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Collagenase Digestion (collagenase + digestion)
Selected AbstractsAcetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptideFEBS JOURNAL, Issue 6 2008Adam Frederick To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChET subunit, which should be able to produce all three globular forms of AChE: monomers (G1), dimers (G2), and tetramers (G4), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G1a) and non-amphiphilic tetramers (G4na) are found. Amphiphilic dimers (G2a) and non-amphiphilic tetramers (G4na) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A12 form of the enzyme. Collagenase digestion of the A12 AChE produces a lytic G4 form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates. [source] Collagenase-Assisted Fat Dissociation for Autologous Fat TransferDERMATOLOGIC SURGERY, Issue 10 2008DAVID K. MOSCATELLO PHD BACKGROUND The quality of fat for autologous transfer procedures has been a major focus of research in the past few years. The primary goal of these efforts is to improve the viability and longevity of the graft in human subjects. One possible factor in the permanence of theses transplants is the size of the adipose tissue grafts. OBJECTIVE This study evaluated the effects of collagenase digestion on the viability of human adipose tissue. MATERIALS AND METHODS Samples of fat were obtained from subjects undergoing tumescent liposuction. The tissue was digested in a variety of concentrations of collagenase using optimized methods of processing. The digested fat was also subjected to mock injections through small bore needles. RESULTS Eight subjects completed the study. The viability of the fat using the optimized methods of collagenase digestion was consistently higher than 79%. During the mock injection trials, the viability of fat was improved from approximately 17% to 84% by collagenase digestion. CONCLUSIONS Our results show increased viability of human adipose tissue when digested by collagenase. These techniques can be applied to human autologous lipoaugmentation procedures in an effort to improve longevity of the transplanted tissue. [source] Effects of zotepine and olanzapine on noradrenaline transporter in cultured bovine adrenal medullary cellsHUMAN PSYCHOPHARMACOLOGY: CLINICAL AND EXPERIMENTAL, Issue 7 2005Reiji Yoshimura Abstract Background Previously, it was demonstrated that the inhibitory effects of atypical antipsychotic drugs such as clozapine and risperidone on noradrenaline transporter (NAT) might in part be associated with their clinical profile. The present study examined the effects of zotepine on NAT in the cells and compared them with those of olanzapine. Materials and Methods Adrenal medullary cells were isolated by a method of collagenase digestion of slices of fresh bovine adrenal medulla and the cells were plated at a density of 4,×,106 cells. Cells were incubated with [3H]noradrenaline (NA) in the presence or absence of zotepine or olanzapine. The amount of radioactivity taken into the cells was counted by a liquid scintillation counter. Plasma membranes of bovine adrenal medulla were prepared, and the binding of [3H]desipramine (DMI) was determined by incubating the membrane suspension in binding buffer together with zotepine or olanzapine. Specific binding of [3H] DMI was defined as that binding which was inhibited by nisoxetine. Results Both zotepine (10,1000,ng/ml) and olanzapine (10,1000,ng/ml) decreased [3H]NA uptake in a concentration-dependent manner. The IC50 values of zotepine and olanzapine on [3H]NA uptake were 10,±,4 and 14,±,8,ng/ml, respectively. Eadie-Hofstee analysis of [3H]NA uptake showed that treatment with zotepine and olanzapine decreased the Vmax of uptake without changing the Km. Both zotepine (10,1000,ng/ml) and olanzapine (30,1000,ng/ml) inhibited [3H]DMI binding in a concentration-dependent manner. The IC50 values of zotepine and olanzapine on [3H]DMI binding were 50,±,18, and 120,±,38,ng/ml, respectively. Scatchard plot analysis of [3H]DMI binding showed that zotepine and olanzapine decreased the Bmax of binding without altering the Kd. Conclusions The inhibitory effects of zotepine and olanzapine might be responsible in part for their clinical profile. Copyright © 2005 John Wiley & Sons, Ltd. [source] Direct and indirect manipulation of the MEK-ERK pathway regulates the formation of a pericellular HA-dependent matrix by chick articular surface cells without modifying CD44 expresssionINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004Edward R. Bastow Introduction Recent evidence suggests that hyaluronan (HA) facilitates the mechano-dependent joint cavity-forming process through the elaboration and retention of a HA-rich pericellular matrix in the developing joint interzone (IZ). The presumptive joint IZ phenotype shows a capacity to bind and synthesize HA and also exhibits elevated activated ERK, prior to synovial joint cavity formation (Lamb et al. 2001; Edwards et al. 1994; Dowthwaite et al. 1998). We have found that immobilization, which induces embryonic joint fusion with loss of the joint IZ phenotype, also reduces ERK activity levels in the IZ. As the signalling events regulating