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)

The ability of fresh human amnion to bind and internalize horseradish peroxidase-labeled IgG (IgG-HRP) was examined in an in vitro Ussing chamber system. The amnion demonstrated unique cell membrane receptors for the Fc portion of IgG molecules (Fc gamma R). The Fc gamma R exhibit exquisite specificity and affinity for IgG monomers as demonstrated by staining with labeled IgG. Labeled IgA, IgM, F(ab')2 fragments of IgG, aggregated IgG, and antigen-antibody complexes all failed to bind to the amnionic epithelial cells. Binding was only minimally affected by changes in ionic strength or pH when viewed at the light microscopic level. The Fc gamma R are located on both the apical and basal cell membranes. The binding of IgG-HRP to the amnion cell membrane was detectable within 1 min, and internalization of the ligand occurred within 5 min. No binding of IgG-HRP was observed following treatment of the membrane with 0.25% trypsin for 30 min at room temperature. Incubation of the amnion at 4 degrees C or in the presence of colchicine or cytochalasin D prevented internalization of the IgG-HRP. These experiments demonstrate Fc gamma R on human amnionic epithelial cells that both bind and internalize IgG, thus allowing the amnion to be used as a model system for studying IgG transport.
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PMID:Human amnion as a model for IgG transport. 295 76

An active form of phosphorylase phosphatase of Mr = 33,000, referred to as the catalytic subunit for over a decade, was purified to near-homogeneity from rabbit skeletal muscle. Repeated immunization of a sheep produced immunoglobulins that blocked the activity of the phosphatase. These immunoglobulins were affinity-purified on columns of immobilized phosphorylase phosphatase and used as macromolecular probes in a "Western" immunoblotting procedure with peroxidase-conjugated rabbit anti-sheep immunoglobulins. Only one protein, of Mr = 33,000, was stained in samples of the immunogen, attesting to the specificity of the probes. However, the Mr = 33,000 phosphatase protein was not detected in muscle extracts or in partially purified preparations. Instead, a single protein of Mr = 70,000 was detected. Limited proteolysis, in particular by Staphylococcus aureus V8 protease and thermolysin, converted the immunoreactive protein from Mr = 70,000 to Mr = 33,000. Coagulation of the phosphatase preparation with 80% ethanol at room temperature rendered the Mr = 70,000 protein insoluble, but allowed extraction of the Mr = 33,000 protein from the precipitate. Thus, we conclude that the immunoreactive protein of Mr = 70,000 is the "catalytic subunit" of phosphorylase phosphatase with a catalytic domain of Mr = 33,000. Previous purification schemes have yielded only the fragment of Mr = 33,000 due to its relative resistance to proteolysis and coagulation. Gel filtration chromatography of the "native" form of phosphorylase phosphatase showed Mr approximately 230,000. Both the Mr = 70,000 catalytic subunit and a Mr = 60,000 protein related to inhibitor-2 were detected by immunoblotting in the same fractions that exhibited activity after treatment with Co2+ and trypsin. Only the Mr = 60,000 protein was degraded during this activation process. We propose that the native phosphorylase phosphatase is an elongated structure with two-fold symmetry, containing one catalytic subunit of Mr = 70,000 and one regulatory subunit of Mr = 60,000.
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PMID:Phosphorylase phosphatase catalytic subunit. Evidence that the Mr = 33,000 enzyme fragment is derived from a native protein of Mr = 70,000. 298

