Search PubMed⌕ Search

Biomedical subjects

M Itakura

Publications and source records attributed to M Itakura.

At least 145 records · Page 8Linked to original sources
PubMed1992

Application of 67Ga for the estimation of reticulocyte production.

In order to estimate the production of reticulocytes, which have a larger number of transferrin receptors than erythrocytes, we used 67Ga which is exclusively bound to transferrin in the blood. The pattern of uptake of 67Ga by reticulocytes was quite similar to the time course of transglutaminase activity which might be involved in receptor-mediated endocytosis. The preinjection of Fe3+ decreased the uptake of 67Ga by reticulocytes. These results suggested that 67Ga in a transferrin-bound form was taken up by reticulocytes via receptor-mediated endocytosis. It was showed that the application of 67Ga is very easy and useful for the estimation of reticulocyte production.

Animals↗
PubMed1992

Physiologic concentrations of inorganic phosphate accelerate fructosamine synthesis.

The effect of physiologic concentrations of inorganic phosphate (Pi) on fructosamine (FRA) synthesis was studied. After 75 g oral glucose administration (OGTT), 'delta FRA/24 h', defined as delta FRA after incubating serum or other specimens at 37 degrees C for 24 h after adding 1000 mg/dl glucose, was significantly decreased in parallel to the decrease of plasma Pi concentrations. 'The FRA index', defined as the FRA value divided by the corresponding glucose concentration, both at fasting, correlated significantly with plasma Pi concentrations. In vitro incubation of serum total protein (TP), albumin (ALB), gamma-globulin (GLB), free lysine (Lys), and free valine (Val) with glucose at different concentrations of Pi showed a Pi-dependent increase of FRA synthesis throughout 48 h of incubation. The accelerating effect of 5 mg/dl Pi on FRA synthesis from TP, ALB, GLB, Lys, and Val at pH 7.4 was, respectively, as great as 48, 20, 24, 13 or 25% of those without Pi. Increase of pH from 6 to 10 logarithmically increased delta FRA/24 h in contrast to a logarithmic decrease of the accelerating effect of Pi on delta FRA/24 h. These data show that physiologic concentrations of Pi accelerate protein glycation by accelerating dehydrogenation during the Amadori rearrangement through the negative charge of Pi. Because this accelerating effect of physiologic Pi presumably exists in vivo, Pi concentration must be taken into account as an accelerating factor for FRA synthesis in evaluating diabetic control, and further studies must be carried out to elucidate whether hyperphosphatemia accelerates glycation-induced diabetic complications.

Adult↗
PubMed1992

Static and kinetic studies of calmodulin and melittin complex.

Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.

Calcium↗
PubMed1992

Structure of the bovine endothelin-B receptor gene and its tissue-specific expression revealed by northern analysis.

We have isolated and characterized the gene encoding the bovine endothelin (ET) receptor ETB with seven putative membrane-spanning domains. The gene exists as a single copy of the bovine genome and extends over 36 kilobases. The coding region is divided into seven exons separated by six introns; the first exon encodes the first and second transmembrane domains and each of the following transmembrane domains is encoded by a separate exon. The 5' region of the transcript was examined by the ribonuclease protection analysis; multiple start sites were deduced with no apparent "TATA" box in the expected upstream region. Northern blot analysis demonstrated a widespread but tissue-specific distribution of the ETB receptor in bovine tissues.

Amino Acid Sequence↗
PubMed1992

ras mutations in endocrine tumors: mutation detection by polymerase chain reaction-single strand conformation polymorphism.

To elucidate the molecular basis for endocrine tumorigenesis, ras mutations in human endocrine tumors were analyzed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Mutations of the H-, K-, N-ras genes were examined in genomic DNAs from 169 successfully amplified primary endocrine tumors out of 189 samples. Four out of 24 thyroid follicular adenomas analyzed contained mutated N-ras codon 61, and one contained the mutated H-ras codon 61. One of the 19 pheochromocytomas revealed mutation of the H-ras codon 13. No mutations of the ras gene were detected in pituitary adenomas, parathyroid tumors, thyroid cancers, endocrine pancreatic tumors, and adrenocortical tumors. Based on these findings we conclude that activation of the ras gene may play a role in the tumorigenesis of a limited number of thyroid follicular adenomas and pheochromocytomas, and that mutation of the ras gene is not frequent in other human endocrine tumors.

Adult↗
PubMed1992

Hereditary adrenocortical unresponsiveness to adrenocorticotropin with a postreceptor defect.

