Urinary glycosaminoglycan excretion in rheumatic diseases.
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We used Alcian Blue (AB) and dimethylmethylene blue (DMB) methods to measure glycosaminoglycan (GAG) excretion in the first morning urine specimens of patients with osteoarthritis (OA), ankylosing spondylitis (AS), and rheumatoid arthritis (RA) in different stages of disease. By the AB method, urinary GAG excretion in patients with RA was not different from healthy control subjects. However, the DMB method showed significant differences (in milligrams of GAG per gram of creatinine) for OA (median 25.4, range 14.3-44.0, P less than 0.01, n = 23) and RA patients (median 33.0; range 10.0-147.6; P less than 0.001, n = 63) in comparison with unaffected individuals (median 20.2; range 8.9-41.4, n = 38). We noted a significant difference in urinary GAG excretion between RA and OA patients (P less than 0.01) and between RA and AS (P less than 0.01) patients. The DMB method was further investigated by clinical decision analysis. The DMB method is simple and rapid and may be useful in diagnosing RA by distinguishing between RA and OA or AS. (+info)
Radiochemical assay to measure the biofilm produced by coagulase-negative staphylococci on solid surfaces and its use to quantitate the effects of various antibacterial compounds on the formation of the biofilm.
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A firmly adherent mass of slime plus organisms (biofilm) accumulates on the sides of culture tubes when some strains of coagulase-negative staphylococci are grown in a chemically-defined medium containing [14C]glucose. This mass was washed (to remove labelled medium) and then counted after adding scintillation fluid. Organisms from the liquid culture were also washed and counted to check that [14C]glucose had been utilised to label the bacteria. Nine strains were examined in this way, and the results were compared with those obtained with four older techniques for recognising slime production or adherent bacteria. The new method is quick, and has advantages of reproducibility and good discrimination between strains; there was a 15-fold difference in counts in the biofilm between slime-producing and non-producing strains respectively. With the new radiolabel assay, the effects of several antibacterial compounds on the build-up of the biofilm were investigated with four slime-producing strains. Tunicamycin, chloramphenicol and 5-fluorouracil, at levels below their minimum growth-inhibitory concentrations, each greatly diminished biofilm formation; several other drugs had less effect. (+info)
Versican/PG-M regulates chondrogenesis as an extracellular matrix molecule crucial for mesenchymal condensation.
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Mesenchymal cell condensation is an essential step for cartilage development. Versican/PG-M, a large chondroitin sulfate proteoglycan, is one of the major molecules expressed in the extracellular matrix during condensation. However, its role, especially as an environment for cells being condensed, has not been elucidated. Here we showed several lines of evidence for essential roles of versican/PG-M in chondrogenic condensation using a new chondrocytic cell line, N1511. Chondrogenic stimuli (treatment with parathyroid hormone, dexamethasone, 10% serum) induced a marked increase in the transcription and protein synthesis of versican/PG-M. Stable antisense clones for versican/PG-M, depending on suppression of the expression of versican/PG-M, showed different capacities for chondrogenesis, as indicated by the expression and deposition of aggrecan, a major chondrocytic cell product. The cells in the early stages of the culture only expressed V0 and V1 forms, having more chondroitin sulfate chains among the four variants of versican/PG-M, and treatment of those cells with chondroitinase ABC suppressed subsequent chondrogenesis. Furthermore, treatment with beta-xyloside, an artificial chain initiator of chondroitin sulfate synthesis to consequently inhibit the synthesis on the core proteins, suppressed chondrogenesis. In addition, forced expression of the variant V3, which has no chondroitin sulfate chain, disrupted the deposition and organization of native versican/PG-M (V0/V1) and other extracellular matrix molecules known to be expressed during the mesenchymal condensation and resulted in the inhibition of subsequent chondrogenesis. These results suggest that versican/PG-M is involved in positively regulating the formation of the mesenchymal matrix and the onset of chondrocyte differentiation through the attached chondroitin sulfate chains. (+info)
The first case of Barrett's oesophagus with chronic inflammation having predominantly (> 50%) Mott cells, i.e. plasma cells with stored immunoglobulins, known as Russell bodies, is reported. Biopsies from two oesophagoscopies revealed similar changes, suggesting that the predominance of Mott cells is not a fortuitous event but a more long-lasting microscopic process. Periodic acid-Schiff (PAS) stain ruled out Candida albicans and immunostains, plasma cell neoplasia. Mott cells were not present in biopsies from the gastric mucosa or the urinary bladder, suggesting that this phenomenon was not widespread but localized to the Barrett's mucosa. The retention of immunoglobulins (Russell bodies) suggests that the mechanism of protein transport in those plasma cells is incompetent, and that the proteins are neither degraded nor secreted, but remain stored in dilated cisternae. Increased awareness of the existence of this subgroup of Barrett's oesophagitis may result in similar cases being reported in the future. (+info)
MNN5 encodes an iron-regulated alpha-1,2-mannosyltransferase important for protein glycosylation, cell wall integrity, morphogenesis, and virulence in Candida albicans.
