HAS2, 3) was investigated in pancreatic cancer growth and the tumor microenvironment. Overexpression of HAS3 increased hyaluronan synthesis in BxPC-3 pancreatic cancer cells. In vivo, overexpression of HAS3 led to faster growing xenograft tumors with abundant extracellular hyaluronan accumulation. Treatment with pegylated human recombinant hyaluronidase (PEGPH20) removed extracellular hyaluronan and dramatically decreased the growth rate of BxPC-3 HAS3 tumors compared to parental tumors. PEGPH20 had a weaker effect on HAS2-overexpressing tumors which grew more slowly and contained both extracellular and intracellular hyaluronan. Accumulation of hyaluronan was associated with loss of plasma membrane E-cadherin and accumulation of cytoplasmic β-catenin, suggesting disruption of adherens junctions. PEGPH20 decreased the amount of nuclear hypoxia-related proteins and induced translocation of E-cadherin and β-catenin to the plasma membrane. Translocation of E-cadherin was also seen in tumors from a transgenic mouse model of pancreatic cancer and in a human non-small cell lung cancer sample from a patient treated with PEGPH20. In conclusion, hyaluronan accumulation by HAS3 favors pancreatic cancer growth, at least in part by decreasing epithelial cell adhesion, and PEGPH20 inhibits these changes and suppresses tumor growth. PMID:25147816

  6. Accumulation of extracellular hyaluronan by hyaluronan synthase 3 promotes tumor growth and modulates the pancreatic cancer microenvironment.

    PubMed

    Kultti, Anne; Zhao, Chunmei; Singha, Netai C; Zimmerman, Susan; Osgood, Ryan J; Symons, Rebecca; Jiang, Ping; Li, Xiaoming; Thompson, Curtis B; Infante, Jeffrey R; Jacobetz, Michael A; Tuveson, David A; Frost, Gregory I; Shepard, H Michael; Huang, Zhongdong

    2014-01-01

    Extensive accumulation of the glycosaminoglycan hyaluronan is found in pancreatic cancer. The role of hyaluronan synthases 2 and 3 (HAS2, 3) was investigated in pancreatic cancer growth and the tumor microenvironment. Overexpression of HAS3 increased hyaluronan synthesis in BxPC-3 pancreatic cancer cells. In vivo, overexpression of HAS3 led to faster growing xenograft tumors with abundant extracellular hyaluronan accumulation. Treatment with pegylated human recombinant hyaluronidase (PEGPH20) removed extracellular hyaluronan and dramatically decreased the growth rate of BxPC-3 HAS3 tumors compared to parental tumors. PEGPH20 had a weaker effect on HAS2-overexpressing tumors which grew more slowly and contained both extracellular and intracellular hyaluronan. Accumulation of hyaluronan was associated with loss of plasma membrane E-cadherin and accumulation of cytoplasmic β-catenin, suggesting disruption of adherens junctions. PEGPH20 decreased the amount of nuclear hypoxia-related proteins and induced translocation of E-cadherin and β-catenin to the plasma membrane. Translocation of E-cadherin was also seen in tumors from a transgenic mouse model of pancreatic cancer and in a human non-small cell lung cancer sample from a patient treated with PEGPH20. In conclusion, hyaluronan accumulation by HAS3 favors pancreatic cancer growth, at least in part by decreasing epithelial cell adhesion, and PEGPH20 inhibits these changes and suppresses tumor growth. PMID:25147816

  7. Hyaluronan and hyaluronan synthases expression and localization in embryonic mouse molars.

    PubMed

    Yang, Guofeng; Jiang, Beizhan; Cai, Wenping; Liu, Shangfeng; Zhao, Shouliang

    2016-08-01

    Hyaluronan (HA) and hyaluronan synthases (HASs) have been shown to play critical roles in embryogenesis and organ development. However, there have not been any studies examining HA and HAS expression and localization during tooth development. The present study was designed to investigate the expression of HA and three isoforms of HASs (HAS1, 2, 3) in embryonic mouse molars. The first mandibular embryonic mouse molars were examined by immunohistochemistry at E11.5, E13.5, E14.5, E16.5, and E18.5. PCR and western blot analyses were performed on RNA and proteins samples from E13.5 to E18.5 tooth germs. At the initial stage (E11.5), HA and HASs were expressed in the dental epithelium but not the underlying dental mesenchyme. HA immunostaining gradually increased in the enamel organ from the bud stage (E13.5) to the late bell stage (E18.5), and HA and HASs were highly expressed in the stellate reticulum and stratum intermedium. HA immunostaining was also enhanced in the dental mesenchyme and its derived tissues, but it was not expressed in the ameloblast and odontoblast regions. The three HAS isoforms had distinct expression patterns, and they were expressed in the dental mesenchyme and odontoblast at various levels. Furthermore, HAS1 and HAS2 expression decreased, while HAS3 expression increased from E13.5 to E18.5. These results suggested that HA synthesized by different HASs is involved in embryonic mouse molar morphogenesis and cytodifferentiation. PMID:27318667

  8. 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.

  9. 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.

  10. The control and importance of hyaluronan synthase expression in palatogenesis

    PubMed Central

    Galloway, Jennifer L.; Jones, Sarah J.; Mossey, Peter A.; Ellis, Ian R.

    2013-01-01

    Development of the lip and palate involves a complex series of events that requires the close co-ordination of cell migration, growth, differentiation, and apoptosis. Palatal shelf elevation is considered to be driven by regional accumulation and hydration of glycosoaminoglycans, principally hyaluronan (HA), which provides an intrinsic shelf force, directed by components of the extracellular matrix (ECM). During embryogenesis, the extracellular and pericellular matrix surrounding migrating and proliferating cells is rich in HA. This would suggest that HA may be important in both shelf growth and fusion. TGFβ3 plays an important role in palatogenesis and the corresponding homozygous null (TGFβ3−/−) mouse, exhibits a defect in the fusion of the palatal shelves resulting in clefting of the secondary palate. TGFβ3 is expressed at the future medial edge epithelium (MEE) and at the actual edge epithelium during E14.5, suggesting a role for TGFβ3 in fusion. This is substantiated by experiments showing that addition of exogenous TGFβ3 can “rescue” the cleft palate phenotype in the null mouse. In addition, TGFβ1 and TGFβ2 can rescue the null mouse palate (in vitro) to near normal fusion. In vivo a TGFβ1 knock-in mouse, where the coding region of the TGFβ3 gene was replaced with the full-length TGFβ1 cDNA, displayed complete fusion at the mid portion of the secondary palate, whereas the anterior and posterior regions failed to fuse appropriately. We present experimental data indicating that the three HA synthase (Has) enzymes are differentially expressed during palatogenesis. Using immunohistochemistry (IHC) and embryo sections from the TGFβ3 null mouse at days E13.5 and E14.5, it was established that there was a decrease in expression of Has2 in the mesenchyme and an increase in expression of Has3 in comparison to the wild-type mouse. In vitro data indicate that HA synthesis is affected by addition of exogenous TGFβ3. Preliminary data suggests that this

  11. Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme

    PubMed Central

    Camenisch, Todd D.; Spicer, Andrew P.; Brehm-Gibson, Tammy; Biesterfeldt, Jennifer; Augustine, Mary Lou; Calabro, Anthony; Kubalak, Steven; Klewer, Scott E.; McDonald, John A.

    2000-01-01

    We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2–/– embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5–10). Heart explants from Has2–/– embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2–/– explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, transformation in Has2–/– explants mediated by exogenous HA is inhibited by dominant-negative Ras. Collectively, our results demonstrate the importance of HA in mammalian embryogenesis and the pivotal role of Has2 during mammalian development. They also reveal a previously unrecognized pathway for cell migration and invasion that is HA-dependent and involves Ras activation. PMID:10930438

  12. Analysis of human hyaluronan synthase gene transcriptional regulation and downstream hyaluronan cell surface receptor mobility in myofibroblast differentiation.

    PubMed

    Midgley, Adam C; Bowen, Timothy

    2015-01-01

    The ubiquitous extracellular glycosaminoglycan hyaluronan (HA) is a polymer composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating β-1,4 and β-1,3 glycosidic bonds. Emerging data continue to reveal functions attributable to HA in a variety of physiological and pathological contexts. Defining the mechanisms regulating expression of the human hyaluronan synthase (HAS) genes that encode the corresponding HA-synthesizing HAS enzymes is therefore important in the context of HA biology in health and disease. We describe here methods to analyze transcriptional regulation of the HAS and HAS2-antisense RNA 1 genes. Elucidation of mechanisms of HA interaction with receptors such as the cell surface molecule CD44 is also key to understanding HA function. To this end, we provide protocols for fluorescent recovery after photobleaching analysis of CD44 membrane dynamics in the process of fibroblast to myofibroblast differentiation, a phenotypic transition that is common to the pathology of fibrosis of large organs such as the liver and kidney. PMID:25325984

  13. Hyaluronan

    PubMed Central

    Bogdani, Marika; Nagy, Nadine; Johnson, Pamela Y.; Wight, Thomas N.

    2015-01-01

    Hyaluronan (HA) is an extracellular matrix (ECM) component that is present in mouse and human islet ECM. HA is localized in peri-islet and intra-islet regions adjacent to microvessels. HA normally exists in a high molecular weight form, which is anti-inflammatory. However, under inflammatory conditions, HA is degraded into fragments that are proinflammatory. HA accumulates in islets of human subjects with recent onset type 1 diabetes (T1D), and is associated with myeloid and lymphocytic islet infiltration, suggesting a possible role for HA in insulitis. A similar accumulation of HA, in amount and location, occurs in non-obese diabetic (NOD) and DORmO mouse models of T1D. Furthermore, HA accumulates in follicular germinal centers and in T-cell areas in lymph nodes and spleen in both human and mouse models of T1D, as compared with control tissues. Whether HA accumulates in islets in type 2 diabetes (T2D) or models thereof has not been previously described. Here we show evidence that HA accumulates in a mouse model of islet amyloid deposition, a well-known component of islet pathology in T2D. In summary, islet HA accumulation is a feature of both T1D and a model of T2D, and may represent a novel inflammatory mediator of islet pathology. PMID:26216136

  14. 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. PMID:15794859

  15. Methods for measuring Class I membrane-bound hyaluronan synthase activity.

    PubMed

    Weigel, Paul H; Padgett-McCue, Amy J; Baggenstoss, Bruce A

    2013-01-01

    Detecting and quantifying hyaluronan (HA) made by Class I HA synthase (HAS) and determining the level of activity of these membrane-bound enzymes is critical in studies to understand the normal biology of HA and how changes in HAS activity and HA levels or size are important in inflammatory and other diseases, tumorigenesis, and metastasis. Unlike the products made by the vast majority of glycosyltransferases, HA products are more complicated since they are made as a heterogeneous population of sizes spanning a broad mass range. Three radioactive and nonradioactive assay methods are described that can give the amount of HA made with or without information about the distribution of product sizes. PMID:23765666

  16. 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.

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

    PubMed

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

    2015-05-01

    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

  18. 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. PMID:26518873

  19. 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

  20. 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

  1. 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. PMID:17705506

  2. 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.

  3. Extracellular UDP-Glucose Activates P2Y14 Receptor and Induces Signal Transducer and Activator of Transcription 3 (STAT3) Tyr705 Phosphorylation and Binding to Hyaluronan Synthase 2 (HAS2) Promoter, Stimulating Hyaluronan Synthesis of Keratinocytes*

    PubMed Central

    Jokela, Tiina A.; Kärnä, Riikka; Makkonen, Katri M.; Laitinen, Jarmo T.; Tammi, Raija H.; Tammi, Markku I.

    2014-01-01

    Hyaluronan, a major matrix molecule in epidermis, is often increased by stimuli that enhance keratinocyte proliferation and migration. We found that small amounts of UDP-sugars were released from keratinocytes and that UDP-glucose (UDP-Glc) added into keratinocyte cultures induced a specific, rapid induction of hyaluronan synthase 2 (HAS2), and an increase of hyaluronan synthesis. The up-regulation of HAS2 was associated with JAK2 and ERK1/2 activation, and specific Tyr705 phosphorylation of transcription factor STAT3. Inhibition of JAK2, STAT3, or Gi-coupled receptors blocked the induction of HAS2 expression by UDP-Glc, the latter inhibitor suggesting that the signaling was triggered by the UDP-sugar receptor P2Y14. Chromatin immunoprecipitations demonstrated increased promoter binding of Tyr(P)705-STAT3 at the time of HAS2 induction. Interestingly, at the same time Ser(P)727-STAT3 binding to its response element regions in the HAS2 promoter was unchanged or decreased. UDP-Glc also stimulated keratinocyte migration, proliferation, and IL-8 expression, supporting a notion that UDP-Glc signals for epidermal inflammation, enhanced hyaluronan synthesis as an integral part of it. PMID:24847057

  4. 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.

  5. Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo- and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3*

    PubMed Central

    Bart, Geneviève; Vico, Nuria Ortega; Hassinen, Antti; Pujol, Francois M.; Deen, Ashik Jawahar; Ruusala, Aino; Tammi, Raija H.; Squire, Anthony; Heldin, Paraskevi; Kellokumpu, Sakari; Tammi, Markku I.

    2015-01-01

    In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1–3 (HAS1–3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647–23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis. PMID:25795779

  6. Hyaluronan Synthesis and Myogenesis

    PubMed Central

    Hunt, Liam C.; Gorman, Chris; Kintakas, Christopher; McCulloch, Daniel R.; Mackie, Eleanor J.; White, Jason D.

    2013-01-01

    Exogenous hyaluronan is known to alter muscle precursor cell proliferation, migration, and differentiation, ultimately inhibiting myogenesis in vitro. The aim of the current study was to investigate the role of endogenous hyaluronan synthesis during myogenesis. In quantitative PCR studies, the genes responsible for synthesizing hyaluronan were found to be differentially regulated during muscle growth, repair, and pathology. Although all Has genes (Has1, Has2, and Has3) were differentially regulated in these models, only Has2 gene expression consistently associated with myogenic differentiation. During myogenic differentiation in vitro, Has2 was the most highly expressed of the synthases and increased after induction of differentiation. To test whether this association between Has2 expression and myogenesis relates to a role for Has2 in myoblast differentiation and fusion, C2C12 myoblasts were depleted of Has2 by siRNA and induced to differentiate. Depletion of Has2 inhibited differentiation and caused a loss of cell-associated hyaluronan and the hyaluronan-dependent pericellular matrix. The inhibition of differentiation caused by loss of hyaluronan was confirmed with the hyaluronan synthesis inhibitor 4-methylumbelliferone. In hyaluronan synthesis-blocked cultures, restoration of the pericellular matrix could be achieved through the addition of exogenous hyaluronan and the proteoglycan versican, but this was not sufficient to restore differentiation to control levels. These data indicate that intrinsic hyaluronan synthesis is necessary for myoblasts to differentiate and form syncytial muscle cells, but the hyaluronan-dependent pericellular matrix is not sufficient to support differentiation alone; additional hyaluronan-dependent cell functions that are yet unknown may be required for myogenic differentiation. PMID:23493399

  7. Localisation and endocrine control of hyaluronan synthase (HAS) 2, HAS3 and CD44 expression in sheep granulosa cells.

    PubMed

    Chavoshinejad, R; Marei, W F A; Hartshorne, G M; Fouladi-Nashta, A A

    2016-04-01

    The aim of the present study was to investigate the hormonal regulation of hyaluronan (HA) components in sheep granulosa cells. HA components are present in the reproductive tract and have a range of physical and signalling properties related to reproductive function in several species. First, abattoir-derived ovaries of sheep were used to determine the localisation of HA synthase (HAS) 1-3 and CD44 proteins in antral follicles. Staining for HAS1-3 and CD44 proteins was most intense in the granulosa layer. Accordingly, the expression of HAS2, HAS3 and CD44 mRNA was measured in cultured granulosa cells exposed to 0-50ngmL(-1) of 17β-oestradiol and different combinations of oestradiol, gonadotropins, insulin-like growth factor (IGF)-1 and insulin for 48-96h (1ngmL(-1) FSH, 10ngmL(-1) insulin, 10ngmL(-1) IGF-1, 40ngmL(-1) E2 and 25ngmL(-1) LH.). mRNA expression was quantified by real-time polymerase chain reaction using a fold induction method. The results revealed that the hormones tested generally stimulated mRNA expression of the genes of interest in cultured granulosa cells. Specifically, oestradiol, when combined with IGF-1, insulin and FSH, stimulated HAS2 mRNA expression. Oestradiol and LH had synergistic effects in increasing HAS3 mRNA expression. In conclusion, we suggest that the hormones studied differentially regulate HAS2, HAS3 and CD44 in ovine granulosa cells in vitro. Further work is needed to address the signalling pathways involved. PMID:25427133

  8. Donor substrate promiscuity of the N-acetylglucosaminyltransferase activities of Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA.

    PubMed

    Li, Yanhong; Yu, Hai; Thon, Vireak; Chen, Yi; Muthana, Musleh M; Qu, Jingyao; Hie, Liana; Chen, Xi

    2014-02-01

    The biological activities of heparan sulfate (HS) and heparin (HP) are closely related to their molecular structures. Both Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA have been used for enzymatic and chemoenzymatic synthesis of HS and HP oligosaccharides and their derivatives. We show here that cloning using the pET15b vector and expressing PmHS2 as an N-His6-tagged fusion protein improve its expression level in E. coli. Investigation of the donor substrate specificity of the N-acetylglucosaminyltransferase activities of P. multocida heparosan synthase 2 (PmHS2) and E. coli K5 KfiA indicates the substrate promiscuities of PmHS2 and KfiA. Overall, both PmHS2 and KfiA can use uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and some of its C2'- and C6'-derivatives as donor substrates for their α1-4-GlcNAcT activities. Nevertheless, PmHS2 has a broader tolerance towards substrate modifications. Other than the UDP-sugars that can be used by KfiA, additional C6'-derivatives of UDP-GlcNAc, UDP-glucose, and UDP-N-acetylgalactosamine (UDP-GalNAc) are tolerable substrates for the α1-4-GlcNAcT activity of PmHS2. The substrate promiscuities of PmHS2 and KfiA will allow efficient chemoenzymatic synthesis of diverse HS and HP oligosaccharide derivatives which may have improved or altered activities compared to their natural counterparts. PMID:23661084

  9. 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. PMID:25583822

  10. 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

  11. Biology of hyaluronan: Insights from genetic disorders of hyaluronan metabolism.

    PubMed

    Triggs-Raine, Barbara; Natowicz, Marvin R

    2015-08-26

    Hyaluronan is a rapidly turned over component of the vertebrate extracellular matrix. Its levels are determined, in part, by the hyaluronan synthases, HAS1, HAS2, and HAS3, and three hyaluronidases, HYAL1, HYAL2 and HYAL3. Hyaluronan binding proteins also regulate hyaluronan levels although their involvement is less well understood. To date, two genetic disorders of hyaluronan metabolism have been reported in humans: HYAL1 deficiency (Mucopolysaccharidosis IX) in four individuals with joint pathology as the predominant phenotypic finding and HAS2 deficiency in a single person having cardiac pathology. However, inherited disorders and induced mutations affecting hyaluronan metabolism have been characterized in other species. Overproduction of hyaluronan by HAS2 results in skin folding and thickening in shar-pei dogs and the naked mole rat, whereas a complete deficiency of HAS2 causes embryonic lethality in mice due to cardiac defects. Deficiencies of murine HAS1 and HAS3 result in a predisposition to seizures. Like humans, mice with HYAL1 deficiency exhibit joint pathology. Mice lacking HYAL2 have variably penetrant developmental defects, including skeletal and cardiac anomalies. Thus, based on mutant animal models, a partial deficiency of HAS2 or HYAL2 might be compatible with survival in humans, while complete deficiencies of HAS1, HAS3, and HYAL3 may yet be recognized. PMID:26322170

  12. Biology of hyaluronan: Insights from genetic disorders of hyaluronan metabolism

    PubMed Central

    Triggs-Raine, Barbara; Natowicz, Marvin R

    2015-01-01

    Hyaluronan is a rapidly turned over component of the vertebrate extracellular matrix. Its levels are determined, in part, by the hyaluronan synthases, HAS1, HAS2, and HAS3, and three hyaluronidases, HYAL1, HYAL2 and HYAL3. Hyaluronan binding proteins also regulate hyaluronan levels although their involvement is less well understood. To date, two genetic disorders of hyaluronan metabolism have been reported in humans: HYAL1 deficiency (Mucopolysaccharidosis IX) in four individuals with joint pathology as the predominant phenotypic finding and HAS2 deficiency in a single person having cardiac pathology. However, inherited disorders and induced mutations affecting hyaluronan metabolism have been characterized in other species. Overproduction of hyaluronan by HAS2 results in skin folding and thickening in shar-pei dogs and the naked mole rat, whereas a complete deficiency of HAS2 causes embryonic lethality in mice due to cardiac defects. Deficiencies of murine HAS1 and HAS3 result in a predisposition to seizures. Like humans, mice with HYAL1 deficiency exhibit joint pathology. Mice lacking HYAL2 have variably penetrant developmental defects, including skeletal and cardiac anomalies. Thus, based on mutant animal models, a partial deficiency of HAS2 or HYAL2 might be compatible with survival in humans, while complete deficiencies of HAS1, HAS3, and HYAL3 may yet be recognized. PMID:26322170

  13. Lentiviral-mediated over-expression of hyaluronan synthase-1 (HAS-1) decreases the cellular inflammatory response and results in regenerative wound repair.

