Biomedical subjects
Publications and source records attributed to Y Yawata.
Multiple cerebral infarctions were observed in a patient with refractory idiopathic thrombocytopenic purpura who was positive for lupus anticoagulant (LAC) when her platelet counts were 2000 microL-1. It is suspected that LAC may have played an important role in the pathogenesis of this patient's cerebral infarctions, although she had severe thrombocytopenia.
The characteristic features of the incidence of hereditary red cell membrane disorders in the Japanese population are described, based on our studies on 610 patients from 353 kindreds during 20 years since 1975. These patients were screened by a protocol on red cell morphology (scanning and transmission electron microscopy), red cell membrane proteins (sodium dodecylsulfate polyacrylamide gel electrophoresis, and kinetics of membrane proteins), membrane lipids, biophysical studies (ektacytometry, mechanical stability, and fluorescence recovery after photobleaching method), and membrane transport (sodium influx and efflux, and anion transport). Hereditary spherocytosis (HS) is most frequent (308 patients from 156 kindreds), hereditary elliptocytosis (HE) is the second (98 patients from 47 kindreds) followed by hereditary stomatocytosis (57 patients from 40 kindreds). Among the molecular abnormalities detected, alpha-spectrin mutation in the Japanese HE patients appeared extremely rare (only one family with spectrin alpha 1/74), despite three novel beta-spectrin mutations were found out of nine world-wide cases. Most of the Japanese HE patients were associated with partial protein 4.1 deficiencies. Ankyrin abnormalities in the Japanese HS patients appeared less common than those in the Western countries. Complete protein 4.2 deficiencies (34 patients from 20 kindreds) were unique in the Japanese population. Membrane lipid abnormalities included hereditary high red cell membrane phosphatidylcholine hemolytic anemia (30 patients from 18 kindreds), congenital beta-lipoprotein deficiency (acanthocytosis: seven patients from five kindreds), and each one patient of congenital lecithin: cholesterol acyltransferase deficiency and of congenital alpha-lipoprotein deficiency (Tangier disease).
Interleukin-6 (IL-6) is one of the most important growth factors for myeloma cells. We examined the effect of recombinant IL-6 on the proliferation of five human myeloma cell lines, which were independently established AT Kawasaki Medical School. Only the KMS-11 cell line among these five lines showed growth enhancement induced by IL-6. Based on the results, a possible contribution of Ca(2+)-phospholipid-dependent protein kinase C (PKC) to the signal transduction in KMS-11 cells during growth enhancement was studied, since PKC may play an important role in malignant transformation or cell proliferation induced by some growth factors, such as IL-6. When exogenous IL-6 was added to KMS-11 culture, we observed (1) reduction of total PKC activity, and (2) translocation of PKC activity from its cytosol fraction to the membrane fraction. These findings may indicate that down regulation of PKC occurred during the myeloma cell proliferation induced by IL-6. However, IL-6 does not appear to be involved in cell proliferation and differentiation in the other cell lines studied.
We studied bovine subjects that exhibited a moderate uncompensated anemia with hereditary spherocytosis inherited in an autosomal incompletely dominant mode and retarded growth. Based on the results of SDS-PAGE, immunoblotting, and electron microscopic analysis by the freeze fracture method, we show here that the proband red cells lacked the band 3 protein completely. Sequence analysis of the proband band 3 cDNA and genomic DNA showed a C --> T substitution resulting in a nonsense mutation (CGA --> TGA; Arg --> Stop) at the position corresponding to codon 646 in human red cell band 3 cDNA. The proband red cells were deficient in spectrin, ankyrin, actin, and protein 4.2, resulting in a distorted and disrupted membrane skeletal network with decreased density. Therefore, the proband red cell membranes were extremely unstable and showed the loss of surface area in several distinct ways such as invagination, vesiculation, and extrusion of microvesicles, leading to the formation of spherocytes. Total deficiency of band 3 also resulted in defective Cl-/HCO3- exchange, causing mild acidosis with decreases in the HCO3- concentration and total CO2 in the proband blood. Our results demonstrate that band 3 indeed contributes to red cell membrane stability, CO2 transport, and acid-base homeostasis, but is not always essential to the survival of this mammal.
