Chisato Namikawa

Duplication of the MHC-linked Xenopus complement factor B gene

We have previously reported the molecular cloning of the mammalian major histocompatibility complex (MHC) class III gene, complement factor B (Bf) from Xenopus laevis, and linkage of the gene to the frog MHC. Here, we estimated the copy number of the Xenopus Bf gene by genomic Southern blotting analysis and demonstrated that Xenopus laevis has two copies of the Bf gene. Both genes co-segregated with the MHC-linked HSP70 genes among 19 offspring of an f/r × f/r cross, indicating a close linkage of the two Bf genes to the frog MHC. Both genes are transcribed and contain open reading frames. When compared with the previously determined cDNA sequence (Xenopus Bf A), the predicted amino acid sequence of the second cDNA species (Xenopus Bf B) shows 82% overall identity. Polymerase chain reaction analysis indicated that all of the partially inbred frogs with the f, r, g, and j MHC haplotypes, as well as 12 outbred frogs tested have both Bf genes, suggesting that the duplicated Bf genes are stable genetic traits in Xenopus laevis.

Human erythrocyte multicatalytic proteinase: Activation and binding to sulfated galacto- and lactosylceramides

Chymotrypsin-like activity of multicatalytic proteinase (MCP) purified from human erythrocytes was selectively activated 2.5--3.5-fold by sulfated glycolipids such as galactosylceramide sulfate (SM4) and lactosylceramide sulfate (SM3) but not by other glycolipids including galactosylceramide (GalCer), lactosylceramide (LacCer), GD1a, GM1 and GM3. Heparin also selectively activated trypsin-like activity 2.5-fold, while other mucopolysaccharides did not. This proteinase molecule bound specifically and with high affinity to both SM4 and SM3, but not to GalCer, LacCer and GM3. The binding of SM4 and SM3 to the enzyme molecule was also confirmed by thin layer chromatography.

Purification and characterization of α1-thiol proteinase inhibitor and its identity with kinin- and fragment 1·2-free high molecular weight kininogen

1. alpha 1-Thiol proteinase inhibitor (alpha 1 TPI) purified from outdated human plasma was a glycoprotein with Mr 83,000 and was composed of heavy and light chains held together with a disulfide bond. 2. The data on amino acid composition, amino terminal sequence of the light chain and carboxyl terminal sequences of the heavy and light chains indicate that alpha 1 TPI is identical with kinin- and fragment 1.2-free HMW kininogen. 3. Purified human plasmin generated a derivative having the same molecular weight (Mr 83,000), same subunit structure (heavy and light chains) and same inhibitory capacity as alpha 1 TPI from HMW kininogen and kinin-free HMW kininogen. This indicated the possibility that alpha 1 TPI is derived from HMW kininogen by plasmin.

Recurrence of osteogenesis imperfecta because of paternal mosaicism: Gly862→Ser substitution in a type I collagen gene (COL1A1)

We determined that two siblings with type III osteogenesis imperfecta (OI) had the same single base substitution that converted the codon for glycine (Gly) 862 to a codon for serine (Ser) in exon 44 of the α1 chain of the type I (α 1(I)) collagen gene (COL1A1). The mutation was also detected in various paternal tissues; the mutant allele accounted for approximately 11% of the COL1A1 alleles in blood, 24% of those in fibroblasts, and 43% of those in sperm determined by allele-specific colony hybridization using amplified genomic sequences. These findings demonstrate that germ-line mosaicism in the phenotypically normal father is responsible for the recurrence. There is a cluster of serine substitutions for Gly (Gly832, Gly844 and Gly901) which is associated with nonlethal phenotypes and which is located between two lethal clusters. In the cases studied here, a Gly862→Ser mutation was identified that is located inside the nonlethal cluster.

A procedure for large scale purification of human plasma amyloid P component

Plasma amyloid P component (PAP) is a glycoprotein with a characteristic pentagonal ultrastructure composed of five identical subunits [l-4]. It is indistinguishable in terms of antigenicity, molecular weight, ultrastructure and the partial amino acid sequence from amyloid P component (AP) which is found in all forms of amyloid [l-6]. PAP also has approximately 60% homology of amino acid sequence with human C-reactive protein (CRP), a classical acute phase reactant [6-81. It is thought that these two proteins diverged from one original protein during evolution. Although the isolation and characterization of PAP from plasma and amyloid protein (AP) from amyloid tissues [2-61 have been studied extensively, and recently, the selective binding of aggregated AP to fibronectin and Cd-binding protein in the presence of calcium ion has been found in human serum [9], their physiological role and pathological significance remain unknown. We describe a new procedure employing metal chelate Sepharose and Red Sepharose column chromatographies for the isolation of PAP in high yield from a large quantity of human plasma, and the characterization of this material.