    PubMed

    Caskey, Robert C; Allukian, Myron; Lind, Robert C; Herdrich, Benjamin J; Xu, Junwang; Radu, Antoneta; Mitchell, Marc E; Liechty, Kenneth W

    2013-01-01

    Fetal wounds have been found to have increased levels of high-molecular-weight hyaluronan (HMW-HA) compared with those of adults. The primary enzyme responsible for producing HMW-HA is hyaluronic acid synthase-1 (HAS-1). We hypothesized that over-expression of HAS-1 in adult dermal wounds would decrease inflammation and promote regenerative healing. To test this hypothesis, the flanks of adult C57Bl/6 mice were treated with a lentiviral construct containing either HAS-1-GFP or GFP transgenes. After 48 h, a 4-mm excisional wound was made at the site of treatment. Wounds were harvested at days 3, 7, or 28 after wounding. Wound phenotype was assessed by histology to examine tissue architecture and immunohistochemistry for CD45. At 7 and 28 days, lenti-HAS-1-treated wounds demonstrated the restoration of the normal dermal elements and organized collagen fiber orientation. In contrast, the lenti-GFP-treated wounds lacked normal dermal architecture and demonstrated a disorganized collagen scar. At 3 and 7 days, wounds treated with lenti-HAS-1 exhibited a significant decrease in the number of inflammatory cells when compared with wounds treated with lenti-GFP. Thus, HAS-1 over-expression promotes dermal regeneration, in part by decreasing the inflammatory response and by recapitulation of fetal extracellular matrix HMW-HA content. PMID:23149717

  14. Natural Antisense Transcript for Hyaluronan Synthase 2 (HAS2-AS1) Induces Transcription of HAS2 via Protein O-GlcNAcylation*

    PubMed Central

    Vigetti, Davide; Deleonibus, Sara; Moretto, Paola; Bowen, Timothy; Fischer, Jens W.; Grandoch, Maria; Oberhuber, Alexander; Love, Dona C.; Hanover, John A.; Cinquetti, Raffaella; Karousou, Eugenia; Viola, Manuela; D'Angelo, Maria Luisa; Hascall, Vincent C.; De Luca, Giancarlo; Passi, Alberto

    2014-01-01

    Changes in the microenvironment organization within vascular walls are critical events in the pathogenesis of vascular pathologies, including atherosclerosis and restenosis. Hyaluronan (HA) accumulation into artery walls supports vessel thickening and is involved in many cardiocirculatory diseases. Excessive cytosolic glucose can enter the hexosamine biosynthetic pathway, increase UDP-N-acetylglucosamine (UDP-GlcNAc) availability, and lead to modification of cytosolic proteins via O-linked attachment of the monosaccharide β-N-GlcNAc (O-GlcNAcylation) from UDP-GlcNAc by the enzyme O-GlcNAc transferase. As many cytoplasmic and nuclear proteins can be glycosylated by O-GlcNAc, we studied whether the expression of the HA synthases that synthesize HA could be controlled by O-GlcNAcylation in human aortic smooth muscle cells. Among the three HAS isoenzymes, only HAS2 mRNA increased after O-GlcNAcylation induced by glucosamine treatments or by inhibiting O-GlcNAc transferase with PUGNAC (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate). We found that the natural antisense transcript of HAS2 (HAS2-AS1) was absolutely necessary to induce the transcription of the HAS2 gene. Moreover, we found that O-GlcNAcylation modulated HAS2-AS1 promoter activation by recruiting the NF-κB subunit p65, but not the HAS2 promoter, whereas HAS2-AS1 natural antisense transcript, working in cis, regulated HAS2 transcription by altering the chromatin structure around the HAS2 proximal promoter via O-GlcNAcylation and acetylation. These results indicate that HAS2 transcription can be finely regulated not only by recruiting transcription factors to the promoter as previously described but also by modulating chromatin accessibility by epigenetic modifications. PMID:25183006

  15. 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. PMID:27072623

  16. Orphan nuclear receptor HNF4G promotes bladder cancer growth and invasion through the regulation of the hyaluronan synthase 2 gene

    PubMed Central

    Okegawa, T; Ushio, K; Imai, M; Morimoto, M; Hara, T

    2013-01-01

    Nuclear receptors (NRs) are a class of transcription factors that are closely involved in the progression of certain types of cancer. We aimed to study the relation between bladder cancer and NRs, with special focus on orphan NRs whose ligands and functions have not been identified. First, we examined the expression levels of 22 genes encoding orphan NRs in clinical bladder cancer and found that hepatocyte nuclear factor 4γ (HNF4G; NR2A2) and NR2F6 were the genes that were upregulated most frequently in cancer tissues compared with their paired normal tissues. Knockdown and overexpression of each of these orphan NRs suppressed and stimulated the growth of bladder cancer cells in vitro, respectively. HNF4G also promoted tumor growth in bladder cancer xenograft models in vivo. Furthermore, HNF4G was both necessary and sufficient for the invasion of bladder cancer cells in vitro. Moreover, using microarray analyses, we identified hyaluronan synthase 2 (HAS2) as one of the genes induced by HNF4G in bladder cancer cells. Transcription was activated by HNF4G in reporter assays using the promoter/enhancer region of the HAS2 gene. The endogenous expression of the HAS2 gene was suppressed by knockdown of HNF4G. In turn, knockdown of HAS2 inhibited the growth and invasion of bladder cancer cells. Taken together, our data suggest that some orphan NRs are involved in bladder cancer progression and that, among them, HNF4G promotes the growth and invasion of bladder cancer, at least in part, via the regulation of the HAS2 gene. PMID:23896584

  17. 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. PMID:20178474

  18. 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

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

    PubMed

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

    2015-11-01

    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 procedure for

  20. Transformation of Pasteurella novicida

    PubMed Central

    Tyeryar, Franklin J.; Lawton, William D.

    1969-01-01

    Deoxyribonucleic acid from a streptomycin-resistant mutant of Pasteurella novicida transformed portions of P. novicida streptomycin-sensitive populations to streptomycin-resistant. Similarly, mutants auxotrophic for tryptophan or purine biosynthesis were also transformed to nutritional independence. PMID:5359612

  1. 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. PMID:26768539

  2. Pasteurella multocida liver abscess.

    PubMed

    Cortez, J C; Shapiro, M; Awe, R J

    1986-08-01

    A previously healthy 61-year-old woman was seen with an abnormal chest roentgenogram and a 3-week history of fever, chills, malaise, and right upper quadrant pain. Blood cultures revealed Pasteurella multocida sensitive to penicillin. Liver spleen radioisotope scan and CT scan revealed space occupying lesions in the right lobe of the liver. The patient was a gardener with no pets or animal exposure. This case illustrates P. multocida septicemia and a liver abscess in a patient without animal exposure. In addition, the possibility of soil as another reservoir of infection is raised. PMID:3487981

  3. Murine abortion is associated with enhanced hyaluronan expression and abnormal localization at the fetomaternal interface.

    PubMed

    Cordo-Russo, R; Garcia, M G; Barrientos, G; Orsal, A S; Viola, M; Moschansky, P; Ringel, F; Passi, A; Alaniz, L; Hajos, S; Blois, S M

    2009-01-01

    The remodelling of the endometrial architecture is fundamental to create a suitable environment for the establishment of pregnancy. During this process, substantial alterations in the composition of maternal extracellular matrix play an important role by providing a prosperous medium for implantation as well as modulating trophoblast invasion leading to the formation of a functional placental unit. Hyaluronan is a conspicuous component of the extracellular matrix, particularly in remodelling tissues undergoing regeneration and repair. During gestation, changes in HA deposition and distribution indicate that this molecule may participate in preparation of the endometrial stroma for reception and implantation of the embryo. However, little is known about the role of hyaluronan at the fetomaternal interface, specially regarding its influence in pregnancy outcome. In the present study we show increased decidual hyaluronan levels in spontaneous abortion compared with normal pregnancy mice on gestation day 7.5. Both in normal and pathologic pregnancies, high molecular size hyaluronan was found at the fetomaternal unit. However, hyaluronan metabolism (which results from the activity of hyaluronan synthases and hyaluronidases) seems to be altered in spontaneous abortion as shown by a decrease in Hyal-3 expression as well as by differences in hyaluronan molecular size spectrum. This alteration in hyaluronan metabolism in spontaneous abortion could explain its increased concentration observed in decidua and the abnormal distribution of hyaluronan around the embryo implantation crypt. Thus, increased decidual hyaluronan levels resulting from abnormal deposition and turn over may contribute to the pathogenesis of pregnancy failure. PMID:19059644

  4. Mesotheliomas show higher hyaluronan positivity around tumor cells than metastatic pulmonary adenocarcinomas.

    PubMed

    Törrönen, Kari; Soini, Ylermi; Pääkkö, Paavo; Parkkinen, Jyrki; Sironen, Reijo; Rilla, Kirsi

    2016-10-01

    Hyaluronan is a unique glycosaminoglycan of the extracellular matrix, abundant in normal connective tissues but highly increased in many pathological conditions like cancer. Mesothelioma, one of the most malignant cancer types, is associated with high content of hyaluronan, with elevated levels of hyaluronan in pleural effusions and serum of the patients. Metastatic lung adenocarcinomas are typically less aggressive and have a better prognosis as compared to mesotheliomas, a reason why it is highly important to find reliable tools to differentiate these cancer types. The main purpose of this study was to evaluate the amount of hyaluronan, hyaluronan producing synthases (HAS's) and hyaluronan receptor CD44, in mesothelioma and metastatic lung adenocarcinomas. Furthermore, we wanted to clarify the role of hyaluronan, CD44 and HAS's as putative markers for differentiating malignant mesothelioma from metastatic lung adenocarcinomas. The main finding of this study was that mesotheliomas are significantly more positive for hyaluronan staining than metastatic adenocarcinomas. Unexceptionally, a trend of CD44 positivity of stromal cells was higher in adenocarcinomas as compared to mesotheliomas. However, no statistically significant differences were found between the staining of any of the HAS isoenzymes either in tumor cells or stromal cells of different groups of cases. The results show that there are significant differences in hyaluronan content between metastatic lung adenocarcinomas and mesotheliomas. However, as previous studies have suggested, hyaluronan alone is not a sufficient independent marker for diagnostic differentiation of these cancer types, but could be utilized as a combination together with other specific markers. PMID:26912058

  5. Pasteurella multocida- and Pasteurella haemolytica-ghosts: new vaccine candidates.

    PubMed

    Marchart, J; Dropmann, G; Lechleitner, S; Schlapp, T; Wanner, G; Szostak, M P; Lubitz, W

    2003-09-01

    Pasteurella multocida is an important animal pathogen. Bacterial ghosts produced by the expression of phage PhiX174 lysis gene E are empty cells devoid of cytoplasmic and genomic material. Lysis of P. multocida 7A and P. haemolytica A1 carrying Pasteurella-specific lysis vectors (pSR2 and pSON2) occurred 140 min after induction of gene E expression induced by temperature upshift. The E-mediated cell lysis and killing activity was the same in both Pasteurella species and no viable cells could be detected after lysis of P. multocida and P. haemolytica. Pasteurella ghosts were used for immunization of rabbits and mice. Rabbits immunized subcutaneously with either P. multocida- or P. haemolytica-ghosts developed antibodies reacting with the immunizating strain, as well as with other Pasteurella strains. The number of proteins in whole cell protein extracts recognized by the sera constantly increased during the observation period of 51 days. In addition, dose-dependent protection against homologous challenge was observed in mice immunized with P. multocida-ghosts. Animals which received 1.15 x 10(8) ghosts and a challenge dose of up to 60 cfu (LD90), showed 100% protection. According to these results, we suggest ghosts of P. multocida and P. haemolytica as new vaccine candidates. PMID:12922135

  6. Serum susceptibility of bovine pasteurellas.

    PubMed Central

    Blau, K A; Ward, A C; Prieur, D J; Corbeil, L B

    1987-01-01

    In this study, the serum sensitivity of 23 P. haemolytica isolates and 18 P. multocida isolates was determined by incubating dilutions of bacteria with equal volumes of fresh or heat-inactivated bovine serum for one, two, or three hours. Clinical isolates of both Pasteurella species were resistant to serum, whereas isolates from asymptomatic cattle varied in serum susceptibility. The classical pathway of complement appeared to be the principal means of complement mediated killing as detected by incubation in the presence or absence of EGTA-MgCl2. Lyzozyme and iron saturation of serum did not greatly affect serum susceptibility with either of the Pasteurella species. PMID:3300919

  7. Comamonas testosteronan synthase, a bifunctional glycosyltransferase that produces a unique heparosan polysaccharide analog

    PubMed Central

    Otto, Nigel J; Solakyildirim, Kemal; Linhardt, Robert J; DeAngelis, Paul L

    2011-01-01

    Glycosaminoglycans (GAGs) are linear hexosamine-containing polysaccharides. These polysaccharides are synthesized by some pathogenic bacteria to form an extracellular coating or capsule. This strategy forms the basis of molecular camouflage since vertebrates possess naturally occurring GAGs that are essential for life. A recent sequence database search identified a putative protein from the opportunistic pathogen Comamonas testosteroni that exhibits similarity with the Pasteurella multocida GAG synthase PmHS1, which is responsible for the synthesis of a heparosan polysaccharide capsule. Initial supportive evidence included glucuronic acid (GlcUA)-containing polysaccharides extracted from C. testosteroni KF-1. We describe here the cloning and analysis of a novel Comamonas GAG synthase, CtTS. The GAG produced by CtTS in vitro consists of the sugars d-GlcUA and N-acetyl-d-glucosamine, but is insensitive to digestion by GAG digesting enzymes, thus has distinct glycosidic linkages from vertebrate GAGs. The backbone structure of the polysaccharide product [-4-d-GlcUA-α1,4-d-GlcNAc-α1-]n was confirmed by nuclear magnetic resonance. Therefore, this novel GAG, testosteronan, consists of the same sugars as the biomedically relevant GAGs heparosan (N-acetyl-heparosan) and hyaluronan but may have distinct properties useful for future medical applications. PMID:21610195

  8. 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

  9. Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds.

    PubMed

    Puperi, Daniel S; O'Connell, Ronan W; Punske, Zoe E; Wu, Yan; West, Jennifer L; Grande-Allen, K Jane

    2016-05-01

    Advanced tissue engineered heart valves must be constructed from multiple materials to better mimic the heterogeneity found in the native valve. The trilayered structure of aortic valves provides the ability to open and close consistently over a full human lifetime, with each layer performing specific mechanical functions. The middle spongiosa layer consists primarily of proteoglycans and glycosaminoglycans, providing lubrication and dampening functions as the valve leaflet flexes open and closed. In this study, hyaluronan hydrogels were tuned to perform the mechanical functions of the spongiosa layer, provide a biomimetic scaffold in which valve cells were encapsulated in 3D for tissue engineering applications, and gain insight into how valve cells maintain hyaluronan homeostasis within heart valves. Expression of the HAS1 isoform of hyaluronan synthase was significantly higher in hyaluronan hydrogels compared to blank-slate poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Hyaluronidase and matrix metalloproteinase enzyme activity was similar between hyaluronan and PEGDA hydrogels, even though these scaffold materials were each specifically susceptible to degradation by different enzyme types. KIAA1199 was expressed by valve cells and may play a role in the regulation of hyaluronan in heart valves. Cross-linked hyaluronan hydrogels maintained healthy phenotype of valve cells in 3D culture and were tuned to approximate the mechanical properties of the valve spongiosa layer. Therefore, hyaluronan can be used as an appropriate material for the spongiosa layer of a proposed laminate tissue engineered heart valve scaffold. PMID:27120017

  10. Hematogenous Pasteurella multocida brain abscess

    SciTech Connect

    Wallace, M.; Lipsky, B.A.

    1985-10-01

    A case of hematogenously acquired brain abscess caused by Pasteurella multocida is described. CT scans of the head revealed the lesions in a 67 year old man with mild alcoholic liver disease and severe chronic obstructive pulmonary disease. Ultrasound examinations of the abdomen and chest and an echocardiogram failed to reveal a source for the abscess. On autopsy examination three encapsulated brain abscesses were found. 34 references, 2 figures, 1 table.

  11. 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.

  12. 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. PMID:26082778

  13. 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

  14. Inhibition of hyaluronan export reduces collagen degradation in interleukin-1 treated cartilage

    PubMed Central

    Deiters, Barthold; Prehm, Peter

    2008-01-01

    Background Osteoarthrosis is characterized by cartilage erosion, proteolysis of aggrecan and collagen, and disturbed rates of synthesis of aggrecan and hyaluronan by chondrocytes, with hyaluronan over-production being an early reaction. We considered that inhibition of hyaluronan export might prevent subsequent proteoglycan loss and collagen degradation. Methods To test this hypothesis, we studied a tissue culture model using bovine cartilages explants activated with IL-1α to induce osteoarthritic reactions using the phosphodiesterase-5 inhibitors tadalafil, zaprinast and vardenafil. Results These drugs inhibited hyaluronan export, but they did not inhibit hyaluronan synthase activity. Simultaneously, they inhibited proteoglycan loss and collagen degradation, but not their synthesis. They also reduced the release of gelatinases into the culture media and diffusion of the indicator protein horseradish peroxidase through the cartilage explants. The mechanism of action of these compounds may be through inhibition of hyaluronan exporter multidrug resistance-associated protein 5 (MRP5), because the effective drug concentrations were much higher than required for phosphodiesterase-5 inhibition and intracellular cGMP accumulation. Conclusion Inhibition of hyaluronan over-production may be an appropriate target to attenuate IL-1-induced reactions in osteoarthritic cartilage. PMID:18205921

  15. Anti-obesity potential of enzymatic fragments of hyaluronan on high-fat diet-induced obesity in C57BL/6 mice.

    PubMed

    Park, Byong-Gon; Park, Yoon-Sun; Park, Joo Woong; Shin, Eunji; Shin, Woon-Seob

    2016-04-22

    Hyaluronan has diverse biological activities depending on its molecular size. The hyaluronan fragments (50 kDa) can decrease adipogenic differentiation in vitro. However, in vivo anti-obesitic effects of hyaluronan fragments have not been elucidated. Therefore, we examined the anti-obesity effects of hyaluronan fragments on high-fat diet induced obesity in C57BL/6 mice. Oral administration of hyaluronan fragments (200 mg/kg for 8 weeks) decreased body weight, adipose tissues, serum lipid (low-density lipoprotein cholesterol, triglyceride), and leptin level. Hyaluronan fragments decreased the hypertrophy of adipose tissue and ameliorated liver steatosis. The mRNA expression of leptin was reduced in adipocyte by treatment with hyaluronan fragments. Additionally, hyaluronan fragments enhanced the mRNA expression of PPAR-α and its target genes UCP-2 and decreased mRNA expression of PPAR- γ and fatty acid synthase in liver. In conclusions, hyaluronan fragments had marked effects on inhibiting the development of obesity in obese mice fed the high-fat diet. It suggested that enhancing PPAR-α and suppressing PPAR-γ expression are two possible mechanisms for the anti-obesitic effect of hyaluronan fragments. PMID:27012203

  16. 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.

  17. 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

  18. Infective Exacerbation of Pasteurella multocida

    PubMed Central

    Hamada, Mayumi; Elshimy, Noha; Abusriwil, Hatem

    2016-01-01

    An 89-year-old lady presented with a one-day history of shortness of breath as well as a cough productive of brown sputum. Her medical history was significant for chronic obstructive pulmonary disease (COPD). She was in severe type one respiratory failure and blood tests revealed markedly raised inflammatory markers; however her chest X-ray was clear. On examination there was bronchial breathing with widespread crepitations and wheeze. She was treated as per an infective exacerbation of COPD. Subsequent blood cultures grew Pasteurella multocida, a common commensal in the oropharynx of domesticated animals. The patient was then asked about any contact with animals, after which she revealed she had a dog and was bitten on her left hand the day before admission. We should not forget to enquire about recent history of injuries or animal bites when patients present acutely unwell. She made a complete recovery after treatment with penicillin. PMID:26942025

  19. Infective Exacerbation of Pasteurella multocida.

    PubMed

    Hamada, Mayumi; Elshimy, Noha; Abusriwil, Hatem

    2016-01-01

    An 89-year-old lady presented with a one-day history of shortness of breath as well as a cough productive of brown sputum. Her medical history was significant for chronic obstructive pulmonary disease (COPD). She was in severe type one respiratory failure and blood tests revealed markedly raised inflammatory markers; however her chest X-ray was clear. On examination there was bronchial breathing with widespread crepitations and wheeze. She was treated as per an infective exacerbation of COPD. Subsequent blood cultures grew Pasteurella multocida, a common commensal in the oropharynx of domesticated animals. The patient was then asked about any contact with animals, after which she revealed she had a dog and was bitten on her left hand the day before admission. We should not forget to enquire about recent history of injuries or animal bites when patients present acutely unwell. She made a complete recovery after treatment with penicillin. PMID:26942025

  20. Restriction of mast cell proliferation through hyaluronan synthesis by co-cultured fibroblasts.

    PubMed

    Takano, Hirotsugu; Furuta, Kazuyuki; Yamashita, Kazuhito; Sakanaka, Mariko; Itano, Naoki; Gohda, Eiichi; Nakayama, Kazuhisa; Kimata, Koji; Sugimoto, Yukihiko; Ichikawa, Atsushi; Tanaka, Satoshi

    2012-01-01

    Appropriate culture models for tissue mast cells are required to determine how they are involved in regulation of local immune responses. We previously established a culture model for cutaneous mast cells, in which bone marrow-derived immature mast cells were co-cultured with Swiss 3T3 fibroblasts in the presence of stem cell factor. In this study, we focused on the roles of hyaluronan, which is produced by the feeder fibroblasts and forms the extracellular matrix during the co-culture period. Hyaluronan synthesis was found to be mediated by hyaluronan synthase 2 (HAS2) expressed in Swiss 3T3 cells. A decreases in the amount of hyaluronan, which was achieved by retroviral expression of short hairpin RNA for Has2 or by addition of hyaluronidase, significantly enhanced the proliferation of the cultured mast cells without any obvious effects on their maturation. Although we previously demonstrated that CD44 is required for proliferation of cutaneous mast cells, the deficiency of hyaluronan did not affect the proliferation of the cultured mast cells that lack CD44. These findings suggest that the extracellular matrix containing hyaluronan may have a potential to restrict proliferation of cutaneous mast cells in a CD44-independent manner. PMID:22382329

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. Regulation of hyaluronan expression during cervical ripening.

    PubMed

    Straach, Kelly J; Shelton, John M; Richardson, James A; Hascall, Vincent C; Mahendroo, Mala S

    2005-01-01

    In preparation for birth, the uterine cervix undergoes a remarkable transformation from a closed, rigid structure to a distensible, remodeled configuration that stretches to allow passage of a fetus. Cervical ripening requires changes in the composition and structure of the extracellular matrix. These include an increase in the glycosaminoglycan hyaluronan (HA) prior to parturition. We show that the increase in cervical HA with advancing gestation correlates with the temporal increase in transcription of hyaluronan synthase 2 (HAS2) in the mouse. On gestation day 18, 1 day prior to birth, HAS2 transcripts are most abundant and begin to decline after birth. The steroid 5alpha-reductase type 1 deficient mouse, which fails to undergo cervical remodeling, has decreased expression of HAS2 mRNA and decreased tissue HA. HAS2 transcripts are expressed by cervical epithelium, and HA is localized to the matrix surrounding the stroma and to a lesser extent around the epithelium. HAS2 expression is suppressed in mice treated with progesterone. The mRNA expression levels of HA metabolizing enzymes hyaluronidase 1 and 2 were unchanged during pregnancy but increased after birth. Thus the net increase in HA content at term correlates with increased transcription of HAS2. Regulation of HA content is conserved in women because HAS2 transcripts are up-regulated in cervices of women in labor as compared to pregnant women not in labor. These results provide insights into the regulation of HA biosynthesis during cervical ripening and underscore the physiological role of HA in this essential process. PMID:15317739

  10. 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).

  11. The first chemical synthesis of novel MeO-3-GlcUA derivative of hyaluronan-based disaccharide to elucidate the catalytic mechanism of hyaluronic acid synthases (HASs)

    PubMed Central

    Wei, Guohua; Kumar, Vipin; Xue, Jun; Locke, Robert D.; Matta, Khushi L.

    2009-01-01

    The first chemical synthesis of MeO-3-GlcUAβ(1→3)GlcNAc-UDP to elucidate the catalytic mechanism of hyaluronic acid synthases (HASs) is described. Construction of the desired β(1→3)-linked disaccharide 10 was achieved very efficiently by coupling MeO-3-GlcUA donor 3 with the suitable protected GlcNTroc acceptor 4 using BF3.Et2O as Lewis acid. Chemoselective removal of anomeric NAP, phosphorylation, hydrogenation, coupling with UMP-morpholidate and finally complete deprotection gave the target compound 1 in good yield. PMID:20161585

  12. Modulation of Hyaluronan Synthesis by the Interaction between Mesenchymal Stem Cells and Osteoarthritic Chondrocytes

    PubMed Central

    Antonioli, Eliane; Piccinato, Carla A.; Nader, Helena B.; Cohen, Moisés; Goldberg, Anna Carla; Ferretti, Mario

    2015-01-01

    Bone marrow mesenchymal stem cells (BM-MSCs) are considered a good source for cellular therapy in cartilage repair. But, their potential to repair the extracellular matrix, in an osteoarthritic environment, is still controversial. In osteoarthritis (OA), anti-inflammatory action and extracellular matrix production are important steps for cartilage healing. This study examined the interaction of BM-MSC and OA-chondrocyte on the production of hyaluronan and inflammatory cytokines in a Transwell system. We compared cocultured BM-MSCs and OA-chondrocytes with the individually cultured controls (monocultures). There was a decrease in BM-MSCs cell count in coculture with OA-chondrocytes when compared to BM-MSCs alone. In monoculture, BM-MSCs produced higher amounts of hyaluronan than OA-chondrocytes and coculture of BM-MSCs with OA-chondrocytes increased hyaluronan production per cell. Hyaluronan synthase-1 mRNA expression was upregulated in BM-MSCs after coculture with OA-chondrocytes, whereas hyaluronidase-1 was downregulated. After coculture, lower IL-6 levels were detected in BM-MSCs compared with OA-chondrocytes. These results indicate that, in response to coculture with OA-chondrocytes, BM-MSCs change their behavior by increasing production of hyaluronan and decreasing inflammatory cytokines. Our results indicate that BM-MSCs per se could be a potential tool for OA regenerative therapy, exerting short-term effects on the local microenvironment even when cell:cell contact is not occurring. PMID:26273306

  13. [Pasteurella multocida meningitis with cerebral abscesses].

