Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

While uterine stromal cells (USC) appear to modify the function of uterine epithelial cells (UEC) under certain conditions in vivo, relatively little is known about the effect of epithelial cells on stromal cell differentiation and function. To determine if UEC modulate USC function in vitro, highly enriched (> 95%) cultures of polarized UEC were first cultured on Matrigel-coated filters in serum-free medium until confluent, then cocultured with USC for up to 120 h. Subsequently, while maintaining both cell types in physically separate compartments, filters containing UEC were removed, and USC phenotypic markers assayed. Coculture with UEC did not affect the expression of two markers of USC differentiation (desmin and laminin), USC DNA content, [35S]methionine uptake, or total protein synthesis or secretion. However, coculture of USC with UEC or medium conditioned by UEC induced the secretion of a 30-kilodalton protein (p30) from USC as early as 24 h of coculture and through 120 h of coculture. In addition, secretion of a 60-kilodalton protein by USC was frequently observed in response to coculture with UEC. Neither the hormonal stage from which uterine cells were recovered, nor the addition of exogenous progesterone or estradiol modulated UEC-induced p30 secretion. Several purified growth factors (transforming growth factor-beta, epidermal growth factor, interleukin-1 alpha, and fibroblast growth factor) added to the serum-free culture medium failed to induce p30 secretion by USC. The p30-inducing activity in UEC-conditioned medium could not be abolished by either heat or trypsin treatment, suggesting that it is not a protein. Purified prostaglandin E2 or F2 alpha or platelet-activating factor did not induce p30 secretion by isolated USC. Of several epithelial and fibroblastic cell lines tested, UEC and a human uterine adenocarcinoma cell line (RL95-2) were the most effective in inducing p30 secretion by USC. Moreover, UEC also were able to modulate protein secretion by nonuterine murine fibroblast cell lines. Collectively, these data demonstrate that UEC can modulate USC function in vitro via a soluble factor(s).
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PMID:Mouse uterine stromal cells secrete a 30-kilodalton protein in response to coculture with uterine epithelial cells. 144

In the developing peripheral nerve, Schwann cells proliferate rapidly and then become quiescent, an essential step in control of Schwann cell differentiation. Cell proliferation is controlled by growth factors that can exert positive or inhibitory influences on DNA synthesis. It has been well established that neonatal Schwann cells divide very slowly in culture when separated from neurons but here we show that when culture was continued for several months some cells began to proliferate rapidly and non-clonal lines of immortalised Schwann cells were established which could be passaged for over two years. These cells had a similar molecular phenotype to short-term cultured Schwann cells, except that they expressed intracellular and cell surface fibronectin. The difference in proliferation rates between short- and long-term cultured Schwann cells appeared to be due in part to the secretion by short-term cultured Schwann cells of growth inhibitory activity since DNA synthesis of long-term, immortalised Schwann cells was inhibited by conditioned medium from short-term cultures. This conditioned medium also inhibited DNA synthesis in short-term Schwann cells stimulated to divide by glial growth factor or elevation of intracellular cAMP. The growth inhibitory activity was not detected in the medium of long-term immortalised Schwann cells, epineurial fibroblasts, a Schwannoma (33B), astrocytes or a fibroblast-like cell-line (3T3) and it did not inhibit serum-induced DNA synthesis in epineurial fibroblasts, 33B cells or 3T3 cells. The activity was apparently distinct from transforming growth factor-beta, activin, IL6, epidermal growth factor, atrial natriuretic peptide and gamma-interferon and was heat and acid stable, resistant to collagenase and destroyed by trypsin treatment. We raise the possibility that loss of an inhibitory autocrine loop may contribute to the rapid proliferation of long-term cultured Schwann cells and that an autocrine growth inhibitor may have a role in the cessation of Schwann cell division that precedes differentiation in peripheral nerve development.
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PMID:Spontaneous immortalisation of Schwann cells in culture: short-term cultured Schwann cells secrete growth inhibitory activity. 176 38

