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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA library was prepared in lambda gt11 bacteriophage from poly(A)+ RNA isolated from primary cultures of endothelial cells from human umbilical vein. Approximately 2.5 million independent recombinants were screened and 2 of those were found to synthesize a fusion protein with beta-galactosidase that reacted with rabbit antibody against human von Willebrand factor. Comparison of the amino acid sequence translated from the cDNA insert of the two clones with the amino acid sequence determined by Edman degradation of the protein established that both phage isolates code for von Willebrand factor. The first clone (lambda HvWF1) contained an insert of 404 nucleotides that corresponded to amino acid residues 1-110 in the mature protein circulating in blood, in addition to a portion (24 amino acids) of a prepro leader sequence. The second cDNA clone (lambda HvWF3) contained an insert of 4.9 kilobases that coded for the carboxyl-terminal 1525 amino acids of von Willebrand factor, a stop codon of TGA, 134 nucleotides of 3' noncoding sequence, and a poly(A) tail of 150 nucleotides. The two clones together code for greater than 80% of the molecule circulating in blood. The same carboxyl-terminal lysine residue was identified in the mature protein as well as in the cDNA, indicating that all of the proteolytic processing that occurs during the biosynthesis and assembly of von Willebrand factor is associated with the amino-terminal portion of the precursor protein. The amino acid sequence of von Willebrand factor indicates the presence of two different internal gene duplications and one triplication. These repetitive amino acid sequences account for about one-half of the amino acids present in the mature protein. The tetrapeptide sequence of Arg-Gly-Asp-Ser, which mediates the cell attachment and platelet binding activity of fibronectin, was also identified in the carboxyl-terminal portion of von Willebrand factor.
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PMID:Cloning and characterization of two cDNAs coding for human von Willebrand factor. 286 88

The collagen-binding domain of human fibronectin has been expressed as a cro/beta-galactosidase fusion protein in Escherichia coli. The hybrid polypeptide was recognized by an anti-(human plasma fibronectin) serum and bound specifically to gelatin-Sepharose. The collagen-binding region was subdivided by constructing a series of overlapping bacterial expression plasmids. The fusion proteins produced by these constructs were analysed for gelatin-binding activity. The results indicate that the binding site lies within an approximately 12.5 kd fragment of fibronectin, and show that the following 14 amino acid sequence is critical for gelatin-binding activity: Ala-Ala-His-Glu-Glu-Ile-Cys-Thr-Thr-Asn-Glu-Gly-Val-Met. This sequence links the second type II homology unit with the adjacent type I repeat in the amino-terminal third of the fibronectin molecule.
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PMID:Mapping the collagen-binding site of human fibronectin by expression in Escherichia coli. 302 62

Two cDNA subfragments containing the cell-attachment site of human fibronectin (FN) were expressed as beta-galactosidase fusion proteins in E. coli. The products were purified to homogeneity by monoclonal antibody affinity chromatography and assayed for activity in a standard cell-adhesion assay. A fusion protein containing an 80 kDa fragment of human FN appeared functionally equivalent to intact FN purified from human plasma, whereas a truncated fusion protein of 33 kDa still containing a previously postulated cell-attachment site was approx. 50-fold less active. Our study establishes a system for analyzing adhesive protein function by DNA manipulation, rules out any major role for eukaryotic post-translational modifications in FN adhesive function, and localizes additional functional activity to a 1.3 kb region.
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PMID:Expression of the cell-binding domain of human fibronectin in E. coli. Identification of sequences promoting full to minimal adhesive function. 310 88