    PubMed

    Nguefack, S; Moifo, B; Chiabi, A; Mah, E; Bogne, J-B; Fossi, M; Fru, F; Mbonda, E; Djientcheu, V-P

    2014-03-01

    Pasteurella multocida is classically responsible for local soft tissue infections secondary to dog bites or cat scratches. It can be responsible for meningitis in infants and elderly persons. We report the case history of a 5-year-old male child admitted to our pediatric unit for meningitis. Cerebrospinal fluid analysis revealed an infection with P. multocida. The suspected mode of contamination was either from the saliva of a pet dog or through an unnoticed skull fracture sustained after an accident 1 year prior to the occurrence of meningitis. In spite of the neurologic complication (cerebral abscess), the progression was favorable after drainage of the abscess, 5 weeks of parenteral treatment, and 3 weeks of oral antibiotic therapy. Meningitis due to Pasteurella sp. is rare and can lead to neurologic complications. The notion of bites or scratches can be absent and the mode of contamination is sometimes difficult to unveil. PMID:24457110

  14. 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

  15. 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

  16. 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

  17. Pasteurella multocida pneumonia complicated by Staphylococcus aureus.

    PubMed

    Martyn, V; Swift, D

    1984-02-01

    A 71-year-old woman presented with acute non-cardiogenic pulmonary oedema. She proved to have a Pasteurella multocida pneumonia, with blood stream invasion by the organism, and required positive pressure ventilation for 53 days. Penicillin G., the drug of choice for this infection, failed to reverse the steady decline in her arterial oxygen-tension, and it was only after treatment with chloramphenicol and prednisolone that she began to improve. Serological tests strongly indicated the presence of a Staphylococcus aureus infection and the delay in giving antibiotics appropriate to this second pathogen may have been the reason for the patient's initial downhill course. PMID:6709548

  18. Regulation of Synthesis and Roles of Hyaluronan in Peritoneal Dialysis

    PubMed Central

    Bowen, Timothy; Meran, Soma; Williams, Aled P.; Newbury, Lucy J.; Sauter, Matthias; Sitter, Thomas

    2015-01-01

    Hyaluronan (HA) is a ubiquitous extracellular matrix glycosaminoglycan composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating β-1,4 and β-1,3 glycosidic bonds. HA is synthesized in humans by HA synthase (HAS) enzymes 1, 2, and 3, which are encoded by the corresponding HAS genes. Previous in vitro studies have shown characteristic changes in HAS expression and increased HA synthesis in response to wounding and proinflammatory cytokines in human peritoneal mesothelial cells. In addition, in vivo models and human peritoneal biopsy samples have provided evidence of changes in HA metabolism in the fibrosis that at present accompanies peritoneal dialysis treatment. This review discusses these published observations and how they might contribute to improvement in peritoneal dialysis. PMID:26550568

  19. Pasteurella multocida Toxin Manipulates T Cell Differentiation

    PubMed Central

    Hildebrand, Dagmar; Heeg, Klaus; Kubatzky, Katharina F.

    2015-01-01

    Pasteurella multocida causes various diseases in a broad range of wild and domestic animals. Toxigenic strains of the serotypes A and D produce an AB protein toxin named Pasteurella multocida toxin (PMT). PMT constitutively activates the heterotrimeric G protein subunits Gαq, Gα13, and Gαi through deamidation of a glutamine residue, which results in cytoskeletal rearrangements as well as increased proliferation and survival of the host cell. In human monocytes, PMT alters the lipopolysaccharide (LPS)-induced activation toward a phenotype that suppresses T cell activation. Here we describe that the toxin also modulates CD4-positive T helper (Th) cells directly. PMT amplifies the expansion of Th cells through enhanced cell cycle progression and suppression of apoptosis and manipulates the differentiation of Th subclasses through activation of Signal Transducers and Activators of Transcription (STAT) family members and induction of subtype-specific master transcription factors. A large population of toxin-treated T cells is double-positive for Foxp3 and RORγt, the transcription factors expressed by Treg and Th17 cells, respectively. This suggests that these cells could have the potential to turn into Th17 cells or suppressive Treg cells. However, in terms of function, the PMT-differentiated cells behave as inflammatory Th17 cells that produce IL-17 and trigger T cell proliferation. PMID:26635744

  20. Hyaluronan-coated extracellular vesicles--a novel link between hyaluronan and cancer.

    PubMed

    Rilla, Kirsi; Siiskonen, Hanna; Tammi, Markku; Tammi, Raija

    2014-01-01

    The synthesis of hyaluronan (HA) on the plasma membrane is a unique and still partly mysterious way of macromolecular biosynthesis. HA forms pericellular coats around many cell types and accumulates in the extracellular matrix (ECM) of growing and renewing tissues. It is secreted to high concentrations in body fluids with antifriction properties like pleural, peritoneal, and synovial fluids, but is also detectable in plasma, saliva, and urine. In pathological states, like cancer and inflammation, the amount of HA is increased around cells, in the ECM, and in the body fluids. HA is an indicator of poor prognosis for cancer patients and creates a favorable environment for cellular growth and motility. The recent finding that HA-coated extracellular vesicles act both as a product of HA synthase activity and as special vehicles for HA, and perhaps carry signals important for malignant growth, provides a novel link between HA and cancer. HA could be carried on the surface of these vesicles in tissues and body fluids, creating beneficial environments by itself, or by associated molecules, for the invasion and metastasis of cancer cells. The HA-coated plasma membrane protrusions and vesicles shed from them are potential biomarkers in cancer and other HA-associated disease states. PMID:25081528

  1. 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

  2. Draft genome sequences of two virulent serotypes of avian Pasteurella multocida

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the draft genome sequences of two virulent avian strains of Pasteurella multocida. Comparative analyses of these genomes were done with the published genome sequence of avirulent Pasteurella multocida strain Pm70....

  3. Early requirement of Hyaluronan for tail regeneration in Xenopus tadpoles.

    PubMed

    Contreras, Esteban G; Gaete, Marcia; Sánchez, Natalia; Carrasco, Héctor; Larraín, Juan

    2009-09-01

    Tail regeneration in Xenopus tadpoles is a favorable model system to understand the molecular and cellular basis of tissue regeneration. Although turnover of the extracellular matrix (ECM) is a key event during tissue injury and repair, no functional studies to evaluate its role in appendage regeneration have been performed. Studying the role of Hyaluronan (HA), an ECM component, is particularly attractive because it can activate intracellular signaling cascades after tissue injury. Here we studied the function of HA and components of the HA pathway in Xenopus tadpole tail regeneration. We found that transcripts for components of this pathway, including Hyaluronan synthase2 (HAS2), Hyaluronidase2 and its receptors CD44 and RHAMM, were transiently upregulated in the regenerative bud after tail amputation. Concomitantly, an increase in HA levels was observed. Functional experiments using 4-methylumbelliferone, a specific HAS inhibitor that blocked the increase in HA levels after tail amputation, and transgenesis demonstrated that the HA pathway is required during the early phases of tail regeneration. Proper levels of HA are required to sustain proliferation of mesenchymal cells in the regenerative bud. Pharmacological and genetic inhibition of GSK3beta was sufficient to rescue proliferation and tail regeneration when HA synthesis was blocked, suggesting that GSK3beta is downstream of the HA pathway. We have demonstrated that HA is an early component of the regenerative pathway and is required for cell proliferation during the early phases of Xenopus tail regeneration. In addition, a crosstalk between HA and GSK3beta signaling during tail regeneration was demonstrated. PMID:19666825

  4. A novel role of low molecular weight hyaluronan in breast cancer metastasis.

    PubMed

    Wu, Man; Cao, Manlin; He, Yiqing; Liu, Yiwen; Yang, Cuixia; Du, Yan; Wang, Wenjuan; Gao, Feng

    2015-04-01

    Low molecular weight hyaluronan (LMW-HA), a degradation fragment of the extracellular matrix component hyaluronan (HA), has been proven to play a crucial role in cancer progression. However, no systematic clinical study of breast cancer has been performed to correlate LMW-HA levels with metastasis. In the present study, we analyzed 176 serum specimens and found for the first time that the serum LMW-HA (but not total HA) level significantly correlated with lymph node metastasis, suggesting that serum LMW-HA represents a better prognostic indicator of breast cancer progression than HA. Similarly, we found that breast cancer cell lines displaying higher invasive potential had a higher LMW-HA concentration than less-invasive cell lines. This higher LMW-HA level was accompanied by the overexpression of hyaluronan synthase (HAS2) and hyaluronidase (both HYAL1 and HYAL2). Of great importance, decreasing LMW-HA production significantly inhibited breast cancer cell migration and invasion. Overall, our results suggest that during cancer progression, cancer cells may actively remodel their microenvironment via an autocrine/paracrine-like process, resulting in elevated LMW-HA levels, which in turn may facilitate cancer progression by promoting the migration and invasion of cancer cells. Therefore, cancer-associated LMW-HA may be a more promising molecular biomarker than total HA for detecting metastasis and may have further applications in breast cancer treatment. PMID:25550464

  5. Hyaluronan and phospholipid association in biolubrication.

    PubMed

    Wang, Min; Liu, Chao; Thormann, Esben; Dėdinaitė, Andra

    2013-12-01

    It is becoming increasingly clear that the outstanding lubrication of synovial joints is achieved by a sophisticated hierarchical structure of cartilage combined with synergistic actions of surface-active components present in the synovial fluid. In this work we focus on the association of two components of the synovial fluid, hyaluronan and dipalmitoyl phosphatidyl choline (DPPC), in bulk solution and at interfaces. We demonstrate that hyaluronan associates with DPPC vesicles and adsorbs to supported DPPC bilayers. The association structures formed at the interface are sufficiently stable to allow sequential adsorption of DPPC and hyaluronan, whereby promoting the formation of thick composite layers of these two components. The lubricating ability of such composite layers was probed by the AFM colloidal probe technique and found to be very favorable with low friction coefficients and high load bearing capacity. With DPPC as the last adsorbed component, a friction coefficient of 0.01 was found up to pressures significantly above what is encountered in healthy synovial joints. Hyaluronan as the last added component increases the friction coefficient to 0.03 and decreases the load bearing capacity somewhat (but still above what is needed in the synovial joint). Our data demonstrate that self-assembly structures formed by hyaluronan and phospholipids at interfaces are efficient aqueous lubricants, and it seems plausible that such self-assembly structures contribute to the exceptional lubrication of synovial joints. PMID:24171653

  6. Surface motion upregulates superficial zone protein and hyaluronan production in chondrocyte-seeded three-dimensional scaffolds.

    PubMed

    Grad, Sibylle; Lee, Cynthia R; Gorna, Katarzyna; Gogolewski, Sylwester; Wimmer, Markus A; Alini, Mauro

    2005-01-01

    A cartilage engineering bioreactor has been developed that provides joint-specific kinematics. This study investigated the effect of articular motion on the gene expression of superficial zone protein (SZP) and hyaluronan synthases (HASs) and on the release of SZP and hyaluronan of chondrocytes seeded onto biodegradable scaffolds. Cylindrical (8 x 4 mm) porous polyurethane scaffolds were seeded with bovine articular chondrocytes and subjected to static or dynamic compression, with and without articulation against a ceramic hip ball. After loading, the mRNA expression of SZP and HASs was analyzed, and SZP immunoreactivity and hyaluronan concentration of conditioned media were determined. Surface motion significantly upregulated the mRNA expression of SZP and HASs. Axial compression alone had no effect on SZP and increased HAS mRNA only at high strain amplitude. SZP was immunodetected only in the media of constructs exposed to surface motion. The release of hyaluronan into the culture medium was significantly enhanced by surface motion. These results indicate that specific stimuli that mimic the kinematics of natural joints, such as articular motion, may promote the development of a functional articular surface-synovial interface. PMID:15738679

  7. Phenotypic characterization of Zimbabwean isolates of Pasteurella multocida.

    PubMed

    Mohan, K; Sadza, M; Madsen, M; Hill, F W; Pawandiwa, A

    1994-02-01

    The phenotypic characteristics of 60 Zimbabwean isolates of Pasteurella multocida sensu stricto, from disease syndromes in different host species were studied. A number of representative strains were also serotyped. Consistent results were obtained in the tests for; catalase, oxidase, urease, indole, acid in glucose, inositol, salicin and sucrose. There was no obvious relationship between serotype, host or disease and the pattern of utilization of certain substrates by an isolate. This has been discussed in the context of recent proposals to reclassify Pasteurella and P. multocida on genotypic and phenotypic studies. It is suggested that notwithstanding the relevance of genetic studies in circumscribing P. multocida, the phenotype and disease significance of the taxon should not be ignored. A case of bronchitis in a dog which was simultaneously colonized by three different strains of Pasteurella is described. Also septicaemic pasteurellosis in a Nile crocodile (Crocodylus niloticus) is reported and for the first time prevalence of various serotypes in pasteurellosis of animals in Zimbabwe. PMID:8160349

  8. Plasmids for heterologous expression in Pasteurella haemolytica.

    PubMed

    Fedorova, N D; Highlander, S K

    1997-02-28

    New cloning and expression vectors that replicate both in Pasteurella haemolytica and in Escherichia coli were constructed based on a native sulfonamide (SuR) and streptomycin (SmR) resistant plasmid of P. haemolytica called pYFC1. Each shuttle vector includes an MCS and a selectable antibiotic resistance marker that is expressed in both organisms. Plasmid pNF2176 carries the P. haemolytica ROB-1 beta-lactamase gene (blaP, ApR) and pNF2214 carries the Tn903 aph3 kanamycin resistance (KmR) element. The expression vector, pNF2176, was created by placing the MCS downstream of the sulfonamide gene promoter (PsulII) on pYFC1; this was used to clone and express the promoterless Tn9 chloramphenicol resistance gene (cat, CmR) in P. haemolytica (pNF2200). A promoter-probe vector (pNF2283) was constructed from pNF2200 by deleting PsulII. PMID:9074498

  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. A transport medium for specimens containing Pasteurella pestis

    PubMed Central

    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. PMID:5301387

  11. 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

  12. 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

  13. Differential activation of noncanonical SMAD2/SMAD3 signaling by bone morphogenetic proteins causes disproportionate induction of hyaluronan production in immortalized human granulosa cells.

    PubMed

    Zhang, Han; Tian, Shen; Klausen, Christian; Zhu, Hua; Liu, Ruizhi; Leung, Peter C K

    2016-06-15

    Successful fertilization depends upon proper cumulus-oocyte complex (COC) expansion. Synthesized by hyaluronan synthases (HASs), hyaluronan forms the backbone of the COC matrix and plays a critical role in COC expansion. This study investigated the effects and mechanisms of ovarian BMPs on HAS expression and hyaluronan production in human granulosa cells. Treatment with BMP4, BMP6, BMP7 or BMP15 induced differing levels of noncanonical SMAD2/3, but equal levels of canonical SMAD1/5/8, phosphorylation which were mirrored by differing levels of HAS2 up-regulation and hyaluronan production. The effects of BMP4 and BMP15 on HAS2 mRNA were partially reversed by knockdown of SMAD3, and blocked by knockdown of SMAD2+SMAD3 or SMAD4. BMP4-induced SMAD2/3 phosphorylation and HAS2 mRNA up-regulation were mediated by both BMP and activin/transforming growth factor-β type I receptors. Our results suggest differential activation of noncanonical SMAD2/SMAD3 signaling by BMPs causes disproportionate induction of HAS2 expression and hyaluronan production in immortalized human granulosa cells. PMID:26992562

  14. 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. 113.69 Section 113.69 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Bacterial Vaccines §...

  15. 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 Isolate. 113.70 Section 113.70 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Bacterial Vaccines §...

  16. 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, Bovine. 113.68 Section 113.68 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Bacterial Vaccines §...

  17. 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. 113.69 Section 113.69 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Bacterial Vaccines §...

  18. Hamster bite peritonitis: Pasteurella pneumotropica peritonitis in a dialysis patient.

    PubMed

    Campos, A; Taylor, J H; Campbell, M

    2000-11-01

    We report the first case of Pasteurella pneumotropica peritonitis in a peritoneal dialysis patient. This rare infection was the result of contamination of the dialysis tubing by a pet hamster. We stress the importance of household pets as a source of infection in the peritoneal dialysis population. PMID:11095007

  19. Identification of Pasteurella multocida CHAPS-soluble outer membrane proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fowl cholera continues to be of concern to the poultry industry, especially for turkey growers. This disease costs the turkey industry millions of dollars annually. In order to gain a better understanding of Pasteurella multocida virulence factors involved in colonization and pathogenesis, the outer...

  20. The CHAPS-soluble outer membrane proteome of Pasteurella multocida

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fowl cholera continues to be of major concern to the poultry industry, especially for turkey growers. This disease costs the turkey industry millions of dollars annually. Fowl cholera is caused primarily by three serotypes of Pasteurella multocida serotypes A:1, A:3, and A:4. a live attenuated vacc...

  1. 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

  2. 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

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

    PubMed

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

    2015-04-15

    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

  4. Formation and properties of hyaluronan/nano Ag and hyaluronan-lecithin/nano Ag films.

    PubMed

    Khachatryan, Gohar; Khachatryan, Karen; Grzyb, Jacek; Fiedorowicz, Maciej

    2016-10-20

    A facile and environmentally friendly method of the preparation of silver nanoparticles embedded in hyaluronan (Hyal/Ag) and hyaluronan-lecithin (Hyal-L/Ag) matrix was developed. Thin, elastic foils were prepared from gels by an in situ synthesis of Ag in an aqueous solution of sodium hyaluronate (Hyal), using aq. d-(+)-xylose solution as a reducing agent. The gels were applied to a clean, smooth, defatted Teflon surface and left for drying in the air. The dry foils were stored in a closed container. UV-vis spectroscopy, transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectra confirmed formation of about 10nm ball-shaped Ag nanoparticles situated within the polysaccharide template. Thermal properties of the composites were characterized involving differential scanning calorimetry (DSC) and thermogravimetric (TGA) analyses, whereas molecular weights of polysaccharide chains of the matrix were estimated with the size exclusion chromatography coupled with multiangle laser light scattering and refractometric detectors (HPSEC-MALLS-RI). An increase in the molecular weight of the hyaluronate after generation of Ag nanoparticles was observed. The foils showed specific properties. The study confirmed that silver nanoparticles can be successfully prepared with environmentally friendly method, using hyaluronan as a stabilizing template. Hyaluronan and hyaluronan-lecithin matrices provide nanocrystals uniform in size and shape. The composites demonstrated a bacteriostatic activity. PMID:27474588

  5. Sepsis-induced purpura fulminans caused by Pasteurella multocida.

    PubMed

    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

  6. 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

  7. 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.

  8. 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

  9. A cryopreservation method for Pasteurella multocida from wetland samples.

    PubMed

    Moore, M 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. PMID:9476245

  10. Prolonged incubation period in neonatal Pasteurella multocida meningitis and bacteremia.

    PubMed

    Yamaguchi, Hiroshi; Tamura, Takuya; Abe, Michiko; Ogiwara, Shigetoshi; Sai, Shuji; Kosugiyama, Kiyotaka; Sugihara, Akemi; Nagumo, Kiyoshi; Iwata, Seido; Kinugawa, Yoshikazu

    2014-12-01

    Pasteurella multocida, often found as part of the human oral flora and in finger/toenails, also exists in many animals, especially cats, dogs, and pigs. Although rare, pasteurella infection in neonates can cause serious systemic disease, such as meningitis. In this article, a 23-day-old girl presented with decreased appetite and irritability for >2 days. Eighteen days previously her pet cat had jumped onto the left side of her head while she was sleeping. On laboratory data C-reactive protein was high, and on cerebrospinal fluid (CSF) analysis leukocyte count was extremely high, with low glucose and high protein. P. multocida grew out of the blood and CSF cultures, and she was successfully treated with antibiotics for 3 weeks. Although pasteurellosis rarely occurs, it can sometimes lead to life-threatening situations, so parents should exercise caution when having pets around their children. PMID:25521988

  11. Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology

    PubMed Central

    Cyphert, Jaime M.; Trempus, Carol S.; Garantziotis, Stavros

    2015-01-01

    Hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. This review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size-dependent differences in hyaluronan signaling as well as their downstream biological effects. PMID:26448754

  12. [Invasive Pasteurella multocida infections: Two clinical cases and literature review].

    PubMed

    Smíšková, Dita; Džupová, Olga

    2015-06-01

    Pasteurella multocida is a common commensal of the gastrointestinal and respiratory tracts of animals, especially cats and dogs. It is transmitted to humans through contact with animals. Bite wound infection is the most common clinical manifestation. Systemic infections are unusual and mainly affect immunocompromised individuals. The article presents two cases of Pasteurella infection. Wound infection in a 75-year-old female following a bite from her pet cat was associated with bacteremia. The disease course was favorable with the initial clindamycin treatment despite in vitro resistance. The other patient was a 62-year-old female diagnosed with acute bacterial meningitis with multiple brain abscesses and transient expressive aphasia. She reported frequent contacts with pets and domestic animals without a recent bite. Hematogenous dissemination of the infection was suspected. Because of poor therapeutic response, cefotaxime was switched to chloramphenicol which was later switched to a combination of cefotaxime with ciprofloxacin due to anemia. Following 6 weeks of intravenous antibiotic therapy and another 10 weeks of oral ciprofloxacin therapy, magnetic resonance imaging showed normal results and the neurological defect resolved. Epidemiological, clinical and therapeutic aspects of Pasteurella infection are discussed and literature is reviewed. PMID:26312375

  13. Hyaluronan Depolymerization by Megakaryocyte Hyaluronidase-2 Is Required for Thrombopoiesis.

    PubMed

    Petrey, Aaron C; Obery, Dana R; Kessler, Sean P; Flamion, Bruno; de la Motte, Carol A

    2016-09-01

    Hyaluronan is the predominant glycosaminoglycan component of the extracellular matrix with an emerging role in hematopoiesis. Modulation of hyaluronan polymer size is responsible for its control over cellular functions, and the balance of hyaluronan synthesis and degradation determines its molecular size. Although two active somatic hyaluronidases are expressed in mammals, only deficiency in hyaluronidase-2 (Hyal-2) results in thrombocytopenia of unknown mechanism. Our results reveal that Hyal-2 knockout mice accumulate hyaluronan within their bone marrow and within megakaryocytes, the cells responsible for platelet generation. Proplatelet formation by Hyal-2 knockout megakaryocytes was disrupted because of abnormal formation of the demarcation membrane system, which was dilated and poorly developed. Importantly, peptide-mediated delivery of exogenous hyaluronidase rescued deficient proplatelet formation in murine and human megakaryocytes lacking Hyal-2. Together, our data uncover a previously unsuspected mechanism of how hyaluronan and Hyal-2 control platelet generation. PMID:27398974

  14. 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 ...

