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)

This study describes an easy 3 step-procedure to prepare rapidly and at low cost, pure myoblast cell cultures from a normal muscle biopsy. Following collagenase and trypsin treatment of the tissue (step 1), dissociated cells were cloned at a density of 10 cells/ml in MCDB 120 medium (0.2 ml/well). Clones that grew were then tested for NCAM cell surface expression by cytofluorometric analysis (CFA) using Coulter CD56-PE monoclonal antibodies (step 2). Only those clones with more than 98% strongly labelled positive cells were expanded (step 3) for further trials in cell transfer therapy for dystrophic patients. Visualization of the pattern of NCAM expression was performed by immunoperoxidase assay, while the potential ability to form myotubes was confirmed by the observation of their formation within a period of 1 to 2 weeks. The 65% of the CD56+ clones in CFA were the same clones that proved to be myogenic with positive immunoperoxidase assay and myotube formation. This method avoids the fastidious and costly approach of cell sorting (whenever available), avoids contamination hazards due to many manipulations of the clones. Moreover this approach leads to a pure myoblast population free of any contaminating fibroblast which could contribute to connective tissue implement already deleterious in dystrophic patients.
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PMID:Rapid selection of donor myoblast clones for muscular dystrophy therapy using cell surface expression of NCAM. 751 May 44

To clarify the neuroendocrine differentiation and CD10 expression in solid-pseudopapillary tumors (SPTs) of the pancreas, we performed immunohistochemical analysis in 19 such tumors, including one solid-pseudopapillary carcinoma (SPC), along with 20 pancreatic neuroendocrine tumors (PNTs), six acinar cell carcinomas (ACCs), and one pancreatoblastoma (PB). We used antisera directed against CD56, synaptophysin, protein gene product 9.5, the alpha-subunit of Go protein, chromogranin A, CD10, trypsin, chymotrypsin, various cytokeratins (CKs), CA19-9, vimentin, and alpha-1-antitrypsin (AAT). All SPTs exhibited immunoreactivity for CD56 and CD10, and 15 expressed other neuroendocrine markers focally with the exception of chromogranin A. Frequent clustering of synaptophysin-positive cells was noted. Two cases contained a peculiar nodule that cytomorphologically and immunohistochemically resembled PNT. CD10-positive cells were scarce in one SPC. PNTs were CD56-positive, but often with faint intensity, and staining for other neuroendocrine markers, including chromogranin A, was diffusely positive. CD10 was detected, mostly in a focal pattern, in five PNTs. Pan-CK, CK8, CK18, and CK19 were more frequently demonstrated in PNT than SPT. Vimentin and AAT were often identified in PNT as well and were not specific for SPT. ACCs were CD56-negative, with the exception of one case designated as a mixed acinar-endocrine carcinoma. PB was focally positive for CD56 at the periphery of the tumor nests. Four ACCs and one PB exhibited focal CD10 reactivity. This study demonstrated the unique immunohistochemical features of SPT. Our results also suggest that SPT exhibits, at least focally, neuroendocrine differentiation, and that these neuroendocrine markers and CD10 are diagnostically useful.
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PMID:Solid-pseudopapillary tumor of the pancreas: immunohistochemical localization of neuroendocrine markers and CD10. 1102 97