Monoclonal mouse hybridoma antibodies were obtained for secreted cellular fibronectin (cFn) from A8387 fibrosarcoma cells. One of them, 52-DH1 (DH), reacted exclusively with cFns but not with plasma Fns (pFns) in immunoblotting and solid-phase EIA. The DH antibody also recognized thermolysin cFn fragments and beta-galactosidase-Fn fusion protein which contained the ED sequence specific to at least some forms of cFns. On the other hand, the DH antibody failed to recognize a fusion protein that was otherwise identical but lacked the ED sequence. Thus, the antigenic determinant for the DH antibody was located to the ED sequence. The DH antibody was then used to study the expression of ED sequence containing cFn (EcFn). For comparisons, another monoclonal antibody, 52BF12 (BF), recognizing equally well both pFns and cFns, was used. Immunoblotting of pFn fragments indicated that this antibody had the antigenic determinant at or close to the cell-binding site of Fn. EcFn was revealed by the DH antibody in embryonic and adult fibroblasts and in a variety of other cultured normal and malignant human cells. In embryonic tissues EcFn was abundant in developing basement membranes, as shown in foetal kidney and muscle, while in adult tissues it was confined only to endothelia of larger blood vessels. Furthermore, in embryonic tissues the capillaries showed bright EcFn-positivity not found any more in adult tissues. Human plasma contained a small quantity of EcFn, which may hence have an endothelial origin. EcFn was also prominent in the stroma of malignant tumours as well as in reactive benign conditions, such as granulation tissue and decidual cells. The results suggest that EcFn is a form of the protein which may have a particular role in developing and reactive tissues in embryos and adults.
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PMID:Differential expression of the ED sequence-containing form of cellular fibronectin in embryonic and adult human tissues. 350

Multiple fibronectin mRNAs arise by alternative splicing of the primary transcript of a single gene. We describe analyses of the contribution of this alternative splicing to fibronectin subunit heterogeneity in three different cell types using antisera directed against specific segments of fibronectin. beta-galactosidase-fibronectin fusion proteins produced with the lambda gt11 bacterial expression vector were used as immunogens. One region of alternative splicing accounts for differences in subunit size, while a second contributes to differences between the fibronectins present in blood plasma and in fibroblastic cells. We also show, however, that these two regions of alternative splicing do not account for all detectable subunits. We have also used these segment-specific antisera to show that blood platelets contain a spectrum of fibronectin subunits distinct from that found in blood plasma.
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PMID:Cell-type-specific fibronectin subunits generated by alternative splicing. 352 52

The following proteins were subjected to electrophoresis in SDS gels and stained with both Coomassie Brilliant Blue R and Coomassie Brilliant Blue G: the pepsin-treated collagen types I, II, III and V, and non-pepsin-treated type IV collagen, and the non-collagens, laminin, fibronectin, myosin, beta-galactosidase, fibrin, phosphorylase b and serum albumin. The Coomassie Brilliant Blue G stain was formulated as in the dye-binding protein assay reagent of Bradford (Anal. Biochem. 72: 248-254, 1976). Coomassie Brilliant Blue R prominently stained all polypeptides, but the collagen chains, including the type IV chains, stained metachromatically (red or pink) while the non-collagens stained orthochromatically (blue-violet). In the Bradford reagent, however, only the non-collagens and the intact type IV chains were prominently stained; the pepsin-treated collagen chains were virtually undetectable provided that detergent had been exhaustively removed prior to immersion in the stain. Metachromatic staining with Coomassie Brilliant Blue R is attributed to the presence of closely-spaced proline and hydroxyproline residues in sequences from triple-helical domains. The staining of type IV chains with the Bradford reagent is attributed to the presence of binding sites in the sequences from the non-triple-helical domains only, since such binding sites are absent from chains derived from the pepsin-treated collagens.
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PMID:Differential staining of collagens and non-collagens with Coomassie Brilliant Blue G and R. 619 10