  15. Cyclic mechanical stretch augments hyaluronan production in cultured human uterine cervical fibroblast cells.

    PubMed

    Takemura, Maki; Itoh, Hiroaki; Sagawa, Norimasa; Yura, Shigeo; Korita, Daizo; Kakui, Kazuyo; Kawamura, Makoto; Hirota, Naoyoshi; Maeda, Hiroshi; Fujii, Shingo

    2005-09-01

    Hyaluronan (HA) a glycosaminoglycan with high affinity for water molecules stimulates local inflammatory reactions. Parturition causes a dramatic increase in the amount of HA fragments in the uterine cervix, thereby contributing to a rapid softening as well as opening of the cervical canal, i.e. cervical ripening. The aim of this study was to investigate the possible involvement of cyclic distension caused by labour in the augmentation of HA production during cervical ripening. Immunohistochemistry and/or RT-PCR detected hyaluronan synthase (HAS)1, 2 and 3 in samples of human cervical tissue obtained from pregnant women. Labour-like cyclic mechanical stretch for 24, 36 and 48 h significantly enhanced the secretion of HA, from cultured human uterine cervical fibroblast (CxF) cells, 128.7, 151.4 and 173.2%, respectively, concomitant with a significant augmentation of HAS1 (36, 48 h), HAS2 (24, 36 and 48 h) and HAS3 (48 h) mRNA expression. Cyclic mechanical stretch for 12, 36 and 48 h increased molecular size of the HA secreted from CxF cells. In conclusion, cyclic mechanical stretch of the uterine cervix caused by the presenting part of the fetus in labour may contribute to the increase in the secretion of HA during the process of cervical ripening. PMID:16199413

  16. Growth promoting effect of hyaluronan synthesis promoting substances on Japanese eel leptocephali.

    PubMed

    Kawakami, Yutaka; Nomura, Kazuharu; Tanaka, Hideki

    2014-01-01

    Hyaluronans (HAs) are glycosaminoglycans produced in the bodies of Anguilliform and Elopiform leptocephali, and play a role in metabolic energy. In mammals, HA synthesis-promoting substances (HASPS) up-regulate the expression of HA synthase (HAS) and increase the amount of HA in the body. In this study, Japanese eel leptocephali were fed a HASPS containing diet. We analyzed HAS1s and HAS2 expression, HA content, and their influence on growth. HASPS extracted from Grifola frondosa promoted HAS1s and HAS2 mRNA and HA content. Other than mammals, these results are first reported in vertebrate. Moreover, HASPS extracted from G. frondosa promoted leptocephalus growth. The relationship between growth and HA in the leptocephali is not yet clear. However, based on our results we hypothesize that HA is involved in the storage of energy, which is metabolized to sugars when needed for metabolic energy. PMID:24896609

  17. 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. PMID:25839341

  18. Hyaluronan and hyaluronidase, which is better for embryo development?

    PubMed

    Marei, Waleed F A; Raheem, Kabir A; Salavati, Mazdak; Tremaine, Tina; Khalid, Muhammad; Fouladi-Nashta, Ali A

    2016-09-01

    Our aim was to examine size-specific effects of Hyaluronan (HA) on preimplantation embryo development. We investigated the effects of Hyalovet (HA, 500-750 kDa; the size produced by HA synthase-3, which is abundant in the oviduct), or HA treated with Hyaluronidase-2 (Hyal2; also expressed in the oviduct that breaks down HA into 20 kDa fragments). In experiment 1 (in vivo), oviducts of synchronized and superovulated ewes (n = 20) were surgically exposed on Day 2 post-mating, ligated, and infused with either Hyalovet, Hyalovet + Hyal2, Hyal2, or PBS (control). Ewes were killed 5 days later for recovery of embryos and oviductal epithelial cells (OEC). Blastocyst rates were significantly higher in Hyal2 and Hyalovet + Hyal2 oviducts. Hyaluronidase-2 infusion resulted in higher blastocyst cell numbers and hatching rates. This was associated with increased HSP70 expression in OEC. In contrast, Hyalovet resulted in the lowest development to blastocyst stage and lowest hatching rates, and decreased IGF2 and IGFBP2 expression in OEC. IGF1 and IL1α expression were not affected. In experiment 2, to rule out indirect effects of oviductal factors, ovine embryos were produced and cultured with the same treatments in vitro from Day 2 to 8. Hyaluronidase-2, but not Hyalovet, enhanced blastocyst formation and reduced inner cell mass apoptosis. Hyalovet inhibited hatching. In conclusion, the presence of large-size HA (500-750 kDa) in the vicinity of developing embryos appears to disturb the oviductal environment and embryo development in vivo and in vitro. In contrast, we show evidence that breakdown of HA into smaller fragments is required to maximize embryo development and blastocyst quality. PMID:27091071

  19. 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.

  20. Cancer Microenvironment and Inflammation: Role of Hyaluronan

    PubMed Central

    Nikitovic, Dragana; Tzardi, Maria; Berdiaki, Aikaterini; Tsatsakis, Aristidis; Tzanakakis, George N.

    2015-01-01

    The role of inflammation in the development of cancer was described as early as the nineteenth century. Abundant evidence supports the preposition that various cancers are triggered by infection and chronic inflammatory disease whereas, evading immune destruction has been proposed as one of the new “hallmarks of cancer.” Changes of the tumor microenvironment have been closely correlated to cancer-mediated inflammation. Hyaluronan (HA), an important extracellular matrices component, has become recognized as an active participant in inflammatory, angiogenic, fibrotic, and cancer promoting processes. This review discusses how HA and specific HA-binding proteins participate in and regulate cancer-related inflammatory processes. PMID:25926834

  1. Intracellular hyaluronan in arterial smooth muscle cells: association with microtubules, RHAMM, and the mitotic spindle.

    PubMed

    Evanko, Stephen P; Parks, W Tony; Wight, Thomas N

    2004-12-01

    Although considered a pericellular matrix component, hyaluronan was recently localized in the cytoplasm and nucleus of proliferating cells, supporting earlier reports that hyaluronan was present in locations such as the nucleus, rough endoplasmic reticulum, and caveolae. This suggests that it can play roles both inside and outside the cell. Hyaluronan metabolism is coupled to mitosis and cell motility, but it is not clear if intracellular hyaluronan associates with cytoskeletal elements or plays a structural role. Here we report the distribution of intracellular hyaluronan, microtubules, and RHAMM in arterial smooth muscle cells in vitro. The general distribution of intracellular hyaluronan more closely resembled microtubule staining rather than actin filaments. Hyaluronan was abundant in the perinuclear microtubule-rich areas and was present in lysosomes, other vesicular structures, and the nucleolus. Partially fragmented fluorescein-hyaluronan was preferentially translocated to the perinuclear area compared with high-molecular-weight hyaluronan. In the mitotic spindle, hyaluronan colocalized with tubulin and with the hyaladherin RHAMM, a cell surface receptor and microtubule-associated protein that interacts with dynein and maintains spindle pole stability. Internalized fluorescein-hyaluronan was also seen at the spindle. Following telophase, an abundance of hyaluronan near the midbody microtubules at the cleavage furrow was also noted. In permeabilized cells, fluorescein-hyaluronan bound to RHAMM-associated microtubules. These findings suggest novel functions for hyaluronan in cellular physiology. PMID:15557208

  2. Hyaluronan and synovial joint: function, distribution and healing

    PubMed Central

    2013-01-01

    Synovial fluid is a viscous solution found in the cavities of synovial joints. The principal role of synovial fluid is to reduce friction between the articular cartilages of synovial joints during movement. The presence of high molar mass hyaluronan (HA) in this fluid gives it the required viscosity for its function as lubricant solution. Inflammation oxidation stress enhances normal degradation of hyaluronan causing several diseases related to joints. This review describes hyaluronan properties and distribution, applications and its function in synovial joints, with short review for using thiol compounds as antioxidants preventing HA degradations under inflammation conditions. PMID:24678248

  3. 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. PMID:23464634

  4. 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

  5. Naturally acquired Pasteurella multocida infection in rabbits: clinicopathological aspects.

    PubMed Central

    DiGiacomo, R F; Xu, Y M; Allen, V; Hinton, M H; Pearson, G R

    1991-01-01

    A cohort of 41 New Zealand White rabbits, 35 to 60 days old, from twelve litters were followed for twelve weeks for development of pasteurellosis. Eleven of 19 rabbits in five litters acquired Pasteurella multocida infection. The incubation period was difficult to determine as P. multocida infection was detected both before and after the onset of rhinitis. The response of rabbits to infection varied from subclinical infection to death from systemic pasteurellosis. Atrophy of the maxilloturbinates of the nares was detected in rabbits with chronic rhinitis associated with P. multocida infection. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1889034

  6. Haemolytic effect of Pasteurella haemolytica on blood from young mammals.

    PubMed

    Smith, G R; Turner, A; Hawkey, C M

    1988-09-01

    On agar plates containing young lamb blood, Pasteurella haemolytica produces a wide outer zone of partial haemolysis in addition to the narrow zone of complete clearing seen on adult sheep blood agar. To determine whether this phenomenon was limited to lamb blood, samples from young animals of 20 mammalian species were examined. Two species--the barbary sheep (Ammotragus lervia) and scimitar horned oryx (Oryx tao)--possessed blood that gave this effect provided that the samples were taken from young animals. The 18 species that gave negative results included an ovine species, the bighorn sheep (Ovis canadensis). PMID:3194597

  7. 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

  8. Attachment of hyaluronan to metallic surfaces.

    PubMed

    Pitt, William G; Morris, Robert N; Mason, Mitchell L; Hall, Matthew W; Luo, Yi; Prestwich, Glenn D

    2004-01-01

    Metal implants are in general not compatible with the tissues of the human body, and in particular, blood exhibits a severe hemostatic response. Herein we present results of a technique to mask the surface of metals with a natural biopolymer, hyaluronan (HA). HA has minimal adverse interactions with blood and other tissues, but attachment of bioactive peptides can promote specific biological interactions. In this study, stainless steel was cleaned and then surface-modified by covalent attachment of an epoxy silane. The epoxy was subsequently converted to an aldehyde functional group and reacted with hyaluronan through an adipic dihydrazide linkage, thus covalently immobilizing the HA onto the steel surface. Fluorescent labeling of the HA showed that the surface had a fairly uniform covering of HA. When human platelet rich plasma was placed on the HA-coated surface, there was no observable adhesion of platelets. HA derivatized with a peptide containing the RGD peptide sequence was also bound to the stainless steel. The RGD-containing peptide was bioactive as exemplified by the attachment and spreading of platelets on this surface. Furthermore, when the RGD peptide was replaced with the nonsense RDG sequence, minimal adhesion of platelets was observed. This type of controlled biological activity on a metal surface has potential for modulating cell growth and cellular interactions with metallic implants, such as vascular stents, orthopedic implants, heart valve cages, and more. PMID:14661254

  9. 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).

  10. Virulence of raptor-origin Pasteurella multocida in domestic chickens.

    PubMed

    Aye, P P; Morishita, T Y; Angrick, E J

    1999-01-01

    Pasteurella multocida belonging to somatic serotype 1 and capsular type A has been known to cause avian cholera in domestic poultry. Pasteurella multocida serotype 1 has also been isolated from raptorial birds. However, the capsular type for these raptorial isolates remains unknown. Moreover, the virulence of these raptorial isolates for domestic poultry has not been determined. The objectives of this study were to determine the capsular type of raptorial P. multocida serotype 1 isolates and to determine if these isolates were virulent for domestic chickens. Study chickens were inoculated with one of three P. multocida isolates. Isolate WESO-1 was obtained from a western screech owl (Otus kennicottii) and isolates RTHA-2 and RTHA-4 were isolated from two red-tailed hawks (Buteo jamaicensis). These isolates were given by either the oral, intravenous, or intraocular route. Control birds were given brain-heart infusion broth. The capsular serotypes of three isolates were also determined. The RTHA-2 and RTHA-4 isolates belonged to P. multocida capsular type A. The WESO-1 isolate belonged to capsular type F. Results also demonstrated that, for the isolates examined, the intraocular route did not cause mortality in chickens. There was mortality in all groups for the intravenous route. However, various mortality patterns were observed when P. multocida was given orally for the three different isolates. The RTHA-4 isolate (serotype 1:A) was the most virulent for domestic chickens. The WESO-1 isolate (serotype 1:F) was the least virulent for chickens among the raptorial isolates examined. PMID:10396641

  11. Enzymatic production of specifically distributed hyaluronan oligosaccharides.

    PubMed

    Yuan, Panhong; Lv, Mengxian; Jin, Peng; Wang, Miao; Du, Guocheng; Chen, Jian; Kang, Zhen

    2015-09-20

    High-molecular-mass hyaluronan (HA) was controllably depolymerized in pure aqueous solution with recombinant leech hyaluronidase (HAase). The HAase concentration per unit HA and hydrolysis time played important roles in molecular mass distribution. By modulating the concentrations of HAase and controlling the hydrolysis time, any molar-mass-defined HA oligomers could be efficiently and specifically produced on a large scale (40 g/L), such as HA oligosaccharides with weight-average molar mass of 4000, 10,000, and 30,000Da and end hydrolysates containing only HA6 and HA4. High performance liquid chromatography-size exclusion chromatography, polyacrylamide gel electrophoresis, capillary zone electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry confirmed low polydispersity of the produced molar-mass-defined HA oligosaccharides. Therefore, large-scale production of defined HA oligosaccharides with narrow molecular mass distribution will significantly promote progress in related research and its potential applications. PMID:26050905

  12. The where, when, how, and why of hyaluronan binding by immune cells.

    PubMed

    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

  13. Septic Arthritis and Osteomyelitis Caused by Pasteurella multocida.

    PubMed

    Vranis, Neil; Paryavi, Ebrahim; Christian, Matthew; Joshi, Manjari; Pensy, Raymond A

    2015-07-01

    This report presents a case of progressive septic arthritis and osteomyelitis caused by a rare pathogen, Pasteurella multocida, thought to be provoked by the use of systemic corticosteroids. Despite initial improvement after antibiotics and surgical procedure, the patient returned with new, associated symptoms 1 month later. This concurrent set of circumstances leading to a life-threatening condition has not been reported, to the best of our knowledge. Physicians aware of such a case will be better prepared to diagnose, treat, and educate their patients. Additionally, the diagnostic challenge presented by this case report emphasizes the need for vigilance and thoroughness in obtaining histories from patients presenting with seemingly benign complaints, especially in vulnerable populations, such as infants, pregnant women, and immunocompromised adults. PMID:26161771

  14. Pasteurella canis Isolation following Penetrating Eye Injury: A Case Report.

    PubMed

    Rashid, Noor-Khairul; Zam, Zarifah; Mdnoor, Siti-Suraya; Siti-Raihan, Ishak; Azhany, Yaakub

    2012-01-01

    A 3-year-old boy presented with history of trauma to the left eye after he accidentally injured his eye with a broom stick made up from coconut skewers. There was history of cats as their pets but not dogs. Ocular examination revealed left superonasal conjunctival laceration and scleral perforation with prolapsed vitreous. Fundus examination showed minimal vitreous haemorrhage and flat retina. Conjunctiva swab at the wound site was sent for gram staining, culture, and sensitivity. He underwent scleral suturing, vitreous tap, and intravitreal injection of Ceftazidime and Amikacin. Vitreous tap was sent for gram stained, culture and sensitivity. Postoperatively, he was started empirically on IV Ciprofloxacin 160 mg BD, Guttae Ciprofloxacin, and Guttae Ceftazidime. Conjunctiva swab grew Pasteurella canis which was sensitive to all Beta lactams, Ciprofloxacin, Chloramphenicol, and Aminoglycoside. Post-operative was uneventful, absent signs of endophthalmitis or orbital cellulitis. PMID:22606491

  15. Response of the ruminant respiratory tract to Mannheimia (Pasteurella) haemolytica.

    PubMed

    Ackermann, M R; Brogden, K A

    2000-07-01

    Pneumonia is a leading cause of loss to the sheep and cattle industry throughout the world. Mannheimia (Pasteurella) haemolytica is one of the most important respiratory pathogens of domestic ruminants and causes serious outbreaks of acute pneumonia in neonatal, weaned and growing lambs, calves, and goats. M. haemolytica is also an important cause of pneumonia in adult animals. Transportation, viral infections with agents such as infectious bovine rhinotracheitis virus, parainfluenza-3 virus or bovine respiratory syncytial virus, overcrowding, housing of neonates and weaned animals together and other stressful conditions predispose animals to M. haemolytica infection [1, 2]. This review assimilates some of the findings key to cellular and molecular responses of the lung from a pathologist's perspective. It includes some of what is known and underscores areas that are not fully understood. PMID:10967288

  16. Pasteurella canis Isolation following Penetrating Eye Injury: A Case Report

    PubMed Central

    Rashid, Noor-Khairul; Zam, Zarifah; MdNoor, Siti-Suraya; Siti-Raihan, Ishak; Azhany, Yaakub

    2012-01-01

    A 3-year-old boy presented with history of trauma to the left eye after he accidentally injured his eye with a broom stick made up from coconut skewers. There was history of cats as their pets but not dogs. Ocular examination revealed left superonasal conjunctival laceration and scleral perforation with prolapsed vitreous. Fundus examination showed minimal vitreous haemorrhage and flat retina. Conjunctiva swab at the wound site was sent for gram staining, culture, and sensitivity. He underwent scleral suturing, vitreous tap, and intravitreal injection of Ceftazidime and Amikacin. Vitreous tap was sent for gram stained, culture and sensitivity. Postoperatively, he was started empirically on IV Ciprofloxacin 160 mg BD, Guttae Ciprofloxacin, and Guttae Ceftazidime. Conjunctiva swab grew Pasteurella canis which was sensitive to all Beta lactams, Ciprofloxacin, Chloramphenicol, and Aminoglycoside. Post-operative was uneventful, absent signs of endophthalmitis or orbital cellulitis. PMID:22606491

  17. Pasteurella pestis detection in fleas by fluorescent antibody staining*

    PubMed Central

    Hudson, Bruce W.; Kartman, Leo; Prince, Frank M.

    1966-01-01

    In an effort to develop a method for the rapid field identification of plague-infected fleas, the authors have studied the feasibility of direct fluorescent antibody staining of the midgut contents of fleas fed on mice infected with Pasteurella pestis. Fluorescent antibodies prepared from antisera derived from rabbits inoculated with the water-soluble P. pestis fraction 1b antigen, the somatic antigen of heat-killed P. pestis (Bryans strain), and live avirulent (strain A1122) or virulent (Yreka strain) plague vaccines were used used in this study. This direct staining method proved to be impracticable, but encouraging results were obtained by fluorescent antibody staining of broth cultures of macerates of infected fleas after 24-48 hours' incubation. The broth enrichment technique has not yet been evaluated in the field, but it is expected to be of value since it is relatively simple to perform and requires only material that can easily be transported to remote areas. PMID:5328902

  18. New solid medium for enhanced growth of Pasteurella tularensis.

    PubMed

    GASPAR, A J; TRESSELT, H B; WARD, M K

    1961-10-01

    Gaspar, Andrew J. (Fort Detrick, Frederick, Md.), Hugh B. Tresselt, and Martha K. Ward. New solid medium for enhanced growth of Pasteurella tularensis. J. Bacteriol. 82:564-569. 1961.-The purpose of this work was to develop a solid medium for more rapid growth of Pasteurella tularensis, especially from small inocula comparable to those generally encountered in clinical materials. AFTER TITRATION OF VARIOUS INGREDIENTS, SEPARATELY AND IN COMBINATION, THE OPTIMAL BASE WAS FOUND TO BE: 2.6% tryptose broth (Difco) with thiamine, 0.5% cysteine-HCl, 0.2% sodium thioglycolate, 1.0% glucose, dissolved by mixing without heat, and adjusted to pH 7.2 +/- 0.03. To this base 1.0% agar (Difco) is added and dissolved by heating in flowing steam for about 5 min prior to autoclaving at 121 C for 20 min. After sterilization and cooling, 5.0% defibrinated rabbit blood is added aseptically. Plates of the completed medium are incubated at 37 C for 24 hr prior to use. Colonies of P. tularensis approximately 1.0 mm in diameter are obtained on this medium after 26 to 29 hr incubation if conditions of high relative humidity (90 to 100% saturation) are maintained.The mechanisms involved in the growth enhancement obtained on this medium are under study. The role of thioglycolate appears to be that of keeping in solution the relatively high concentration of cysteine-HCl required. The specific methods of preparation described, the incubation of plates prior to use, and final incubation under conditions of high humidity all are important for optimal results. This developmental work has employed only pure cultures. Attempts are being made to develop a selective medium, using this new preparation as base, for the direct isolation of P. tularensis from a variety of clinical materials. PMID:13897160

  19. 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

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

    PubMed

    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

  1. Hyaluronan deposition and correlation with inflammation in a murine ovalbumin model of asthma

    PubMed Central

    Cheng, Georgiana; Swaidani, Shadi; Sharma, Manisha; Lauer, Mark E.; Hascall, Vincent C.; Aronica, Mark A.

    2011-01-01

    Asthma is a chronic inflammatory disease of the airways characterized by airway remodeling, which includes changes in the extracellular matrix (ECM). However the role of the ECM in mediating these changes is poorly understood. Hyaluronan (HA), a major component of the ECM, has been implicated in asthma as well as in many other biological processes. Our study investigates the processes involved in HA synthesis, deposition, localization and degradation during an acute and chronic murine model of ovalbumin (OVA)-induced allergic pulmonary inflammation. Mice were sensitized, challenged to OVA and sacrificed at various time points during an 8-week challenge protocol. Bronchoalveolar lavage (BAL) fluids, blood, and lung tissue were collected for study. RNA, HA, protein and histopathology were analyzed. Analyses of lung sections and BAL fluids revealed an early deposition and an increase in HA levels within 24 hours of antigen exposure. HA levels peaked at day 8 in BAL, while inflammatory cell recovery peaked at day 6. Hyaluronan synthase (HAS)1 and HAS2 on RNA levels peaked within 2 hours of antigen exposure, while hyaluronidase (HYAL)1 and HYAL2 on RNA levels decreased. Both inflammatory cell infiltrates and collagen deposition co-localized with HA deposition within the lungs. These data support a role for HA in the pathogenesis of inflammation and airway remodeling in a murine model of asthma. HA deposition appears largely due to up regulation of HAS1 and HAS2. In addition, HA appears to provide the scaffolding for inflammatory cell accumulation as well as for new collagen synthesis and deposition. PMID:21251977

  2. 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. PMID:23783513

  3. Pulmonary surfactant adsorption is increased by hyaluronan or polyethylene glycol.

    PubMed

    Taeusch, H William; Dybbro, Eric; Lu, Karen W

    2008-04-01

    In acute lung injuries, inactivating agents may interfere with transfer (adsorption) of pulmonary surfactants to the interface between air and the aqueous layer that coats the interior of alveoli. Some ionic and nonionic polymers reduce surfactant inactivation in vitro and in vivo. In this study, we tested directly whether an ionic polymer, hyaluronan, or a nonionic polymer, polyethylene glycol, enhanced adsorption of a surfactant used clinically. We used three different methods of measuring adsorption in vitro: a modified pulsating bubble surfactometer; a King/Clements device; and a spreading trough. In addition we measured the effects of both polymers on surfactant turbidity, using this assay as a nonspecific index of aggregation. We found that both hyaluronan and polyethylene glycol significantly increased the rate and degree of surfactant material adsorbed to the surface in all three assays. Hyaluronan was effective in lower concentrations (20-fold) than polyethylene glycol and, unlike polyethylene glycol, hyaluronan did not increase apparent aggregation of surfactant. Surfactant adsorption in the presence of serum was also enhanced by both polymers regardless of whether hyaluronan or polyethylene glycol was included with serum in the subphase or added to the surfactant applied to the surface. Therefore, endogenous polymers in the alveolar subphase, or exogenous polymers added to surfactant used as therapy, may both be important for reducing inactivation of surfactant that occurs with various lung injuries. PMID:18065212

  4. Pathophysiology of the Peritoneal Membrane during Peritoneal Dialysis: The Role of Hyaluronan

    PubMed Central

    Yung, Susan; Chan, Tak Mao

    2011-01-01

    During peritoneal dialysis (PD), constant exposure of mesothelial cells to bioincompatible PD solutions results in the denudation of the mesothelial monolayer and impairment of mesothelial cell function. Hyaluronan, a major component of extracellular matrices, is synthesized by mesothelial cells and contributes to remesothelialization, maintenance of cell phenotype, and tissue remodeling and provides structural support to the peritoneal membrane. Chronic peritoneal inflammation is observed in long-term PD patients and is associated with increased hyaluronan synthesis. During inflammation, depolymerization of hyaluronan may occur with the generation of hyaluronan fragments. In contrast to native hyaluronan which offers a protective role to the peritoneum, hyaluronan fragments exacerbate inflammatory and fibrotic processes and therefore assist in the destruction of the tissue. This paper will discuss the contribution of mesothelial cells to peritoneal membrane alterations that are induced by PD and the putative role of hyaluronan in these processes. PMID:22203782

  5. Hyaluronan-induced VEGF-C promotes fibrosis-induced lymphangiogenesis via Toll-like receptor 4-dependent signal pathway.

