Subject(s)
Critical Illness , Eczema , Infant , Humans , Child , Multiple Organ Failure/etiology , Intensive Care Units, Pediatric , Eczema/complicationsABSTRACT
Transcription factors of the NFAT family are thought to play a major role in regulating the expression of cytokine genes and other inducible genes during the immune response. The role of NFAT1 was investigated by targeted disruption of the NFAT1 gene. Unexpectedly, cells from NFAT1 -/- mice showed increased primary responses to Leishmania major and mounted increased secondary responses to ovalbumin in vitro. In an in vivo model of allergic inflammation, the accumulation of eosinophils and levels of serum immunoglobulin E were increased in NFAT1 -/- mice. These results suggest that NFAT1 exerts a negative regulatory influence on the immune response.
Subject(s)
DNA-Binding Proteins/physiology , Hypersensitivity/immunology , Immunity , Lymphocyte Activation , Nuclear Proteins , Transcription Factors/physiology , Amino Acid Sequence , Animals , Antigens, Protozoan/immunology , Cell Line , Cytokines/biosynthesis , DNA-Binding Proteins/genetics , Eosinophils/immunology , Gene Targeting , Immunoglobulin E/biosynthesis , Immunologic Memory , Leishmania major/immunology , Mice , Molecular Sequence Data , NFATC Transcription Factors , Ovalbumin/immunology , T-Lymphocytes/immunology , Transcription Factors/geneticsABSTRACT
In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR1 allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NR1-/-). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1-/- mice, demonstrating that there is an interdependence of subunit expression. NR1-/- mice died 8-15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1-/- mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death.
Subject(s)
Mice, Knockout/anatomy & histology , N-Methylaspartate/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Animals , Calcium/physiology , Cells, Cultured , Cerebellum/physiology , Gene Expression , Glutamates/pharmacology , Hippocampus/physiology , Mice , Mutagenesis, Insertional , Nervous System/embryology , RNA, Messenger/genetics , Synaptic TransmissionABSTRACT
Oxidation of ribosomal protein L12 with hydrogen peroxide converts the three methionine residues to methionine sulfoxide. The oxidized protein has a decreased ability to bind to ribosomes, interact with ribosomal protein L10, be precipitated by L12 antiserum, and serve as substrate for the acetylating enzyme that converts L12 to L7. Full activity of L12 is regained when the protein is reduced with 2-mercaptoethanol. Sedimentation equilibrium analysis shows that oxidation of the methionine residues in L12 causes the conversion of the protein from the dimer to the monomer form, and the results indicate that the dimer is the active form of the protein in the above reactions.
Subject(s)
Escherichia coli/metabolism , Hydrogen Peroxide/pharmacology , Methionine , Ribosomal Proteins/metabolism , Alkylation , Iodoacetates/pharmacology , Mercaptoethanol/pharmacology , Molecular Weight , Oxidation-Reduction , Precipitin Tests , Protein Biosynthesis/drug effectsABSTRACT
A procedure is described for the isolation of the nervous system-specific protein designated 14-3-2 from rat brain. The methods utilized are salt precipitation, DEAE-cellulose ion exchange chromatography, Sephadex G-150 gel filtration, and column isoelectric focusing. The native 14-3-2 protein has an isoelectric point of 4.7 in the absence of denaturing agents and 5.0 in the presence of 2.0 M urea. The protein, as isolated, appears homogeneous since it migrates as a single band on Tris-glycine (pH 8.9), sodium dodecyl sulfate (pH 7.2), and 8 M urea (pH 4.0) polyacrylamide gels. Sedimentation velocity and equilibrium data indicate a homogeneous component of molecular weight 78,000. Sedimentation of 14-3-2 in 6 M guanidine HCl containing 0.02% glutathione yielded a molecular weight of 39,000, indicating the dimeric nature of the protein as isolated. The rat brain protein seems to be composed of one subunit type, since polyacrylamide gel electrophoresis in 8 M urea yields a single protein component. Sodium dodecyl sulfate gel electrophoresis of rat brain 14-3-2 produced one sharp band with a relative mobility corresponding to a molecular weight of 48,000. Specific anti-14-3-2 serum has been prepared from both New Zealand white rabbits and goats. Rat 14-3-2 is very similar in amino acid composition to the beef brain protein and to antigen alpha. The antigenic properties of rat and beef 14-3-2 are also similar, since beef 14-3-2 antiserum reacts well with rat 14-3-2 and vice versa. Electrophoretic mobilities of denatured rat and beef 14-3-2 (0.1% sodium dodecyl sulfate and 8 M urea) are identical. Despite these similarities the two proteins are completely resolved on Tris-glycine gels. The sedimentation behavior of the beef and rat proteins are also different, indicating a difference in the association state and conformation of the two preparations.