We report two cases in one pedigree with hereditary adrenocortical unresponsiveness to ACTH (HACUA) where it is suggested that the pathogenic defect occurs after cAMP generation. Although the patients showed increased plasma ACTH, decreased plasma cortisol and dehydroepiandrosterone, and no steroidogenic response to exogenous ACTH, they responded normally to both furosemide administration and to a low sodium diet by showing increases in plasma aldosterone. The peripheral blood mononuclear leukocytes (MNLs) from these patients possessed ACTH receptors similar to adrenocortical ones, which was in contrast to a previously reported case with a deficiency of ACTH receptors in the MNLs. Furthermore, ACTH receptors in the patients' MNLs were functionally coupled to adenylate cyclase. Dibutyryl cAMP infusion did not, however, increase plasma cortisol nor aldosterone in these patients in a sharp contrast to its remarkable increase in a normal control subject. These results suggest that these patients represent a new subtype of HACUA with a failure of intracellular reception of the cAMP message in adrenocortical cells. We propose to classify our patients with a postreceptor defect as HACUA type II using an analogy to pseudohypoparathyroidism type II.

Adenylyl Cyclases↗
PubMed1992

Somatic gene therapy for diabetes with an immunological safety system for complete removal of transplanted cells.

To develop somatic gene therapy for diabetes, we studied an animal model with proinsulin-producing fibroblasts with an immunological safety system. Cultured mouse fibroblasts of the Ltk- cell line were transfected first with the efficient human proinsulin expression vector pBMG-Neo-Ins. Initially, 2 x 10(6) cells with a proinsulin-production rate of 91 ng.24 h-1.10(6) cells-1 were transplanted i.p. into streptozocin-induced diabetic C3H mice. The blood glucose concentrations improved between the first and the 28th day, but the animals died of hypoglycemia between the 29th and 46th days. The proinsulin-producing Ltk- cells were further transfected with a second plasmid, pHEBo-CD8.2, encoding BALB/c mouse T-cell differentiation antigen. The CD8.2 allotype is different from CD8.1 allotype by only one amino acid substitution and should be only slightly antigenic to the recipient C3H mice. Somatic gene therapy with these doubly transfected cells followed by the consecutive administration of a monoclonal antibody to CD8.2 resulted in an initial decrease of blood glucose concentrations followed by the permanent recurrence of hyperglycemia, thus proving the complete removal of the transplanted cells. Cultured fibroblasts were thus proven capable of supplying sufficient proinsulin to lower the blood glucose concentrations in diabetic animals. The immunological safety system with a combination of artificial expression of cell surface antigen and the administration of the specific monoclonal antibody was an effective safety system for somatic gene therapy.

Animals↗
PubMed1992

Molecular and genetic analysis of a compound heterozygote for dysprothrombinemia of prothrombin Tokushima and hypoprothrombinemia.

The molecular and genetic basis of a compound heterozygote for dys- and hypoprothrombinemia was analyzed. Abnormal nucleotide sequences of the human prothrombin gene were screened by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion and mutated primer-mediated PCR-RFLP. A single nucleotide substitution responsible for dysprothrombinemia of prothrombin Tokushima was detected, as were three polymorphisms. The mutation for hypoprothrombinemia was detected by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion in exon 6, near MboII-RFLP and NcoI-RFLP. Sequencing of PCR-amplified genomic DNA revealed a single base insertion of thymine (T) at position 4177. The resulting frameshift mutation caused both an altered amino acid sequence from codon 114 and a premature termination codon (i.e., TGA) at codon 174 in exon 7. Because exon 7 encodes the kringle 2 domain preceding the thrombin sequence, this frameshift leads to the null prothrombin phenotype. The inheritance of the hypoprothrombinemia gene from the father to the proband was proved by PCR-SSCP with endonuclease digestion and mutated primer-mediated PCR-RFLP.

Amino Acid Sequence↗
PubMed1992

Amelioration of dermal lesions in streptozotocin-induced diabetic rats by aminoguanidine.

As aminoguanidine (AG) is known to prevent non-enzymatic glycosylation in various tissues, we have histologically and biochemically evaluated AG effects on the skin in control, SZ-diabetic and AG-treated (25 mg/kgbw/day, 10w) diabetic rats. HbA1c and plasma glucose levels in diabetic and AG-treated diabetic rats were maintained about two times higher than those in control rats during the 10 weeks of the experiment. Histological findings revealed that the dermis in diabetic rats was thin and edematous, associated with swelling and degeneration of collagen fibers. Necrobiotic changes were seen in the lower dermis. These changes were greatly improved in AG-treated diabetic rats. Skin glucose contents in diabetic and AG-treated diabetic rats were about 10 times higher than those in the controls, whereas there was no difference in the sorbitol contents between three groups. Dry weight of the skin and collagen content was well correlated (r = 0.9044) and collagen represented 78.0 +/- 2.3% of the dry weight. By SDS-PAGE analysis of cyanogen bromide digests it was shown that high molecular weight peptides were increased in diabetic rats, but were decreased in AG-treated diabetic rats. The mean of glycosaminoglycan (GAG) contents of diabetic skin was 54% of that in the controls (1.58 +/- 0.09 vs. 2.94 +/- 0.39 micrograms/mg dry weight, P < 0.0025), which increased significantly in AG-treated diabetic rats (1.75 +/- 0.07 microgram/mg dry weight, P < 0.01 vs. diabetic).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Detection of a single base substitution of the gene for prothrombin Tokushima. The application of PCR-SSCP for the genetic and molecular analysis of dysprothrombinemia.

