Tatehiko Tanaka

Differential expression of phosphatidylethanolamine-binding protein in rat hepatoma cell lines: Analyses of tumor necrosis factor-α-resistant cKDH-8/11 and -sensitive KDH-8/YK cells by two-dimensional gel electrophoresis

To determine intracellular factors influencing the sensitivity of cancer cells to tumor necrosis factor‐α (TNF‐α), we studied the expression of intracellular proteins in TNF‐α‐resistant cKDH‐8/11 and ‐sensitive KDH‐8/YK rat hepatoma cell lines using the technique of two‐dimensional gel electrophoresis (2‐DE). From the 2‐DE patterns, it was demonstrated that TNF‐α‐resistant cKDH‐8/11 cells had increased levels of protein of molecular weight (Mr) 22 500 and isoelectric point (pI) 5.2, compared with TNF‐α‐sensitive KDH‐8/YK cells. Therefore, we excised cyanogen bromide (CNBr) fragments of proteins in the spot for N‐terminal sequencing. Microsequencing for the CNBr fragments identified the protein as rat phosphatidylethanolamine‐binding protein. These findings suggest that the intracellular phosphatidylethanolamine‐binding protein could be one of the factors responsible for the resistance of cKDH‐8/11 cells to TNF‐α‐induced cell death.

A New Krüppel-Like Factor 1 Mutation (c.947G > A or p.C316Y) in Humans Causes β-Thalassemia Minor

Abstract Here we describe a Japanese patient with mild β-thalassemia (β-thal) with an intact β-globin gene but a new missense mutation of c.947G > A or p.C316Y in the erythroid Krüppel-Like Factor (KLF1) gene which is strongly associated with the expression of the β-globin gene. The association of the KLF1 mutation with β-thal, is here described. The p.C316Y mutation occurred at one of the cysteines that constitute the second zinc finger motif of KLF1, and would have changed the zinc finger conformation to impair the DNA binding properties or the promoter function of the β-globin gene. Our expression study found that the mutant KLF1 gene had a markedly negative effect on the β-globin gene expression, or 7.0% of that of its normal counterpart. A presumed heterozygous state, or equimolar presence of the mutant and normal KLF1s reduced the expression rate to 70.0% of the normal alone. This degree of the decrease may explain the very mild phenotype of the patient’s β-thal. Furthermore, the patient’s whole-exome analysis using next-generation sequencing revealed that the β-thal defect is caused by only this KLF1 gene mutation. The Hb A2 and Hb F levels that are frequently elevated in KLF1 mutations were elevated by 4.1 and 1.3%, respectively, in this case. The contribution to their elevation by KLF1: p.C316Y is uncertain.

Nondenaturing two‐dimensional electrophoretic analysis of loop‐sheet polymerization of serpin, squamous cell carcinoma antigen‐2

Two homologous serine proteinase inhibitors (serpins), squamous cell carcinoma (SCC) antigen‐1 and ‐2 were separated by nondenaturing two‐dimensional electrophoresis combined with immunostaining to acquire further information on these proteins under physiological conditions. Polymers of SCC antigen‐2 were detected in cytosolic extracts prepared from tumor tissues. The polymer formation of SCC antigen‐2 was apparently decreased and the SCC antigen‐2‐synthetic peptide binary complexes were newly formed by the addition of synthetic peptide with sequences corresponding to residues from P14 to P2 in the reactive center loop of SCC antigen‐2. On the other hand, the incubation with synthetic peptides having the sequence of the reactive center loop of SCC antigen‐1 or antithrombin had no effect on polymerization of SCC antigen‐2. These data suggest that the polymerization of SCC antigen‐2 may occur spontaneously in vivo by the loop‐sheet mechanism of serpin.

Characteristics of Japanase Thalassemia

Abstract Thalassemia is relatively rare in Japan. Of 387 β-thalassemia cases of Japanese, homozygotes for β+- and β0-thalassemias were twenty-two (5.7%) and one (0.26%), respectively. The rest (94%) were of β- thalassemia trait. Ten different kinds of β-thalassemia mutations comprised about 80% of all cases. Half of these “common” mutations were unique to Japanese and another half were possibly from abroad. Sixty-nine α-thalassemia cases were analyzed, in which HbH and α- thalassemia trait comprised nineteen and fifty, respectively. Most of the αo- and α+-thalassemia chromosomes were of Southeast Asian type (--SEA) and –α3.7 type, respectively. The frequency of α+- thalassemia as well as triplication of α-globin gene was high in northern Japan. Recent increase in the number of immigrants from Southeast Asia seems to raise the number of α-thalassemia found in Japan. They comprise at least 20% of the α-thalassemia. Six mutants classified into dominant-type β-thalassemia were found. All of them exhibited moderate anemia and marked anisopoikiocytosis. Heinz body varied in degree from copious to rare or even absent. Any mutation at initiation codon demonstrated marked microcytosis and erythremia. The breakpoint determination for large deletion-type thalassemia became feasible by estimation of gene dosage and PCR. Thus, the precise breakpoints for Filipino-type αo-thalassemia (--FIL) and Japanese type-2 δβ-thalassemia were disclosed. The genetic diagnosis for these thalassemias are now readily conducted by gap PCR. The characterization of several new large deletions is being carried out.