    PubMed

    Jung, Yu Jin; Lee, Ae Sin; Nguyen-Thanh, Tung; Kang, Kyung Pyo; Lee, Sik; Jang, Kyu Yun; Kim, Myung Ki; Kim, Sun Hee; Park, Sung Kwang; Kim, Won

    2015-10-23

    Hyaluronan (HA), a component of the extracellular matrix, modulates cellular behavior including angiogenesis. However, little is known about the effect of HA on lymphangiogenesis in fibrosis model. In this study, we investigated the roles of HA in lymphangiogenesis of unilateral ureteral obstruction (UUO). We found that HA cooperated synergistically with vascular endothelial cell growth factor-C to stimulate capillary-like tube formation and increase migration of cells in a haptotaxis assay. Accumulation of HA in the cortical interstitial space was positively correlated with the number of lymphatic vessels after UUO. Depletion of macrophages with clodronate decreased UUO-induced HA accumulation and lymphangiogenesis. Additionally, hyaluronan synthase (HAS) mRNA expression and HA production were increased in bone marrow-derived macrophages upon stimulation with TGF-β1. Transfer of mHAS2 and mHAS3 knock-down CD11b-positive macrophages to SCID mice resulted in a partial decrease in UUO-induced lymphangiogenesis. HA increased expression of vascular endothelial cell growth factor-C in macrophages. Vascular endothelial cell growth factor-C expression and LYVE-1-positive lymphatic area was significantly lower in the UUO-kidney from TLR4 null mice than that from TLR4 wild-type mice. Collectively, these results suggest that HA increases lymphangiogenesis in renal fibrosis model and also stimulates vascular endothelial cell growth factor-C production from macrophages through Toll-like receptor 4-dependent signal pathway. PMID:26362177

  6. 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

  7. 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.

  8. Hypotheses on the evolution of hyaluronan: a highly ironic acid.

    PubMed

    Csoka, Antonei B; Stern, Robert

    2013-04-01

    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

  9. 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

  10. Hyaluronan: More than just a wrinkle filler.

    PubMed

    Maytin, Edward V

    2016-06-01

    Dermatology is a field that strives not only to alleviate skin disease (therapeutics) but also to improve the perception of wellness (cosmetics). Thus, in this special issue of Glycobiology, it seems appropriate to discuss the biology of a glycosaminoglycan, called hyaluronic acid (hyaluronan, or HA), that has become the most popular agent today for intradermal injections to improve wrinkles and other cosmetic defects. HA is a simple linear polymer in which a simple disaccharide is repeated thousands of time, thereby creating a huge hydrophilic molecule that confers a large volume of hydration and contributes to the turgor and flexibility of healthy skin. Beyond cosmetic considerations, however, HA also has important biological and physiological functions that were largely under-appreciated until recently. New research has confirmed that HA is dynamically produced by most skin cells, not only fibroblasts (the cells that make most of the skin's extracellular matrix) but also by keratinocytes in the outer protective layer (epidermis). For both fibroblasts and keratinocytes, HA plays a regulatory role in controlling cell physiology through interaction of extracellular HA with a major cell-surface receptor, CD44. This interaction mediates intracellular signaling both directly and indirectly, through CD44 interactions with the cytoskeleton and with EGF and TGFβ receptors. Furthermore, degradation of HA by specific hyaluronidase enzymes produces HA fragments that can help to regulate inflammatory processes. In this review, current knowledge about the role of HA in skin inflammation and wound healing are reviewed and possible future applications of such knowledge discussed. PMID:26964566

  11. Interaction of Hyaluronan with Cationic Nanoparticles.

    PubMed

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

    2015-08-01

    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. PMID:26146006

  12. 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.

  13. Sepsis puerperalis caused by a genotypically proven cat-derived Pasteurella multocida strain.

    PubMed

    Voss, A; van Zwam, Y H; Meis, J F; Melchers, W; Steegers, E A

    1998-01-01

    We report a disseminated intrauterine Pasteurella multocida infection in a puerperal woman who could not remember any traumatic exposure to her cat. An oral swab taken from the cat, just 2 days after the patient's admission, grew Pasteurella multocida, with an PCR-fingerprinting pattern identical to the patient's isolate. Hand-washing after every contact with cats and dogs and if feasible separation of in-house pets from mother and infant should be applied to prevent this uncommon but serious occurrence of post-partum infections. To our knowledge this is the first case of Pasteurella multocida 'child-bed fever', with a genotypically identical strain isolated from the in-house cat. PMID:9481551

  14. Survival of toxigenic Pasteurella multocida in aerosols and aqueous liquids.

    PubMed Central

    Thomson, C M; Chanter, N; Wathes, C M

    1992-01-01

    The survival of toxigenic Pasteurella multocida in air and liquids was studied to identify possible risk factors in the etiology of atrophic rhinitis. In aerosols, at low relative humidity (28%), the viability of toxigenic P. multocida 5 min after aerosolization was at least 22% of its initial value. Viability at low relative humidity declined to 8% after 45 min. Viability at high relative humidity (79%) was 69% after 5 min and declined to 2% after 45 min. Survival of toxigenic P. multocida in liquids depended on storage and constituents in the liquid. Toxigenic P. multocida became nonculturable 1 to 14 days after inoculation in water and artificial seawater, depending on the storage temperature. Toxigenic P. multocida stored at 37 degrees C could be detected for up to 6 days in pig slurry and more than 36 days in Bacto Tryptose broth and nasal lavages. However, in Bacto Tryptose broth and nasal lavages stored at 4 degrees C, P. multocida was detected for up to 14 days whereas at 15 and 37 degrees C it was detected for more than 49 days. These results suggest that aerosols and fomites can play a role in the transmission of atrophic rhinitis. PMID:1575496

  15. Spontaneous bacterial peritonitis by Pasteurella multocida under treatment with rifaximin.

    PubMed

    Lutz, P; Parcina, M; Bekeredjian-Ding, I; Hoerauf, A; Strassburg, C P; Spengler, U

    2014-02-01

    Spontaneous bacterial peritonitis (SBP) is a life-threatening complication of liver cirrhosis. Recently, rifaximin, a non-absorbable antibiotic which is used to prevent recurrent hepatic encephalopathy, has been proposed as effective prophylaxis for SBP. Here, we present an unusual case of SBP under treatment with rifaximin. A 50-year-old woman with liver cirrhosis was admitted because of tense ascites and abdominal pain. She was under long-term oral prophylaxis with rifaximin due to hepatic encephalopathy. Paracentesis revealed SBP caused by Pasteurella multocida, which was sensitive to multiple antibiotics, including rifaximin. Treatment with ceftriaxone resulted in rapid resolution of the peritonitis and restoration of the patient. Since P. multocida is usually transmitted from pets, the patient's cat was tested and could be identified as the most likely source of infection. This case should elicit our awareness that uncommon pathogens and unusual routes of transmission may lead to SBP, despite antibacterial prophylaxis with non-absorbable antibiotics. Nevertheless, such infections may still remain sensitive to systemic therapy with conventional antibiotics. PMID:23526308

  16. Binding of Pasteurella haemolytica leukotoxin to bovine leukocytes.

    PubMed Central

    Brown, J F; Leite, F; Czuprynski, C J

    1997-01-01

    Pasteurella haemolytica is the principal bacterial pathogen in the bovine respiratory disease complex. This organism produces an exotoxin (referred to as leukotoxin) during logarithmic-phase growth that is a potent leukocyte-modulating agent. At low concentrations, it activates neutrophils and mononuclear phagocytes to release inflammatory mediators, while at the same time making these cells destined to undergo apoptotic cell death. At higher concentrations, the toxin causes rapid swelling and loss of cell viability. In this study, we demonstrated that toxin binding can be directly evaluated by flow cytometry with biologically active biotinylated leukotoxin. Leukotoxin binding was blocked by the addition of a neutralizing anti-leukotoxin monoclonal antibody and was not detected when bovine leukocytes were incubated with culture filtrates from a mutant strain of P. haemolytica that does not produce biologically active leukotoxin. In addition, treatment of bovine leukocytes with protease K eliminated subsequent binding of leukotoxin, suggesting that there is a protein on the leukocyte surface that is either a leukotoxin binding site or is required for stabilization of leukotoxin binding. We did not detect binding of biotinylated leukotoxin to porcine or human leukocytes, which have been reported previously to be resistant to the lytic effects of the leukotoxin. These findings suggest that there may be a specific binding site for P. haemolytica leukotoxin on bovine but not on porcine or human leukocytes and that it might be involved in the activation and lytic activities of the leukotoxin. PMID:9284143

  17. Pasteurella pestis detection in Fleas by fluorescent antibody staining.

    PubMed

    Hudson, B W; Kartman, L; Prince, F M

    1966-01-01

    In an effort to develop a method for the rapid field identification of plague-infected fleas, the authors have studied the feasibility of direct fluorescent antibody staining of the midgut contents of fleas fed on mice infected with Pasteurella pestis. Fluorescent antibodies prepared from antisera derived from rabbits inoculated with the water-soluble P. pestis fraction 1b antigen, the somatic antigen of heat-killed P. pestis (Bryans strain), and live avirulent (strain A1122) or virulent (Yreka strain) plague vaccines were used used in this study.This direct staining method proved to be impracticable, but encouraging results were obtained by fluorescent antibody staining of broth cultures of macerates of infected fleas after 24-48 hours' incubation.The broth enrichment technique has not yet been evaluated in the field, but it is expected to be of value since it is relatively simple to perform and requires only material that can easily be transported to remote areas. PMID:5328902

  18. 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.

  19. Chemically Modified N-Acylated Hyaluronan Fragments Modulate Proinflammatory Cytokine Production by Stimulated Human Macrophages*

    PubMed Central

    Babasola, Oladunni; Rees-Milton, Karen J.; Bebe, Siziwe; Wang, Jiaxi; Anastassiades, Tassos P.

    2014-01-01

    Low molecular mass hyaluronans are known to induce inflammation. To determine the role of the acetyl groups of low molecular mass hyaluronan in stimulating the production of proinflammatory cytokines, partial N-deacetylation was carried out by hydrazinolysis. This resulted in 19.7 ± 3.5% free NH2 functional groups, which were then acylated by reacting with an acyl anhydride, including acetic anhydride. Hydrazinolysis resulted in bond cleavage of the hyaluronan chain causing a reduction of the molecular mass to 30–214 kDa. The total NH2 and N-acetyl moieties in the reacetylated hyaluronan were 0% and 98.7 ± 1.5% respectively, whereas for butyrylated hyaluronan, the total NH2, N-acetyl, and N-butyryl moieties were 0, 82.2 ± 4.6, and 22.7 ± 3.8%, respectively, based on 1H NMR. We studied the effect of these polymers on cytokine production by cultured human macrophages (THP-1 cells). The reacetylated hyaluronan stimulated proinflammatory cytokine production to levels similar to LPS, whereas partially deacetylated hyaluronan had no stimulatory effect, indicating the critical role of the N-acetyl groups in the stimulation of proinflammatory cytokine production. Butyrylated hyaluronan significantly reduced the stimulatory effect on cytokine production by the reacetylated hyaluronan or LPS but had no stimulatory effect of its own. The other partially N-acylated hyaluronan derivatives tested showed smaller stimulatory effects than reacetylated hyaluronan. Antibody and antagonist experiments suggest that the acetylated and partially butyrylated lower molecular mass hyaluronans exert their effects through the TLR-4 receptor system. Selectively N-butyrylated lower molecular mass hyaluronan shows promise as an example of a novel semisynthetic anti-inflammatory molecule. PMID:25053413

  20. Viscoelastic Properties of Hyaluronan in Physiological Conditions

    PubMed Central

    Cowman, Mary K.; Schmidt, Tannin A.; Raghavan, Preeti; Stecco, Antonio

    2015-01-01

    Hyaluronan (HA) is a high molecular weight glycosaminoglycan of the extracellular matrix (ECM), which is particularly abundant in soft connective tissues. Solutions of HA can be highly viscous with non-Newtonian flow properties. These properties affect the movement of HA-containing fluid layers within and underlying the deep fascia. Changes in the concentration, molecular weight, or even covalent modification of HA in inflammatory conditions, as well as changes in binding interactions with other macromolecules, can have dramatic effects on the sliding movement of fascia. The high molecular weight and the semi-flexible chain of HA are key factors leading to the high viscosity of dilute solutions, and real HA solutions show additional nonideality and greatly increased viscosity due to mutual macromolecular crowding. The shear rate dependence of the viscosity, and the viscoelasticity of HA solutions, depend on the relaxation time of the molecule, which in turn depends on the HA concentration and molecular weight. Temperature can also have an effect on these properties. High viscosity can additionally affect the lubricating function of HA solutions. Immobility can increase the concentration of HA, increase the viscosity, and reduce lubrication and gliding of the layers of connective tissue and muscle. Over time, these changes can alter both muscle structure and function. Inflammation can further increase the viscosity of HA-containing fluids if the HA is modified via covalent attachment of heavy chains derived from Inter-α-Inhibitor. Hyaluronidase hydrolyzes HA, thus reducing its molecular weight, lowering the viscosity of the extracellular matrix fluid and making outflow easier. It can also disrupt any aggregates or gel-like structures that result from HA being modified. Hyaluronidase is used medically primarily as a dispersion agent, but may also be useful in conditions where altered viscosity of the fascia is desired, such as in the treatment of muscle stiffness

  1. Collagen VI and Hyaluronan: The Common Role in Breast Cancer

    PubMed Central

    Karousou, Evgenia; D'Angelo, Maria Luisa; Kouvidi, Katerina; Vigetti, Davide; Viola, Manuela; Passi, Alberto

    2014-01-01

    Collagen VI and hyaluronan are widely distributed extracellular matrix macromolecules that play a crucial role in tissue development and are highly expressed in cancers. Both hyaluronan and collagen VI are upregulated in breast cancer, generating a microenvironment that promotes tumour progression and metastasis. A growing number of studies show that these two molecules are involved in inflammation and angiogenesis by recruiting macrophages and endothelial cells, respectively. Additionally, collagen VI induces epithelial-mesenchymal transition that is correlated to increased synthesis of hyaluronan in mammary cells. Hyaluronan has also a specific role in cellular functions that depends mainly on the size of the polymer, whereas the effect of collagen VI in tumour progression may be the result of the intact molecule or the C5 peptide of α3(VI) chain, known as endotrophin. Collectively, these findings strongly support the parallel role of these molecules in tumour progression and suggest that they may be used as prognostic factors for the breast cancer treatment. PMID:25126569

  2. Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor

    PubMed Central

    Bono, Petri; Rubin, Kristofer; Higgins, Jonathan M. G.; Hynes, Richard O.

    2001-01-01

    The actin cytoskeleton plays a significant role in changes of cell shape and motility, and interactions between the actin filaments and the cell membrane are crucial for a variety of cellular processes. Several adaptor proteins, including talin, maintain the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. To facilitate studies of layilin's function, we have generated a layilin-Fc fusion protein comprising the extracellular part of layilin joined to human immunoglobulin G heavy chain and used this chimera to identify layilin ligands. Here, we demonstrate that layilin-Fc fusion protein binds to hyaluronan immobilized to Sepharose. Microtiter plate-binding assays, coprecipitation experiments, and staining of sections predigested with different glycosaminoglycan-degrading enzymes and cell adhesion assays all revealed that layilin binds specifically to hyaluronan but not to other tested glycosaminoglycans. Layilin's ability to bind hyaluronan, a ubiquitous extracellular matrix component, reveals an interesting parallel between layilin and CD44, because both can bind to cytoskeleton-membrane linker proteins through their cytoplasmic domains and to hyaluronan through their extracellular domains. This parallelism suggests a role for layilin in cell adhesion and motility. PMID:11294894

  3. Diffusion of Particle in Hyaluronan Solution, a Brownian Dynamics Simulation

    NASA Astrophysics Data System (ADS)

    Takasu, Masako; Tomita, Jungo

    2004-04-01

    Diffusion of a particle in hyaluronan solution is investigated using Brownian dynamics simulation. The slowing down of diffusion is observed, in accordance with the experimental results. The temperature dependence of the diffusion is calculated, and a turnover is obtained when the temperature is increased.

  4. Getting a grip: hyaluronan-mediated cellular adhesion

    NASA Astrophysics Data System (ADS)

    Curtis, Jennifer E.; Spatz, Joachim P.

    2004-10-01

    Holographic optical tweezers (HOTs) techniques are further developed to study hyaluronan-mediated adhesion of chondrocyte cells. We present a calibration scheme and address fundamental issues concerning the use of HOTs for quantitative force measurements. Influence of SLM pixelation on trap stiffness is observed and can be utilized to design calibrated HOTs more effectively. It is also shown that the HOTs trapping stiffness can vary significantly over short distances. Then we use HOTs cell adhesion assays investigate the viscoelastic and adhesive nature of chondrocytes' pericellular matrix (PCM) at two different time steps (30 minute and 24 hour incubation periods). Surprisingly, no physical influence of the large, presumably gel-like PCM is observed. However, a difference is discerned in the adhesiveness of the two sets of cells. The early-stage cells have reversible adhesion with negatively-charged and fibronectin-coated microspheres even after they are held at the cell surface for 10 seconds. In contrast, late stage cells stick irreversibly to all types of beads: positive, negative, fibronectin and hyaluronan-coated. Additionally, only the late stage cells produce membrane tethers. These observations suggest that the late-stage chondrocytes have less surface-associated hyaluronan and have interesting implications for the role of hyaluronan in the early stages of cell adhesion.

  5. A Chronic Respiratory Pasteurella multocida Infection Is Well-Controlled by Long-Term Macrolide Therapy.

    PubMed

    Seki, Masafumi; Sakata, Tomomi; Toyokawa, Masahiro; Nishi, Isao; Tomono, Kazunori

    2016-01-01

    A 57-year-old woman with severe bronchiectasis frequently received antibiotics, including penicillin, for acute exacerbations due to Pasteurella multocida. Although the bacteria showed a decrease in antibiotic susceptibility, her symptoms and X-ray findings became stable, and severe exacerbations were not observed for the last few years after a low-dose erythromycin treatment was started. The development of a respiratory infection with Pasteurella multocida is relatively uncommon, but it can be controlled by immunomodulation which is associated with long-term macrolide therapy. PMID:26831030

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

    SciTech Connect

    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.

  7. Stilbene Synthase and Chalcone Synthase 1

    PubMed Central

    Rolfs, Claus-Henning; Kindl, Helmut

    1984-01-01

    Cultured cells of Picea excelsa capable of forming stilbenes and flavanoids have been established. Unlike needles of intact plants containing piceatannol (3,3′,4′,5-tetrahydroxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into resveratrol monomethyl ether (3,4′-dihydroxy-5-methoxystilbene) and naringenin. Partially purified enzyme preparations were assayed for chalcone synthase as well as for stilbene synthase activity converting malonyl-CoA plus p-coumaroyl-CoA into 3,4′,5-trihydroxystilbene (resveratrol). Although stilbene synthase and chalcone synthase use the same substrates and exhibit similar molecular properties, i.e. molecular weight and subunit molecular weight, they are two different proteins. This difference was demonstrated by gel electrophoresis and by means of monospecific antibodies. PMID:16663649

  8. The role of hyaluronan in the pathobiology and treatment of respiratory disease.

    PubMed

    Garantziotis, Stavros; Brezina, Martin; Castelnuovo, Paolo; Drago, Lorenzo

    2016-05-01

    Hyaluronan, a ubiquitous naturally occurring glycosaminoglycan, is a major component of the extracellular matrix, where it participates in biological processes that include water homeostasis, cell-matrix signaling, tissue healing, inflammation, angiogenesis, and cell proliferation and migration. There are emerging data that hyaluronan and its degradation products have an important role in the pathobiology of the respiratory tract. We review the role of hyaluronan in respiratory diseases and present evidence from published literature and from clinical practice supporting hyaluronan as a novel treatment for respiratory diseases. Preliminary data show that aerosolized exogenous hyaluronan has beneficial activity against airway inflammation, protects against bronchial hyperreactivity and remodeling, and disrupts the biofilm associated with chronic infection. This suggests a role in airway diseases with a predominant inflammatory component such as rhinosinusitis, asthma, chronic obstructive pulmonary disease, cystic fibrosis, and primary ciliary dyskinesia. The potential for hyaluronan to complement conventional therapy will become clearer when data are available from controlled trials in larger patient populations. PMID:26747781

  9. A novel epigenetic mechanism regulating hyaluronan production in pancreatic cancer cells.

    PubMed

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

    2016-03-01

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma enriched with hyaluronan (HA), a major component of extracellular matrix known to play a critical role in tumor progression. The mechanisms that regulate HA synthesis in PDAC are poorly understood. To investigate whether DNA methylation and HA production from PDAC cells are associated, we studied the effect of 5-aza-2'-deoxycitidine (5-aza-dC), an inhibitor of DNA methylation, or DNA methyltransferase 1 (DNMT1) knockdown by small interfering RNA, on the HA production from PDAC cells. HA production into the conditioned medium was evaluated in PDAC cells treated with 5-aza-dC or DNMT1 knockdown. mRNA expression of HA synthase (HAS) genes was investigated by real-time RT-PCR. Treatment of PDAC cells with 5-aza-dC led to a significant increase in the HA production (up to 2.5-fold increase) in all 4 cell lines tested. This enhanced HA production by 5-aza-dC treatment was accompanied by increased mRNA expression of HAS2 and HAS3. Furthermore, increased HA production and HAS2/HAS3 mRNA expression was also observed in PDAC cells by knockdown of DNMT1. These findings provide evidence, for the first time, that epigenetic mechanism is involved in the regulation of HA synthesis in PDAC cells. PMID:26589701

  10. Adiponectin resides in mouse skin and upregulates hyaluronan synthesis in dermal fibroblasts.

    PubMed

    Akazawa, Yumiko; Sayo, Tetsuya; Sugiyama, Yoshinori; Sato, Takashi; Akimoto, Noriko; Ito, Akira; Inoue, Shintaro

    2011-01-01

    Adipose tissue is a hormonally active tissue that produces adipokines that influence the activity of other tissues. Adiponectin is an adipocyte-specific adipokine involved in systemic metabolism. We detected the expression of adiponectin receptors (AdipoR1 and AdipoR2) mRNA in cultured dermal fibroblasts. The full-length adiponectin (fAd), but not the globular adiponectin (gAd), increased hyaluronan (HA) production and upregulated HA synthase (HAS) 2 mRNA expression. AdipoR1 and AdipoR2 mRNAs were also expressed in keratinocytes, though neither fAd nor gAd had any effect on HA synthesis. In mouse skin, we found that adiponectin was present and decreased markedly with aging. The age-dependent pattern of adiponectin decrease in skin, correlated well with that of HA in skin. Our experiments were also the first to identify adiponectin production in cultured mouse sebocytes, a finding that suggests that skin adiponectin may derive not only from plasma and/or subcutaneous adipose tissue, but also from the sebaceous gland. These results indicated that adiponectin plays an important role in the HA metabolism of skin. PMID:21117904

  11. Induction of hyaluronan production by oncogenic KSHV and the contribution to viral pathogenesis in AIDS patients.

    PubMed

    Dai, Lu; Chen, Yihan; Bonstaff, Karlie; Doyle, Lisa; Toole, Bryan; Parsons, Chris; Qin, Zhiqiang