The genetic and molecular basis of a mutant prothrombin of 'prothrombin Tokushima' was studied by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses. The abnormal gene was detected by altered migration by PCR-SSCP and by the loss of an MspI site by PCR-RFLP. The gene for prothrombin Tokushima was shown to be inherited from the mother of the proband. Sequencing analysis using PCR-amplified genomic DNA clarified a substitution of thymine (T) for cytosine (C) at position 9,490, changing arginine (Arg) to tryptophan (Trp) at position 418 of the polypeptide chain. This point mutation is assumed to be the molecular basis of prothrombin Tokushima, firstly, because of the absence of distinct changes in Southern blot analysis of the proband's DNA (using a full-length human prothrombin cDNA as a probe), secondly, because it has the same molecular weight as the abnormal gene product, and, thirdly, because of the absence of other amino acid abnormalities in the proteolytic peptide-fragments. It is concluded that PCR-SSCP and PCR-RFLP were useful for detecting the abnormal gene and for directly diagnosing the carrier status of dysprothrombinemia. This is the first report of gene analysis of dysprothrombinemia.

Amino Acid Sequence↗
PubMed1991

Primary structure of bovine endothelin ETB receptor and identification of signal peptidase and metal proteinase cleavage sites.

A cDNA clone corresponding to the entire coding region of the bovine ETB endothelin receptor mRNA was isolated from a lung cDNA library and sequenced. The cDNA encodes 441 amino acids: 26 constituting an NH2-terminal signal peptide and 415 constituting the mature receptor. The signal peptidase cleavage site was determined by direct amino acid sequencing of purified receptor. A comparison of the predicted amino acid sequence with the available bovine ETA and rat ETB endothelin receptor sequences revealed 63 and 85% homology, respectively. Endothelin receptors of various species are known to be very sensitive to a certain metal proteinase(s) and have been shown to be converted to a lower Mr form in the absence of EDTA. The metal proteinase cleavage site was also determined by direct protein sequencing of the proteolysis product. The amino acid sequence (Ala-Gly-X-Pro-Pro-Arg) surrounding the cleavage site (between Ala-79 and Gly-80) is conserved among the ETB endothelin receptors, explaining the above mentioned proteolytic conversion from the higher to lower Mr forms observed in various species.

Amino Acid Sequence↗
PubMed1991

Aminoguanidine decreases urinary albumin and high-molecular-weight proteins in diabetic rats.

The effect of aminoguanidine (AG) on diabetic proteinuria was studied in control rats ([C]), streptozotocin (SZ)-induced diabetic rats ([DM]), control rats treated with AG [( C + AG]), or diabetic rats treated with AG [( DM + AG]). Increased glycation of hemoglobin (HbA1C), and glomerular basement membrane (GBM) type IV collagen (IV-C) at 10 wk of stable diabetes were associated with the appearance of high-molecular-weight (HMW) cross-linked type I collagen and HMW proteinuria of 62 kD, 69 kD albumin and 77 kD proteins to the levels of 362, 381, and 408%, while 9.9, 13.5, 17, 18, and 23 kD proteins were decreased, respectively, to non-detectable, 37, 16, and 13%. AG decreased cross-linkage of type I collagen and significantly decreased urinary 62 kD protein to 54%, 69 kD albumin to 40%, and 77 kD protein to 49% at 10 wk in [DM + AG] compared to [DM] without changing diabetic control. It is suggested that glycation-derived late-stage protein modification is etiologically important for diabetic proteinuria, and that AG can potentially prevent diabetic HMW proteinuria.

Albuminuria↗
PubMed1991

Correction of fructosamine value for serum albumin and globulin concentrations.

