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 47,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Excessive production and deposition of extracellular matrix proteins are characteristic features of diabetic nephropathy. This study tests the hypothesis that cells from diabetic patients who develop nephropathy have a disturbance in collagen metabolism compared with cells from diabetic patients without complications. Kinetics of overall collagen metabolism and total protein synthesis were examined in serially passaged, subconfluent, quiescent skin fibroblasts cultured in either normal (5 mM) or high (25 mM) glucose concentrations from 14 insulin-dependent diabetic (IDDM) patients with nephropathy; 14 IDDM patients without nephropathy matched for age, diabetes duration, and body mass index; and 14 healthy subjects. Fibroblasts were incubated in the presence of 2 microCi/ml [3H]proline, and after labeling the incorporation of [3H]proline into total protein, collagen (collagenase-sensitive material), and noncollagen proteins (collagenase-resistant material) was determined at different time points. Collagen degradation was determined in pulse-chase experiments by following the residual collagen-bound radioactivity after incubation for 8 h with 10 microCi/ml [3H]proline. In high glucose concentrations (25 mM), overall collagen synthesis (measured as [3H]proline incorporation into extracellular and intracellular collagenase-sensitive material) was significantly greater in the patients with nephropathy (mean +/- SEM after a 24-h labeling period: 7189 +/- 671 dpm/10(6) cells) than in the patients without (4341 +/- 267 dpm/10(6) cells; P < 0.01) or healthy control subjects (3836 +/- 234 dpm/10(6) cells; P < 0.01). No significant differences were observed in noncollagen protein production or in collagen degradation rates among the three groups of subjects. In the presence of normal glucose concentrations (5 mM), collagen synthesis was lower in all groups studied, but the differences between IDDM patients with nephropathy and those without remained unaltered. These results suggest that long-term cultured fibroblasts derived from diabetic patients with nephropathy exhibit an abnormality in collagen metabolism. Cells from long-standing diabetic patients without nephropathy have normal collagen metabolism. The increased collagen synthesis is likely to be intrinsic to those diabetic patients susceptible to nephropathy and may play an important role in the sclerotic processes that occur in the kidneys, arteries, and heart.
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PMID:Enhanced collagen synthesis in cultured skin fibroblasts from insulin-dependent diabetic patients with nephropathy. 921 63

In this study we determined the efficiency of magnetization transfer magnetic resonance imaging (MT-MRI) to differentiate native and enzymatically degraded cartilage, using bovine sesamoid bones from the metacarpophalangeal joint as a model system. Gradual proteoglycan (PG) depletion was achieved by increasing incubation periods with testicular hyaluronidase. For native cartilage a Ms/Mo ratio of 0.303 +/- 0.09 (mean +/- SEM) was measured. Biochemically determined PG diminution up to 50% correlated strongly (r = 0.953) with changes in the Ms/Mo ratio. Further PG loss is not reflected in an equally drastic Ms/Mo increase, whereas subsequent treatment of PG-depleted cartilage samples with collagenase led to an additional rise in the Ms/Mo ratio. Proteoglycan depletion and the beginning destruction of the collagen structure were also assessed histochemically. Our study confirms that collagen contributes to the baseline MT effect observed in articular cartilage. However, the changes in the MT ratio in gradually PG-depleted cartilage with a largely intact collagen network indicate that PG contributes to the MT effect as well. Therefore MT-MRI might become a sensitive technique for the monitoring of subtle degradational changes in articular cartilage, the still inaccessible process in osteoarthritis.
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PMID:Can magnetization transfer magnetic resonance imaging follow proteoglycan depletion in articular cartilage? 921 83

