Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes.
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During feeding by infected mosquitoes, malaria sporozoites are injected into the host's bloodstream and enter hepatocytes within minutes. The remarkable target cell specificity of this parasite may be explained by the presence of receptors for the region II-plus of the circumsporozoite protein (CS) on the basolateral domain of the plasma membrane of hepatocytes. We have now identified these receptors as heparan sulfate proteoglycans (HSPG). The binding of CS to the receptors is abolished by heparitinase treatment, indicating that the recognition of region II-plus is via the glycosaminoglycan chains. We have purified and partially characterized the CS-binding HSPGs from HepG2 cells. They have a molecular weight of 400,000-700,000, are tightly associated with the plasma membrane, and are released from the cell surface by very mild trypsinization, a property which the CS receptors share with the syndecan family of proteoglycans.Keywords:
Trypsinization
Cell surface receptor
Internalization
Circumsporozoite protein
The aim of this study was to investigate the optimal conditions for trypsinization in the culture of cerebral cortical neurons. Methods: The cerebral cortical region was dissected out from newborn rats,and then was treated with different concentration of trypsin at different time,in which the effects of trypsinization on cell viability were compared by cell morphology and cell count.Results: The cortical neurons with higher production and viability were obtained under the trypsinization condition of a final concentration of 0.25% tripsin at 37℃ for 5 min.The results show that 0.25% tripsin at 37℃ for 5 min made more monolayer cells and improved the viability of neurons.
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Viability assay
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Versican
Biglycan
Syndecan 1
Dermatan sulfate
Chondroitin sulfate proteoglycan
Heparanase
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We show here that the endothelial cell-line ECV 304 expresses the heparan sulfate proteoglycan glypican-1. The predominant cellular glycoform carries truncated side-chains and is accompanied by heparan sulfate oligosaccharides. Treatment with brefeldin A results in accumulation of a glypican proteoglycan with full-size side-chains while the oligosaccharides disappear. During chase the glypican proteoglycan is converted to partially degraded heparan sulfate chains and chain-truncated proteoglycan, both of which can be captured by treatment with suramin. The heparan sulfate chains in the intact proteoglycan can be depolymerized by nitrite-dependent cleavage at internally located N-unsubstituted glucosamine moieties. Inhibition of NO-synthase or nitrite-deprivation prevents regeneration of intact proteoglycan from truncated precursors as well as formation of oligosaccharides. In nitrite-deprived cells, formation of glypican proteoglycan is restored when NO-donor is supplied. We propose that, in recycling glypican-1, heparan sulfate chains are cleaved at or near glucosamines with unsubstituted amino groups. NO-derived nitrite is then required for the removal of short, nonreducing terminal saccharides containing these N-unsubstituted glucosamine residues from the core protein stubs, facilitating re-synthesis of heparan sulfate chains.
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본 연구는 체세포의 종류, 세포 제공 개체, 계대배양 수 및 세포의 trypsin 처리시간이 소 체세포 핵이식란의 체외발육에 미치는 영향을 검토하였다. 세 종류의 체세포(피부, 근육 및 난구세포) 와 암소 3 개체를 실험에 공시하였으며, 한 개체 유래의 피부세포는 5∼30회 계대배양하였고, 핵이식 전에 1∼3분간 trypsin처리하여 핵이식에 사용하였다. 핵이식과정은 상법에 따라 전기융합법을 이용하였다. 핵이식란의 배반포 발육율은 세포의 종류(16.5∼23.9%)나 개체 간(16.4∼19.5%)에 차이가 없으나, 30회 계대 배양한 세포를 사용한 경우(5.8%)에는 5회(25.3%) 또는 15회(23.5%) 계대 배양한 세포를 사용한 경우에 비해 유의적으로 낮았다(P＜0.05). 또한, 1분간 trypsinization 한 세포를 사용한 경우(30.7%)는 3분간 trypsinization 한 경우에 비해 배반포 발육율이 유의적으로 높게 나타났다(P＜0.05). 본 연구의 결과는 소 체세포 핵이식란의 발육이 체세포의 종류 및 세포 제공 개체에 따라서는 영향을 받지 않으나, passage 수 및 체세포의 trypsinization 시간에 따라서는 영향을 받을 수 있음을 시사한다.