The extent and the specificity of the initial cell attachment induced by various proteins coated on plastic surfaces have been studied with the following results: (a) Cell adhesion on the surfaces coated with sialidase and beta-galactosidase was as strong as on concanavalin A and limulus lectin-coated surfaces and the reactions were strongly inhibited by glycosidase inhibitors or by competitive substrates. The adhesion on sialidase was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid and by polysialoganglioside (GT1b) at low concentration (0.05-0.1 mM). The cell adhesion on beta-galactosidase coat was inhibited by 1,4-D-galactonolactone and beta-methylgalactoside but not by alpha-methylgalactoside. Thus, the initiation of cell adhesion on glycosidase surfaces could be mediated through the interactions of the specific binding sites of the enzyme surface with the cell surface substrates under physiological conditions. (b) Cell adhesion on various lectins could be blocked by various competing monosaccharides at the concentrations similar to the inhibitory concentrations for binding of lectins from solution to the cells. (c) Cell adhesion on fibronectin surfaces as well as on gelatin-coated surfaces was equally inhibited by GT1b at relatively high concentrations (0.25-0.5 mM). Lower concentrations of GT1b (0.05-0.1 mM) inhibited the cell adhesion on surfaces of Limulus lectin and sialidase. It is suggested that the cell adhesion mediated by fibronectin is based on yet unknown interactions in contrast to a specific cell adhesion through glycosidases and lectins.
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PMID:Studies on cell adhesion and recognition. I. Extent and specificity of cell adhesion triggered by carbohydrate-reactive proteins (glycosidases and lectins) and by fibronectin. 678 7

Ultrastructural, histochemical and immunohistochemical features of porcine intestinal lamina propria macrophages (LPMs), peripheral blood fibronectin-adherent cells (FACs) and splenic-adherent cells (SPACs) were compared. Freshly isolated FACs and SPACs were small and showed small cytoplasmic processes, little evidence of endocytic vacuoles, few lysosomes and sparse rough endoplasmic reticulum (RER). Fresh FACs were negative for acid phosphatase, non-specific esterase (NSE) and beta-galactosidase activity. Of the SPACs, 20-40% were positive for acid phosphatase, < 5% for NSE and 5-10% for beta-galactosidase. Pre-cultured FACs and SPACs were large and showed an abundance of endocytic vacuoles; they possessed dilated and prominent RER and > 95% were positive for the three enzyme activities. LPMs exhibited abundant endocytic vacuoles or vesicles and lysosomes but sparse RER, and > 85% were positive for the three enzymes. LPMs (24%), FACs (49%) and SPACs (40%) expressed MHC (major histocompatibility complex) class II glycoproteins. Macrophage-granulocyte antigens were detected in LPMs (14%), FACs (50%) and SPACs (33%). The results thus suggest that freshly isolated FACs differ from LPMs morphologically and in enzymic features, and the differences may represent part of the cell maturation process.
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PMID:Ultrastructural, histochemical and immunohistochemical features of porcine intestinal lamina propria macrophages, peripheral blood monocytes and splenic adherent cells. 772 9

In this report a method for transfection and selection of mammalian cells in serum-free medium is described. Chinese hamster ovary (CHO) cells were grown in serum-free medium in plastic dishes coated with one of the following attachment factors: poly-D-lysine, Cell-Tak (polyphenolic proteins extracted from the marine mussel Mytilus edulis), fibronectin or laminin. Cells grown to 80% confluence were transfected with an expression vector encoding the hygromycin resistance gene as a selectable marker and beta-galactosidase as the reporter gene. Transfectants were selected using hygromycin at a concentration of 500 micrograms/ml. Both fibronectin and laminin supported colony formation following selection in serum-free medium. However, poly-D-lysine and Cell-Tak did not. This method can, thus, be used to select for clones producing a recombinant product in cells that are growing in serum-free medium from the onset to provide a better system from which to purify proteins.
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PMID:Serum-free transfection and selection in Chinese hamster ovary (CHO) cells. 826 82

Rats were immunized with a fusion protein (gal-FnBP) encompassing beta-galactosidase and the domains of fibronectin binding protein from Staphylococcus aureus responsible for binding to fibronectin. Antibodies against gal-FnBP were shown to block the binding of S. aureus to immobilized fibronectin in vitro. Endocarditis in immunized and non-immunized control rats was induced by catheterization via the right carotid artery, resulting in damaged aortic heart valves which became covered by fibrinogen and fibronectin. The catheterized rats were then infected intravenously with 1 x 10(5) cells of S. aureus. The number of bacteria associated with aortic valves was determined 1 1/2 days after the challenge infection and a significant difference in bacterial numbers between immunized and non-immunized groups was then observed (p < 0.05).
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PMID:Immunization with fibronectin binding protein from Staphylococcus aureus protects against experimental endocarditis in rats. 827 22


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