Yasuhiro Yamashiro

Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix

The previously isolated cDNA encoding human adenylate kinase (AK) isozyme 3 was recently renamed AK4. Consequently, human AK3 cDNA remains to be identified and we have little information about the functional relationship between human AK3 and AK4. In pursuit of the physiological roles of both the AK3 and AK4 proteins, we first isolated an authentic human AK3 cDNA and compared their expression. Nucleotide sequencing revealed that the cDNA encoded a 227-amino-acid protein, with a deduced molecular mass of 25.6 kDa, that shares greater homology with the AK3 cDNAs isolated from bovine and rat than that from human. We named the isolated cDNA AK3. Northern-blot analysis revealed that AK3 mRNA was present in all tissues examined, and was highly expressed in heart, skeletal muscle and liver, moderately expressed in pancreas and kidney, and weakly expressed in placenta, brain and lung. On the other hand, we found that human AK4 mRNA was highly expressed in kidney, moderately expressed in heart and liver and weakly expressed in brain. Western-blot analysis demonstrated expression profiles of AK3 and AK4 that were similar to their mRNA expression patterns in each tissue. Over expression of AK3, but not AK4, in both Escherichia coli CV2, a temperature-sensitive AK mutant, and a human embryonic kidney-derived cell line, HEK-293, not only produced significant GTP:AMP phosphotransferase (AK3) activity, but also complemented the CV2 cells at 42 degrees C. Subcellular and submitochondrial fractionation analysis demonstrated that both AK3 and AK4 are localized in the mitochondrial matrix.

Characterization of β-Thalassemia Mutations Among the Japanese

Characterization of beta-thalassemia mutations were attempted for 29 Japanese families clinically diagnosed as having beta-thalassemia. Following the identification of a mutation by cloning and sequencing, all families were screened for this particular mutation, using biotinylated allele-specific oligonucleotide probes. Seven different mutations were detected in 17 families: Six families had the frameshift mutation at codons 41/42, resulting from a 4 nucleotide deletion (TTCTTT----TT); four had the deletion at codons 127/128 (CAGGCT----CCT); and three had the TATA box mutation at nucleotide -31 (A----G). Four additional families had mutations at codon 24 (GGT----GGA), codon 26 (GAG----AAG), IVS-II-654 (C----T) and codon 110 (GTG----CCG), respectively. The newly discovered deletion mutation at codons 127/128, and mutations at nucleotide -31, and at codon 110 are peculiar to Japanese, and have not been found in any other ethnic group. The haplotypes of the beta-globin gene cluster were also determined. Some of the haplotypes and beta-thalassemia mutations are identical to those reported in the Chinese population. However, it is noteworthy that nearly half of the beta-thalassemia mutations were unique to Japanese.

Two β-Thalassemia Mutations in Japan: Codon 121 (Gaa→Taa) and IVS-I-130 (G→C)

(1992). Two β-Thalassemia Mutations in Japan: Codon 121 (Gaa→Taa) and IVS-I-130 (G→C) Hemoglobin: Vol. 16, No. 4, pp. 295-302.

Quantitative analysis of telomerase activity and telomerase reverse transcriptase expression in renal cell carcinoma

Abstract To investigate the relationship between the telomerase activity levels and clinicopathological features of tumors, we quantified the telomerase activities of 23 renal cell carcinomas (RCCs) and four non-cancerous tissues, using a modified telomeric repeat amplification protocol assay, and assessed the hTERT mRNA levels of these samples by reverse transcription-polymerase chain reaction analysis. Elevated levels of telomerase activity had correlation with tumor stages as well as the degree of nuclear grades. Our findings suggested that telomerase activity is a useful indicator for tumor aggressiveness in RCCs. However, hTERT mRNA levels in RCCs had no correlation with nuclear grades and tumor stages. The telomerase activities and the hTERT mRNA levels in cancer cells were not always in parallel. These results suggested that telomerase activity is regulated in a posttranscriptional manner as well as a post-translational manner in tumor cells.

Molecular cloning and characterization of the gene encoding human NeuroD

NeuroD is a basic helix-loop-helix transcription factor that binds to an E-box sequence, CANNTG, in the target gene promoter region. The gene encoding NeuroD is expressed specifically in brain, intestine, and pancreatic cells and plays a pivotal role in tissue-specific differentiation. To investigate the regulation of human NeuroD gene expression, we isolated and sequenced a genomic DNA containing two exons and flanking regions of the NeuroD gene. The sizes of exons 1 and 2 were 168 and 2404 bp, respectively. CAT reporter analysis showed that the elements responsible for basal promoter activity lay in the proximal 408-bp region. Cotransfection of the CAT reporter plasmids with a NeuroD expression plasmid resulted in 5-fold enhancement of the CAT activity, indicating an autoregulatory mechanism is involved in human NeuroD gene expression.

cDNA cloning and chromosomal mapping of the gene encoding adenylate kinase 2 from Drosophila melanogaster.

As a step toward understanding of the role of adenylate kinase (AK) in energy metabolism, we analyzed this enzyme in Drosophila melanogaster. The enzyme activities of all three AK isozymes were determined in cell-free extracts of flies, and their proteins were detected by Western blot analysis using polyclonal antibodies against the mammalian isozymes. A cDNA encoding adenylate kinase was isolated from D. melanogaster cDNA library. The cDNA encodes a 240-amino acid protein, which shows high similarity to bovine, human and rat AK2, and hence was named DAK2. Preliminary subcellular fractionation analysis indicated that DAK2 is localized in both cytoplasm and mitochondria. In situ hybridization to salivary gland polytene chromosomes revealed that the Dak2 gene is located at 60B on the right arm of the second chromosome.