To obtain direct evidence of impaired intramembrane particles (IMPs) and a deranged cytoskeletal network in situ in human red cells of band 4.2 deficiency, electron microscopic studies were performed utilizing the freeze fracture method for IMPs and the quick-freeze deep-etching method for the cytoskeletal network. Three patients with three different previously identified mutations of the band 4.2 gene, i.e., band 4.2 Komatsu (homozygous; codon 175 GAT --> TAT), band 4.2 Nippon (homozygous; codon 142 GCT --> ACT), and band 4.2 Shiga (compound heterozygous; codon 317 CGC --> TGC and codon 142 GCT --> ACT), were selected for this study. The decrease in the number of IMPs with increase in their size was most marked in band 4.2 Komatsu, which was clinically most severe with no band 4.2 protein. In this regard, in band 4.2 Nippon, which showed moderate severity in clinical hematology with a nearly missing band 4.2 protein, increased sizing was less marked. The abnormalities in IMPs were the least in band 4.2 Shiga, which demonstrated compensated hemolysis with band 4.2 protein in a trace amount. The extent of the impairment of IMPs may be reflected by the total absence or the presence of band 4.2 protein even in a trace amount and/or by the specific site(s) of the mutation of the band 4.2 gene. Derangement of the cytoskeletal network was also observed in these three patients. It was most abnormal in band 4.2 Komatsu, and less so in band 4.2 Nippon and in band 4.2 Shiga. These results clearly indicate that 1) band 4.2 plays an important role not only in its binding to band 3 but also to the skeletal network (mostly to spectrins) vertically, and 2) its deficiency produces critical abnormality in maintenance of the structural and functional integrity of the integral proteins (such as band 3), as well as the cytoskeletal network.
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Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes compared to control cells. In contrast, the lateral mobility of neither glycophorins nor a fluorescent phospholipid analog was altered in band 4.2-deficient cells. Compared to controls, band 4.2-deficient erythrocytes manifested a decreased ratio of band 3 to spectrin, and band 4.2-deficient membrane skeletons had decreased extractability of band 3 under low-salt conditions. Normal band 4.2 was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. We conclude that band 4.2 provides low-affinity binding sites for both band 3 oligomers and spectrin dimers on the human erythrocyte membrane. Band 4.2 may serve as an accessory linking protein between the membrane skeleton and the overlying lipid bilayer.
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Five cases of hereditary high red cell membrane phosphatidylcholine hemolytic anemia in three families were described. All cases were clinically manifested by jaundice and splenomegaly. Hemolysis was evident from indirect hyperbilrubinemia, reticulocytosis and decrement of serum haptoglobin. Red blood cells showed morphological abnormalities such as poikylocytosis, anisocytosis and target cells on blood smears. Both direct and indirect Coombs' tests were negative. Ham test, sugar water test and hemoglobin electrophoresis showed no abnormalities. Osmotic fragility test showed decreased membrane fragility. Lipid analysis of red cell membrane showed increment of phosphatidylcholine content and decrement of sphingomyelin content, although plasma lipids were essentially normal. Influx and efflux of sodium through the red cell membrane were both increased. Splenectomy was performed without effect on one patient and the mother of other patients.
