Makoto Iwane

Stabilizing basic fibroblast growth factor using protein engineering

Using site directed mutagenesis, each of the four cysteines present at amino acid residues 26, 70, 88, and 93 of the mature protein of human basic fibroblast growth factor (bFGF) was individually changed to serine. The biological activity and heparin binding ability was retained when the serine was substituted for the cysteine residue at either 70 or 88 of the bFGF protein. This finding indicates that the cysteines at these positions are not essential for expressing biological activity. The substitution of the residues at these positions, especially at position 88, reduced the heterogeneity recognized as several peaks of bFGF eluted from a heparin affinity column, even after oxidation with hydrogen peroxide, suggesting that the cysteines at these positions are exposed to the surface of the molecule to form disulfide bonds that induce heterologous conformations. Furthermore, under acidic conditions, these modified bFGFs are revealed to be more stable in maintaining their activity. These facts suggest that this protein has been successfully modified by protein engineering.

Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA.

The expression of human basic fibroblast growth factor (bFGF) cDNA in mouse BALB/c 3T3 clone A31 cells induced morphological transformation. These transformed cells grew well and reached more than a sixfold-higher saturation density than parental A31 cells even in serum-free medium. They were able to form colonies in soft agar. The phenotypic alteration in the transformed cells was reversed by the addition of anti-human bFGF antibodies to the medium. These results suggest that the cellular transformation mediated by bFGF is caused by autocrine stimulation with secreted bFGF molecules.

Increase of basic fibroblast growth factor immunoreactivity and its mRNA level in rat brain following transient forebrain ischemia

SummaryWe examined the time course of basic fibroblast growth factor (bFGF) immunoreactivity and its mRNA level mainly in the hippocampus after transient forebrain ischemia using immunohistochemistry, enzyme immunoassay (EIA), Western blot analysis and in situ hybridization. Neuronal death in the hippocampal CA1 subfield was observed 72 h after 20 min of ischemia. The number of bFGF-immunoreactive(IR) cells increased 48 h–5 days after ischemia in all hippocampal regions. At 10 and 30 days, the bFGF-IR cells in the CA1 subfield had further increased in numbers and altered their morphology, enlarging and turning into typical reactive astrocytes with the advancing neuronal death in that area. In contrast, the number of bFGF-IR cells in other hippocampal regions had decreased 30 days after ischemia. The EIA study showed a drastic increase in bFGF levels in the hippocampus 48 h after ischemia (150% of that in normal rat) which was followed by further increases. In Western blot analysis, three immunoreactive bands whose molecular weights correspond to 18, 22 and 24 kDa were observed in normal rat and ischemia increased all their immunoreactivities. In the in situ hybridization study of the hippocampus, bFGF mRNA positive cells were observed in the CA1 subfield in which many bFGF-IR cells existed after ischemia. These data demonstrate that transient forebrain ischemia leads to an early and strong induction of bFGF synthesis in astrocytes, suggesting that the role of bFGF is related to the function of the reactive astrocytes which appear following brain injury.

Expression of cDNA encoding human basic fibroblast growth factor in E. coli

The cDNA encoding human basic fibroblast growth factor was expressed in E. coli under the control of trp promoter. Bacterially synthesized hbFGF was highly purified using a heparin affinity HPLC column. By this chromatography, hbFGF was eluted as four distinct forms, which were indistinguishable by SDS polyacrylamide gel electrophoresis, amino acid composition, and partial terminal sequence analysis. These molecules stimulated the growth of fibroblasts and endothelial cells although their specific activities varied. The angiogenesis activity of these molecules was also confirmed.

TAK-147, an acetylcholinesterase inhibitor, increases choline acetyltransferase activity in cultured rat septal cholinergic neurons

TAK-147, a potent acetylcholinesterase (AChE) inhibitor, potentiated choline acetyltransferase (ChAT) activity in cultured rat septal cholinergic neurons in a concentration-dependent manner with an EC50 value of 4.47 nM. Donepezil, another potent AChE inhibitor, also increased ChAT activity although its potency was less than that of TAK-147. Other AChE inhibitors (rivastigmine, tacrine, physostigmine and neostigmine) showed no effect. The effects of TAK-147 were greater in the presence of NGF, suggesting a synergistic action of TAK-147 and NGF. TAK-147 and donepezil showed high affinity for sigma receptors, whereas tacrine and physostigmine did not. Haloperidol and ifenprodil, high-affinity sigma ligands, potently enhanced ChAT activity in the septal neurons. These results suggest that TAK-147 may have neurotrophic activity on central cholinergic neurons, not via AChE inhibition but possibly via an effect on tau receptors.