Oxidation Status of β-Thalassemia Minor and Hb H Disease, and Its Association with Glycerol Lysis Time (GLT50)

Abstract β-Thalassemia (β-thal), especially β-thalassemia major (β-TM), is reported to be related to reactive oxygen species (ROS) and enhanced oxidation status. It is reflected by increased malondialdehyde (MDA), by membrane lipid peroxidation and decreased by the newly developed total antioxidant capacity (TAC). However, there is less evidence for β-thal minor and Hb H (β4) disease on its association with oxidation status. On the other hand, hemolysis by glycerol lysis time (GLT50) is invariably prolonged in thalassemia. The reason for the prolongation of GLT50 is not well understood. The aim of this study was to investigate the oxidation state in β-thal minor and Hb H disease and to find out the association of the oxidation with the prolongation of GLT50. Blood samples from 39 subjects (33 with β-thal minor, six with Hb H disease) were collected from individuals living in Japan. The clinical screening tests and molecular identification of the thalassemias were performed. Malondialdehyde and TAC were measured using spectrophotometric analyses. In β-thal minor and Hb H disease, the plasma MDA level was significantly elevated and the TAC reduced. A highly reversed correlation between MDA and TAC was noted. Their GLT50 levels were evidently prolonged, and the GLT50 has significant correlations with MDA and TAC. β-Thalassemia minor and mild Hb H disease are evidently in a milieu of reduced redox state, and GLT50 prolongation in β-thal minor and Hb H disease has a close correlation with the oxidation state, possibly by oxidative impairment of the membrane protein of the red cell.

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.

A New β0-Thalassemia Mutation (codon 102, AAC>ATCAC) in Coexistence with a Heterozygous P4.2 Nippon Gene

A new β-thalassemia (β-thal) frameshift mutation was found at codon 102 (AAC>ATCAC) in a 17-year-old Japanese male and his 14-year-old sister. Both demonstrated a more severe phenotype than the usual β-thal minor with mild hemolytic involvement. No mRNA derived from the thalassemic allele, or βTmRNA, was detected in the sequencing analysis of the whole mRNA (cDNA). However, the βTmRNA from the whole βmRNA was specifically amplified by amplification refractory mutation system (ARMS), and was actually found to be present. Furthermore, quantitative polymerase chain reaction (q-PCR) demonstrated a negligible amount of βTmRNA. Thus, their more severe phenotype was not caused by the “dominant type” β-thal in which a considerable amount of the βTmRNA would be expected. In fact, our proband had a total βmRNA level that was mostly normal. Thus, the cause of a β-thal phenotype by the frameshift mutation was ascribed to the reduced amount of mRNA. We further searched for the cause of their severe phenotype. However, factors that exacerbated the phenotype of β-thal, such as α-globin gene triplication, coexisting iron deficiency and infection were not found. Finally, we noticed that the red cell morphology revealed ovalocytosis and small numbers of stomatocytes that were seen in the hereditary spherocytosis (HS), especially by P4.2 mutations. The sequence of the P4.2 gene disclosed heterozygous P4.2 Nippon, or missense mutation at codon 142 (GCT>ACT) on exon 3, the most common mutation of Japanese HS. Frequent mutations of other membrane proteins, Band 3 and ankyrin that are common cause of HS in the Japanese population, other than P4.2, were not detected. When HS by P4.2 Nippon develops it is homozygous, and no P4.2 protein is observed in sodium dodecilsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), while in our case the amount of the P4.2 was almost normal in the SDS-PAGE. However, there are several reports that revealed more severe phenotypes of β-thal by the coexisting abnormality of membrane protein. It is uncertain, but the presence of heterozygous P4.2 Nippon may be associated with the exacerbation of the phenotype of β-thal minor.

A patient with acute lymphoblastic leukaemia presenting with an extremely high level (21.0%) of HbA1c:

A 52-year-old Japanese woman was referred to our hospital because of fever and coxalgia. She had a white blood cell count of 241 × 102/μL with 59.6% blasts, which had a high nuclear/cytoplasmic ratio and variably condensed nuclear chromatin. Flow cytometry and chromosomal analysis of bone marrow cells indicated positive findings of CD10, CD19, CD34, HLA-DR antigens and t(9; 22)(q34; q11.2), respectively. No rearrangements of bcr/abl in peripheral blood neutrophils were found by fluorescence in situ hybridization, suggesting that she had B-acute lymphoblastic leukaemia with Ph chromosome. Blood glucose and HbA1c (glycated haemoglobin) levels on admission were 23.4 mmol/L and 21.0%, respectively. The results of 1.5 anhydro-d-glucitol and glycoalbumin tests revealed that she certainly had diabetes mellitus (DM). Insulin therapy was initiated. Her high level of HbA1c also suggested the possibility that the patient suffered from haemoglobinopathies in addition to DM. Sequencing analyses of α1-, α2- and β-globin genes were all normal. The patient achieved complete remission (CR) by one month after her first course of chemotherapy, and the HbA1c level decreased to 10.4% following insulin therapy and chemotherapy, which were initiated when she attained CR. Her extremely high HbA1c level was due mainly to DM. Also, suppression of erythropoiesis by proliferation of leukaemic cells and latent iron deficiency might have partially contributed to the increased HbA1c. This could result in a transient but extremely high HbA1c level. To our knowledge, this is the first report of an acute leukaemia patient who expressed an extremely high level of HbA1c.