In an attempt to identify novel diagnostic markers for mast cell (MC)-proliferative disorders, serial bone marrow (bm) sections of 22 patients with mastocytosis (systemic indolent mastocytosis, n = 19; mast cell leukemia [MCL], n = 1; isolated bm mastocytosis, n = 2) were analyzed by immunohistochemistry using antibodies against CD2, CD15, CD29, CD30, CD31, CD34, CD45, CD51, CD56, CD68R, CD117, HLA-DR, bcl-2, bcl-x(L), myeloperoxidase (MPO), and tryptase. Staining results revealed expression of bcl-x(L), CD68R, and tryptase in neoplastic MCs (focal dense infiltrates) in all patients. Mastocytosis infiltrates were also immunoreactive for CD45, CD117 (Kit), and HLA-DR. In most cases, the CD2 antibody produced reactivity with bm MCs in mastocytosis, whereas in control cases (reactive bm, immunocytoma, myelodysplastic syndrome), MCs were consistently CD2 negative. Expression of bcl-2 was detectable in a subset of MCs in the patient with MCL, whereas no reactivity was seen in patients with SIM or bm mastocytosis. Mastocytosis infiltrates did not react with antibodies against CD15, CD30, CD31, CD34, or MPO. In summary, our data confirm the diagnostic value of staining for tryptase, Kit, and CD68R in mastocytosis. Apart from these, CD2 may be a novel useful marker because MCs in mastocytosis frequently express this antigen, whereas MCs in other pathologic conditions are CD2 negative.
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PMID:Immunohistochemical properties of bone marrow mast cells in systemic mastocytosis: evidence for expression of CD2, CD117/Kit, and bcl-x(L). 1138 74

UVB irradiation can cause considerable changes in the composition of cells in the skin and in cutaneous cytokine levels. We found that a single exposure of normal human skin to UVB induced an infiltration of numerous IL-4(+) cells. This recruitment was detectable in the papillary dermis already 5 h after irradiation, reaching a peak at 24 h and declining gradually thereafter. The IL-4(+) cells appeared in the epidermis at 24 h postradiation and reached a plateau at days 2 and 3. The number of IL-4(+) cells was markedly decreased in both dermis and epidermis at day 4, and at later time points, the IL-4 expression was absent. The IL-4(+) cells did not coexpress CD3 (T cells), tryptase (mast cells), CD56 (NK cells), and CD36 (macrophages). They did coexpress CD15 and CD11b, showed a clear association with elastase, and had a multilobed nucleus, indicating that UVB-induced infiltrating IL-4(+) cells are neutrophils. Blister fluid from irradiated skin, but not from control skin, contained IL-4 protein as well as increased levels of IL-6, IL-8, and TNF-alpha. In contrast to control cultures derived from nonirradiated skin, a predominant type 2 T cell response was detected in T cells present in primary dermal cell cultures derived from UVB-exposed skin. This type 2 shift was abolished when CD15(+) cells (i.e., neutrophils) were depleted from the dermal cell suspension before culturing, suggesting that neutrophils favor type 2 T cell responses in UVB-exposed skin.
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PMID:Ultraviolet B radiation induces a transient appearance of IL-4+ neutrophils, which support the development of Th2 responses. 1193 23