Using the data of 131 patients with non-insulin dependent diabetes mellitus (NIDDM), the correction formulas of fructosamine value ([FRA]) were devised to standardize the uncorrected [FRA] to serum albumin concentrations ([ALB]) of 4 g/dl, globulin concentrations ([GLB]) of 3 g/dl, and total protein concentrations ([TP]) of 7 g/dl. The following formula was derived for its maximum correlation coefficient (r) between corrected [FRA] ([FRAc]) and fasting blood glucose concentration ([G]); [FRAc] = [FRA]x33.3/(7.6 [ALB]+[GLB]). In these 131 diabetic patients, r between uncorrected [FRA] and [G] at 2 weeks ago was 0.562. When corrected by [FRA]x33.3/(7.6 [ALB]+[GLB]), [FRA]x4/[ALB], [FRA]+30 (4-[ALB]), [FRA]+23 (4-[ALB]), [FRA]+30 (7-[TP]), [FRA]x7/[TP], and [FRA]x3/[GLB], r was, respectively, 0.616, 0.612, 0.595, 0.589, 0.582, 0.581 and 0.478. In 24 patients with NIDDM whose [ALB] is either above 4.5 g/dl or below 3.5 g/dl, r between uncorrected [FRA] and [G] was as low as 0.389 without positive correlation. By using our correction formulas of [FRA]x33.3/(7.6 [ALB]+[GLB]) or [FRA] x 4/[ALB], r was statistically increased, respectively, to 0.769 or 0.788 (P less than 0.05 in both cases) in contrast to no significant increase of r by other formulas being at 0.598, 0.556, 0.540, 0.562 and 0.121. Based on these analyses, it is concluded that our correction formula of [FRA] by [FRAc]=[FRA]x33.3/(7.6 [ALB]+[GLB]) accurately reflects [G] in NIDDM even with hypo- or hyper-albuminemia, and [FRAc]=[FRA]x4/[ALB] is useful for practical application for its simplicity.

Diabetes Mellitus, Type 2↗
PubMed1991

9-(Dicyanovinyl)julolidine binding to bovine brain calmodulin.

A molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ), is a fluorescent dye whose intramolecular rotation determines its fluorescence yield [Kung, C.E. & Reed, J.K. (1989) Biochemistry 28, 6678-6686]. DCVJ binds to bovine brain calmodulin and emits strong fluorescence. In fluorescence titration experiments, the dissociation constant and the number of binding sites were determined to be 20 +/- 10 microM and 0.7 +/- 0.5 in the presence of Ca2+, and 22 +/- 10 microM and 0.6 +/- 0.5 in the absence of Ca2+, respectively. The fluorescence intensity of bound DCVJ increased 10-fold in the presence of Ca2+ compared to that in the absence of Ca2+. The fluorescence titration curve of DCVJ-calmodulin showed a transition at pCa 6.5. Over the same Ca2+ range, a decrease in molecular ellipticity at 222 nm and an increase in tyrosine fluorescence of calmodulin were observed. These results mean that the conformational change of calmodulin due to the Ca2+ binding induces the microenvironmental change of the DCVJ binding site from the flexible to the rigid state, resulting in inhibition of the intramolecular rotation of DCVJ and an increase in its fluorescence.

Binding Sites↗
PubMed1991

Allele loss on chromosome 11 in a pituitary tumor from a patient with multiple endocrine neoplasia type 1.

We have examined the allele loss of chromosome 11 in a pituitary tumor from a patient with familial multiple endocrine neoplasia type 1 (MEN 1). The extensive loss of chromosome 11, including loci of D11S149, HRAS1 and F2, was detected by the loss of heterozygosity. All of the lost alleles of these loci were transmitted from the unaffected father and not from an affected mother. This is the first evidence of allele loss of chromosome 11 in a pituitary tumor of MEN 1 and supports the idea that similar allelic deletion of MEN1 locus on chromosome 11 is the common genetic basis for tumorigenesis in the pituitary, endocrine pancreas, and parathyroid gland in MEN 1.

Adult↗
PubMed1990

Chondroma of the diaphragm--report of a case.

A rare case of chondroma of the diaphragm is reported. The patient was a 23-year-old female who had had recurrent upper abdominal pain for 4 years. Her symptoms and physical findings on admission suggested an intra-abdominal tumor. Roentgenological study disclosed a mass with a thick calcified wall in the upper abdomen. Laparoscopy revealed a multinodular white mass protruding from the left hemidiaphragm. The mass was surgically enucleated from the diaphragm. The resected tumor had a thick, calcified wall and a narrow lumen containing colorless fluid. Microscopic examination disclosed a hyaline chondroid matrix with mature chondrocytes, as well as extensive ossification. Primary tumor of the diaphragm is an uncommon disease. Only one case of chondroma of the diaphragm has been reported in the literature. Laparoscopy was useful in confirming the tumor's location and in speculating on its nature.

Adult↗