Oxidative Modification of Apolipoprotein E3 and Its Biological Significance

Apolipoprotein E (apoE), in very low density lipoprotein (VLDL), formed aggregates and lost its heparin-binding activity with lipid peroxidation by an oxidation system consisting of 101.μM ferrous sulfate in saline under aerobic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis of the aggregated apoE indicated the oxidation of basic amino acid residues and the intermolecular cross-linking of apoE may be caused by the formation of 4-hydroxy-2-nonenal (HNE), which reacts with e-amino groups of lysyl residues on apoE molecules. The presence of 1% heparin inhibited the oxidative modification of apoE to restore its heparin-binding activity. These findings suggest that the oxidative modification of apoE in VLDL causes the accumulation of lipid peroxides by the decrease in the rate of VLDL uptake via binding to heparin on the surface of cells. This maybe a possible mechanism of the deposit of oxidized lipids in the vascular system and of oxidized apoE in senile plaques in the brain of Alzheimer’s disease.

Two-Dimensional Affinity Electrophoresis: Its Application to Separation of Individual Immunoglobulins from Heterogeneous Antibodies

Abstract We have developed a two-dimensional affinity electrophoresis carrying in the first direction by isoelectric focusing (IEF) and in the second direction by affinity electrophoresis (AEP). Using Dnp- and Tnp-polyacrylamide conjugates as affinity ligands for AEP, rabbit anti-Dnp antibodies were separated. They consisted of several groups of monoclonal IgG families. Dissociation constants of the individual IgG spots to the Dnp- and Tnp-haptens calculated from the affinity patterns ranged from 2.0 × 10 -6 M to 1.1 × 10 -4 M and from 1.4 × 10 -5 M to 1.3 × 10 -4 M, respectively.

A Novel GγAγ(δβ)0-Thalassemia with a 27 kb Deletion

A new GγAγ(δβ)0-thalassemia (thal) was found in six unrelated Japanese individuals, and characterized by a method employing only polymerase chain reaction (PCR) and direct sequencing. This GγAγ(δβ)0-thal mutation has removed a fragment of about 27 kb of DNA, that starts approximately 2.8 kb downstream of the Aγ-globin gene and ends in the L1 repeat sequence, 7.0 kb downstream of the β-globin gene. The 5′ breakpoint is similar to that of the previously reported Japanese GγAγ(δβ)0-thal (called here Jpn type 1 for convenience). However, the 3′ endpoint is quite different. This new Japanese δβ-thal, designated as Japanese type 2 (Jpn type 2), shows a deletion rather similar to Turkish type 3 δβ-thal but with 5′ and 3′ breakpoints located inside the deletion of Turkish type 3. A mutation-specific gap PCR was designed to diagnose patients with the Jpn type 2 GγAγ(δβ)0-thal. The identified carriers exhibited a thalassemia minor.

Nucleotide sequence for cDNA of bovine mitochondrial ATP-dependent protease and determination of N-terminus of the mature enzyme from the adrenal cortex

We have determined the cDNA sequence encoding bovine mitochondrial ATP-dependent Lon protease. Since the 5′-end region of the cDNA was highly GC-rich and thus could not be amplified by the 5′-RACE method, a genomic DNA fragment containing an in-frame ATG was isolated and sequenced. The translated amino acid sequence contained 961 amino acids with a calculated molecular weight 106,665. Sequence similarities of the bovine enzyme to human and E. coli orthologs were 92 and 27%, respectively. The N-terminal amino acid sequence seemed to be a mitochondrial targeting signal. To determine the cleavage site of the signal sequence we analyzed the mature enzyme purified from bovine adrenocortical mitochondria. Analysis of CNBr-digested peptides revealed that the N-terminus was heterogeneous. We suggest that nonspecific aminopeptidase might remove several amino acids from the N-terminus after mitochondrial processing peptidase has cleaved Gly67–Leu68 or Leu68–Trp69.

A Coincidental Discovery of a New Stable Variant (Hb Hachioji or HBB: c.187C>T) in a Patient with Chronic Hemolytic Anemia of Unexplained Origin

Abstract We report a new hemoglobin (Hb) variant, Hb Hachioji (HBB: c.187C>T), which was detected in a 32-year-old male with hemolytic anemia. The proband had undergone splenectomy in his childhood after being diagnosed with hereditary spherocytosis (HS) with no clinical improvement. A recent study showed that Heinz bodies were frequently observed in his red cells, however, no abnormal band was separated by isoelectric focusing (IEF), and the isopropanol (instability) test was negative. Direct sequencing revealed that the proband was a heterozygous carrier of a novel mutation (GCT>GTT) at codon 62 of the β-globin gene, leading to an alanine to valine substitution. This variant was named Hb Hachioji. Characterization at the mRNA level by cDNA sequencing detected βHachioji mRNA, as well as βA mRNA. Subsequently, study of the proband’s family indicated that his father was a carrier of this Hb variant, although unexpectedly, the father was asymptomatic and clinically healthy. Oxygen affinity measurement of total Hb showed no alteration in the P50 and oxygen equilibrium curve. The presence of Hb Hachioji was confirmed by mass spectrometry (MS). Hb Hachioji comprised approximately 50.0% of the total Hb and was a stable variant. The phenotypic discrepancy between these two carriers suggests that Hb Hachioji may not be associated with the hemolytic involvement in the proband. P4.2Nippon, which is the primary cause of most cases of Japanese HS, was absent in the proband’s parents. The coexistence of glucose-6-phosphate dehydrogenase (G6PD) deficiency was ruled out. Thus, the cause of hemolytic involvement in this patient remains unclear.