Tomoyuki Yamasaki

Phosphofructokinase Deficiency Past, Present and Future

Phosphofructokinase deficiency (Tarui disease, glycogen storage disease VII, GSD VII) stands out among all the GSDs. PFK deficiency was the first recognized disorder that directly affects glycolysis. Ever since the discovery of the disease in 1965, a wide range of biochemical, physiological and molecular studies of the disorder have greatly expanded our understanding of the function of normal muscle, general control of glycolysis and glycogen metabolism. The studies of PFK deficiency vastly enriched the field of glycogen storage diseases, as well as the field of metabolic and neuromuscular disorders. This article cites a historical overview of this clinical entity and the progress that has been made in molecular genetic area. We will also present the results of a search in-silico, which allowed us to identify a previously unknown sequence of the human platelet PFK gene (PFK-P). In addition, we will describe phylogenetic analysis of evolution of PFK genes.

Cloning of human muscle phosphofructokinase cDNA

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3′‐untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5′‐untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C‐ and N‐halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).

Three new mutations in the hepatocyte nuclear factor-1α gene in Japanese subjects with diabetes mellitus: clinical features and functional characterization

Aims/hypothesis. Mutations in the hepatocyte nuclear factor-1α gene are a common cause of the type 3 form of maturity-onset diabetes of the young. We examined the clinical features and molecular basis of hepatocyte nuclear factor-1α (HNF-1α) diabetes. Methods. Thirty-seven Japanese subjects with early onset Type II (non-insulin-dependent) diabetes mellitus and 45 with Type I (insulin-dependent) diabetes mellitus were screened for mutations in this gene. Functional properties of mutant HNF-1α were also investigated. Results. Three new mutations [G415R, R272C and A site of the promoter ( + 102G-to-C)] were found. Insulin secretion was impaired in the three subjects. Insulin and glucagon secretory responses to arginine in the subject with the R272C mutation were also diminished. Molecular biological studies indicated that the G415R mutation generated a protein with about 50 % of the activity of wild-type HNF-1α. The R272C mutation had no transactivating or DNA binding activity and acted in a dominant negative manner. The + 102 G-to-C mutation in the A site of the promoter activity was associated with an increase in promoter activity and it had 42–75 % more activity than the wild-type sequence. Conclusion/interpretation. Mutations in the HNF-1α gene may affect the normal islet function by different molecular mechanisms. [Diabetologia (1999) 42: 621–626]

Structure of the entire human muscle phosphofructokinase-encoding gene: a two-promoter system

We have recently shown that three types (A,B, and C) of mRNA species are transcribed from a single gene encoding human muscle phosphofructokinase (hPFK-M) through alternative splicing [Nakajima et al., Biochem. Biophys. Res. Commun. 166 (1990) 637-641]. To determine its complete structure and elucidate the mechanism of alternative RNA splicing, we isolated the hPFK-M gene, which spans about 30 kb, and contains 24 exons. Transcription start points were observed for both exon 1 and exon 2 by S1 nuclease protection assay and primer extension. Motifs of an Sp1-binding site were observed in the upstream region of exon 1 (promoter 1). A TATA-box-like sequence and a CAAT-box-like sequence were identified in the upstream region of exon 2 (promoter 2). Reporter assay revealed that the promoter 1 region was functional both in HeLa cells and myoblastic clonal cells, and that the promoter 2 region was active only in myoblastic cells. Motifs of M-CAT known as a muscle-specific enhancer, were observed in the promoter 2 region. These results indicated that the hPFK-M gene contains at least two promoter regions, facilitating the expression of the heterogeneous gene transcripts in a cell-type-specific manner.

Tissue specificity in expression and alternative RNA splicing of human phosphofructokinase-M and -L genes

Mode of the expression of phosphofructokinase (PFK) -M and -L genes was examined in various human tissues including muscle, placenta, liver, kidney, pancreas, stomach and reticulocytes. The gross level of mRNA expression of PFK-M and -L genes was estimated by Northern analysis. Polymerase chain reaction was used to detect mRNA expressed at low levels in these tissues. Tissue-specific expression of alternatively spliced PFK-M gene transcripts was also determined by polymerase chain reaction. The results indicated that alternative splicing of PFK-M gene transcripts was controlled in a tissue-specific manner.

Risk factors for the progression of microalbuminuria in Japanese type 2 diabetic patients--a 10 year follow-up study.

To clarify risk factors for the progression of microalbuminuria in Japanese type 2 diabetic patients, the longitudinal study for 10 years was conducted on 67 outpatients with type 2 diabetes, who had shown no overt proteinuria at baseline. The urinary albumin index (UAI) has been determined based on the mean of at least two random urine samples each year. Categories were defined as normoalbuminuria (UAI < 30.0 mg/g x Cr.), microalbuminuria (30.0 < or = UAI < 300.0), and macroalbuminuria (UAI > or = 300.0). Progression was defined as worsening of the category and/or more than doubling of the baseline UAI value. Multiple logistic regression analysis was performed using age, duration of diabetes, HbA1c, blood pressure, BMI, serum lipids, smoking habits, and alcohol consumption as independent variables and the progression of microalbuminuria as a dependent variable. Age and HbA1c were estimated as significant and independent variables. Furthermore, genetic polymorphisms of angiotensin I-converting enzyme (ACE) and angiotensinogen were analyzed to evaluate the genetic contribution. The D/D genotype of ACE was significantly more common in progressors than in non-progressors. These results suggest that glycemic control and age are important risk factors and the D/D genotype of ACE acts as a risk factor for the progression of microalbuminuria in Japanese type 2 diabetic patients.