The interaction between organ-resident cells from the anterior uvea of the eye and T helper (Th) cells was investigated. Cells from Lewis rat ciliary body processes (CB cells), grown in tissue culture using an explant technique, could be induced to express major histocompatibility complex class II (Ia) antigens by incubation with rat interferon-gamma. Ia+ CB cells only poorly functioned as antigen-presenting cells (APC) for a syngeneic, uveitogenic Th cell line specific for the retinal soluble antigen (SAg). Moreover, if added to an Ag-driven lymphocyte proliferation assay in the presence of conventional APC, the rat CB cells had an inhibiting effect on Th proliferation. This inhibitory activity was not species specific, since similar effects were observed with bovine and human ciliary epithelial cells. The suppressive activity of CB cells was composed of a soluble factor, as well as a membrane-associated inhibitor. The soluble activity did not appear to be related to transforming growth factor-beta (TGF-beta), since no reversal of inhibition by a neutralizing antibody to TGF-beta was found. Part of the soluble inhibitory activity could be reversed by indomethacin treatment. The membrane-associated component was trypsin sensitive, suggesting a protein molecule. After abrogation of the inhibitory capacity by trypsin treatment and fixation by glutaraldehyde, CB cells effectively presented SAg to Th cells. These data suggest that CB cells are capable of mediating both Ag presentation and inhibition of Th cell proliferation.
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PMID:Dual effect of ciliary body cells on T lymphocyte proliferation. 214 48

We have previously reported the presence of a high molecular weight polypeptide growth factor in the plasma of normal human or rat serum which stimulates DNA synthesis in primary cultures of normal rat hepatocytes. We referred to this activity as hepatopoietin A (HPTA) (Michalopoulos, G., Houck, K. A., Dolan, M. L., and Luetteke, N. C. Control of hepatocytes replication by two serum factors. Cancer Res., 44: 4414-4419, 1984; Thaler, J., and Michalopoulos, G. Hepatopoietin A. Partial characterization and trypsin activation of a hepatocyte growth factor. Cancer Res., 45: 2545-2549, 1985). At that time, however, complete purification of this growth factor had not been achieved. In the present report we describe the steps required for complete purification of HPTA from human plasma or rabbit serum. The purification involved sequential ammonium sulfate precipitation, heparin-affinity chromatography, anion-exchange high-performance liquid chromatography (HPLC), and reversed phase HPLC. The final purified product is a heterodimer consisting of a heavy and a light polypeptide chain with molecular weights of 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Under nonreducing conditions, however, the purified HPTA migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 69,000. The mitogenic activity of HPTA was associated with this band when it was eluted from unstained sodium dodecyl sulfate-polyacrylamide gels. Gel filtration HPLC under neutral isotonic conditions indicated that HPTA tends to form aggregates with molecular weights of greater than 300,000. Chromatofocusing indicated that HPTA is an acidic protein with an isoelectric point value of about 5.5. The mitogenic activity of HPTA was sensitive to heat, trypsin, and 2-mercaptoethanol, but relatively resistant to exposure to 1 N acetic acid, 2 M guanidine-HCl, and 0.1% sodium dodecyl sulfate. The stimulation of DNA synthesis induced by HPTA was totally abrogated by transforming growth factor-beta and markedly reduced in the presence of heparin. We present biochemical as well as biological evidence that HPTA is a hepatocyte growth factor distinct from other known polypeptide mitogens such as epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor, fibroblast growth factor, and thrombin.
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PMID:Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes. 252 51

We determined whether transforming growth factor-beta (TGF-beta) could be encapsulated in phospholipid liposomes and then would mediate antiproliferative activity against the sensitive, human breast cancer cell line, MDA-MB-435. TGF-beta was encapsulated in multilamellar liposomes consisting of phosphatidylcholine (PC) or PC and phosphatidylserine (PS) at a 7:3 molar ratio. It was captured in both the aqueous phase and the bilayer lipid (hydrophilic and lipophilic association) and was stable for at least 24 hr of incubation at 37 degrees C in medium that contained 5% fetal bovine serum. In calcium- and magnesium-free Hanks' balanced salt solution, TGF-beta in the internal aqueous compartment was stable for at least five days, even in the presence of trypsin and ethylenediamine tetraacetic acid. TGF-beta (type 1 or 2) in liposomes was active as free-form TGF-beta in mediation of antiproliferative effects. The lipophilic nature of TGF-beta, which resulted in a high capture ratio in liposomes, coupled with exceptional stability, suggested that liposomes could be a carrier for the in vivo use of TGF-beta.
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PMID:Antiproliferative activity of liposome-encapsulated transforming growth factor-beta against MDA-MB-435 human breast carcinoma cells. 270 39