The development of more effective means to separate pancreatic islets from the unwanted exocrine tissue would greatly advance the field of clinical islet allotransplantation in the treatment of insulin-dependent diabetes mellitus. Recent experiments with hamster islets have demonstrated a selective destruction of dissociated single exocrine cells when exposed to hypotonic conditions. It was the aim of this study to extend these observations to the canine model with collagenase dissociated pancreatic tissue and to evaluate the treatment's effect on islet function. Pancreases from five mongrel dogs were digested using an automated protocol of intraductal delivery of collagenase, and gentle dissociation. Duplicate samples of pancreatic digest were removed for insulin and amylase determination prior to and immediately following exposure to 50 mOsm/kg salt solution for a period of 30, 60, or 300 s before returning the digest to isoosmotic conditions. The remaining digest was cultured for a period of 48 h at 37 degrees C before the tissue was recombined, washed, and a third sample removed for insulin and amylase. In vitro viability was then assessed using a static incubation assay with insulin content measured using a double-antibody radioimmunoassay, and amylase was determined using a colorimetric assay system. No difference in the insulin or amylase levels between the experimental groups was observed immediately following the hypotonic exposure; however, a significant decrease in the amylase content was observed following the 48-h culture period in digest that had been hypoosmotically exposed for 60 or 300 s compared with the pretreatment group (2.83 +/- 0.41 IU amylase/mg pancreas vs. 1.29 +/- 0.21 and 0.83 +/- 0.12, mean +/- SEM, p < 0.05). Insulin content was also significantly reduced in the 300-s exposure group compared with nontreated controls (3.2 +/- 0.6 mU insulin/mg pancreas vs. 2.0 +/- 0.2). The insulin/ amylase ratio (I/A), a measure of islet and exocrine content, was 1.1 +/- 0.13 following pancreas dissociation and 1.34 +/- 0.21 for control tissue cultured for 48 h. The I/A ratio increased following hypoosmotic exposure to 1.50 +/- 0.31 for tissue exposed for 30 s, 1.77 +/- 0.19 for 60-s exposure, and 2.54 +/- 0.13 for tissue exposed for 300 s (p < 0.05, vs. pretreatment group). In vitro insulin secretion was equivalent with the exception of the tissue exposed for 300 s, which had an increased basal level of insulin resulting in a significantly decreased stimulation index (3.8 +/- 0.5 vs. 8.1 +/- 1.2 for the purified islet control group, p < 0.05). These results suggest that a brief hypotonic exposure to pancreatic digest can alter the insulin/amylase ratio; however, there is a functional impairment on subsequent islet function after a period of in vitro tissue culture.
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PMID:Hypoosmotic exposure of canine pancreatic digest as a means to purify islet tissue. 925 16

In order to evaluate the effect of prolonged muscle inactivity on sarcolemmal lactate/H+ transport in humans, the lactate/H+ transport capacity was determined in the thigh muscle of spinal-cord-injured (SCI) individuals. The lactate transport rate was measured in sarcolemmal giant vesicles produced by collagenase treatment of muscle biopsies obtained from the vastus lateralis muscle. Six SCI subjects with total loss of motor and sensory functions of their lower limbs participated in the study. The duration of the injury ranged from 2 to 15 years. The total lactate transport rate in the muscle of SCI patients was 46.5 +/- 2.6 pmol.cm-2.s-1 (mean +/- SEM), which corresponds to a 17% lower (P < 0.05) transport rate than that found in healthy, untrained subjects. The estimated carrier-mediated lactate/H+ transport capacity was approximately 26% lower in the SCI patients than in healthy, untrained subjects. The observed muscle lactate/H+ transport capacity of SCI individuals is in accordance with a positive correlation between the capacity of the lactate/H+ transporters and the percentage occurrence of slow-twitch fibres in a muscle, although there seems to be a wide range of transport capacities within each fibre type. The present results show that the sarcolemmal lactate/H+ transport capacity is lower in SCI individuals than in normally physically active subjects, which indicates that prolonged muscle inactivity reduces the lactate/H+ transport capacity of human muscle.
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PMID:Lactate/H+ transport in skeletal muscle from spinal-cord-injured patients. 956 14

Density gradient separation of islets from exocrine tissue is usually performed with Ficoll. However, this reagent adds significantly to the cost of the isolation. The aim of this study was to evaluate the performance of Dextran as a potential low-cost substitute for Ficoll and to evaluate the effects of cold storage of the pancreatic digest prior to purification. Pancreases were procured from mongrel dogs, loaded with collagenase and mechanically dissociated. Washed pancreatic digest was collected and divided into two fractions that were purified using discontinuous gradients on the Cobe 2991 processor using identically prepared EuroFicoll (EF) or EuroDextran (ED) gradients. Alternate groups were suspended in EC and stored on ice, while the other fraction were resuspended in the 1.108-g/mL gradient layer (either EF or ED) and loaded into the COBE. This tissue layer was overlaid with layers of densities 1.096 and 1.037 g/mL and a HBSS cap, and centrifuged for 5 min at 800 x g. Purified islets were collected from the interface between the 1.037 and 1.096 layers and islet recovery, purity, and function were assessed. From a series of eight isolations, 72.9 +/- 8.2% (mean +/- SEM) of the islets were recovered from the EF purified gradients compared with 62.6 +/- 8.3% from ED gradients (p = NS, paired t-test). Gradients of ED that were run following hypothermic storage of the digest in cold EC solution (stored ED) had reduced islet recovery when compared with islet recovery from gradients prepared in EF(stored EF) (51.6 +/- 9.6% for ED stored vs. 72.9 +/- 11.9 for EF stored, p < 0.05). Islet recovery from EF gradients was equivalent between the stored and nonstored groups. The purity of preparations from the stored ED gradients was also reduced (71.3 +/- 4.3%) when compared with islets that were immediately purified after dissociation (82.5 +/- 4.8%, p < 0.05). Static glucose stimulation assay showed equivalence between the islets from ED and EF gradients. The stimulation index (SI) was 9.3 +/- 0.9 for EF islets compared with 7.9 +/- 1.4 for ED islets for digest purified immediately. However, if the digest was hypothermically stored in EC solution, a decrease in functional viability was observed in both the EF and the ED groups (7.7 +/- 1.4 and 5.9 +/- 0.8, respectively). Out of five alloxan-induced diabetic nude mice transplanted under the kidney capsule with 2000 islets isolated from the nonstored groups, three remained euglycemic >50 days posttransplant with either EF or ED islets. These experiments demonstrate effective recovery of equivalent numbers of canine islets using discontinuous gradients of ED or EF immediately following enzymatic digestion. However, following storage of the digest in cold EC solution results in a reduction in both islet recovery and function when gradients of ED are utilized.
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PMID:A prospective comparison of discontinuous EuroFicoll and EuroDextran gradients for islet purification. 978 68

