ABSTRACT
We have used directed evolution methods to express a fungal enzyme, galactose oxidase (GOase), in functional form in Escherichia coli. The evolved enzymes retain the activity and substrate specificity of the native fungal oxidase, but are more thermostable, are expressed at a much higher level (up to 10.8 mg/l of purified GOase), and have reduced negative charge compared to wild type, all properties which are expected to facilitate applications and further evolution of the enzyme. Spectroscopic characterization of the recombinant enzymes reveals a tyrosyl radical of comparable stability to the native GOase from Fusarium.
Subject(s)
Directed Molecular Evolution/methods , Escherichia coli/genetics , Galactose Oxidase/genetics , Galactose Oxidase/metabolism , Amino Acid Motifs , Amino Acid Substitution , Electrochemistry , Enzyme Stability , Escherichia coli/enzymology , Fusarium/enzymology , Galactose Oxidase/chemistry , Galactose Oxidase/isolation & purification , Gene Expression , Genetic Variation , Hot Temperature , Hydrogen Bonding , Kinetics , Models, Molecular , Mutagenesis , Plasmids , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrophotometry , Transformation, BacterialABSTRACT
Intestinal goblet cells of patients with ulcerative colitis and Crohn's disease highly express a binding protein of the Fc portion of IgG (FcgammaBP), which is entirely different from the Fcgamma receptors I,II, and III on neutrophils and macrophages. In this study, we proved the qualitative existence of FcgammaBP antigen in the sera of patients with rheumatoid arthritis and systemic lupus erythematosus, and, further, established a highly sensitive and quantitatively reproducible assay for FcgammaBP antigen in order to prevent the cross-reactivity of FcgammaBP with von Willebrand factor which has about 30% homology. This assay revealed a higher level of FcgammaBP antigen in the blood stream of patients with autoimmune diseases, especially progressive systemic sclerosis. This would suggest that abnormal production of autoantibodies reflects increased generation of FcgammaBP in goblet cells and its secretion into the circulation by an unknown mechanism.
Subject(s)
Autoantigens/immunology , Autoimmune Diseases/immunology , Carrier Proteins/immunology , Animals , Antibodies, Monoclonal/immunology , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/immunology , Autoantigens/blood , Autoimmune Diseases/blood , CHO Cells , Carrier Proteins/blood , Cell Adhesion Molecules , Cricetinae , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay , Haplorhini , Humans , Immune Sera/immunology , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/immunology , Membrane Proteins , Precipitin Tests , Rabbits , Reproducibility of Results , Sensitivity and Specificity , von Willebrand Factor/immunologyABSTRACT
A glutamate racemase gene of Lactobacillus brevis ATCC 8287 was cloned into Escherichia coli TM93 by the phenotypic complementation of a phosphoenolpyruvate carboxylase deficiency on minimum agar medium containing D-glutamate. The gene was localized to a 1.4-kb HindIII-EcoRI DNA fragment and the total nucleotide sequence of the fragment was analyzed. The gene has typical promoter and SD sequences which appeared to function in E. coli. The deduced amino acid sequence of the enzyme had 276 amino acids and the molecular weight was calculated as 29,426. Two cysteine residues and their surrounding regions of the enzyme are homologous to those of other cofactor-independent racemases. The glutamate racemase was purified from recombinant E. coli to homogeneity and characterized. The enzyme required no cofactors for the activity, and retained its activity even in 2 M (300 g/l) L-glutamate.
Subject(s)
Amino Acid Isomerases/genetics , Coenzymes/metabolism , Lactobacillus/enzymology , Amino Acid Isomerases/isolation & purification , Amino Acid Isomerases/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Molecular Sequence DataABSTRACT
Twelve cDNA clones of Japanese hepatitis C virus (HCV) have been isolated from liver tissue of a single non-A, non-B hepatitis patient. These clones represented the non-structural domains of HCV. The degree of substitution in the nucleotide sequences and deduced amino acid sequences between these clones was 9.5 and 7.7%, respectively. This high level of substitution suggested that repeated infections of different HCVs may have occurred in the patient.
Subject(s)
Genes, Viral , Genetic Variation , Hepacivirus/genetics , Hepatitis C/microbiology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Viral , Genomic Library , Hepacivirus/isolation & purification , Humans , Japan , Liver/microbiology , Molecular Sequence Data , Sequence Homology, Nucleic AcidABSTRACT
In order to study the sperm-egg recognition mechanism on the surface of the plasma membrane, zonae were removed from mouse eggs by exposure to acidic conditions. Sperm binding to denuded eggs was then observed in the presence of various sugars. Among several carbohydrates tested, only glucosamine (GlcN) was found to increase the number of sperm bound to eggs while inhibiting sperm-egg fusion. The inhibition was reversible; when denuded eggs were transferred to a GlcN free medium, a high rate of polyspermy was observed.
Subject(s)
Glucosamine/pharmacology , Sperm-Ovum Interactions/drug effects , Animals , Carbohydrates/pharmacology , Cell Membrane/physiology , Female , Male , Mice , Ovum/physiology , Zona Pellucida/physiologyABSTRACT
Mouse eggs freed from zonae by chymotrypsin were mixed with sperm and pronuclear formation was observed. When anti-mouse sperm monoclonal antibody (OBF 13) from ascites fluid was added to the medium (at a final concentration of 0.05%), fertilization was significantly inhibited (9.7 +/- 4.3% compared to control 56.7 +/- 7.4%, P less than 0.01). This was based on the inhibition of sperm binding to the egg. However, when similar experiments were performed using zona-free hamster eggs, addition of the OBF 13 antibody caused no significant reduction in fertilization rate (91 +/- 7.1% compared to control 97 +/- 3.2%). It was also observed that binding of mouse sperm to hamster eggs was not inhibited by the antibody. It is therefore suggested that mouse sperm and mouse egg recognize each other in a species-specific manner.
Subject(s)
Antibodies, Monoclonal , Sperm-Ovum Interactions , Spermatozoa/immunology , Animals , Cricetinae , Female , In Vitro Techniques , Male , Mesocricetus , Mice , Species Specificity , Zona Pellucida/immunologyABSTRACT
The anti-mouse sperm monoclonal antibody OBF13 did not react with fresh epididymal sperm. However, when sperm were incubated in a culture medium capable of inducing capacitation, the entire head of the sperm began to react with this antibody. This change of reactivity was not observed when sperm were incubated in a Ca2+-free medium. The change of the reactivity to the antibody was studied in relation to the fertilizing ability of sperm as measured in an in vitro fertilization system; a significant correlation was observed between the appearance of head-stained sperm and fertilization rate.