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The cell walls of microbial pathogens mediate physical interactions with host cells and hence play a key role in infection. Mannosyltransferases have been shown to determine the cell wall properties and virulence of the pathogenic fungus Candida albicans. We previously identified a C. albicans alpha-1,2-mannosyltransferase, Mnn5, for its novel ability to enhance iron usage in Saccharomyces cerevisiae. Here we have studied the enzymatic properties of purified Mnn5 and characterized its function in its natural host. Mnn5 catalyzes the transfer of mannose to both alpha-1,2- and alpha-1,6-mannobiose, and this activity requires Mn2+ as a cofactor and is regulated by the Fe2+ concentration. An mnn5Delta mutant showed a lowered ability to extend O-linked, and possibly also N-linked, mannans, hypersensitivity to cell wall-damaging agents, and a reduction of cell wall mannosylphosphate content, phenotypes typical of many fungal mannosyltransferase mutants. The mnn5Delta mutant also exhibited some unique defects, such as impaired hyphal growth on solid media and attenuated virulence in mice. An unanticipated phenotype was the mnn5Delta mutant's resistance to killing by the iron-chelating protein lactoferrin, rendering it the first protein found that mediates lactoferrin killing of C. albicans. In summary, MNN5 deletion impairs a wide range of cellular events, most likely due to its broad substrate specificity. Of particular interest was the observed role of iron in regulating the enzymatic activity, suggesting an underlying relationship between Mnn5 activity and cellular iron homeostasis. (+info)
Ultrastructural localisation of anionic sites at the dermo-epidermal junction in normal human skin.
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Glycosaminoglycans have been demonstrated throughout the cutaneous BMZ at the ultrastructural level. Colloidal iron and cationised ferritin proved of limited value, whilst staining with Alcian blue and application of the critical electrolyte concentration principle has provided evidence for the presence of sulphated GAGs at the lamina lucida and lamina reticularis. Digestions with chondroitin ABC lyase and heparin lyase have confirmed the existence of chondroitin and/or dermatan sulphates and heparan sulphates, although the results obtained with hyaluronate lyase have indicated that hyaluronates are also present. (+info)
Impaired posterior frontal sutural fusion in the biglycan/decorin double deficient mice.
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Biglycan (Bgn) and decorin (Dcn) are highly expressed in numerous tissues in the craniofacial complex. However, their expression and function in the cranial sutures are unknown. In order to study this, we first examined the expression of biglycan and decorin in the posterior frontal suture (PFS), which predictably fuses between 21 and 45 days post-natal and in the non-fusing sagittal (S) suture from wild-type (Wt) mice. Our data showed that Bgn and Dcn were expressed in both cranial sutures. We then characterized the cranial suture phenotype in Bgn deficient, Dcn deficient, Bgn/Dcn double deficient, and Wt mice. At embryonic day 18.5, alizarin red/alcian blue staining showed that the Bgn/Dcn double deficient mice had hypomineralization of the frontal and parietal craniofacial bones. Histological analysis of adult mice (45-60 days post-natal) showed that the Bgn or Dcn deficient mice had no cranial suture abnormalities and immunohistochemistry staining showed increased production of Dcn in the PFS from Bgn deficient mice. To test possible compensation of Dcn in the Bgn deficient sutures, we examined the Bgn/Dcn double deficient mice and found that they had impaired fusion of the PFS. Semi-quantitative RT-PCR analysis of RNA from 35 day-old mice revealed increased expression of Bmp-4 and Dlx-5 in the PFS compared to their non-fusing S suture in Wt tissues and decreased expression of Dlx-5 in both PF and S sutures in the Bgn/Dcn double deficient mice compared to the Wt mice. Failure of PFS fusion and hypomineralization of the calvaria in the Bgn/Dcn double deficient mice demonstrates that these extracellular matrix proteoglycans could have a role in controlling the formation and growth of the cranial vault. (+info)
Identification of superficial zone articular chondrocyte stem/progenitor cells.
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Identification of progenitor/stem cell populations that differentiate specifically towards superficial zone articular chondrocytes is an unmet challenge for cartilage tissue engineering. Using fluorescence activated cell sorting (FACS) analysis we found a characteristic pattern of "side population" (SP) stem cells identified by the Hoechst 33342 dye. We established micromass cultures from this population of cells and tested their chondrogeneic potential. Control (untreated) cultures were minimally stained for Alcian blue - a marker of chondrogenesis. However, with BMP-7 treatment, Alcian blue staining was increased. Superficial zone protein - a specific marker for articular cartilage superficial zone chondrocytes - increased with BMP-7 and/or TGF-beta1 treatment in SP micromass cultures. Our results demonstrate the presence of stem/progenitor cells in the SP fraction isolated from the surface zone of bovine cartilage and have the ability to specifically differentiate towards the superficial zone articular chondrocyte. (+info)