Recent studies suggest that mast cell-derived neutral proteases can activate matrix-degrading metalloproteinases (MMPs). We have investigated the role of the mast cell proteases tryptase and chymase in the activation of MMPs in human carotid endarterectomy specimens (atherosclerotic, n=32) and postmortem carotid arteries (control, n=17). In vitro degranulation of mast cells in atherosclerotic carotid arteries by compound 48/80 caused a significant increase in MMP activity. Addition of the nonselective tryptase inhibitor antipain, the specific trypsinlike protease inhibitor 4-amidinophenylmethanesulfonyl fluoride, and the chymase inhibitor chymostatin reduced this increase in MMP activity by 30+/-6%, 23+/-6%, and 9+/-2%, respectively. Immunocytochemistry identified significantly higher numbers of tryptase-containing cells (mast cells) and cells expressing MMP-1 and MMP-3 in the "shoulder" regions of atherosclerotic artery lesions compared with the tunica media of control arteries. Dual immunocytochemistry showed collocation of MMP-1 and MMP-3 with mast cells in the shoulder regions. Degranulation was observed in 78+/-5% (mean+/-SEM) of mast cells in this area, whereas nonactivated mast cells were observed in all other areas. In situ zymography revealed caseinolytic and gelatinolytic activity in these areas. In conclusion, in vitro mast cell degranulation, which releases mast cell proteases, in carotid arteries increases MMP activity. Furthermore, elevated MMP-1 and MMP-3 expression is collocated with increased numbers of degranulated mast cells and with greater MMP activity in the shoulder regions of atherosclerotic plaques. Activation of MMPs by mast cell-derived proteases may be an important mechanism in atherosclerotic plaque destabilization.
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PMID:Activation of matrix-degrading metalloproteinases by mast cell proteases in atherosclerotic plaques. 981 8

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4 degrees C) Liberase-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35 degrees C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28-32 degrees C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 +/- 2.6 g vs. 4.6 +/- 2.1 g, mean +/- SEM, p < 0.05). Postdigestion recovery of islets was 471 +/- 83 x 10(3) IE in the perfusion group compared with 391 +/- 57 x 10(3) IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 +/- 45 vs. 251 +/- 28 x 10(3) IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 +/- 0.6 for the perfusion group and 4.2 +/- 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.
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PMID:Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion. 1044 41

The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA) as well as mercuric chloride (HgCl(2)) and methylmercury chloride (MeHgCl) on gluconeogenesis was investigated in isolated rat kidney tubules. From starved rats kidney tubules were prepared and isolated by digestion with collagenase. Every 10 min up to 60 min 1-ml samples were drawn from the cell suspension for quantitating the glucose content. Glucose formation in controls was 3.3 +/- 0.2 nmol/mg. per min (mean +/- SEM, n=21). Relative rates of glucose formation were obtained by expressing individual rates as a percentage of the corresponding control. X-Y concentration curves (effective concentration, EC) of the substances were calculated by fitting a four-parametric sigmoid function to the relative rates of glucose formation at various test concentrations. At the end of the incubation period cell viability was assessed by trypan blue exclusion. Cell viability decreased within the 60 min interval from 90 to approx. 80% (controls), <25 (HEMA), <20 (TEGDMA), <10 (MeHgCl), and <10% (HgCl(2)). Values of 50% effective concentration (EC(50)) were calculated from fitted curves. EC(50) values were (mmol; mean +/- SEM; n=4): HEMA, 17.7 +/- 2.9; TEGDMA, 1.8 +/- 0.2; MeHgCl, 0.018 +/- 0.0005; and HgCl(2), 0. 0016 +/- 0.0005. The toxic effect of HgCl(2) was approximately 1000 or 10 000 higher than that of the dental composite components TEGDMA or HEMA, respectively.
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PMID:Effect of dental materials on gluconeogenesis in rat kidney tubules. 1055 Apr 80