Portal blood leukocytes play an important, but still poorly defined, role in the immune processes of the liver. In our previous studies, we showed that certain leukocyte subsets are selectively halted in the liver. These cells marginate in sinusoids and, together with resident Kupffer and endothelial sinusoidal cells, participate in antiviral and antitumor processes. The molecular mechanisms of margination and cooperation with resident sinusoidal cells require clarification. However, in vivo harvesting of portal blood leukocytes is associated with cumbersome cannulation of portal and hepatic veins and manipulation of the liver, causing major disturbances in splanchnic blood flow and liver blood supply, totally distorting sinusoidal blood perfusion and leukocyte margination. To overcome these difficulties, we have developed an in situ normothermic rat liver perfusion model permitting quantitative observations of blood leukocyte extraction in sinusoids. First, liver was flushed through the portal vein and the effluent leukocytes, named liver-associated leukocytes (LAL1), were collected from hepatic veins. Then, the liver was perfused for 60 min with 50 ml of blood using a semiclosed perfusion system. Upon completion of perfusion, the liver portal vasculature was flushed again to retrieve the leukocytes extracted from the perfusing blood (LAL2). These cells were characterized with respect to their phenotype and cytotoxicity. The mean leukocyte count of the washout before perfusion was 1.04+/-0.2x10(6)/g of liver tissue and 0.9+/-0.1x10(6)/g after 60 min of perfusion, indicating retention by the perfused liver, the live leukocyte extracting capacity. To further evaluate the efficiency of perfusion, FITC-labelled leukocytes were added to the perfusing leukocyte-free blood. Around 95% of the postperfusion washout LALs were FITC(+). Heat-killed leukocytes did not marginate in sinusoids. Preincubation of leukocytes with substances able to lower adhesion capacity, such as lidocaine, trypsin and AAGM1, significantly decreased the postperfusion LAL2 washout population. The numbers of extracted postperfusion LAL2 CD5(+), CD4(+), CD8(+), CD56(+) and class II(+) subsets did not differ statistically from those of preperfusion LAL1. Moreover, the cytotoxicity of LAL2 and LAL1 against CC531 and K562 remained at a similar level. Thus, perfused liver also retained its selective leukocyte extraction capacity. This model shows that the process of selective margination of portal blood cell subsets in the liver can be studied in an artificially perfused liver subject to physiological blood flow parameters, temperature, oxygenation and minimal ischemic time before connection to the perfusion device. Furthermore, it is suitable for studies of the selective recruitment of blood cells in sinusoids in a wide large of situations including liver tumors, infections, rejection after transplantation, graft vs. host disease, as well as in the investigation of the effect of drugs on these processes.
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PMID:A liver perfusion model for studies of selective adherence and transient halting of portal blood leukocytes in sinusoids. 1250 17

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform-ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.
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PMID:Identification of N-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry. 1465 30

Protein phosphorylation is a crucial post-translational modification mechanism in the regulation of synaptic organization and function. Here, we analyzed synaptosome fractions from human cerebral cortex obtained during therapeutic surgery. To minimize changes in the phosphorylation state of proteins, the tissue was homogenized within two minutes of excision. Synaptosomal proteins were digested with trypsin and phosphopeptides were isolated by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The method allowed the detection of residues on synaptic proteins that were presumably phosphorylated in the intact cell, including synapsin 1, syntaxin 1, and SNIP, PSD-93, NCAM, GABA-B receptor, chaperone molecules, and protein kinases. Some of the residues identified are the same or homologous to sites that had been previously described to be phosphorylated in mammals whereas others appear to be novel sites which, to our knowledge, have not been reported previously. The study shows that new phosphoproteomic strategies can be used to analyze subcellular fractions from small amounts of tissue for the identification of phosphorylated residues for research and potentially for diagnostic purposes.
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PMID:Phosphoproteomic analysis of synaptosomes from human cerebral cortex. 1582 5

Although flow cytometry is useful for studying neural lineage relationships, the method of dissociation can potentially bias cell analysis. We compared dissociation methods on viability and antigen recognition of mouse central nervous system (CNS) tissue and human CNS tumor tissue. Although nonenzymatic dissociation yielded poor viability, papain, purified trypsin replacement (TrypLE), and two purified collagenase/neutral protease cocktails (Liberase-1 or Accutase) each efficiently dissociated fetal tissue and postnatal tissue. Mouse cells dissociated with Liberase-1 were titrated with antibodies identifying distinct CNS precursor subtypes, including CD133, CD15, CD24, A2B5, and PSA-NCAM. Of the enzymes tested, papain most aggressively reduced antigenicity for mouse and human CD24. On human CNS tumor cells, CD133 expression remained highest after Liberase-1 and was lowest after papain or Accutase treatment; Liberase-1 digestion allowed magnetic sorting for CD133 without the need for an antigen re-expression recovery period. We conclude that Liberase-1 and TrypLE provide the best balance of dissociation efficiency, viability, and antigen retention. One implication of this comparison was confirmed by dissociating E13.5 mouse cortical cells and performing prospective isolation and clonal analysis on the basis of CD133/CD24 or CD15/CD24 expression. Highest fetal expression of CD133 or CD15 occurred in a CD24(hi) population that was enriched in neuronal progenitors. Multipotent cells expressed CD133 and CD15 at lower levels than did these neuronal progenitors. We conclude that CD133 and CD15 can be used similarly as selectable markers, but CD24 coexpression helps to distinguish fetal mouse multipotent stem cells from neuronal progenitors and postmitotic neurons. This particular discrimination is not possible after papain treatment. Disclosure of potential conflicts of interest is found at the end of this article.
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PMID:Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24. 1733 13