The activation of 14C-labeled estradiol by "true" and "pseudo" peroxidases to form conjugates and other products was compared in four model systems using H2O2, glutathione, Mn2+ or irradiated riboflavin. Albumin was used as acceptor except in the glutathione system. The binding of estradiol to glutathione in the presence of the true peroxidases, lacto- or uterine peroxidase (no H2O2 added), was also examined and the conditions shown to differ from those required with the pseudoperoxidases, microperoxidase or trypsin-digested cytochrome c. The conjugates were purified by chromatography after elution from Amberlite XAD-2 and the relative amounts of these products assessed by autoradiography. The ratio of steroid to glutathione in the main water-soluble metabolite formed with lactoperoxidase was found to be approx 1:1 in a double label experiment with [14C]estradiol and [3H]glutathione. It was also shown, using estradiol labeled with 3H in different positions of the steroid molecule, that lactoperoxidase acts non-specifically in catalyzing the formation of glutathionyl conjugates as indicated by the release of 3H2O. The possible role of peroxidase and glutathione in the metabolism of estrogens and in the formation of artifactual products is discussed.
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PMID:Metabolism of estradiol by true and pseudoperoxidases. 299 58

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

At the present time, the molecular nature of the fragile site at Xq27.3 is not well understood. To examine the sensitivity of this region to DNAase I, in situ nick translation was performed on metaphase chromosomes from a fragile X (fra(X] positive individual. In this technique DNAase I is used to nick regions of chromosomal DNA that are in "open" conformation. Biotinylated dUTP was incorporated by nick translation at these sites. The incorporation was identified by double antibody labeling and avidin-horseradish peroxidase staining. Spreads, which had been stained with this technique, were photographed and subsequently trypsin-Giemsa G banded (post-GTG banded) for chromosome identification. In 36 of 44 (82%) fra(X) positive male cells, the region distal to fra(X) (q27.3) was prominently stained in contrast to its light staining appearance in GTG preparations. The fragile site itself was outlined more clearly than can be achieved by GTG or homogeneous staining. When autosomal fragile sites were induced by the addition of 1.5 microM aphidicolin 17 hours prior to harvest, 24 of 27 (89%) fragile sites on the ends of autosomes were prominently stained in regions distal to the break. Because the fra(X) and autosomal fragile regions behaved similarly, this suggests that they have a similar conformation. Thus, while autosomal and Xq27.3 fragile sites are strongly induced by different means, the organization of these sites and the regions distal to them appear to be similar.
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PMID:In situ nick translation of the fragile X region. 305 67

Because the correct diagnosis of indeterminate leprosy (IL) requires the finding of acid-fast bacilli in skin lesions from clinically and histopathologically suggestive cases, it is necessary to develop a reliable method for this purpose. This paper presents a simple procedure, available to every general laboratory, which consists in obtaining 2 suspensions: SI, by mincing and grinding the tissue in phosphate-buffered saline; and SII, after treating SI with NaOH solution and digesting with trypsin. In 22 IL skin biopsies, bacilli were directly observed in only 3 with the Ziehl-Neelsen (ZN) stain; and with the peroxidase-antiperoxidase method it was impossible to differentiate between nonspecific precipitate and true positive reactions. In contrast, 18 positive results from the same 22 samples were obtained when both SI and SII were evaluated with ZN stain. The logarithmic bacterial index was also increased in at least 7 cases.
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PMID:Demonstration of acid-fast bacilli in skin biopsies from indeterminate leprosy cases. 306 61

Immunolocalization of type III collagen and procollagen in cirrhotic human liver was studied using monoclonal antibody specific for the helical determinant of type III collagen extracted from human placenta. Deparaffinized, trypsin-treated cirrhotic liver sections from 8 autopsy cases were examined by the unlabeled peroxidase-antiperoxidase and immunofluorescence techniques. These techniques revealed the localization of this epitope shared by type III collagen and procollagen not only in the extracellular matrix of hepatocytes and sinusoidal cells but also in the cytoplasm. In hepatocellular carcinoma concurrent with cirrhosis, neoplastic cells were shown to react with this antibody as well. These results are consistent with data obtained using antiserum specific for bovine type III procollagen aminopeptide which appeared in our previous report.
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PMID:Immunolocalization of type III collagen and procollagen in cirrhotic human liver using monoclonal antibodies. 308 39

