Masahiro Kawabata

Presence of macrophage migration inhibitory factor (MIF) in ependyma, astrocytes and neurons in the bovine brain☆

We investigated the immunohistochemical localization of a cytokine macrophage migration inhibitory factor (MIF) in the bovine brain. MIF was present in the ependymal cell linings of the cerebral ventricles throughout. Double immunostaining of the section with anti-glial fibrillary acidic protein (GFAP) antibody and with anti-MIF antibody showed that the astrocytes present in subependymal layer were immunoreactive for MIF. In the hippocampus, the pyramidal cells in the CA3 and CA4 subfields and the granule cells of the dentate gyrus were immunoreactive. The bundles of mossy fibers were stained along their projections to CA3 and CA4 regions. The nuclei of the subpopulation of these MIF-immunoreactive cells were also immunostained. These results indicated the widespread distribution of a cytokine, MIF, in the bovine brain and suggested the possibility that MIF might play additional roles than a proinflammatory mediator role in the brain.

Sequence analysis and expression of a novel mouse homolog of Escherichia coli recA gene.

Escherichia coli recA and its yeast homologs RAD51 and DMC1 play crucial roles in mitotic and/or meiotic recombination and in repair of double-strand DNA breaks. We have identified a murine novel recA-like gene (MmTRAD). The predicted 329 amino acid protein showed significant homology to mouse Rec2, Rad51, Dmc1 (or Lim15) and E. coli RecA. Northern blot analysis revealed that MmTRAD was ubiquitously transcribed in various tissues.

A novel serpin-like protein, B-43, exists in both neurons and astrocytes: an immunohistochemical study in the parietal region of the bovine brain

The presence of a novel member of serine proteinase inhibitor, B-43, was immunohistochemically indicated in both neurons and astrocytes in the parietal region of the bovine brain. B-43-like immunoreactivity was detected in pyramidal cells in the cortex and GFAP-positive astroglial cells in the white matter. The processes of B-43 may play a cooperative role with glia-derived nexin/protease nexin-1 and alpha 1-antichymotrypsin in the brain.

Cloning of a serine proteinase inhibitor from bovine brain: expression in the brain and characterization of its target proteinases.

A cDNA encoding of the serine proteinase inhibitor (serpin), B-43, was cloned from the cDNA library of the bovine brain. It encoded 378 amino acids, and the MW of the protein was estimated to be 42.6 kDa, which is consistent with that of the native B-43 purified from the bovine brain. The homology search revealed that B-43 belongs to the ovalbumin branch of the serpin superfamily. Among them, B-43 was most homologous to human placental thrombin inhibitor (PI-6) and its murine counterpart, with the amino acid identity of 76% and 71%, respectively. Northern blot analysis showed that the size of the transcript was 1.4 kb, and that the expression of B-43 in the bovine brain varied depending on the brain regions, i.e. a lower level of expression was observed in the cerebral cortex and the hippocampus compared to the level of expression that was observed in the medulla oblongata. [35S]-labeled B-43 protein was synthesized in vitro by using a rabbit reticulocyte lysate system, which formed complexes with proteinases such as thrombin, trypsin, alpha-chymotrypsin, and 7S nerve growth factor (NGF), but not with urokinase or plasmin. These results, together with the immunohistochemical localization of B-43 in astrocytes and in some neurons which was observed in the previous study suggest that B-43 may be involved in the regulation of serine proteinases present in the brain or extravasated from the blood.

Purification of a novel serpin-like protein from bovine brain

We purified a novel serine proteinase inhibitor (serpin)-like protein from the bovine brain and named it B-43 from its molecular mass, 43 kDa. A cleaved peptide from B-43 was copurified with the native B-43. Partial amino acid sequencing of the purified B-43 showed that this protein was homologous to glia-derived nexin/protease nexin-1 (GDN/PN-1), plasminogen activator inhibitor 2, leukocyte elastase inhibitor (LEI) and placental thrombin inhibitor (PTI) among the serpins. Although B-43 had a similar amino acid composition to these serpins, the biochemical features of B-43 were different from them. B-43 did not form sodium dodecyl sulfate (SDS)-resistant serpin-proteinase complexes with thrombin, urokinase, pancreatic elastase and plasmin, suggesting that these proteinases were not the targets of B-43. In contrast to GDN/PN-1, B-43 did not have an affinity for heparin. B-43, having different biochemical properties from GDN/PN-1, appears to be an additional serpin expressed in the brain.