    2015-07-01

    Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), malignancies arising primarily in immunocompromised patients particularly AIDS-patients, which still lack effective therapy. Hyaluronan (HA) is a large glucuronic acid and has been found closely related to multiple functions in cancer cells, although its role in viral oncogenesis remains largely unknown. Here we provide first evidence that KSHV de novo infection induces HA production from primary endothelial cells through upregulation of HA synthase gene 1 (Has1) and a multifunctional glycoprotein, CD147. Further data demonstrate that KSHV-induced HA production requires viral latent protein, LANA (in particular functional domain A) and MAPK/ERK signaling activities. In functions, HA production is necessary for KSHV/LANA-induced primary endothelial cell invasion, a hallmark feature for KS development. For clinical relevance, our data indicate that the KSHV+ group has higher levels of HA and Has1 activities in its plasma than the KSHV- group of cohort HIV-infected patients. Together, our findings provide innovative insights into the mechanisms of oncogenic virus activation of HA production and its role in virus-associated malignancy pathogenesis, which may help to develop novel therapeutic strategies by targeting HA and related signaling. PMID:25837851

  12. Biotinylated hyaluronan: a versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure.

    PubMed

    Melrose, J; Numata, Y; Ghosh, P

    1996-01-01

    Hyaluronan influences cellular proliferation and migration in developing, regenerating and remodelling tissues and in tissues undergoing malignant tumour-cell invasion. The widespread occurrence of hyaluronan-binding proteins indicates that the recognition of hyaluronan is important to tissue organisation and the control of cellular behaviour. A number of extracellular matrix and cellular proteins, which have been termed the hyaladherins, have specific affinities for hyaluronan. These include cartilage link-protein, hyaluronectin, neurocan, versican and aggrecan, which all bind to HA within the extracellular matrix. Cellular receptors for hyaluronan such as CD44 and RHAMM (receptor for hyaluronate-mediated motility) have also been identified. In the present study biotinylated hyaluronan (bHA) was prepared by reacting adipic dihydrazide with a 170 kDa hyaluronan sample using the bifunctional reagent 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide. The resultant free amine moeity of the hydrazido-hyaluronan was then reacted with biotin succinimidyl ester (sulfo-NHS-biotin) to prepare the bHA. After 4-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotting to nitrocellulose membranes, bHA and avidin alkaline phosphatase conjugate could be used in conjunction with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates to specifically visualise with high sensitivity (> or = 2 ng), bovine nasal cartilage link-protein, aggrecan hyaluronan binding region, and human fibroblast hyaluronan receptors such as CD-44. Conventional Western blotting using specific monoclonal antibodies to these proteins was also used to confirm the identities of these proteins. PMID:8907541

  13. Extracellular Processing of the Cartilage Proteoglycan Aggregate and Its Effect on CD44-mediated Internalization of Hyaluronan*

    PubMed Central

    Danielson, Ben T.; Knudson, Cheryl B.; Knudson, Warren

    2015-01-01

    In many cells hyaluronan receptor CD44 mediates the endocytosis of hyaluronan and its delivery to endosomes/lysosomes. The regulation of this process remains largely unknown. In most extracellular matrices hyaluronan is not present as a free polysaccharide but often is found in complex with other small proteins and macromolecules such as proteoglycans. This is especially true in cartilage, where hyaluronan assembles into an aggregate structure with the large proteoglycan termed aggrecan. In this study when purified aggrecan was added to FITC-conjugated hyaluronan, no internalization of hyaluronan was detected. This suggested that the overall size of the aggregate prevented hyaluronan endocytosis and furthermore that proteolysis of the aggrecan was a required prerequisite for local, cell-based turnover of hyaluronan. To test this hypothesis, limited C-terminal digestion of aggrecan was performed to determine whether a size range of aggrecan exists that permits hyaluronan endocytosis. Our data demonstrate that only limited degradation of the aggrecan monomer was required to allow for hyaluronan internalization. When hyaluronan was combined with partially degraded, dansyl chloride-labeled aggrecan, blue fluorescent aggrecan was also visualized within intracellular vesicles. It was also determined that sonicated hyaluronan of smaller molecular size was internalized more readily than high molecular mass hyaluronan. However, the addition of intact aggrecan to hyaluronan chains sonicated for 5 and 10 s reblocked their endocytosis, whereas aggregates containing 15-s sonicated hyaluronan were internalized. These data suggest that hyaluronan endocytosis is regulated in large part by the extracellular proteolytic processing of hyaluronan-bound proteoglycan. PMID:25733665

  14. 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

  15. Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles

    PubMed Central

    Roier, Sandro; Fenninger, Judith C.; Leitner, Deborah R.; Rechberger, Gerald N.; Reidl, Joachim; Schild, Stefan

    2013-01-01

    Pasteurella multocida is able to cause disease in humans and in a wide range of animal hosts, including fowl cholera in birds, atrophic rhinitis in pigs, and snuffles in rabbits. Together with Mannheimia haemolytica, P. multocida also represents a major bacterial causative agent of bovine respiratory disease (BRD), which is one of the most important causes for economic losses for the cattle backgrounding and feedlot industry. Commercially available vaccines only partially prevent infections caused by P. multocida and M. haemolytica. Thus, this study characterized the immunogenicity of P. multocida and M. haemolytica outer membrane vesicles (OMVs) upon intranasal immunization of BALB/c mice. Enzyme-linked immunosorbent assays (ELISA) revealed that OMVs derived from P. multocida or M. haemolytica are able to induce robust humoral and mucosal immune responses against the respective donor strain. In addition, also significant cross-immunogenic potential was observed for both OMV types. Colonization studies showed that a potential protective immune response against P. multocida is not only achieved by immunization with P. multocida OMVs, but also by immunization with OMVs derived from M. haemolytica. Immunoblot and immunoprecipitation analyses demonstrated that M. haemolytica OMVs induce a more complex immune response compared to P. multocida OMVs. The outer membrane proteins OmpA, OmpH, and P6 were identified as the three major immunogenic proteins of P. multocida OMVs. Amongst others, the serotype 1-specific antigen, an uncharacterized outer membrane protein, as well as the outer membrane proteins P2 and OmpA were found to be the most important antigens of M. haemolytica OMVs. These findings are useful for the future development of broad-spectrum OMV based vaccines against BRD and other infections caused by P. multocida or M. haemolytica. PMID:23731905

  16. Distribution of hyaluronan in human endometrium across the menstrual cycle. Implications for implantation and menstruation.

    PubMed

    Salamonsen, L A; Shuster, S; Stern, R

    2001-11-01

    Hyaluronan is a molecule with many known roles in cellular physiology and is often associated with tissue remodeling. The human endometrium undergoes dramatic remodeling during the course of the normal menstrual cycle but its regulation is not well understood. This study examined the levels of deposition of hyaluronan in human cycling endometrium by histochemical localization, using a highly specific hyaluronan-binding peptide. This was facilitated by avoiding conventional formalin fixation, which results in serious loss of the water-soluble hyaluronan. Peaks of hyaluronan deposition were observed in the stromal compartment during the mid-proliferative (days 5-10) and the mid-secretory phase (days 19-23) of the cycle. In physiological terms, the first phase corresponds to the time of rapid cellular proliferation of undifferentiated cells, whereas the second phase coincides with the time when the implantation of the conceptus would be initiated in a fertile cycle. By the time menstruation starts, very little hyaluronan remains in the stroma. In contrast with the stromal staining, hyaluronan deposition around blood vessels was constant throughout the cycle. The dramatic changes in hyaluronan deposition and their correlation with the cyclic growth and remodeling in the human endometrium suggest a major role for hyaluronan in the physiology of this tissue. PMID:11702245

  17. 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

  18. 9 CFR 113.118 - Pasteurella Multocida Bacterin, Avian Isolate, Type 3.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Pasteurella Multocida Bacterin, Avian Isolate, Type 3. 113.118 Section 113.118 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS...

  19. 9 CFR 113.117 - Pasteurella Multocida Bacterin, Avian Isolate, Type 1.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Pasteurella Multocida Bacterin, Avian Isolate, Type 1. 113.117 Section 113.117 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS...

  20. 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...

  1. 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...

  2. Immune response in mice and swine to DNA vaccines derived from the Pasteurella multocida toxin gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA vaccines were constructed with either a 5’-truncated or full-length, genetically detoxified toxin gene from Pasteurella multocida and two different DNA vaccine vectors, distinguished by the presence or absence of a secretion signal sequence. Optimal PMT-specific antibody responses and spleen cel...

  3. 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

  4. Cellulose synthase interacting protein

    PubMed Central

    Somerville, Chris

    2010-01-01

    Cellulose is the most abundant biopolymer on earth. The great abundance of cellulose places it at the forefront as a primary source of biomass for renewable biofuels. However, the knowledge of how plant cells make cellulose remains very rudimentary. Cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes. The only known components of cellulose synthase complexes are cellulose synthase (CESA) proteins until the recent identification of a novel component. CSI1, which encodes CESA interacting protein 1 (CSI1) in Arabidopsis. CSI1, as the first non-CESA proteins associated with cellulose synthase complexes, opens up many opportunities. PMID:21150290

  5. A case of intravenous leiomyomatosis with high levels of hyaluronan.

    PubMed

    Yaguchi, Chizuko; Oi, Hidekazu; Kobayashi, Hiroshi; Miura, Katsutoshi; Kanayama, Naohiro

    2010-04-01

    Intravenous leiomyomatosis (IVL) is a rare benign tumor. The clinical behavior can be life-threatening due to extension through the pelvic veins. A 70-year-old woman was referred to our hospital with IVL originating from a uterine leiomyoma and extending to the inferior vena cava. The patient was diagnosed on the basis of the results of various studies, and the tumor was resected completely through a single-stage approach. The intravascular tumor was 20 cm long, multinodular and rubbery. Microscopic findings showed benign smooth muscle that was partly hyalinized and fibrous. Immunohistochemical studies revealed that hyaluronan was expressed more prominently in IVL than in uterine leiomyomas. IVL has viscoelastic properties and contains a large amount of hyaluronan, which may promote invasion during pathogenesis. PMID:20492407

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

    PubMed

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

    2016-01-01

    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

  7. 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

  8. Hyaluronan-binding properties of human serum hemopexin.

    PubMed

    Hrkal, Z; Kuzelová, K; Muller-Eberhard, U; Stern, R

    1996-03-25

    Hemopexin, the heme-binding serum glycoprotein, exhibits a complex electrophoretic pattern on two-dimensional immunoelectrophoresis on agarose gels into which hyaluronic acid is incorporated in the first and monospecific anti-hemopexin in the second dimension. This heterogeneity reflects a range of interactions of hemopexin isoforms with hyaluronic acid. Electrophoretic patterns of individual human sera greatly differ in their contents of hyaluronan-interacting hemopexin species. Hemopexin itself has no hyaluronidase activity. PMID:8612795

  9. Role of hyaluronan and hyaluronan-binding proteins in lung pathobiology.

    PubMed

    Lennon, Frances E; Singleton, Patrick A

    2011-08-01

    Hyaluronan (HA) has diverse functions in normal lung homeostasis and pulmonary disease. HA constitutes the major glycosaminoglycan in lung tissue, with HA degradation products, produced by hyaluronidase enzymes and reactive oxygen species, being implicated in several lung diseases, including acute lung injury, asthma, chronic obstructive pulmonary disease, and pulmonary hypertension. The differential activities of HA and its degradation products are due, in part, to regulation of multiple HA-binding proteins, including cluster of differentiation 44 (CD44), Toll-like receptor 4 (TLR4), HA-binding protein 2 (HABP2), and receptor for HA-mediated motility (RHAMM). Recent research indicates that exogenous administration of high-molecular-weight HA can serve as a novel therapeutic intervention for lung diseases, including lipopolysaccharide (LPS)-induced acute lung injury, sepsis/ventilator-induced lung injury, and airway hyperreactivity. This review focuses on the regulatory role of HA and HA-binding proteins in lung pathology and discusses the capacity of HA to augment and inhibit various lung diseases. PMID:21571904

  10. Structural basis of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase

    PubMed Central

    Li, Songlin; Kelly, Stephen J.; Lamani, Ejvis; Ferraroni, Marta; Jedrzejas, Mark J.

    2000-01-01

    Streptococcus pneumoniae hyaluronate lyase (spnHL) is a pathogenic bacterial spreading factor and cleaves hyaluronan, an important constituent of the extra– cellular matrix of connective tissues, through an enzymatic β–elimination process, different from the hyaluronan degradation by hydrolases in animals. The mechanism of hyaluronan binding and degradation was proposed based on the 1.56 Å resolution crystal structure, substrate modeling and mutagenesis studies on spnHL. Five mutants, R243V, N349A, H399A, Y408F and N580G, were constructed and their activities confirmed our mechanism hypothesis. The important roles of Tyr408, Asn349 and His399 in enzyme catalysis were proposed, explained and confirmed by mutant studies. The remaining weak enzymatic activity of the H399A mutant, the role of the free carboxylate group on the glucuronate residue, the enzymatic behavior on chondroitin and chondroitin sulfate, and the small activity increase in the N580G mutant were explained based on this mechanism. A possible function of the C–terminal β–sheet domain is to modulate enzyme activity through binding to calcium ions. PMID:10716923

  11. The immunological effect of hyaluronan in tumor angiogenesis

    PubMed Central

    Spinelli, Fiorella M; Vitale, Daiana L; Demarchi, Gianina; Cristina, Carolina; Alaniz, Laura

    2015-01-01

    The relationship between the immune system and angiogenesis has been described in several contexts, both in physiological and pathological conditions, as pregnancy and cancer. In fact, different types of immune cells, such as myeloid, macrophages and denditric cells, are able to modulate tumor neovascularization. On the other hand, tumor microenvironment also includes extracellular matrix components like hyaluronan, which has a deregulated synthesis in different tumors. Hyaluronan is a glycosaminoglycan, normally present in the extracellular matrix of tissues in continuous remodeling (embryogenesis or wound healing processes) and acts as an important modulator of cell behavior by different mechanisms, including angiogenesis. In this review, we discuss hyaluronan as a modulator of tumor angiogenesis, focusing in intracellular signaling mediated by its receptors expressed on different immune cells. Recent observations suggest that the immune system is an important component in tumoural angiogenesis. Therefore, immune modulation could have an impact in anti-angiogenic therapy as a new therapeutic strategy, which in turn might improve effectiveness of treatment in cancer patients. PMID:26719798

  12. Hyaluronan microspheres for sustained gene delivery and site-specific targeting.

    PubMed

    Yun, Yang H; Goetz, Douglas J; Yellen, Paige; Chen, Weiliam

    2004-01-01

    Hyaluronan is a naturally occurring polymer that has enjoyed wide successes in biomedical and cosmetic applications as coatings, matrices, and hydrogels. For controlled delivery applications, formulating native hyaluronan into microspheres could be advantageous but has been difficult to process unless organic solvents are used or hyaluronan has been modified by etherification. Therefore, we present a novel method of preparing hyaluronan microspheres using adipic dihydrazide mediated crosslinking chemistry. To evaluate their potential for medical applications, hyaluronan microspheres are incorporated with DNA for gene delivery or conjugated with an antigen for cell-specific targeting. The results show that our method, originally developed for preparing hyaluronan hydrogels, generates robust microspheres with a size distribution of 5-20mum. The release of the encapsulated plasmid DNA can be sustained for months and is capable of transfection in vitro and in vivo. Hyaluronan microspheres, conjugated with monoclonal antibodies to E- and P-selectin, demonstrate selective binding to cells expressing these receptors. In conclusion, we have developed a novel microsphere preparation using native hyaluronan that delivers DNA at a controlled rate and adaptable for site-specific targeting. PMID:14580918

  13. 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

  14. Candidate vaccine antigens and genes in Pasteurella multocida.

    PubMed

    Adler, B; Bulach, D; Chung, J; Doughty, S; Hunt, M; Rajakumar, K; Serrano, M; van Zanden, A; Zhang, Y; Ruffolo, C

    1999-08-20

    Pasteurella multocida is the causative agent of fowl cholera and other diseases of production animals. Isolates are classified into five groups based on capsular antigens and into 16 serotypes based on LPS antigens. Strains causing fowl cholera are most frequently designated A:1, A:3 or A:4. Whole cell bacterins can provide some degree of protection, but only against the homologous LPS serotype. There is good evidence that cross-protective antigens are expressed only under in vivo conditions. Empirically derived, live, attenuated vaccines can protect against heterologous serotypes, but because the basis for attenuation is undefined, reversion to virulence is not uncommon. Work in our laboratory is aimed at using a variety of approaches to identify potential protective antigens or virulence genes to be used as candidates for attenuating mutations or as the basis for vaccine antigen delivery systems. The gene encoding an outer membrane protein, Oma87, which is a homologue of the D15 protective antigen of Haemophilus influenzae, was cloned and sequenced. Rabbit antiserum prepared against recombinant Oma87 could passively protect mice against infection. Type 4 fimbriae form the basis of vaccines against ovine footrot and bovine keratoconjunctivitis. We have identified type 4 fimbriae on the surface of P. multocida, purified the fimbrial subunit protein, PtfA, and determined its N-terminal amino acid sequence. Subsequent cloning of the ptfA gene and its inactivation will now be used to assess the importance of type 4 fimbriae in virulence. There has long been anecdotal evidence for the importance of capsule in virulence, but unequivocal genetic evidence for such a role is lacking. We have cloned and characterised the capsule biosynthetic locus in P. multocida A:1 and identified four bex genes involved in capsule transport and genes encoding enzymes involved in the biosynthesis and transfer of the N-acetyl glucosamine and glucuronic acid components of the capsule. It has

  15. Partial characterization of the leukotoxin of Pasteurella haemolytica-like bacteria isolated from swine enteritis.

    PubMed

    DeSilva, R T; Chengappa, M M; Oberst, R D; Staats, J J

    1995-08-01

    Pasteurella haemolytica-like (PHL) strains isolated from diarrheic pigs are known to produce a leukotoxin that is lethal to ruminant leukocytes. In the present study, 12 PHL strains were screened for leukotoxin production using a tetrazolium dye-reduction assay. Sterile culture supernatant from strain 6213A, the maximum leukotoxin producer, was used as the crude leukotoxin for characterization studies. The leukotoxin was inactivated by heat at 60 degrees C and by trypsin, protease, and amylase. Toxicity was retained over a pH range of 3.0-11.0. The leukotoxin was lethal to polymorphoneutrophils (PMNs) of cattle, sheep, goat, and swine. Chromosomal DNA of all 12 PHL strains hybridized with a 3.9 kb Pasteurella haemolytica A1 leukotoxin probe, indicating similarities between the leukotoxin genes of P. haemolytica and PHL strains. PMID:7483245

  16. Serological tests as indicators of immunity against Pasteurella multocida infection in sheep.

    PubMed Central

    Dua, S K; PandurangaRao, C C

    1978-01-01

    Five serological tests, i.e. single tube agglutination, doubling dilution tube agglutination, agar agglutination, passive hemagglutination and passive mouse protection tests were evaluated for their efficacy in predicting the fate of vaccinated and unvaccinated sheep on challenge with an ovine strain of Pasteurella multocida. The passive hemagglutination test predicted the fate of unvaccinated sheep while the agar agglutination test indicated the immune status of vaccinated sheep. PMID:743601

  17. Cloning and expression of the dermonecrotic toxin gene of Pasteurella multocida ssp. multocida in Escherichia coli.

    PubMed

    Kamps, A M; Kamp, E M; Smits, M A

    1990-01-15

    A DNA library of Pasteurella multocida ssp. multocida strain CVI 47459 was constructed in the Lambda GEM-11 vector. Recombinant clones that encoded dermonecrotic toxin (DNT) were identified immunologically with antiserum raised against purified DNT. By comparing the DNA restriction maps of the immunoreactive recombinants, we located the DNT gene. Hybridization studies with 10 strains of P. multocida ssp. multocida suggested that strains that do not produce the DNT do not contain sequences homologous to the DNT gene. PMID:2328908

  18. 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%.

  19. New sites of localisation of Pasteurella multocida B:2 in buffalo surviving experimental haemorrhagic septicaemia

    PubMed Central

    2014-01-01

    Background Haemorrhagic septicaemia (HS) is an acute septicaemic disease of buffalo and cattle caused by Pasteurella multocida B:2 and E:2. Field outbreaks of HS are known to result in localisation of bacteria in the tonsils of surviving buffalo, confirming that animals can become carriers and the role of respiratory tract in the transmission of the disease. This report describes additional sites of localisation of P. multocida B:2 in surviving buffalo following experimental induction of HS. Results Following P. multocida B:2 infection, all calves in group 1 and one calf in group 2 that was allowed to commingle with infected calves from group 1 were euthanised within 48 h. Pasteurella multocida B:2 was detected from the nasal and rectal swab samples on days 5 and 6 from the remaining calves in group 2. The first injection of dexamethasone into the carrier animals resulted in reemergence in samples from the nose, rectum and vagina. However, subsequent dexamethasone injections failed to re-activate P. multocida B:2. When surviving carrier calves in group 2 were euthanised at the end of the experiment, P. multocida B:2 was detected in the lungs and various organs of the respiratory, gastrointestinal and urinary tracts. Conclusions Commingling naive buffalo calves with calves acutely infected with P. multocida B:2 resulted in carriers among surviving buffalo. Pasteurella was found in various organs of the respiratory, gastrointestinal and urinary tracts, suggesting their role in the pathogenesis of HS. PMID:24721163

  20. Relaxin regulates hyaluronan synthesis and aquaporins in the cervix of late pregnant mice.

    PubMed

    Soh, Yu May; Tiwari, Anjana; Mahendroo, Mala; Conrad, Kirk P; Parry, Laura J

    2012-12-01

    Cervical ripening is associated with loss of structural integrity and tensile strength, thus enabling the cervix to dilate at term. It is characterized by changes in glycosaminoglycan composition, increased water content, and a progressive reorganization of the collagen network. The peptide hormone relaxin via interaction with its receptor, relaxin family peptide receptor 1 (RXFP1), promotes tissue hydration and increases cervical hyaluronan (HA) concentrations, but the mechanisms that regulate these effects are not known. This study in relaxin mutant (Rln(-/-)) mice tested the hypothesis that relaxin regulates HA synthase and aquaporin (AQP) expression in the cervix. We also assessed expression of the RXFP1 protein by immunohistochemistry. Pregnant Rln(-/-) mice had lower Has2 and Aqp3 expression on d 18.5 of pregnancy and decreased cervical HA compared with wild-type Rln(+/+) mice. Chronic infusion of relaxin for 4 or 6 d in pregnant Rln(-/-) mice reversed these phenotypes and increased Has2 and Aqp3 compared with placebo controls. Relaxin-treated mice also had lower Has1 and Aqp5. Changes in gene expression were paralleled by increases in cervical HA and variations in AQP3 and AQP5 protein localization in epithelial cells of Rln(-/-) cervices. Our findings demonstrate that relaxin alters AQP expression in the cervix and initiates changes in glycosaminoglycan composition through increased HA synthesis. These effects are likely mediated through RXFP1 localized to subepithelial stromal cells and epithelial cells. We suggest these actions of relaxin collectively promote water recruitment into the extracellular matrix to loosen the dense collagen fiber network. PMID:23087172

  1. Regulation of the hyaluronan system in ovine endometrium by ovarian steroids.

    PubMed

    Raheem, Kabir A; Marei, Waleed F; Mifsud, Karen; Khalid, Muhammad; Wathes, D Claire; Fouladi-Nashta, Ali A