Integral proteins of human red cell membranes are reviewed from the standpoint of genetic and phenotypic expressions. Band 3 plays an important role in anion exchange transport at its membrane domain and in its binding to cytoskeletal membrane proteins and cytosolic components. Several possible mechanisms for band 3 gene expression are also discussed. The abnormalities of band 3 were detected in hereditary elliptocytosis, especially Southeast Asian ovalocytosis, and also in hereditary spherocytosis associated with deficiency of band 3 protein. A complete deficiency of band 3 has recently been discovered in cattle, which demonstrated a markedly increased hemolysis, impaired anion transport, and striking instability of cytoskeletal network with extensive microspherocytosis. Glycophorins are the other major integral proteins, and consisted of the two major categories; i.e., glycophorin A (GPA)-glycophorin B (GPB)-glycophorin E (GPE) and glycophorin C (GPC)-glycophorin D (GPD). Genetically, GPB gene appears to be derived from the duplication of GPA gene and insertion of the Alu-Alu structure. GPE gene appears to be formed basically by duplication of GPB gene. Several mutation of the GPA-GPB-GPE system have been reported; En(a-) phenotype by skipping GPA gene, S-s-U-phenotype with by skipping GPB gene, and MKMK phenotype by skipping both GPA and GPB genes. Hybrid forms of GPA and GPB have also been known as Miltenberger V and Sta. GPD appears to be a truncated form of GPC. Several mutation have also been known as Melanesian (Ge: -1), Yus (Ge: -2, 3, 4), Gerbich (Ge: -2, -3, 4), and Leach (Ge: -2, -3, -4).
The complete band4.2 deficiency with a point mutation (GCT-->ACT at codon142: Nippon type) in red cell membrane protein4.2 gene is specific for the Japanese population. The biochemical properties of band4.2 protein have been studied extensively. However, the physiolosical functions of this protein in situ have been unknown. Therefore, we tried to clarify the physiological role of band4.2 by biochemical, biophysical and electron microscopic studies in the patient with the Nippon type mutation. Band4.2 deficient red cells demonstrated significant abnormalities in the function of both the cytoskeletal network and band3 protein, as summarized in our results. 1 Deformability of red cell membrane was decreased when heat-treated up to 48 degrees C. 2 Under the these conditions, the cytoskeletal network became markedly disrupted, and the number of intramembrane particles was reduced with a shift to larger sizes, indicating the possibility of increased oligomerization of band3 molecules in the red cells. 3 The lateral and rotational mobility of band3 in the red cells was substantially increased. 4 Band4.2 protein was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. These results indicate the possibility that band4.2 may play a role in connecting the cytoskeletal network to band3 protein as a kind of anchoring protein.
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We describe a 30-month-old boy with multiple anomalies and mental retardation with hereditary spherocytic anemia. His karyotype was 46,XY,del(8)(p11.23p21.1). Genes for ankyrin and glutathione reductase (GSR) were localized to chromosome areas 8p11.2 and 8p21.1, respectively. Six patients with spherocytic anemia and interstitial deletion of 8p- have been reported. In these patients, severe mental retardation and multiple anomalies are common findings. This is a new contiguous gene syndrome. Lux et al. [1990: Nature 345:736-739] established that ankyrin deficiency and associated deficiencies of spectrin and protein 4.2 were responsible for spherocytosis in this syndrome. We reviewed the manifestations of this syndrome. Patients with spherocytic anemia and multiple congenital anomalies should be investigated by high-resolution chromosomal means to differentiate this syndrome.