the synthesis and binding of HA have yet to be determined, we hypothesize that ERK activation plays a pivotal role in determining the presumptive joint IZ phenotype through HA synthetic and binding capacity. Materials and methods Chick articular surface (AS) cells were harvested from proximal tibiotarsal joints of embryos by collagenase digestion. Pericellular coat formation was assessed using the erythrocyte exclusion assay and cell-coat area ratios determined. ERK activity was modulated by transient transfection of GFP constructs of constitutively active (CA-) or dominant negative (DN-) forms of MEK, the direct upstream regulator of ERK or by treatment with the MEK inhibitor PD98059 (50 µm). ERK activation was monitored by immunochemistry. CD44 expression and ERK activation in PD98059-treated cells were monitored by immunoblotting and medium HA concentrations by ELISA. Results AS cells form large pericellular coats that are lost following hyaluronidase treatment and thus dependent upon HA for their construction. Treatment with PD98059 significantly reduced pericellular coat formation after 6 h. In parallel, we confirmed that PD98059 diminished active ERK expression without modifying overall levels of ERK, suggesting that the elaboration of large HA-pericellular coats is dependent upon MEK's activation of ERK. Western blot analysis of PD98059-treated cells showed that loss of pericellular coats was not, however, associated with any decreased levels of the cell surface HA receptor CD44. Although treatment with PD98059 did not change medium HA concentration after short times of exposure, at times (up to 6 h) during which coat loss was evident, prolonged treatment over 24 h significantly decreased medium HA concentration. Consistent with a role for ERK in pericellular coat formation, transfection with DN-MEK diminished, while CA-MEK increased, both active ERK expression and coat formation efficiency. We also found that, commensurate with this modification in coat forming efficiency, cells expressing DN-MEK exhibited a significant reduction in labelling of free HA on the cell surface. Discussion These studies extend our recent work to indicate that: (i) direct modulation of ERK activation by transfection with its endogenous upstream regulator modifies cell surface-associated HA (ii) PD98059-induced blockade of ERK activation restricts medium HA release and (iii) ERK-mediated changes in pericellular coat elaboration are independent of changes in cellular CD44 expression. These findings suggest an intimate relationship between ERK activation and the formation/retention of HA-rich pericellular matrices in vitro and highlight the role for ERK activation in regulating joint line-related differentiation. [source] Beer But Not Wine, Hard Liquors, or Pure Ethanol Stimulates Amylase Secretion of Rat Pancreatic Acinar Cells In VitroALCOHOLISM, Issue 9 2009Andreas Gerloff Background:, In contrast to pure ethanol, the effect of alcoholic beverages on the exocrine pancreas is greatly unknown. Besides ethanol, alcoholic beverages contain numerous nonalcoholic constituents which might have pathophysiological effects on the pancreas. The aim of the present study was to investigate whether some commonly used alcoholic beverages and pure ethanol influence the main function of rat pancreatic acinar cells, i.e., enzyme output in vitro. Methods:, Rat pancreatic AR4-2J cells were differentiated by dexamethasone treatment for 72 hours and freshly isolated pancreatic acini were prepared from Sprague,Dawley rats using collagenase digestion. After incubation of cells in the absence or presence of 1 to 10% (v/v) beer (containing 4.7% v/v ethanol), 10% (v/v) wine (containing 10.5 to 12.5% v/v ethanol), 10% (v/v) hard liquor (such as whisky, rum, and gin), or of the corresponding ethanol concentrations (4.03 to 80.6 mM) for 60 minutes, protein secretion was measured using amylase activity assay. Results:, Incubation of AR4-2J cells with beer caused a dose-dependent stimulation of basal amylase secretion that was significant at doses of beer above 0.5% (v/v). Stimulation with 10% (v/v) beer induced 92.7 ± 25.2% of maximal amylase release in response to the most effective cholecystokinin (CCK) concentration (100 nM). In contrast, ethanol (up to 80.6 mM) did neither stimulate nor inhibit basal amylase release. Lactate dehydrogenase measurement after treatment of AR4-2J cells with beer for 24 hours indicated that the increase of amylase release was not due to cell membrane damage. Wine and hard liquor had no effect on basal amylase secretion neither diluted to the ethanol concentration of beer nor undiluted. In freshly isolated rat pancreatic acinar cells beer dose-dependently stimulated amylase secretion in a similar manner as in AR4-2J cells. Conclusions:, Our data demonstrate that beer dose-dependently increases amylase output. Since neither ethanol nor the other alcoholic beverages tested caused stimulation of amylase release, our findings indicate that nonalcoholic constituents specific for beer are responsible for this increase. These as yet unknown compounds have to be identified and considered in further studies of ethanol-induced pathological and functional