The transforming growth factor-beta (TGF-beta) receptor type III is a low abundance cell surface component that binds TGF-beta 1 and TGF-beta 2 with high affinity and specificity, and is present in many mammalian and avian cell types. Type III TGF-beta receptors affinity-labeled with 125I-TGF-beta migrate in sodium dodecyl sulfate-polyacrylamide electrophoresis gels as diffuse species of 250-350 kDa. Here we show that type III receptors deglycosylated by the action of trifluoromethanesulfonic acid yield affinity-labeled receptor cores of 110-130 kDa. This marked decrease in molecular weight is also achieved by combined treatment of type III receptors with heparitinase and chondroitinase ABC. Digestion of receptor-linked glycosaminoglycans by treatment of intact cell monolayers with heparitinase and chondroitinase does not prevent TGF-beta binding to the type III receptor core polypeptide and does not release the receptor polypeptide from the membrane. The type III TGF-beta receptor binds tightly to DEAE-Sephacel and coelutes with cellular proteoglycans at a characteristically high salt concentration. Thus, the type III TGF-beta receptor has the properties of a membrane proteoglycan that carries heparan and chondroitin sulfate glycosaminoglycan chains. The binding site for TGF-beta appears to reside in the 100-120-kDa core polypeptide of this receptor. The type III receptor is highly sensitive to cleavage by trypsin. Trypsin action releases the glycosaminoglycan-containing domain of the receptor leaving a 60-kDa membrane-associated domain that contains the cross-linked ligand. A model for the domain structure of the TGF-beta receptor type III is proposed based on these results.
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PMID:The transforming growth factor-beta receptor type III is a membrane proteoglycan. Domain structure of the receptor. 290 57

Neoplastic intercalated duct cells that originated from a human submandibular salivary gland release transforming growth factor and human epidermal growth factor into serum-free medium. The transforming growth factor with the soft agar colony-forming activity when assayed in the presence of mouse epidermal growth factor on normal rat kidney clone 49F indicator cells, but without mitogenic action to quiescent 3T3 cells, does not compete with epidermal growth factor for receptor binding, is heat and acid resistant but trypsin and dithiothreitol sensitive, and therefore is of the transforming growth factor-beta class. The immunoreactive human epidermal growth factor is detected by radioimmunoassay on certain fractions obtained by the Bio-Gel P-60 chromatography of neoplastic epithelial duct cells originated from a human submandibular salivary gland-conditioned medium and is biologically very similar to mouse epidermal growth factor. Moreover, the presence of human epidermal growth factor in cultured neoplastic epithelial duct cells originated from a human submandibular salivary gland is demonstrated by the peroxidase:antiperoxidase method.
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PMID:Expression of epidermal growth factor and transforming growth factor-beta in human salivary gland adenocarcinoma cell line. 299 94