The ability to cryopreserve pancreatic islets has allowed the development of low-temperature banks that permit pooling of islets from multiple donors and allows time for sterility and viability testing. However, previous studies have shown that during cryopreservation and thawing there is a loss of islet mass and a reduction in islet function. The aim of this study was to measure and compare insulin secretion from cultured nonfrozen and frozen-thawed canine islets and beta-cells. Canine islets were isolated from mongrel dogs using intraductal collagenase distention, mechanical dissociation, and EuroFicoll purification. One group of purified islets was cultured overnight before dissociation into single cells and subsequent analysis. Remaining islets were cultured overnight (22 degrees C) and then cryopreserved in 2 M dimethyl sulfoxide (DMSO) solution using a slow stepwise addition protocol with slow cooling to -40 degrees C before storage in liquid nitrogen (-196 degrees C). Frozen islets were rapidly thawed (200 degrees C/min) and the DMSO removed using a sucrose dilution. From a series of seven consecutive canine islet isolations, islet recovery following postcryopreservation tissue culture was 81.5 +/- 4.8% compared to precryopreservation counts. In vitro islet function was equivalent between cultured nonfrozen and frozen-thawed islets with a calculated stimulation index of 10.4 +/- 1.5 (mean +/- SEM) for the frozen-thawed islets, compared with 12.4 +/- 1.2 for the cultured nonfrozen controls (p = ns, n = 7 paired experiments). Amperometric detection of secretion from single beta-cells in vitro has the sensitivity and temporal resolution to detect single exocytotic events and allows secretion to be monitored from single beta-cells in real time. Secretion from single beta-cells elicited by chemical stimulation was detected using a carbon fiber microelectrode. The frequency of exocytosis events was equivalent between the cultured nonfrozen and frozen-thawed beta-cells with an average of 7.0 +/- 1.32 events per stimulation for the cultured nonfrozen group compared with 6.0 +/- 1.45 events from the frozen then thawed preparations (minimum of 10 cells per run per paired experiment, p = ns) following stimulation with tolbutamide. The average amount of insulin released per individual exocytosis event was equivalent for the cultured nonfrozen and frozen-thawed islets. In addition, beta-cells responded to both tolbutamide and muscarinic stimulation following cryopreservation. It was determined that beta-cells recovered following cryopreservation are capable of secreting insulin at levels and frequencies comparable to those of cultured nonfrozen islet preparations.
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PMID:Secretion from islets and single islet cells following cryopreservation. 1070 97

Antiinflammatory mechanisms are important in ovulation and may be regulated by cortisol (F). We previously showed that after administration of human (h)CG for ovulation induction, luteinized granulosa cells (LGC) abundantly express 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) messenger RNA but not 11betaHSD type 2 (11betaHSD2) messenger RNA. 11ssHSD1 is responsible for the reversible formation of antiinflammatory F from its inactive precursor cortisone (E), whereas 11betaHSD2 unidirectionally converts F to E through 11-oxidation. This pattern of gene expression predicts that LGC from periovulatory follicles would show increased activation of E to F, compared with granulosa cells from immature follicles (IGC), and that follicular fluid concentrations of E and F would alter accordingly. To test this hypothesis, we isolated IGC, thecal cells (TC), and follicular fluid, from ovaries of cyclic women, removed during surgery for benign gynecological disease. LGC and follicular fluid were aspirated from periovulatory follicles, 35 h after hCG injection, in patients undergoing in vitro fertilization treatment. In an 11betaHSD assay based on interconversion of tritiated E and F by cell suspensions in vitro, IGC (% conversion, 0.6 +/- 0.4, mean +/- SEM) and collagenase-dispersed TC (0.2 +/- 0.1%) were unable to convert E to F, whereas LGC (36.3 +/- 3.7%) were highly efficient at this reaction. Immature granulosa cells, LGC, and (to a lesser extent) TC were all able to convert F to E. Correspondingly, follicular fluid concentrations of total F and F:E ratios were significantly higher in periovulatory follicles, compared with immature follicles. Culturing IGC for 48 h in the presence of hFSH resulted in increased 11betaHSD1 reductase activity, paralleling stimulation of estrogen (aromatase activity) and progesterone biosynthesis. Similar treatment with hLH did not influence 11betaHSD1 reductase activity, except in a patient with more mature IGC, which also showed a significant increase in E-to-F conversion, as well as progesterone synthesis in response to hLH. These data confirm that 11betaHSD activity in the human ovary is developmentally regulated and gonadotropin responsive, favoring metabolism of F to E in immature follicles and E to F in periovulatory follicles. Increased formation of F by LGC in periovulatory follicles is consistent with an antiinflammatory function for this glucocorticoid at ovulation.
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PMID:Development-related increase in cortisol biosynthesis by human granulosa cells. 1113 35


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