Although significant progresses were made in the field of molecular biology of malignant cerebral gliomas, the prognostic of these tumors continues to be reserved. One of the therapeutic failure reasons is the incomplete knowledge regarding the origin of these tumors and cells features, which in fact represent an obstacle in developing a cell and molecular therapy guided against malignant cells responsible for the tumor development and for the therapeutic resistance. Initiation and characterization of glioblastoma cell lines represents an essential step in order to obtain a better in vitro and in vivo experimental model for glioblastoma. We describe here a new glioblastoma line, named T11, which was successfully isolated in our laboratories starting with a tumor sample obtained intraoperative from a 58 years-old female patient. The histopathological evaluation showed a grad IV WHO glioma (glioblastoma). The sample was prepared by manual fragmentation, followed by enzymatic digestions using different concentration of trypsin. The cell line has been cultivated for more than 150 passages. The characterization of the glioblastoma line consisted in the evaluation of cells proliferation capacity (growth curve), morphological features, karyotyping and identification of specific markers. We found that T11 expressed specific markers for glial progenitors and astrocytes (glial fibrillary acidic protein-GFAP); oligodendrocites (A2B5; O4), and microglia (CD45, CD 11b). Cells were negative for neuronal lineage markers like beta3-tubulin and NCAM. In order to evaluate the differentiation grade of T11 cell line, the presence of stem cell markers (nestin, CD133) was explored. T11l cells expressed higher level of nestin and lower level of CD133 comparing with standard glioblastoma cell line U87. T11 cell line expressed VEGF and Bcl-2, but not EGFR and Mdrl and Bax. This new line has distinct and unique characteristics when compared with standard glioblastoma cell line (e.g., U87) and may become a new and useful in vitro model for glioblastoma.
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PMID:Isolation and partial characterization of a new human glioblastoma cell line. 1988 54

Acinar cell carcinoma of pancreatic type rarely occurs at extra-pancreatic sites. We report four primary liver tumors with features of pancreatic acinar cell carcinoma. The patients were two males and two females with a mean age of 65 years (range, 49-72 years). They had upper abdominal pain, weight loss and/or an incidentally discovered liver mass. None had evidence of a primary pancreatic tumor. Grossly, the tumors were large (mean size, 12 cm), well circumscribed and showed a lobulated cut surface. Histologically, they showed a predominantly microacinar pattern, with occasional trabecular, solid and microcystic areas. Cellular atypia and mitotic activity varied within the same tumor and from tumor to tumor. Immunohistochemically, the tumor cells were positive for cytokeratin 18 and at least one acinar cell marker (ie, trypsin, amylase or lipase), but were negative for cytokeratins 7, 19 and 20, HepPar-1, AFP, CD10, carcinoembryonic antigen, CD56, Islet-1 and CDX2. Two tumors stained focally for synaptophysin and chromogranin A. Adjacent liver parenchyma displayed no evidence of cirrhosis. During a mean follow-up of 22 months (range, 3-38 months) no metastases occurred, but one patient developed local recurrence. Our study demonstrates that acinar cell carcinoma of pancreatic type may also originate from the liver and can be readily distinguished from other primary liver neoplasms by its distinct histological and immunohistochemical features. Because our cases were observed within a rather short period, it is likely that this tumor type is so far underrecognized and has been mistaken as a variant of hepatocellular carcinoma, cholangiocarcinoma or any other liver tumor.
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PMID:Pancreatic-type acinar cell carcinoma of the liver: a clinicopathologic study of four patients. 2184 71


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