Rat carcinoma cells in long-term, serum-free culture provide a continuing supply of collagenase
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Abstract:
Neoplastic, epithelial cells derived from a spontaneously-arising rat mammary carcinoma have been cultured in a defined medium, in the absence of serum, continuously, for over 2 years. The medium is a mixture of Ham's F12 and Dulbecco's Modified Eagle's media supplemented with insulin, transferrin and bovine serum albumin. The cells have retained their potential to produce tumours and, in culture, a true vertebrate collagenase. This system provides a continuing supply of vertebrate collagenase through the application of recently developed methods.Keywords:
Bovine serum albumin
Transferrin is an important factor that is responsible for the transport of iron in the blood. It has got several polymorphisms, out of which there are three major isotypes and the polymorphism vary with different species. Transferrin is also distributed in various living cells and body fluids. Normal plasma concentration is necessary to be maintained and if it found less, then the susceptibility to various infections are more. Transferrin also has the ability to modulate differentiation and growth of cells. Apo-transferrin, a synthetic form of transferrin is highly recommended for the treatment of various clinical conditions. The present review helps in the overviewing of the structure of transferrin, polymorphism, functions of transferrin polymorphism and its clinical applications.
Transferrin receptor
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Transferrin receptor
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The effect of iron on the exocytosis of transferrin by K562 cells was studied by first allowing the cells to endocytose apotransferrin or diferric transferrin. Subsequent release of the apotransferrin was very rapid with a t 1/2 of 3.01 min, compared with 5.5 min for diferric transferrin. Release of apotransferrin was slowed by the weak base methylamine, t 1/2 8.0 min, but the effect of this agent was substantially greater when iron-transferrin was used, t 1/2 18.65 min, suggesting that methylamine affects both iron removal and receptor recycling. Release of iron-transferrin could be accelerated to a rate comparable with that of apotransferrin by addition of the permeant iron-chelator desferrioxamine. The difference in the rates of release of different forms of the protein could be explained by the re-endocytosis of the iron-rich protein, a process detected by the accelerated release of transferrin when the cells were washed in medium at pH 5.5 containing an iron-chelator or treated with a protease-containing medium to digest transferrin accessible at the cell surface. It appears that in cells incubated under control conditions, re-endocytosis of transferrin, which is incompletely depleted of iron, occurs and that a transferrin molecule may make two passes through the cell before all the iron is removed. This mechanism helps to explain why very little iron-transferrin is released from cells and why the efficiency of the iron uptake process is so high.
Transferrin receptor
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Analysis of human serum transferrin on gel isoelectric focusing resolved this iron-transport protein into two iron-containing components which were identified by the use of radioactively labelled iron and iron-specific stain. These two components were found to be monoferric transferrin (Fe-transferrin) and diferric transferrin (Fe2-transferrin) with isoelectric points of 5.6 and 5.2 respectively. The estimation of the relative proportions of these two components in serum did not correspond to the calculated theoretical ratio based on random binding of iron to the two binding sites of transferrin. However, the analysis of partially resatured apotransferrin gave a ratio corresponding to a random distribution of iron. The significance of these results is discussed.
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Objective To study the method of hepatocyte isolation.Methods Using two defferent collagenases (0.05%,type Ⅰ and Ⅳ) the rat hepatocytes were isolated. The effects of defferent types of collagenases and different duration of liver perfusion(5,10 and 15 min) on viability and outputs of hepatocytes were investigated and the effect of culture on viability in 0.02% collagenase solution studied. Results The best results of isolation were accessed by collagenase typeⅣ.Cell viability of two groups(Ⅳ and Ⅰ) was (89.5±3.5)% and (58.0±14.0)%,output (2.5±0.2)×10 11 cell/L and (0.9±0.5)×10 11 cell/L following 10 min perfusion (P0.01) respectively. The structure and functions were normal by culture and biochemical assay.The study also showed that cell viability was increased by the method of cultue in low concentration of collagenase solution.Conclusion Collagenase type Ⅳ and 10 min liver perfusion were the best conditions for rat hepatcyte isolation.
Liver perfusion
Viability assay
Isolation
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A prospective study was undertaken to evaluate the utility of calculating transferrin from total iron-binding capacity in the nutritional assessment of burned patients. Regression analysis was used to compare total iron-binding capacity with radial immunodiffusion transferrin determinations. The method used for calculating transferrin (0.8 TIBC – 43) is a frequently published conversion formula for deriving transferrin. One hundred twenty-five data sets were obtained from 45 burned patients. Values for derived transferrin ranged from 39 to 235 mg/dl, averaging 121 mg/dl. Actual transferrin averaged 162 mg/dl, ranging from 41 to 320 mg/dl. Forty-eight actual serum transferrin samples were normal (greater than 172 mg/dl) whereas only 17 derived transferrin values were normal. While there is a correlation between total iron-binding capacity and serum transferrin (r = 0.85), to calculate transferrin according to the formula above would have resulted in significant error in the clinical assessment of the patients' nutritional status (p < 0.001). From our studies, the formula for conversion of total iron-binding capacity to transferrin was found to be (0.68 TIBC + 21). These results suggest that the development of a universal conversion factor is not feasible. Modification of the formula may be necessary at each institution if clinically useful evaluations of serum transferrins are to be derived from iron-binding capacity for use in nutritional assessment.
Radial immunodiffusion
Total iron-binding capacity
Ceruloplasmin
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Granulation tissue
Microbial collagenase
Type I collagen
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Transferrin receptor
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The binding and uptake of 59 Fe‐loaded 3 H‐labelled rat transferrin by cultured rat hepatocytes was investigated. At 4°C, there is no evidence for a specific binding of transferrin which could be related to the association of neo‐synthesized transferrin with plasma membrane receptors. At 37°C, iron uptake is much more important than transferrin uptake; it proceeds linearly over the time of incubation, is largely proportional to the extracellular transferrin concentration, and is compatible with uptake by fluid phase endocytosis. The difference observed between iron and transferrin uptake implies the existence of a mechanism allowing the reutilization of transferrin after iron delivery.
Transferrin receptor
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