Presence of minute cancer cell dissemination in peritoneal lavage fluid detected by reverse transcription PCR is an independent prognostic factor in patients with resectable pancreatic cancer.

BACKGROUND Presence of minute cancer cell dissemination in peritoneal lavage fluid detected by reverse transcription polymerase chain reaction (RT-PCR) has been reported to be a reliable predictor of the prognosis in several kinds of cancers, but has not been determined in pancreatic cancer. METHODS Peritoneal lavage fluid was harvested just after a laparotomy in 83 patients with adenocarcinoma of the pancreas. Half of the fluid was examined by cytology and the remaining half was used to measure carcinoembryonic antigen/beta-2-microglobulin (beta2M) mRNA expression. Patients were followed after surgery to evaluate its clinical significance. RESULTS Among 83 patients, 3 were cytologically positive (CY+), while 23 were positive by RT-PCR (PCR+). Seventy-one patients underwent a surgical resection whereas 12 were unresectable. Because 2 were CY+ among the 71 operated patients, the remaining 69 CY- patients were further investigated. Among those 69 patients, PCR+ was observed in 15 patients, whose incidence of postoperative peritoneal recurrence was significantly higher than that in PCR- patients (21% vs 4% at 3 years; P = .039). Moreover, both the recurrence-free rate in the abdominal cavity (peritoneal or local recurrence, excluding liver metastases) and the overall survival rate were better in PCR- patients than PCR+ patients (78% vs 33%, P = .0045 and 67% vs 46%, P = .0151). A multivariate analysis revealed positive lymph node metastases (hazard ratio; 5.18) and positive RT-PCR (hazard ratio; 3.65) were independent prognostic factors. CONCLUSION The RT-PCR-based cancer cell detection was an independent prognostic factor in patients with resectable adenocarcinoma of the pancreas and had close association with local or peritoneal recurrence.

A genetic defect in muscle phosphofructokinase deficiency, a typical clinical entity presenting myogenic hyperuricemia.

Myogenic hyperuricemia is a common pathophysiologic feature of muscle glycogenoses1. Type VII glycogenosis is the one which lacks catalytic activity of phosphofructokinase, a key enzyme of glycolysis, in muscle (PFK-M)2. Energy crisis in the exercising muscles of the patient results in the abnormal purine degradation leading to a typical manifestation of myogenic hyperuricemia1,3. As a step to clarify the molecular basis of this abnormal purine metabolism, we have reported the cloning of full-length human PFK-M cDNA4. The sequence data enabled us to reconfirm the gene duplication mechanism as hypothesized in rabbit PFK-M gene5,6,7. We have also elucidated the existence of the alternative splicing and the alternative promoter system of human PFK-M gene8,9. In addition, we have recently reported the complete structure of human PFK-M gene10. In this paper, we report on the genetic defect in a patient with PFK-M deficiency as the typical clinical entity presenting myogenic hyperuricemia.

Molecular Aspect of Myogenic Hyperuricemia: Cloning of Human Muscle Phosphofructokinase cDNA

In the last decade, molecular biology has made a breakthrough in our understanding of gene regulations, protein structures and mutant gene constitutions. Inborn errors of metabolism would be representatives of the fields of interest in which molecular biological techniques are expected to be the most powerful tools for analysis.

Myogenic Hyperuricemia: A Comparative Study between Type V and Type VII Glycogenosis

Glycolysis subsequent to glycogen breakdown is one of the major energy(ATP)-generating systems necessary for muscle exercise. The metabolic process of glycolysis depends on the functional integrity of many enzymes. Glycogenosis types V and VII are genetic errors resulting in deficiencies of muscle phosphorylase and muscle phosphofructokinase, respectively. Patients with these diseases have common muscle symptoms such as easy fatigability, stiffness and pain during exercise. Hyper-uricemia or gout has been documented in some patients with glycogenosis types V and VII. We recently showed that excess purine degradation in exercising muscles due to impaired glycolysis or glycogen breakdown causes hyperuricemia (myogenic hyperuricemia) in these patients (Kono et al., 1986; Mineo et al., 1987). Interestingly, the incidence of hyperuricemia seems to be far greater in type VII than in type V. At least 9 of 26 patients with glycogenosis type VII have been reported to be hyperuricemic (Agamanolis et al., 1980; Hays et al., 1981; Zanella et al., 1982; Vora et al., 1983; Mineo et al., 1985; Fogelfeld et al., 1985; Kono et al., 1986). However, only a few among more than 100 patients with type V have been reported to be hyperuricemic (Hardiman et al., 1987; Kono et al., 1987). In order to elucidate the metabolic basis for the different incidence of hyperuricemia, we compared purine degradation in exercising muscles between type V and type VII glycogenosis.