A novel mutation of 523 GAT-->TAT (175 Asp-->Tyr) in exon 4 of the band 4.2 gene was detected in a 37-year-old Japanese patient with total lack of band 4.2 protein, designated as allele 4.2 Komatsu. In this patient, moderate uncompensated hemolytic anemia (red cell count 3.38 x 10(6)/microliters, hemoglobin 10.8 g/dl, hematocrit 30.9%, reticulocytes 12.4%, indirect bilirubin 1.84 mg/dl) with ovalostomatocytosis and increased osmotic fragility had been noted since birth. Family studies revealed no overt hemolytic anemia in other family members, essentially normal red cell morphology, and a normal profile of red cell membrane proteins including band 4.2. Genetic studies proved that the proband was homozygous and all the family members studied were heterozygous with respect to the mutation of 523 GAT-->TAT of the band 4.2 gene. Although band 4.2 was completely absent in the proband, trace amounts of 72 kDa and 74 kDa peptides were detected in the red cells of all the family members, in which the mutation of 424 GCT-->ACT at exon 3 of the band 4.2 gene (Nippon type) was not present. Electron microscopic studies with the surface replica method and the quick-freeze deep-etching method showed the most marked disorganization of the cytoskeletal network in the patient's red cells in situ among the cases of band 4.2 deficiencies we have studied. This suggests that the amino acid of the band 4.2 protein, which was affected by the present mutation in exon 4, is much more crucial for the functioning of band 4.2 protein than that at codon 142 in exon 3. The cytoplasmic domain of band 3 in the proband's red cells was essentially normal in protein chemistry and in gene analysis with single-stranded conformation polymorphism (SSCP).
Allele alpha LELY is a low-expression allele of erythroid spectrin alpha-chain. It carries mutations both in exon 40 and intron 45 and is associated with partial skipping of exon 46. Allele alpha LELY remains asymptomatic by itself. In contrast, it enhances the expression level of deleterious alpha-alleles occurring in trans, and as such has clinical importance. The aim of this study was to evaluate the incidence of allele alpha LELY in various ethnic groups, i.e. Caucasians, African Blacks, Japanese and Chinese. Allele alpha LELY occurred in all groups investigated with a fairly uniform frequency: 31%, 21%, 20% and 22%, respectively. Mutations in exon 40 and intron 45 appeared linked to one another without exception. Partial skipping of exon 46 or the low-expression feature, whenever they could be assessed, were invariably observed. Allele alpha LELY appears to be an ancient and stable allele.
We present a patient with beta-thalassemia minor diagnosed on the basis of the incidental findings of marked hypochromic and microcytic erythrocytosis. Hemoglobin analysis revealed increased hemoglobin (Hb) A2 levels and decreased beta/alpha synthesis ratio in both the propositus and his mother. Further molecular studies identified a single base substitution of TCA to TAA within codon 35 in heterozygous state, which creates a premature terminator resulting in a defect of effective beta globin synthesis. This is the first report of beta-thalassemia due to a nonsense mutation at codon 35 of beta-thalassemia gene in the Japanese population.
Deoxygenation-induced red blood cell (RBC) sickling probably activates multiple cation leak pathways. In an attempt to model this, we examined the net passive K efflux ("K leak") from normal and sickle RBCs undergoing elliptical deformation in hypotonic media (200 mOsmol/L). This hypotonic deformation activates two deformation-dependent K leak pathways that are not detectable during the balanced leak (Kefflux = Nainflux) resulting from deformation of RBCs in isotonic medium. These are (1) a calcium-dependent leak component and (2) a novel leak pathway that is inhibited by substitution of bromide (but not sulfamate) for chloride, which converts the unbalanced K leak (Kefflux > Nainflux) of hypotonic deformation to a residual balanced leak. This dramatic effect of hypotonic deformation is reversible, is detected in both normal and sickle RBCs, and is inhibited significantly by 4,4'-diisothiocyano-2,2'-stilbene disulfonate. Remarkably, bromide also inhibits by 55% the K leak resulting from authentic deoxygenation-induced RBC sickling and, thereby, blunts the imbalance of accompanying monovalent cation leaks. The unique effect of bromide is not readily explainable on the basis of known behaviors of known ion leak/transport pathways. The mechanical threshold for triggering K leak during hypotonic deformation is at applied shear stress of 164 dyne/cm2, a value similar to the abnormal susceptibility we previously found for oxygenated sickle RBCs during isotonic deformation. These data suggest that membrane stretch accompanying hypotonic deformation activates the same multiple leak pathways that contribute to net K leak during authentic RBC sickling, including a previously unknown bromide-sensitive leak.