We used a conjugate of transferrin and horseradish peroxidase (Tf/HRP) to label the intracellular transferrin receptor route in the human hepatoma cell line HepG2. The recycling kinetics of [125I]Tf/HRP were similar to those of unmodified [125I]Tf, implying identical routes for both ligands. 3,3'Diaminobenzidine (DAB)-cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were incubated with both Tf/HRP and [125I]Tf, and caused two different effects: (a) the equilibrium density of [125I]Tf containing microsomes in a Percoll density gradient was increased, and (b) the amount of immunoprecipitable [125I]Tf from density-shifted lysed microsomes was only 20% of that of nonDAB treated microsomes. The whole biosynthetic route of alpha 1-antitrypsin (AT), a typical secretory glycoprotein in HepG2 cells, was labeled during a 60-min incubation with [35S]methionine. DAB cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were also incubated with Tf/HRP. DAB cytochemistry caused approximately 40% of microsome-associated "complex" glycosylated [35S]alpha 1-antitrypsin ([35S]c-AT) to shift in a Percoll density gradient. Only part of the density shifted [35S]c-AT could be recovered by immunoprecipitation. A maximum effect was measured already after 10 min of Tf/HRP uptake. The density distribution of the "high mannose" glycosylated form of 35S-alpha 1-anti-trypsin [( 35S]hm-AT) was not affected by Tf/HRP. If in addition to Tf/HRP also an excess of non-conjugated transferrin was present in the medium, [35S]c-AT was not accessible for Tf/HRP, showing the involvement of the transferrin receptor (TfR) in the process. Furthermore, we show that if Tf/HRP and [35S]c-AT were located in different vesicles, the density distribution of [35S]c-AT was not affected by DAB-cytochemistry. Pulse-labeling with [35S]methionine was used to show that [35S]c-AT became accessible to endocytosed Tf/HRP minutes after acquirement of the complex configuration. A common intracellular localization of endocytosed Tf/HRP and secretory protein could be confirmed by immuno-electron microscopy: cryosections labeled with anti-albumin (protein A-colloidal gold) as well as DAB reaction product showed double-labeling in the trans-Golgi reticulum.
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PMID:The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum. 326 Feb 38

Formalin fixed, paraffin embedded tissue from 100 consecutive cases of breast carcinoma were studied for binding with Helix pomatia (HPA) and Ulex Europeus (UEA1) lectins. Serial sections were pretreated with trypsin or neuraminidase to determine the effect of these enzymes on lectin binding. The lectins were visualized by the peroxidase antiperoxidase technique and the cell staining proportion assessed in a semi-quantitative manner under the light microscope. Correlating staining with prognostic factors and patient follow-up details showed that UEA1 related to disease-free interval and survival, and HPA to lymph node stage, time to loco regional recurrence and to survival. Relationships with both lectins were abolished by pretreatment with neuraminidase. The study demonstrates that a simple assessment of lectin binding can provide prognostic information in breast cancer. This may be useful particularly when conservational surgical practice restricts the amount of nodal tissue for staging.
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PMID:Helix pomatia and Ulex europeus lectin binding in human breast carcinoma. 330 30

In this work the reactivity of 16 monoclonal antibodies raised against different HLA class I specificities was tested with human skin of healthy donors of known HLA typing. By indirect immunofluorescence, six antibodies reacted strongly with keratinocytes carrying the corresponding alloantigens. The reactivity of 3 other antibodies which was weak or absent using indirect immunofluorescence, was enhanced by various amplification systems such as avidin-biotin-peroxidase method, biotin-streptavidin-fluorescein complex and especially preliminary trypsin treatment that revealed alloantigens masked in the epidermis. The immunostaining of 4 antibodies was negative regardless of the method used. Some of the antibodies we tested cross-reacted with cytoplasmic antigens of keratinocytes. This study has allowed to select a battery of monoclonal antibodies which can specifically detect alloantigens on keratinocytes and will be useful for the recognition the cell origin in allografting experiments.
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PMID:Reactivity of anti-HLA class I polymorphic monoclonal antibodies with normal human skin. 331 66


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