    2013-05-01

    In this study, we investigated steroid regulation of the hyaluronan (HA) system in ovine endometrium including HA synthases (HAS), hyaluronidases, and HA receptor-CD44 using 30 adult Welsh Mountain ewes. Eight ewes were kept intact and synchronized to estrous (day 0). Intact ewes were killed on day 9 (luteal phase; LUT; n=5) and day 16 (follicular phase; FOL; n=3). The remaining ewes (n=22) were ovariectomized and then treated (i.m.) with vehicle (n=6) or progesterone (n=8) for 10 days, or estrogen and progesterone for 3 days followed by 7 days of progesterone alone (n=8). Estradiol and progesterone concentrations in plasma correlated with the stage of estrous or steroid treatment. Our results showed trends (P<0.1) and statistically significant effects (P<0.05, by t-test) indicating that LUT had lower HAS1 and HAS2 and higher HAS3 and CD44 mRNA expression compared with FOL. This was reflected in immunostaining of the corresponding HAS proteins. Similarly, in ovariectomized ewes, progesterone decreased HAS1 and HAS2 and increased HAS3 and CD44, whereas estradiol tended to increase HAS2 and decrease CD44. Sometimes, HAS mRNA expression did not follow the same trend observed in the intact animals or the protein expression. HA and its associated genes and receptors were regulated by the steroids. In conclusion, these results show that the level of HA production and the molecular weight of HA in the endometrium are regulated by ovarian steroids through differential expression of different HAS both at the gene and at the protein levels. PMID:23630333

  2. 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

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

    PubMed

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

    2016-05-01

    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

  4. Esophageal Squamous Cell Carcinoma Cells Modulate Chemokine Expression and Hyaluronan Synthesis in Fibroblasts.

    PubMed

    Kretschmer, Inga; Freudenberger, Till; Twarock, Sören; Yamaguchi, Yu; Grandoch, Maria; Fischer, Jens W

    2016-02-19

    The aim of this study was to characterize the interaction of KYSE-410, an esophageal squamous cell carcinoma cell line, and fibroblasts with respect to the extracellular matrix component hyaluronan (HA) and chemokine expression. KYSE-410 cells induced the mRNA expression of HA synthase 2 (Has2) in normal skin fibroblasts (SF) only in direct co-cultures. Parallel to Has2 mRNA, Has2 antisense RNA (Has2os2) was up-regulated in co-cultures. Knockdown of LEF1, a downstream target of Wnt signaling, abrogated Has2 and Has2os2 induction. After knockdown of Has2 in SF, significantly less α-smooth muscle actin expression was detected in co-cultures. Moreover, it was investigated whether the phenotype of KYSE-410 was affected in co-culture with SF and whether Has2 knockdown in SF had an impact on KYSE-410 cells in co-culture. However, no effects on epithelial-mesenchymal transition markers, proliferation, and migration were detected. In addition to Has2 mRNA, the chemokine CCL5 was up-regulated and CCL11 was down-regulated in SF in co-culture. Furthermore, co-cultures of KYSE-410 cells and cancer-associated fibroblasts (CAF) were investigated. Similar to SF, Has2 and Ccl5 were up-regulated and Ccl11 was down-regulated in CAF in co-culture. Importantly and in contrast to SF, inhibiting HA synthesis by 4-methylumbelliferone abrogated the effect of co-culture on Ccl5 in CAF. Moreover, HA was found to promote adhesion of CD4(+) but not CD8(+) cells to xenogaft tumor tissues. In conclusion, direct co-culture of esophageal squamous cell carcinoma and fibroblasts induced stromal HA synthesis via Wnt/LEF1 and altered the chemokine profile of stromal fibroblasts, which in turn may affect the tumor immune response. PMID:26699196

  5. Mechanisms involved in enhancement of the expression and function of aggrecanases by hyaluronan oligosaccharides

    PubMed Central

    Ariyoshi, Wataru; Takahashi, Nobunori; Hida, Daisuke; Knudson, Cheryl B.; Knudson, Warren

    2011-01-01

    Objective Small hyaluronan (HA) oligosaccharides serve as competitive receptor antagonists to displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and induces stimulation of genes involved in matrix degradation such as matrix metalloproteinases as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2. The objective of this study was to determine changes in the expression and function of aggrecanases after disruption of chondrocyte CD44-HA interactions. Methods Bovine articular chondrocytes or bovine cartilage tissue were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in aggrecanase were monitored by real time reverse transcriptase-polymerase chain reaction and western blot analysis of ADAMTS4, ADAMTS5 and aggrecan proteolytic fragments. To test the interactions between ADAMTS4 and MT4-MMP, protein lysates purified from stimulated chondrocytes were subjected to co-immunoprecipitation. Results Disruption of chondrocyte CD44-HA interactions with HA oligosaccharides induced the transcription of ADAMTS4 and ADAMTS5 in time- and dose-dependent manner. The association of GPI-anchored MT4-MMP with ADAMTS4 was also induced in articular chondrocytes by HA oligosaccharides. Inhibition of the NF-κB pathway blocked HA oligosaccharides-mediated stimulation of aggrecanases. Conclusions Disruptive changes in chondrocyte-matrix interactions by HA oligosaccharides induce matrix degradation and elevate aggrecanases via the activation of the NF-κB signaling pathway. PMID:21905012

  6. Influence of hyaluronan accumulation during cumulus expansion on in vitro porcine oocyte maturation.

    PubMed

    Yokoo, Masaki; Kimura, Naoko; Abe, Hiroyuki; Sato, Eimei

    2008-11-01

    During oocyte maturation, the cumulus-oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process. PMID:18838022

  7. Anticancer Therapeutics: Targeting Macromolecules and Nanocarriers to Hyaluronan or CD44, a Hyaluronan Receptor

    PubMed Central

    Platt, Virginia M.; Szoka, Francis C.

    2009-01-01

    The complex system involved in the synthesis, degradation, and binding of the high molecular weight glycosaminoglycan hyaluronic acid (hyaluronan or HA) provides a variety of structures that can be exploited for targeted cancer therapy. In many cancers of epithelial origin there is an up-regulation of CD44, a receptor that binds HA. In other cancers, HA in the tumor matrix is over-expressed. Both CD44 on cancer cells and HA in the matrix have been targets for anti-cancer therapy. Even though CD44 is expressed in normal epithelial cells and HA is part of the matrix of normal tissues, selective targeting to cancer is possible. This is because macromolecular carriers predominantly extravasate into the tumor and not normal tissue; thus CD44-HA targeted carriers administered intravenously localize preferentially into tumors. Anti-CD44 antibodies have been used in patients to deliver radioisotopes or mertansine for treatment of CD44 expressing tumors. In early phase clinical trials, patients with breast or head and neck tumors treated with anti-CD44 conjugates experienced stabilized disease. A dose-limiting toxicity was associated with distribution of the antibody-drug conjugate to the skin, a site in the body with a high level of CD44. HA has been used as a drug carrier and a ligand on liposomes or nanoparticles to target drugs to CD44 over-expressing cells. Drugs can be attached to HA via the carboxylate on the glucuronic acid residue, the hydroxyl on the N-acetylglucosamine, or the reducing end which are located on a repeating disaccharide. Drugs delivered in HA-modified liposomes exhibited excellent anti-tumor activity both in vitro and in murine tumor models. The HA matrix is also a potential target for anti-cancer therapies. By manipulating the interaction of HA with cell surface receptors, either by degrading it with hyaluronidase or by interfering with CD44-HA interactions using soluble CD44 proteins, tumor progression was blocked. Finally, cytotoxic drugs or pro

  8. 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

  9. Concentration dependence of interstitial flow buffering by hyaluronan in synovial joints.

    PubMed

    Scott, D; Coleman, P J; Mason, R M; Levick, J R

    2000-05-01

    Hyaluronan concentration in synovial fluid spans a 20-fold range, from as low as 0.2 mg ml(-1) in synovitis to as high as 4 mg ml(-1) in healthy joints. The aim was to determine the effect of this on fluid drainage from the joint cavity. The study extends the finding of P. J. Coleman, D. Scott, R. M. Mason, and J. R. Levick (1999, J. Physiol. 514, 265-282) that dissolved hyaluronan at 3.6-4.0 mg ml(-1) (the concentration in young human and rabbit joints) causes the opposition to interstitial fluid drainage to increase with pressure. Hyaluronan was infused into rabbit knees at 0, 0.2, 2.0, and 4.0 mg ml(-1) over a range of intraarticular pressures. Hyaluronan at 2 mg ml(-1) (as in healthy elderly joints and some osteoarthritis) greatly reduced drainage rates and generated a flattening (convex) pressure-flow relation, as observed previously with 4 mg ml(-1). Drainage rates were greater at 2 mg ml(-1) than at 4 mg ml(-1) hyaluronan (P < 0.0001, ANOVA, n = 7). The opposition to outflow (pressure required to drive unit outflow) increased with pressure, but less markedly than with 4 mg ml(-1) hyaluronan. Hyaluronan at 0.2 mg ml(-1) reduced outflow by approximately 50% relative to Ringer solution (P < 0.0001, ANOVA, n = 7) but the pressure-flow relation no longer flattened out with increasing pressure, because there was no significant increase in opposition to outflow with pressure. At 0 mg ml(-1) hyaluronan, outflow opposition decreased with pressure. Viscometry showed a marked transition in the hyaluronan state at >/=1.35 mg ml(-1), indicating that this is the critical concentration for molecular domain overlap and intermolecular coupling. The results broadly supported the concentration-polarization hypothesis, which predicts significant osmotic buffering of drainage at >/=1 mg ml(-1) hyaluronan; at 0.2 mg ml(-1) other factors may predominate. It is inferred that hyaluronan at physiological concentrations can conserve synovial fluid when pressures are raised (e

  10. Interactions between CD44 and Hyaluronan in Leukocyte Trafficking

    PubMed Central

    McDonald, Braedon; Kubes, Paul

    2015-01-01

    Recruitment of leukocytes from the bloodstream to inflamed tissues requires a carefully regulated cascade of binding interactions between adhesion molecules on leukocytes and endothelial cells. Adhesive interactions between CD44 and hyaluronan (HA) have been implicated in the regulation of immune cell trafficking within various tissues. In this review, the biology of CD44–HA interactions in cell trafficking is summarized, with special attention to neutrophil recruitment within the liver microcirculation. We describe the molecular mechanisms that regulate adhesion between neutrophil CD44 and endothelial HA, including recent evidence implicating serum-derived hyaluronan-associated protein as an important co-factor in the binding of HA to CD44 under flow conditions. CD44–HA-mediated neutrophil recruitment has been shown to contribute to innate immune responses to invading microbes, as well as to the pathogenesis of many inflammatory diseases, including various liver pathologies. As a result, blockade of neutrophil recruitment by targeting CD44–HA interactions has proven beneficial as an anti-inflammatory treatment strategy in a number of animal models of inflammatory diseases. PMID:25741341

  11. Characterization of Hyaluronan-Degrading Enzymes from Yeasts.

    PubMed

    Smirnou, Dzianis; Krčmář, Martin; Kulhánek, Jaromír; Hermannová, Martina; Bobková, Lenka; Franke, Lukáš; Pepeliaev, Stanislav; Velebný, Vladimír

    2015-10-01

    Hyaluronidases (HAases) from yeasts were characterized for the first time. The study elucidated that hyaluronate 4-glycanohydrolase and hyaluronan (HA) lyase can be produced by yeasts. Six yeasts producing HAases were found through express screening of activities. The extracellular HAases from two of the yeast isolates, Pseudozyma aphidis and Cryptococcus laurentii, were characterized among them. P. aphidis HAase hydrolyzed β-1,4 glycosidic bonds of HA, yielding even-numbered oligosaccharides with N-acetyl-D-glucosamine at the reducing end. C. laurentii produced hyaluronan lyase, which cleaved β-1,4 glycosidic bonds of HA in β-elimination reaction, and the products of HA degradation were different-sized even-numbered oligosaccharides. The shortest detected HA oligomer was dimer. The enzymes' pH and temperature optima were pH 3.0 and 37-45 °C (P. aphidis) and pH 6.0 and 37 °C (C. laurentii), respectively. Both HAases showed good thermostability. PMID:26239444

  12. Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury.

    PubMed

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

    2015-05-01

    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 Ca(2+), and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca(2+), 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 Ca(2+) channels of airway smooth muscle cells, increasing their contractility and thus causing AHR. PMID:25747964

  13. 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

  14. Effect of Carboxymethylation on the Rheological Properties of Hyaluronan.

    PubMed

    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

  15. An Arabidopsis callose synthase.

    PubMed

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole; Mundy, John

    2002-08-01

    Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially complements a yeast beta-1,3-glucan synthase mutant. AtGsl5 is developmentally expressed at highest levels in flowers, consistent with flowers having high beta-1,3-glucan synthase activities for deposition of callose in pollen. A role for AtGsl5 in callose synthesis is also indicated by AtGsl5 expression in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant. PMID:12081364

  16. Granulocyte plasma membrane damage by leukotoxic supernatant from Pasteurella haemolytica A1 and protection by immune serum.

    PubMed

    Styrt, B; Walker, R D; White, J C; Dahl, L D; Baker, J C

    1990-01-01

    Bovine respiratory disease caused by Pasteurella haemolytica may be partially mediated by a leukotoxin secreted by the microorganism. We examined the effect of leukotoxic Pasteurella supernatants on leakage of the cytosol enzyme lactate dehydrogenase and the lysosomal enzyme arylsulfatase from bovine granulocytes. Lactate dehydrogenase release (94%) was much higher than arylsulfatase release (38%) over 30 minutes of incubation. The Pasteurella supernatants inhibited superoxide production by stimulated granulocytes at concentrations which also caused substantial cell death as measured by failure to exclude trypan blue. Both toxic effects were prevented by serum from aerosol-immunized calves, and protection appeared to be antibody-specific by comparison with fetal bovine serum or with serum absorbed against intact P. haemolytica. These findings suggest that the leukotoxin may selectively disrupt the granulocyte plasma membrane, and that antibody directed against a surface component of the microorganism is also capable of protecting against the leukotoxin effect. PMID:2306664

  17. Hyaluronan in cancer - from the naked mole rat to nanoparticle therapy.

    PubMed

    Rankin, Kenneth S; Frankel, Daniel

    2016-05-01

    Hyaluronan, a glycosaminoglycan, abundant in the tumour microenvironment, is a key player in many processes associated with cancer. Recently the cancer resistance of the naked mole rat has been attributed to the presence of an ultra-high molecular weight form of this molecule. The physical properties of this multifunctional biopolymer have been extensively studied in the context of synovial joints. However, relatively little has been reported with regard to the soft matter properties of hyaluronan in relation to cancer. In this review we examine the role of hyaluronan in cancer, paying particular attention to its mechanical interactions with malignant cells and its soft matter properties. In addition we discuss the use of hyaluronan based gels to study cancer invasion as well as nanoparticle based strategies for disease treatment. PMID:27079782

  18. Endogenous hyaluronan: a cytokine-like factor present in rabbit uterine cervix during pregnancy.

    PubMed

    Uchiyama, Taro; Matsumoto, Tetsunori; Suzuki, Yoshiharu; Ishida, Masami; Obara, Takeo; Kanayama, Toshiji

    2004-12-01

    The effects of endogenous hyaluronan on cervical ripening during pregnancy were examined in rabbits. Hyaluronan of approximately 620 kilodalton (kDa) was detected in the uterine cervix on the 25th and 29th days of pregnancy, while it was not detected in cervix of non-pregnant animals. In addition, low-molecular-weight (less than 191 kDa) hyaluronan was present in cervix at the 29th day of pregnancy. Hyaluronan level in the cervix was lower on the 29th day than on the 25th day of pregnancy, whereas that in serum was significantly higher on the 29th day than on the 25th day of pregnancy. To clarify the physiological functions of endogenous hyaluronan, the effects of hyaluronan (HA600-700), which had approximately equal to endogenous hyaluronan, on uterine cervical tissues and uterine cervical fibroblasts were examined. Rabbits at the 23rd day of pregnancy were administered a vaginal suppository of HA600-700 (20 mg) daily for three days. Promotion of cervical ripening was observed, as well as detachment of collagen fiber bundles, and a reduced density of collagen fiber distribution. Total collagenolytic activity was increased significantly by HA600-700 (1.0 mg/ml) treatment in cultured uterine cervical explants of pregnant rabbits as compared with the untreated group. Moreover, very similar effects of HA600-700 treatment (0.1, 1.0 mg/ml) were observed in cultured uterine cervical fibroblasts. Further, in tissue cultures, but not cell cultures, of pregnant rabbit cervix, prostaglandin E2 (PGE2) production was enhanced by HA600-700 treatment. Therefore, it appears that endogenous hyaluronan is closely concerned with cervical ripening and dilatation in uterine cervix of pregnant rabbits. PMID:15577204

  19. Evaluation of emulsion electrospun polycaprolactone/hyaluronan/epidermal growth factor nanofibrous scaffolds for wound healing.

    PubMed

    Wang, Zhenbei; Qian, Yuna; Li, Linhao; Pan, Lianhong; Njunge, Lucy W; Dong, Lili; Yang, Li

    2016-01-01

    Wound healing scaffolds provide cells with structural integrity and can also deliver biological agents to establish a skin tissue-specific microenvironment to regulate cell functions and to accelerate the healing process. In this study, we fabricated biodegradable nanofibrous scaffolds with an emulsion electrospinning technique. The scaffolds were composed of polycaprolactone, hyaluronan and encapsulating epidermal growth factor. The morphology and core-sheath structure of the nanofibers were characterized by scanning electron microscopy and transmission electron microscopy. The scaffolds were also characterized for chemical composition and hydrophilicity with a Fourier-transform infrared analysis, energy dispersive spectroscopy and the water contact angle. An in vitro model protein bovine serum albumin and epidermal growth factor release study was conducted to evaluate the sustained release potential of the core-sheath structured nanofibers with and without the hyaluronan component. Additionally, an in vitro cultivation of human skin keratinocytes (HaCaT) and fibroblasts on polycaprolactone/hyaluronan and polycaprolactone/hyaluronan-epidermal growth factor scaffolds showed a significant synergistic effect of hyaluronan and epidermal growth factor on cell proliferation and infiltration. Furthermore, there was an up-regulation of the wound-healing-related genes collagen I, collagen III and TGF-β in polycaprolactone/hyaluronan/epidermal growth factor scaffolds compared with control groups. In the full-thickness wound model, the enhanced regeneration of fully functional skin was facilitated by epidermal regeneration in the polycaprolactone/hyaluronan/epidermal growth factor treatment group. Our findings suggest that bioactivity and hemostasis of the hyaluronan-based nanofibrous scaffolds have the capability to encapsulate and control the release of growth factors that can serve as skin tissue engineering scaffolds for wound healing. PMID:26012354

  20. Hyaluronan with dextran added to therapeutic lung surfactants improves effectiveness in vitro and in vivo.

    PubMed

    Lu, Karen W; Taeusch, H William; Clements, John A

    2013-01-01

    Surfactants in current clinical use are largely ineffective in treating acute lung injury (ALI)/ acute respiratory distress syndrome. In part, this ineffectiveness is due to inactivation of surfactant by serum leakage into the alveoli. Previously, we reported that adding hyaluronan and some nonionic polymers to synthetic lipids combined with native SP-B and SP-C enhanced surface activity. In this study, we first tested two therapeutic lung surfactants and then retested after adding hyaluronan, polyethylene glycol or dextran alone or in two-polymer combinations including hyaluronan in the absence or presence of serum. Surface activities were measured in a modified bubble surfactometer. Results indicate that the inhibition threshold (defined as the amount of serum required to produce a minimum surface tension above 10 mN/m after 5 minutes of cycling) was 35 times higher with hyaluronan plus dextran added to Infasurf than with Infasurf alone, and better than all other mixtures tested. The threshold for Survanta with hyaluronan plus polyethylene glycol was 7 times higher than Survanta alone. We next tested selected surfactant mixtures in an animal model that mimicked ALI. All measurements of lung function showed significant improvement (P ≤ .05) with hyaluronan, or with hyaluronan and dextran added to Infasurf compared to Infasurf alone. Also, for these two groups, lung function was still improving at the end of the experiment. We conclude that certain polymers added to clinical surfactants can greatly increase resistance to inactivation in vitro, while in vivo, both Infasurf mixtures containing hyaluronan tended to normalize measures of lung function unlike other mixtures tested. PMID:23638643

  1. Geranyl diphosphate synthase from mint

    SciTech Connect

    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.

  2. Geranyl diphosphate synthase from mint

    SciTech Connect

    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.

  3. A hyaluronan-based scaffold for the in vitro construction of dental pulp-like tissue.

    PubMed

    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

  4. Cellular Uptake and Internalization of Hyaluronan-based Doxorubicin and Cisplatin Conjugates

    PubMed Central

    Cai, Shuang; Alhowyan, Adel Ali B; Yang, Qiuhong; Forrest, W.C. Melanie; Shnayder, Yelizaveta; Forrest, M. Laird

    2015-01-01

    Background Hyaluronan (HA) is a ligand for the CD44 receptor which is crucial to cancer cell proliferation and metastasis. High levels of CD44 expression in many cancers have encouraged the development of HA-based carriers for anti-cancer therapeutics. Purpose The objective of this study was to determine whether HA conjugation of anticancer drugs impacts CD44-specific HA-drug uptake and disposition by human head and neck cancer cells. Methods The internalization and cellular disposition of hyaluronan-doxorubicin (HA-DOX), hyaluronan-cisplatin (HA-Pt), and hyaluronan-cyanine7 (HA-Cy7) conjugates were investigated by inhibiting endocytosis pathways, and by inhibiting the CD44–mediated internalization pathways that are known to mediate hyaluronan uptake in vitro. Results Cellular internalization of HA was regulated by CD44 receptors. In mouse xenografts, HA conjugation significantly enhanced tumor cell uptake compared to unconjugated drug. Discussion The results suggested that the main mechanism of HA-based conjugate uptake may be active transport via CD44 in conjunction with a clathrin–dependent endocytic pathway. Other HA receptors, hyaluronan–mediated motility receptor (RHAMM) and lymphatic vessel endothelial hyaluronan receptor (LYVE-1), did not play a significant role in conjugate uptake. Conclusions HA conjugation significantly increased CD44 mediated drug uptake and extended the residence time of drugs in tumor cells. PMID:24892741

  5. Regulation of plasminogen activator inhibitor-1 and urokinase by hyaluronan fragments in mouse macrophages.

    PubMed

    Horton, M R; Olman, M A; Bao, C; White, K E; Choi, A M; Chin, B Y; Noble, P W; Lowenstein, C J

    2000-10-01

    Pulmonary inflammation and fibrosis are characterized by increased turnover and production of the extracellular matrix as well as an impairment of lung fibrinolytic activity. Although fragments of the extracellular matrix component hyaluronan induce macrophage production of inflammatory mediators, the effect of hyaluronan on the fibrinolytic mediators plasminogen activator inhibitor (PAI)-1 and urokinase-type plasminogen activator (uPA) is unknown. This study demonstrates that hyaluronan fragments augment steady-state mRNA, protein, and inhibitory activity of PAI-1 as well as diminish the baseline levels of uPA mRNA and inhibit uPA activity in an alveolar macrophage cell line. Hyaluronan fragments alter macrophage expression of PAI-1 and uPA at the level of gene transcription. Similarly, hyaluronan fragments augment PAI-1 and diminish uPA mRNA levels in freshly isolated inflammatory alveolar macrophages from bleomycin-treated rats. These data suggest that hyaluronan fragments influence alveolar macrophage expression of PAI-1 and uPA and may be a mechanism for regulating fibrinolytic activity during lung inflammation. PMID:11000131

  6. The effects of hyaluronan and its fragments on lipid models exposed to UV irradiation.

    PubMed

    Trommer, Hagen; Wartewig, Siegfried; Böttcher, Rolf; Pöppl, Andreas; Hoentsch, Joachim; Ozegowski, Jörg H; Neubert, Reinhard H H

    2003-03-26

    The effects of hyaluronan and its degradation products on irradiation-induced lipid peroxidation were investigated. Liposomal skin lipid models with increasing complexity were used. Hyaluronan and its fragments were able to reduce the amount of lipid peroxidation secondary products quantified by the thiobarbituric acid (TBA) assay. The qualitative changes were studied by mass spectrometry. To elucidate the nature of free radical involvement electron paramagnetic resonance (EPR) studies were carried out. The influence of hyaluronan and its fragments on the concentration of hydroxyl radicals generated by the Fenton system was examined using the spin trapping technique. Moreover, the mucopolysaccharide's ability to react with stable radicals was checked. The quantification assay of 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) showed no concentration changes of the stable radical caused by hyaluronan. Hyaluronan was found to exhibit prooxidative effects in the Fenton assay in a concentration dependent manner. A transition metal chelation was proposed as a mechanism of this behavior. Considering human skin and its constant exposure to UV light and oxygen and an increased pool of iron in irradiated skin the administration of hyaluronan or its fragments in cosmetic formulations or sunscreens could be helpful for the protection of the human skin. PMID:12623198

  7. 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

  8. Priming Adipose-Derived Mesenchymal Stem Cells with Hyaluronan Alters Growth Kinetics and Increases Attachment to Articular Cartilage

    PubMed Central

    Succar, Peter; Medynskyj, Michael; Breen, Edmond J.; Batterham, Tony; Molloy, Mark P.; Herbert, Benjamin R.