A novel gelatin-binding 21 kDa protein was identified in the culture medium of fibroblastic and sarcoma cells by affinity chromatography on gelatin-Sepharose. Its affinity for gelatin was lower than that of the other gelatin-binding proteins, fibronectin and the 70 kDa protein, as judged by stepwise elution by urea and arginine. The protein bound also to spermine and to some extent to heparin but not to staphylococcal protein A, bovine serum albumin, concanavalin A or plain Sepharose 4B. In gel filtration chromatography the protein eluted in fractions differing from those of fibronectin and the Mr 70,000 protein and retained its ability to bind to gelatin-Sepharose, indicating that the binding was not mediated by the two other gelatin-binding proteins. It contains intrachain disulfide bridges, as judged by analysis under nonreducing and reducing conditions. The protein is composed of two major subtypes with pI values of 5.85-6.10 and 6.55-6.75. It was sensitive to trypsin but not to collagenase or thrombin. Antiserum was raised in rabbits against the gelatin-binding proteins isolated from serum-free conditioned fibroblast culture medium. The antiserum reacted with fibronectin, the Mr 70,000 protein and the Mr 21,000 protein in immunoprecipitation experiments. Absorption of the antiserum with human plasma fibronectin did not decrease its reactivity with the Mr 70,000 and 21,000 proteins. However, absorption with the Mr 70,000 protein abolished also the reactivity against the Mr 21,000 protein, suggesting immunological cross-reactivity. The protein was synthesized independently from the Mr 70,000 protein, as shown by pulse-chase labeling experiments of cells. The production of the Mr 21,000 protein in cultured cells was enhanced by transforming growth factor-beta.
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PMID:Characterization of a novel gelatin-binding 21 kDa protein secreted by cultured adherent cells. 301 29

The transforming growth factor-beta (TGF-beta) family of proteins exert diverse and potent effects on proliferation, differentiation, and extracellular matrix synthesis. However, relatively little is known about the stability or processing of endogenous TGF-beta activity in vitro or in vivo. Our previous work indicated that 1) TGF-beta 1 has strong heparin-binding properties that were not previously recognized because of neutralization by iodination, and 2) heparin, and certain other polyanions, could block the binding of TGF-beta 1 to alpha 2-macroglobulin (alpha 2-M). The present studies investigated the influence of heparin-like molecules on the stability of the TGF-beta 1 signal in the pericellular environment. The results indicate that heparin and fucoidan, a naturally occurring sulfated L-fucose polymer, suppress the formation of an initial non-covalent interaction between 125I-TGF-beta 1 and activated alpha 2-M. Electrophoresis of 125I-TGF-beta 1 showed that fucoidan protects TGF-beta 1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF-beta derived from vascular smooth muscle cells (SMC), plasmin degraded acid-activated TGF-beta, and purified TGF-beta 1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half-life of 125I-TGF-beta 1 and doubled the amount of cell-associated 125I-TGF-beta 1. Consistent with this protective effect, heparin- and fucoidan-treated SMC demonstrated elevated levels of active, but not latent, TGF-beta activity.
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PMID:Protection of transforming growth factor-beta 1 activity by heparin and fucoidan. 751 Nov 46

The LIM 1863 colon carcinoma cell line grows in suspension as morphologically and functionally organized organoids in serum-containing medium. Addition of TGF-beta caused the organoids to adhere and inhibited DNA synthesis. A 20 min incubation with TGF-beta was sufficient to induce adherence and this could be inhibited by cycloheximide. The adhesion and DNA synthesis inhibition were demonstrated to be separate events. We were not able to detect any changes in matrix or cell membrane antigens. Similarly there were no changes in synthesized proteins (by two-dimensional gel electrophoresis), and no upregulation of proteoglycan. When adhered organoids were lysed from the tissue culture plastic surface, untreated organoids adhered to this surface. This 'conditioned' surface was destroyed by trypsin but not collagenase or medium from normal LIM 1863 cultures. However, the adherent phenotype was prevented when organoids were treated with transforming growth factor-beta (TGF-beta) in the presence of medium conditioned by normal LIM 1863 cultures rather than in fresh medium. The adhesion process was inhibited by an antibody (QE2E5) against beta 1 integrin although no quantitative changes in integrins were observed (by immunoprecipitation or RNA analysis). A second anti-beta 1 integrin antibody (61.2C4) inhibited LIM 1863 adhesion to collagen but not TGF-beta induced adhesion, implying that TGF-beta induced a specific conformational change or interaction of a beta 1 integrin. In this morphologically structured system TGF-beta induced a number of subtle effects including formation of new extracellular matrix and conformational change of a beta 1 integrin, rather than the major quantitative changes in cell/matrix molecules reported previously.
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PMID:Effect of TGF-beta on differentiated organoids of the colon carcinoma cell line LIM 1863. 759 Aug 99


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