changes of the pancreas. [source] The Phosphodiesterase III Inhibitor Olprinone Decreases Sensitivity of Rat Kupffer Cells to EndotoxinALCOHOLISM, Issue 2004Nobuyuki Enomoto Background: Sensitivity of Kupffer cells to endotoxin [lipopolysaccharide (LPS)] and overproduction of tumor necrosis factor-, (TNF-,) are critical for progression of alcoholic liver injury. Therefore, suppression of TNF-, should prove useful for treatment of alcoholic liver injury. However, a transient increase of intracellular calcium ([Ca2+]i) is required for LPS-induced TNF-, production by the macrophage cell line. The phosphodiesterase III inhibitor olprinone has been shown to suppress [Ca2+]i level in vascular smooth muscle cells. Accordingly, the purpose of this study was to determine whether olprinone could prevent sensitization of Kupffer cells to endotoxin. Methods: Kupffer cells were isolated by collagenase digestion and differential centrifugation. LPS was added to Kupffer cells 24 hr after incubation with or without olprinone (0.1 ,mol/liter). After addition of LPS (10 ,g/ml) to culture media, [Ca2+]i was measured using a fluorescent indicator, fura-2. Results: LPS increased [Ca2+]i of Kupffer cells in control rats from basal levels (28 ± 4 nmol/liter) to 280 ± 14 nmol/liter. This increase was blunted by olprinone (91 ± 8 nmol/liter). Similarly, olprinone diminished the LPS (1 ,g/ml)-induced TNF-, production by Kupffer cells by 30% (2220 ± 116 vs. 1386 ± 199 pg/ml; p < 0.05). Conclusions: These results indicate that olprinone decreases sensitivity of Kupffer cells to endotoxin. [source] Size Distribution of Dispersed Luteal Cells During Oestrous Cycle in Angora GoatsREPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2007H Kalender Contents The present study examines the size distribution of the goat steroidogenic luteal cells throughout the oestrous cycle. Corpora lutea (CL) were collected after laparatomy on days 5, 10 and 16 of the oestrous cycle. Luteal cells were isolated from CL by collagenase digestion. Steriodogenic luteal cells were identified by staining of the cells for 3, -hydroxysteroid dehydrogenase activity, a marker for steroidogenic cells. Luteal cells having steroidogenic capacity covered a wide spectrum of sizes, ranging from 5 to 37.5 ,m in diameter. There was a significant increase in mean cell diameters (p < 0.01) as CL aged. The mean cell diameter on day 5 was 11.55 ± 0.12 ,m, which was significantly increased and reached up to 19.18 ± 0.24 ,m by day 16 of the oestrous cycle. The ratio of large to small luteal cells was 0.06:1.0 on day 5 of the oestrous cycle. This ratio increased to 0.78:1.0 by day 16 of the oestrous cycle. Luteal cell size on days 5, 10 and 16 of the oestrous cycle reached its maximum at 7.5, 10 and 35 ,m in diameter, respectively. Development of CL is associated with an increase in luteal cell size in goats. It is likely that small luteal cells could develop into large luteal cells as CL becomes older during oestrous cycle in goats. [source] ORIGINAL ARTICLE: Placental Fas/Fas Ligand Expression in Early Pregnancy LossesAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 1 2008Emine Seda Guvendag Guven Problem, The aim of this study was to compare the expression levels of Fas and Fas ligand (FasL) in first-trimester placentas obtained from spontaneous abortions in patients with antiphospholipid antibody syndrome (APS) or factor V (FV) Leiden mutation, compared with values in placentas from induced abortions in patients negative for these conditions. Method of study, We studied explants from 6- to 10-week-old placentas that had been prepared by collagenase digestion from 10 spontaneous abortions from APS-positive patients, nine spontaneous abortions in patients positive for FV Leiden mutation, and 10 induced abortions. All tissues were analyzed by flow cytometry for expression of Fas and FasL. Results, Flow cytometric analysis showed that placental FasL expression was significantly lower in abnormal pregnancies than in normal ones. However, no such difference was observed for Fas expression. Conclusion, FasL on placental cells may be involved in the maintenance of immune privilege, thereby ensuring the safety and growth of placental tissues. Dysregulation of apoptotic mechanisms may play a critical role in spontaneous abortions. [source] Modified Two-Layer Preservation Method (M-Kyoto/PFC) Improves Islet Yields in Islet IsolationAMERICAN JOURNAL OF TRANSPLANTATION, Issue 3 2006H. Noguchi Islet allotransplantation can achieve insulin independence in patients with type I diabetes. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and UW solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. However, UW solution has several disadvantages, including the inhibition of Liberase activity. In this study, we investigated the features of a new solution, designated M-Kyoto solution. M-Kyoto solution contains trehalose and ulinastatin as distinct components. Trehalose has a cytoprotective effect against stress, and ulinastatin inhibits trypsin. In porcine islet isolation, islet yield was significantly higher in the M-Kyoto/PFC group compared with the UW/PFC group. There was no significant difference in ATP content in the pancreas between the two groups, suggesting that different islet yields are not due to their differences as energy sources. Compared with UW solution, M-Kyoto solution significantly inhibited trypsin activity in the digestion step; moreover, M-Kyoto solution inhibited collagenase digestion less than UW solution. In conclusion, the advantages of M-Kyoto solution are trypsin inhibition and less collagenase inhibition. Based on these data, we now use M-Kyoto solution for clinical islet transplantation from nonheart-beating donor pancreata. [source] Heterogeneity of response of rheumatoid synovium cell subsets to interleukin-18 in relation to differential interleukin-18 receptor expressionARTHRITIS & RHEUMATISM, Issue 3 2003Masanori Kawashima Objective To examine the differential response of rheumatoid arthritis (RA) synovium cell subsets to interleukin-18 (IL-18), the effect of IL-18 on Th1-cytokine production, and the regulation of IL-18 by IL-18 binding protein (IL-18BP). Methods RA fibroblast-like synoviocytes were stimulated with IL-1,, IL-12, and IL-18, and levels of IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). Expression of IL-18 receptor , and , chains (IL-18R, and IL-18R,, respectively), interferon-, (IFN,), and IL-17 messenger RNA (mRNA) by peripheral blood mononuclear cells, by total RA synovium cells containing T cells obtained after collagenase digestion, and by RA fibroblast-like synoviocytes was determined by reverse transcription,polymerase chain reaction. Levels of IFN, were measured by ELISA. Results IL-1, and, less effectively, IL-12 could induce RA fibroblast-like synoviocytes to produce IL-6, but IL-18 failed to have an effect. Although IL-18R, mRNA was constitutively expressed by RA fibroblast-like synoviocytes, IL-18R, could not be detected, either with or without stimulation with IL-1 or IL-12. Total RA synovium cells containing T cells showed a strong expression of both IL-18R, and IL-18R, mRNA, and only IL-18R, was up-regulated by IL-12. The combination of IL-12 and IL-18 synergistically up-regulated IFN, mRNA expression by total RA synovium cells containing T cells, but down-regulated that of IL-17. IL-12,induced IFN, production by total RA synovium cells containing T cells was increased by additional IL-18 and decreased by IL-18BP. Conclusion These results indicate that IL-18 plays an important role in RA inflammation and joint destruction via T cells and macrophages, but it does not have a direct effect on fibroblast-like synoviocytes. IL-18BP may be a tool for RA therapy because of its ability to neutralize endogenous IL-18. [source] Entrapment of dispersed pancreatic islet cells in CultiSpher-S macroporous gelatin microcarriers: Preparation, in vitro characterization, and microencapsulationBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2001S. Del Guerra Abstract Immunoprotection of pancreatic islets for successful allo- or xenotransplantation without chronic immunosuppression is an attractive, but still elusive, approach for curing type 1 diabetes. It was recently shown that, even in the absence of fibrotic overgrowth, other factors, mainly insufficient nutrition to the core of the islets, represent a major barrier for long-term survival of intraperitoneal microencapsulated islet grafts. The use of dispersed cells might contribute to solve this problem due to the conceivably easier nutritional support to the cells. In the present study, purified bovine islets, prepared by collagenase digestion and density gradient purification, and dispersed bovine islet cells, obtained by trypsin and DNAsi (viability > 90%), were entrapped into either 2% (w/v) sodium alginate (commonly used for encapsulation purposes) or (dispersed islet cells only) macroporous gelatin microcarriers (CulthiSpher-S, commonly used for the production of biologicals by animal cells). Insulin release studies in response to glucose were performed within 1 week and after 1 month from preparation of the varying systems and showed no capability of dispersed bovine islet cells within sodium alginate microcapsules to sense glucose concentration changes. On the contrary, bovine islet cells entrapped in CulthiSpher-S microcarriers showed maintained capacity of increasing insulin secretion upon enhanced glucose concentration challenge. In this case, insulin release was approximately 60% of that from intact bovine islets within sodium alginate microcapsules. MTT and hematoxylineosin staining of islet cell-containing microcarriers showed the presence of viable and metabolically active cells throughout the study period. This encouraging functional data prompted us to test whether the microcarriers could be immunoisolated for potential use in transplantation. The microcarriers were embedded within 3% sodium alginate, which was then covered with a poly-L-lysine layer and a final outer alginate layer. Maintained insulin secretion function of this system was observed, which raises the possibility of using microencapsulated CulthiSpher-S microcarriers, containing dispersed pancreatic islet cells, in experimental transplantation studies. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 741,744, 2001. [source] | ||||||||||