    2016-01-01

    Background. Biological therapeutics such as adipose-derived mesenchymal stem cell (MSC) therapy are gaining acceptance for knee-osteoarthritis (OA) treatment. Reports of OA-patients show reductions in cartilage defects and regeneration of hyaline-like-cartilage with MSC-therapy. Suspending MSCs in hyaluronan commonly occurs in animals and humans, usually without supporting data. Objective. To elucidate the effects of different concentrations of hyaluronan on MSC growth kinetics. Methods. Using a range of hyaluronan concentrations, we measured MSC adherence and proliferation on culture plastic surfaces and a novel cartilage-adhesion assay. We employed time-course and dispersion imaging to assess MSC binding to cartilage. Cytokine profiling was also conducted on the MSC-secretome. Results. Hyaluronan had dose-dependent effects on growth kinetics of MSCs at concentrations of entanglement point (1 mg/mL). At higher concentrations, viscosity effects outweighed benefits of additional hyaluronan. The cartilage-adhesion assay highlighted for the first time that hyaluronan-primed MSCs increased cell attachment to cartilage whilst the presence of hyaluronan did not. Our time-course suggested patients undergoing MSC-therapy for OA could benefit from joint-immobilisation for up to 8 hours. Hyaluronan also greatly affected dispersion of MSCs on cartilage. Conclusion. Our results should be considered in future trials with MSC-therapy using hyaluronan as a vehicle, for the treatment of OA. PMID:26981136

  9. Priming Adipose-Derived Mesenchymal Stem Cells with Hyaluronan Alters Growth Kinetics and Increases Attachment to Articular Cartilage.

    PubMed

    Succar, Peter; Medynskyj, Michael; Breen, Edmond J; Batterham, Tony; Molloy, Mark P; Herbert, Benjamin R

    2016-01-01

    Background. Biological therapeutics such as adipose-derived mesenchymal stem cell (MSC) therapy are gaining acceptance for knee-osteoarthritis (OA) treatment. Reports of OA-patients show reductions in cartilage defects and regeneration of hyaline-like-cartilage with MSC-therapy. Suspending MSCs in hyaluronan commonly occurs in animals and humans, usually without supporting data. Objective. To elucidate the effects of different concentrations of hyaluronan on MSC growth kinetics. Methods. Using a range of hyaluronan concentrations, we measured MSC adherence and proliferation on culture plastic surfaces and a novel cartilage-adhesion assay. We employed time-course and dispersion imaging to assess MSC binding to cartilage. Cytokine profiling was also conducted on the MSC-secretome. Results. Hyaluronan had dose-dependent effects on growth kinetics of MSCs at concentrations of entanglement point (1 mg/mL). At higher concentrations, viscosity effects outweighed benefits of additional hyaluronan. The cartilage-adhesion assay highlighted for the first time that hyaluronan-primed MSCs increased cell attachment to cartilage whilst the presence of hyaluronan did not. Our time-course suggested patients undergoing MSC-therapy for OA could benefit from joint-immobilisation for up to 8 hours. Hyaluronan also greatly affected dispersion of MSCs on cartilage. Conclusion. Our results should be considered in future trials with MSC-therapy using hyaluronan as a vehicle, for the treatment of OA. PMID:26981136

  10. Overexpression and immunogenicity of the Oma87 outer membrane protein of Pasteurella multocida.

    PubMed

    Mitchison, M; Wei, L; Kwang, J; Wilkie, I; Adler, B

    2000-03-01

    The outer membrane protein of Oma87 from Pasteurella multocida A:1 has significant similarity to the D15 protective antigen of Haemophilus influenzae (Ruffolo and Adler, 1996). Four fragments of Oma87 from a P. multocida serotype D strain were cloned into a pGEX expression vector and transformed into E. coli JM105. Western blot analysis revealed that convalescent chicken sera reacted with only GST-F1 fusion protein which contained amino acids 18 through to 130 of Oma87 fused to the GST protein. Vaccination with the GST-F1 protein failed to protect chickens against challenge with a virulent P. multocida serotype A. PMID:10699506

  11. 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.

  12. Microbial synthesis of hyaluronan and chitin: New approaches.

    PubMed

    Yamada, Takashi; Kawasaki, Takeru

    2005-06-01

    Hyaluronan (HA) is an important structural element in the vitreous humor of the eye, synovial fluid, and skin of vertebrates. Moreover, HA interacts with proteins such as CD44, RHAMM, and fibrinogen, thereby influencing many natural processes such as angiogenesis, cancer, cell motility, wound healing, and cell adhesion. Reflecting such a variety of functions, HA has attracted attention from a wide range of application fields such as medicine (including surgery), cosmetics, and health foods. Traditionally HA was extracted from rooster combs, but nowadays is produced by the fermentation of streptococci. At present, quality issues such as purity and molecular weight distribution, rather than quantity, have been the focus of strain and process development in HA production. To meet ever-increasing public demand, novel systems that can yield sufficient amounts of high-quality of HA and related materials are required. PMID:16233827

  13. Rheological properties of aqueous solutions of biopolymeric hyaluronan

    NASA Astrophysics Data System (ADS)

    Szwajczak, Elzbieta

    2004-09-01

    Aqueous solutions of hyaluronic acid (hyaluronan, HA) were studied. The HA compound is a natural polysaccharide, bipolymer. It plays an important role in numerous biological processes as a component of the extracellular matrix, connective tissues and, especially, human and animal synovial joints. Natural and artificial solutions of the HA have demonstrated the viscoelastic nature. These properties are shown to be related to the microstructure parameters (bulk concentration, molecular weight) and external parameters (temperature, stress, shear rate). We emphasize the role of the flow properties of polymeric systems. It is found a liquid crystalline "order" can be "induced" during the material flow. The dynamic properties, such as the elastic shear modulus and viscous shear modulus, are given. These results are discussed in relation to the postulated function of hyaluronic acid in synovial joint and with respect to possibilities o their application in medicine and pharmacology.

  14. 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

    2013-10-01

    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. PMID:23950179

  15. Human Milk Hyaluronan Enhances Innate Defense of the Intestinal Epithelium*

    PubMed Central

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

    2013-01-01

    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. PMID:23950179

  16. Hybrid polyketide synthases

    DOEpatents

    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.

  17. First High-Quality Draft Genome Sequence of Pasteurella multocida Sequence Type 128 Isolated from Infected Bone.

    PubMed

    Kavousi, Niloofar; Eng, Wilhelm Wei Han; Lee, Yin Peng; Tan, Lian Huat; Thuraisingham, Ravindran; Yule, Catherine M; Gan, Han Ming

    2016-01-01

    We report here the first high-quality draft genome sequence of Pasteurella multocida sequence type 128, which was isolated from the infected finger bone of an adult female who was bitten by a domestic dog. The draft genome will be a valuable addition to the scarce genomic resources available for P. multocida. PMID:26941132

  18. Identification of clinical Pasteurella isolates by MALDI-TOF -- a comparison with VITEK 2 and conventional microbiological methods.

    PubMed

    Zangenah, Salah; Güleryüz, Gülay; Boräng, Stina; Ullberg, Måns; Bergman, Peter; Ozenci, Volkan

    2013-10-01

    The aim of this study was to compare the performance of four methods that are widely used in the clinical microbiology laboratory for identification of Pasteurella species. The 4 methods evaluated were VITEK2, VITEK MS (BioMerieux), and Bruker Biotyper MS (Bruker) as well as traditional biochemical tests. Sequencing of the sodA gene was used as the reference method. Sixty-five isolates of Pasteurella spp. from 65 patients were analyzed. One Pasteurella multocida isolate from American Type Culture Collection (Manassas, VA, USA) was used as a reference. Traditional biochemical tests accurately identified 62/66 (94%) isolates. Both Bruker and Vitek matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) identified 59/66 (89%) strains, but VITEK2 could only identify 32/66 (48.5%) isolates correctly. The mean time to identification using biochemical tests was 20 hours; VITEK2 took 6 hours and MALDI-TOF approximately 10 minutes. In conclusion, MALDI-TOF is a quick method, which accurately identified most isolates of Pasteurella to the species level. Thus, MALDI-TOF constitutes a valuable diagnostic tool in the clinical laboratory. PMID:23886788

  19. Draft Genome Sequences of Two Pasteurella multocida Strains Isolated from Buffaloes in India with Hemorrhagic Septicemia Disease

    PubMed Central

    Abrahante, J. E.; Veeregowda, B. M.; Hogtapur, S. S.; Briggs, R. E.; Maheswaran, S. K.

    2014-01-01

    Pasteurella multocida serotype B:2 is the causative agent of hemorrhagic septicemia in cattle and buffaloes in Asia. It is an acute fatal disease and is considered one of the most economically important diseases in this region of the world. We present here the draft genome sequences of strains 2213 and 3213 of P. multocida. PMID:25103770

  20. The effect of hypovitaminosis A on the pathogenesis of Pasteurella multocida in turkeys.

    PubMed

    Aye, P P; Morishita, T Y; Saif, Y M; Jonas, M

    2000-01-01

    It has been proposed that Pasteurella multocida can invade the host tissues via the mucous membrane. Vitamin A (VitA) deficiency has been associated with mucous membrane damage, such as squamous metaplasia. The objective of this study was to determine the early stages in the pathogenesis of P. multocida in VitA-deficient turkeys and clinically healthy turkeys. Fifteen-week-old VitA-deficient and clinically healthy turkeys were inoculated with P. multocida P-1059, a virulent strain, and the portal of entry, invasion, and localization of P. multocida were studied by microbial examination of the trachea, liver, and lung and histologic examinations of internal organs. Higher mortality was found in VitA-deficient turkeys. Pasteurella multocida was first reisolated from the trachea, secondarily from the liver and blood, and finally from the lung in both groups. Invasion of P. multocida into tissues occurred between 3 hr and 24 hr postinoculation in both groups. Our findings suggest that altered membrane integrity in VitA-deficient birds did not appear to change the time course of the systemic spread of P. multocida infection in turkeys and that the increased mortality seen in the VitA-deficient turkeys may be associated with immune system impairment. PMID:11195636

  1. Ceftaroline versus isolates from animal bite wounds: comparative in vitro activities against 243 isolates, including 156 Pasteurella species isolates.

    PubMed

    Goldstein, Ellie J C; Citron, Diane M; Merriam, C Vreni; Tyrrell, Kerin L

    2012-12-01

    More than 5 million Americans are bitten by animals, usually dogs, annually. Bite patients comprise ∼1% of all patients who visit emergency departments (300,000/year), and approximately 10,000 require hospitalization and intravenous antibiotics. Ceftaroline is the bioactive component of the prodrug ceftaroline fosamil, which is FDA approved for the treatment of acute bacterial skin and skin structure infections (ABSSSIs), including those containing methicillin-resistant Staphylococcus aureus (MRSA). There are no in vitro data about the activity of ceftaroline against Pasteurella multocida subsp. multocida and Pasteurella multocida subsp. septica, other Pasteurella spp., or other bite wound isolates. We therefore studied the in vitro activity of ceftaroline against 243 animal bite isolates. MICs were determined using the broth microdilution method according to CLSI guidelines. Comparator drugs included cefazolin, ceftriaxone, ertapenem, ampicillin-sulbactam, azithromycin, doxycycline, and sulfamethoxazole-trimethoprim (SMX-TMP). Ceftaroline was the most active agent against all 5 Pasteurella species, including P. multocida subsp. multocida and P. multocida subsp. septica, with a maximum MIC of ≤0.008 μg/ml; more active than ceftriaxone and ertapenem (MIC(90)s, ≤0.015 μg/ml); and more active than cefazolin (MIC(90), 0.5 μg/ml) doxycycline (MIC(90), 0.125 μg/ml), azithromycin (MIC(90), 0.5 μg/ml), ampicillin-sulbactam (MIC(90), 0.125 μg/ml), and SMX-TMP (MIC(90), 0.125 μg/ml). Ceftaroline was also very active against all S. aureus isolates (MIC(90), 0.125 μg/ml) and other Staphylococcus and Streptococcus species, with a maximum MIC of 0.125 μg/ml against all bite isolates tested. Ceftaroline has potential clinical utility against infections involving P. multocida, other Pasteurella species, and aerobic Gram-positive isolates, including S. aureus. PMID:23027193

  2. Ceftaroline versus Isolates from Animal Bite Wounds: Comparative In Vitro Activities against 243 Isolates, Including 156 Pasteurella Species Isolates

    PubMed Central

    Citron, Diane M.; Merriam, C. Vreni; Tyrrell, Kerin L.

    2012-01-01

    More than 5 million Americans are bitten by animals, usually dogs, annually. Bite patients comprise ∼1% of all patients who visit emergency departments (300,000/year), and approximately 10,000 require hospitalization and intravenous antibiotics. Ceftaroline is the bioactive component of the prodrug ceftaroline fosamil, which is FDA approved for the treatment of acute bacterial skin and skin structure infections (ABSSSIs), including those containing methicillin-resistant Staphylococcus aureus (MRSA). There are no in vitro data about the activity of ceftaroline against Pasteurella multocida subsp. multocida and Pasteurella multocida subsp. septica, other Pasteurella spp., or other bite wound isolates. We therefore studied the in vitro activity of ceftaroline against 243 animal bite isolates. MICs were determined using the broth microdilution method according to CLSI guidelines. Comparator drugs included cefazolin, ceftriaxone, ertapenem, ampicillin-sulbactam, azithromycin, doxycycline, and sulfamethoxazole-trimethoprim (SMX-TMP). Ceftaroline was the most active agent against all 5 Pasteurella species, including P. multocida subsp. multocida and P. multocida subsp. septica, with a maximum MIC of ≤0.008 μg/ml; more active than ceftriaxone and ertapenem (MIC90s, ≤0.015 μg/ml); and more active than cefazolin (MIC90, 0.5 μg/ml) doxycycline (MIC90, 0.125 μg/ml), azithromycin (MIC90, 0.5 μg/ml), ampicillin-sulbactam (MIC90, 0.125 μg/ml), and SMX-TMP (MIC90, 0.125 μg/ml). Ceftaroline was also very active against all S. aureus isolates (MIC90, 0.125 μg/ml) and other Staphylococcus and Streptococcus species, with a maximum MIC of 0.125 μg/ml against all bite isolates tested. Ceftaroline has potential clinical utility against infections involving P. multocida, other Pasteurella species, and aerobic Gram-positive isolates, including S. aureus. PMID:23027193

  3. PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines

    PubMed Central

    KATONA, ÉVA; JUHÁSZ, TAMÁS; SOMOGYI, CSILLA SZŰCS; HAJDÚ, TIBOR; SZÁSZ, CSABA; RÁCZ, KÁLMÁN; KÓKAI, ENDRE; GERGELY, PÁL; ZÁKÁNY, RÓZA

    2016-01-01

    Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment. PMID:26717964

  4. PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.

    PubMed

    Katona, Éva; Juhász, Tamás; Somogyi, Csilla Szűcs; Hajdú, Tibor; Szász, Csaba; Rácz, Kálmán; Kókai, Endre; Gergely, Pál; Zákány, Róza

    2016-03-01

    Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment. PMID:26717964

  5. Interaction of hyaluronan binding peptides with glycosaminoglycans in poly(ethylene glycol) hydrogels.

    PubMed

    Roberts, Justine J; Elder, Robert M; Neumann, Alexander J; Jayaraman, Arthi; Bryant, Stephanie J

    2014-04-14

    This study investigates the incorporation of hyaluronan (HA) binding peptides into poly(ethylene glycol) (PEG) hydrogels as a mechanism to bind and retain hyaluronan for applications in tissue engineering. The specificity of the peptide sequence (native RYPISRPRKRC vs non-native RPSRPRIRYKC), the role of basic amino acids, and specificity to hyaluronan over other GAGs in contributing to the peptide-hyaluronan interaction were probed through experiments and simulations. Hydrogels containing the native or non-native peptide retained hyaluronan in a dose-dependent manner. Ionic interactions were the dominating mechanism. In diH2O the peptides interacted strongly with HA and chondroitin sulfate, but in phosphate buffered saline the peptides interacted more strongly with HA. For cartilage tissue engineering, chondrocyte-laden PEG hydrogels containing increasing amounts of HA binding peptide and exogenous HA had increased retention and decreased loss of cell-secreted proteoglycans in and from the hydrogel at 28 days. This new matrix-interactive hydrogel platform holds promise for tissue regeneration. PMID:24597474

  6. Interaction of Hyaluronan Binding Peptides with Glycosaminoglycans in Poly(ethylene glycol) Hydrogels

    PubMed Central

    2015-01-01

    This study investigates the incorporation of hyaluronan (HA) binding peptides into poly(ethylene glycol) (PEG) hydrogels as a mechanism to bind and retain hyaluronan for applications in tissue engineering. The specificity of the peptide sequence (native RYPISRPRKRC vs non-native RPSRPRIRYKC), the role of basic amino acids, and specificity to hyaluronan over other GAGs in contributing to the peptide–hyaluronan interaction were probed through experiments and simulations. Hydrogels containing the native or non-native peptide retained hyaluronan in a dose-dependent manner. Ionic interactions were the dominating mechanism. In diH2O the peptides interacted strongly with HA and chondroitin sulfate, but in phosphate buffered saline the peptides interacted more strongly with HA. For cartilage tissue engineering, chondrocyte-laden PEG hydrogels containing increasing amounts of HA binding peptide and exogenous HA had increased retention and decreased loss of cell-secreted proteoglycans in and from the hydrogel at 28 days. This new matrix-interactive hydrogel platform holds promise for tissue regeneration. PMID:24597474

  7. Characterization of esterified hyaluronan-gelatin polymer composites suitable for chondrogenic differentiation of mesenchymal stem cells*

    PubMed Central

    Angele, Peter; Müller, Rainer; Schumann, Detlef; Englert, Carsten; Zellner, Johannes; Johnstone, Brian; Yoo, Jung; Hammer, Joachim; Fierlbeck, Johann; Angele, Martin K.; Nerlich, Michael; Kujat, Richard

    2008-01-01

    Composite scaffolds of homogeneously mixed esterified hyaluronan (HY) and gelatin (G) were manufactured with variable component compositions (HY100%; HY95%/G5%; HY70%/G30%). The goals of this study were to analyze the produced composite scaffolds using physical and chemical methods, e.g., scanning electron microscopy, IR-spectroscopy, water contact angle, protein assay, and tensile testing as well as to assess the effects of adding gelatin to the composite scaffolds on attachment, proliferation and chondrogenic differentiation of human mesenchymal stem cells. Numbers of attached cells were significantly higher on the composite material compared to pure hyaluronan at different time points of two-dimensional or three-dimensional cell culture (p < 0.02). In composite scaffolds, a significantly greater amount of cartilage-specific extracellular matrix components was deposited after 28 days in culture (glycosaminoglycan: p < 0.001; collagen: p < 0.001) as compared with 100% hyaluronan scaffolds. Additionally, gelatin containing composite scaffolds displayed stronger promotion of collagen type II expression than pure hyaluronan scaffolds. The mechanism, by which gelatin influences cell adhesion, was examined. The effect was inhibited by collagenase treatment of the composites or by addition of α5β1-integrin blocking antibodies to the cell suspension. In summary, the results describe the establishment of a class of composite polymer scaffolds, consisting of esterified hyaluronan and gelatin, which are potentially useful for cell-based tissue engineering approaches using mesenchymal stem cells for chondrogenic differentiation. PMID:18985778

  8. Characterization of esterified hyaluronan-gelatin polymer composites suitable for chondrogenic differentiation of mesenchymal stem cells.

    PubMed

    Angele, Peter; Müller, Rainer; Schumann, Detlef; Englert, Carsten; Zellner, Johannes; Johnstone, Brian; Yoo, Jung; Hammer, Joachim; Fierlbeck, Johann; Angele, Martin K; Nerlich, Michael; Kujat, Richard

    2009-11-01

    Composite scaffolds of homogeneously mixed esterified hyaluronan (HY) and gelatin (G) were manufactured with variable component compositions (HY100%; HY95%/G5%; HY70%/G30%). The goals of this study were to analyze the produced composite scaffolds using physical and chemical methods, for example, scanning electron microscopy, IR-spectroscopy, water contact angle, protein assay, and tensile testing as well as to assess the effects of adding gelatin to the composite scaffolds on attachment, proliferation, and chondrogenic differentiation of human mesenchymal stem cells. Numbers of attached cells were significantly higher on the composite material compared to pure hyaluronan at different time points of two-dimensional or three-dimensional cell culture (p< 0.02). In composite scaffolds, a significantly greater amount of cartilage-specific extracellular matrix components was deposited after 28 days in culture (glycosaminoglycan: p < 0.001; collagen: p < 0.001) as compared with 100% hyaluronan scaffolds. Additionally, gelatin-containing composite scaffolds displayed stronger promotion of collagen type II expression than pure hyaluronan scaffolds. The mechanism, based on which gelatin influences cell adhesion, was examined. The effect was inhibited by collagenase treatment of the composites or by addition of alpha5beta1-integrin blocking antibodies to the cell suspension. In summary, the results describe the establishment of a class of composite polymer scaffolds, consisting of esterified hyaluronan and gelatin, which are potentially useful for cell-based tissue engineering approaches using mesenchymal stem cells for chondrogenic differentiation. PMID:18985778

  9. Intra-articular hyaluronan injections in the treatment of osteoarthritis of the knee: a randomised, double blind, placebo controlled multicentre trial. Hyaluronan Multicentre Trial Group.

    PubMed Central

    Lohmander, L S; Dalén, N; Englund, G; Hämäläinen, M; Jensen, E M; Karlsson, K; Odensten, M; Ryd, L; Sernbo, I; Suomalainen, O; Tegnander, A

    1996-01-01

    OBJECTIVE: To assess the effects of intra-articular injections of hyaluronan on symptoms of knee osteoarthritis (OA). METHODS: Two hundred and forty patients with symptomatic, radiological knee OA were randomly assigned to treatment with weekly injections for five weeks with either 25 mg of high molecular weight hyaluronan or vehicle. Results were evaluated at weeks 1, 2, 3, 4, 5, 13, and 20 by visual analogue scales (pain, function, motion, activity), algofunctional index, and global evaluation by patient and investigator. Analysis was by "intention to treat', "per protocol', and area under the curve principles on unstratified patient groups and for patients stratified into four groups of equal size by age and baseline algofunctional index. RESULTS: No serious side effects were reported. At 20 weeks both treatment groups were improved compared with baseline, with no difference between unstratified groups treated with placebo or hyaluronan. Comparison of treatment groups stratified by age and baseline algofunctional index revealed a significant difference in favour of hyaluronan over placebo (pain, activity, algofunctional index, global evaluations by patient and investigator) for patients older than 60 years and with a baseline algofunctional index greater than 10. There was no clinically relevant difference between the two treatments for the other three stratified subgroups of younger age or fewer symptoms. Similar results were obtained by area under the curve, intention to treat, and per protocol analysis. CONCLUSIONS: Patients older than 60 years with knee osteoarthritis and with significant symptoms corresponding to an index of severity of knee disease of 10 or more, comprise the group most likely to benefit from treatment with intra-articular hyaluronan injections. PMID:8774159

  10. 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.