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BACKGROUND Noncultured Epidermal Cell Suspension (NCECS) is a surgical modality used in treating stable vitiligo. Trypsinization of the epidermis may be done either at 4°C overnight (cold) or at 37°C for 30 to 50 minutes (warm). Recently, trypsinization was done at room temperature (25°C) in an in vitro trial. OBJECTIVE To compare different trypsinization techniques in NCECS regarding cell viability and clinical outcome. METHODS This comparative multicenter study was conducted on 20 patients with stable nonsegmental vitiligo. In each patient, 3, nonacral vitiligo lesions were randomly assigned for treatment by NCECS prepared by warm, room temperature, and cold trypsinization techniques, respectively. A perilesional biopsy was taken from each of the 3 treated lesions as an objective measure of disease stability. After transplantation, all patients received narrow-band ultraviolet B twice weekly for 6 months. Cell viability was assessed in each technique, as well as clinical outcome in all treated lesions. RESULTS Warm and room temperature trypsinization techniques were comparable with each other. Both were significantly better than the cold technique regarding viability and repigmentation. CONCLUSION Room temperature trypsinization can be used as a convenient substitute to warm trypsinization. Cold trypsinization is not recommended because of its poor results and poor patient satisfaction.
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Abstract The distribution and rate of appearance of newly synthesized receptors on the surface of Burkitt lymphoma cells were determined by membrane immunofluorescence tests on trypsinized cell lines growing as continuous cultures in a medium containing gamma globulins with specificity for the receptors. An attempt was made to determine the fate of the „surface receptor‐serum globulin”︁ complex by examining serial samples of cultures of stained positive cells for membrane immunofluorescence. There is partial evidence to suggest that observed fluctuations in the numbers of receptor‐producing cells are partly due to active shedding of receptors from the cell surfaces.
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A method was developed for maintaining isolated rat adipocytes in primary culture in a viable state for several days. Using this method, the kinetics of insulin receptor biosynthesis and membrane incorporation were assessed by inactivating (>95%) cell-surface insulin receptors with trypsin and then incubating cells in medium at 37°C for various times up to 44 h. The insertion of nascent receptors, into the plasma membrane was monitored by measuring the recovery of specific 125l-insulin binding activity. After trypsinization, insulin binding activity recovered at a linear rate with half of the original cell-surface receptor complement replenished by 24 h (6000 receptors or 2% of total surface receptors/h). This recovery was most probably mediated by de novo receptor synthesis, since no recovery of specific insulin binding was seen when cells were pretreated with cycloheximide for 3 h. Under conditions where protein synthesis was blocked immediately after trypsinization, however, a lag of 2–3 h was observed before receptor incorporation was inhibited. This lag suggests that insulin receptors are processed and routed within the cell for 2–3 h before being inserted into the plasma membrane. In support of this hypothesis it was found that the intracellular pool of insulin receptors (10% of the total receptor complement) could be depleted by about 80% by treating cells with cycloheximide for 3 h. Thus, it appears that most, if not the entire, intracellular receptor pool in adipocytes consists of newly synthesized receptors en route to the cell surface. Insulin, after binding to cell-surface receptors, triggers various biologic actions in adipocytes such as glucose transport and endocytosis of insulin-receptor complexes. To determine whether nascent receptors are functional immediately on insertion, we compared the recovery of insulin binding activity after trypsinization with the ability of these cells to respond to insulin with an increase in glucose uptake and an augmented rate of insulin internalization. These data revealed a high correlation between recovery of binding and biologic responsiveness. Thus, these data indicate that immediately upon insertion, or shortly thereafter, receptors become functional and are able to mediate the biologic actions of insulin.
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Journal Article Trypsinization Frequency and Loss of Proliferative Capacity in WI-38 Cells Get access Evan C. Hadley, MD, Evan C. Hadley, MD 2NIA, Clinical Physiology Branch, Gerontology Research Ctr., Baltimore City HospitalsBaltimore, MD 21224. Search for other works by this author on: Oxford Academic PubMed Google Scholar E. Donald Kress, MA, E. Donald Kress, MA Search for other works by this author on: Oxford Academic PubMed Google Scholar Vincent J. Cristofalo, PhD Vincent J. Cristofalo, PhD 3The Wistar Inst. of Anatomy and Biology, 36th St. at Spruce.Philadelphia, PA 19104. Search for other works by this author on: Oxford Academic PubMed Google Scholar Journal of Gerontology, Volume 34, Issue 2, March 1979, Pages 170–176, https://doi.org/10.1093/geronj/34.2.170 Published: 01 March 1979
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The theoretical background and the experimental feasibility of an automatic control for the continuous trypsinization process are discussed. An automatic apparatus is described with experimental evidence that the optimal mean residence time of monkey kidney cells in the trypsinization flask is between 5 and 8 min, in a volume of fluid approximately ten times that of the tissue processed. A temperature of 36 C and a pH of 7.8 provide optimal conditions for cell viability.
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