Upregulation of fibroblast growth factor-receptor messenger RNA expression in rat brain following transient forebrain ischemia

Recently, we demonstrated that transient forebrain ischemia in rats leads to an early and strong induction of basic fibroblast growth factor (bFGF) synthesis in astrocytes in the injured brain regions. In this study, in order to clarify the targets of such raised endogenous bFGF levels, the messenger RNA (mRNA) expression of its receptors (flg and bek) at in the hippocampus following transient forebrain ischemia induced by four-vessel occlusion for 20 min was investigated using an in situ hybridization technique. Transient forebrain ischemia induced an increase in the number of flg mRNA-positive cells from an early stage (24 h after ischemia) in the hippocampal CA1 subfield where delayed neuronal death occurred later (48–72 h after ischemia). This increase became more marked with the progression of neuronal death and was still evident in the same area 30 days later. The time course of the appearance and distribution pattern of flg mRNA-positive cells in the CA1 subfield were quite similar to those of bFGF mRNA-positive cells. On the other hand, in situ hybridization for bek mRNA showed only slight and transient (observed 72 h and 5 days after ischemia) increases in the number of mRNA-positive cells in the CA1 subfield following ischemia. The use of in situ hybridization and glial fibrillary acidic protein immunohistochemistry in combination demonstrated that the cells in the CA1 subfield that exhibited ischemia-induced flg or bek mRNA expression were astrocytes. These data indicate that transient forebrain ischemia induces upregulation of fibroblast growth factor-receptor expression, accompanied by increased bFGF expression in astrocytes, and suggest that the increased astrocytic bFGF levels in injured brain regions act on the astrocytes via autocrine systems and are involved in the development and maintenance of astrocytosis.

Production, purification and characterization of biologically active recombinant human nerve growth factor

The human NGF gene was isolated and inserted downstream from murine leukemia virus LTR in a plasmid having dihydrofolate reductase cDNA. The expression plasmid was introduced into CHO cells. Selection of the transformants for the resistance to methotrexate gave a CHO cell line which produced human NGF at a level of 4 mg/L in the culture medium. The recombinant human NGF was purified to near homogeneity from the culture supernatant. The NH2-terminal amino acid sequence, the COOH-terminal amino acid (Ala), and the amino acid composition of the human NGF were identical to those deduced from the nucleotide sequence of the human NGF gene. The recombinant human NGF was composed of 120 amino acid residues. Three disulfide linkages were determined to be Cys15-Cys80, Cys-58-Cys108, and Cys68-Cys110; the locations were identical to those in the mouse 2.5S NGF molecule. The specific biological activity of the recombinant human NGF was comparable with that of authentic mouse 2.5S NGF as determined by stimulation of neurite outgrowth from PC12 cells.

Purification and characterization of biologically active recombinant human neurotrophin-3 produced by expression of a chimera gene in Chinese hamster ovary cells

In order to obtain high-level expression of recombinant human neurotrophin-3 (NT-3), we constructed several types of expression plasmids and examined several cell lines for expression of the human NT-3 gene. The highest level production of the recombinant protein was attained in Chinese hamster ovary cells transfected with an expression plasmid that contains a chimera gene encoding the human nerve growth factor (NGF) prepro-region and human NT-3 mature-region under control of a murine leukemia virus-derived long terminal repeat (MuLV-LTR). This cell line can produce more than 1 mg recombinant human NT-3/1 conditioned medium. The recombinant protein was purified to apparent homogeneity with a cation exchange column, a gel filtration column and a reversed-phase HPLC column with a recovery of about 30%. The purified NT-3, at a concentration as low as 0.2 ng/ml, induced neurite out-growth in neurons prepared from 8-day-old chick embryonic dorsal root ganglia; however, it showed little neurotrophic effect on rat PC12 pheochromocytoma cells, which are known to be NGF-responding cells. In addition, this protein promoted colony formation by human peripheral blood lymphocytes in soft agar culture.