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Biomedical subjects

H Kadowaki

Publications and source records attributed to H Kadowaki.

At least 91 records · Page 5Linked to original sources
PubMed1990

Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance.

The nucleotide sequence was determined for all 22 exons of the insulin receptor gene from three patients with genetic syndromes associated with extreme insulin resistance. In all three patients, insulin resistance was caused by decreased insulin binding to the cell surface. The patient with leprechaunism (leprechaun/Winnipeg) came from a consanguineous pedigree and was homozygous for a missense mutation substituting arginine for His209 in the alpha-subunit of the insulin receptor. The other two patients were both compound heterozygotes with a nonsense mutation in one allele of the insulin receptor gene, and a missense mutation in the other allele. In the patient with the Rabson-Mendenhall syndrome (patient RM-1), the missense mutation substituted lysine for Asn15 in the alpha-subunit. In the patient with type A extreme insulin resistance (patient A-1), the missense mutation substituted serine for Asn462 in the alpha-subunit. Both nonsense mutations markedly reduced the levels of insulin receptor mRNA transcribed from the alleles with the nonsense mutation as compared to the transcripts from the other allele. The reduction in the level of mRNA would be predicted to greatly reduce the rate at which the truncated receptors would be synthesized. Furthermore, the truncated receptors would be severely impaired in their ability to mediate insulin action.

Adult↗
PubMed1990

The amino acid sequence of the insulin receptor is normal in an insulin-resistant Pima Indian.

Insulin resistance is an early predictor of development of noninsulin-dependent diabetes mellitus (NIDDM) in Pima Indians, a population with the highest reported prevalence of NIDDM. The insulin receptor plays a central role in mediating insulin action, and previous studies have demonstrated that mutations in the insulin receptor gene may cause insulin resistance. Therefore, we have cloned the insulin receptor cDNA from an insulin-resistant Pima Indian to determine if there is a mutation in the patient's insulin receptor gene. We obtained nine cDNA clones spanning exons 4-10 and 12-22 of the patient's insulin receptor gene. Polymorphisms in the nucleotide sequences for codons 523 (Ala), 1058 (His), and 1062 (Leu) provided useful markers to differentiate the patient's two alleles of the insulin receptor gene. These substitutions were silent, in that they did not alter the predicted amino acid sequence. The sequence of exons 1-3 and 11 was determined directly from genomic DNA that had been amplified using the polymerase chain reaction catalyzed by Taq DNA polymerase. Other investigators have reported defects in insulin binding and insulin receptor tyrosine kinase activity in diabetic Pima Indians. However, we did not detect any mutations in this patient's insulin receptor gene. Thus, these observations are consistent with the interpretation that the defects in insulin receptor function are acquired rather than derived from defects in the primary structure of the receptor.

Adult↗
PubMed1990

Mutations in insulin-receptor gene in insulin-resistant patients.

Defects in insulin-receptor function have been associated with insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). Several types of mutations in the insulin-receptor gene have been identified in patients with genetic syndromes of extreme insulin resistance. In some patients, insulin resistance results from a decrease in the number of insulin receptors on the cell surface. In one patient with leprechaunism (leprechaun/Minn-1), there is greater than 90% decrease in the levels of insulin-receptor mRNA. This patient is a compound heterozygote for two mutations in the insulin-receptor gene, both of which act in a cis-dominant fashion to decrease levels of mRNA transcribed from that allele. In one allele, there is a nonsense mutation at codon 897. All 22 exons of the other allele have a normal sequence, so that the mutation in this allele appears to map outside the coding sequence of the gene. Impaired insertion in the plasma membrane also causes insulin resistance. In two sisters (patients A-5 and A-8) with type A extreme insulin resistance, there is an 80-90% decrease in the number of insulin receptors expressed on the surface of their cells. Both sisters, whose parents are first cousins, are homozygous for a point mutation in which valine is substituted for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. This mutation retards the posttranslational processing of the receptor and impairs the transport of receptors to the cell surface. Another patient with leprechaunism (leprechaun/Ark-1) is a compound heterozygote with two different mutant alleles of the insulin-receptor gene. In the allele derived from the father, there is a nonsense mutation at codon 672 that truncates the insulin receptor by deleting the COOH-terminal of the alpha-subunit and the entire beta-subunit. This truncated receptor, lacking a transmembrane domain, appears not to be expressed at the plasma membrane. In leprechaun/Ark-1, there is a missense mutation in the allele of the insulin-receptor gene derived from the mother. This point mutation results in substitution of glutamic acid for lysine at position 460 in the COOH-terminal half of the alpha-subunit. This mutation increases receptor affinity and impairs the ability of acid pH to dissociate insulin from the receptor within the endosome. There is a defect in recycling the receptor back to the plasma membrane associated with this defect. This results in an accelerated rate of receptor degradation and a consequent decrease in the number of receptors on the cell surface in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1989

Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis.

The polymerase chain reaction catalyzed by Taq DNA polymerase has been used for site-specific mutagenesis. The amplification was primed by two oligodeoxyribonucleotides complementary to insulin receptor cDNA. To direct the synthesis of mutant DNA, mismatches were introduced into one of the primers. Six different mutations were constructed by this technique. Of twelve clones whose sequences were determined, ten (83%) had the correct sequence. This technique, which does not require the use of single-stranded DNA templates, provides a simple and efficient approach to site-specific mutagenesis.

Base Sequence↗
PubMed1989

Lactosylceramide molecular species specificity of rat liver CMP-N-acetylneuraminate:lactosylceramide sialyltransferase.

Six naturally occurring and three synthetic molecular species of lactosylceramide (LacCer) were used to examine the molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase in a Golgi-rich fraction of rat liver. The enzyme molecular species specificity was determined either in the presence of nonspecific lipid transfer protein or in the presence of detergents. Assays performed in the presence of transfer protein showed that for those lactosylceramide molecular species with either d18:1 or d18:0 long chain base the enzyme activity decreased linearly as the effective carbon number of the fatty acid increased. An increase in the carbon number of the long chain base decreased the activity of the enzyme twice as much as a corresponding increase in the carbon number of the fatty acid. On the other hand, when the enzyme activity was assayed in the presence of detergents, there was no significant difference in activity among the various molecular species of lactosylceramide based upon the carbon number of the fatty acid or on the presence of a double bond in the long chain base. However, the decrease in enzyme activity with an increase in the carbon number of the long chain base persisted. These results demonstrate that sialyltransferase has binding specificity with respect to the long chain base, but not the fatty acid. The apparent molecular species towards the fatty acid is related to the aqueous solubility of the various LacCer molecular species.

Animals↗
PubMed1989

Separation of derivatized glycosphingolipids into individual molecular species by high performance liquid chromatography.

The high performance liquid chromatography separation of the perbenzoyl derivatives of the neutral glycosphingolipids (GlcCer, LacCer, GbOse3Cer, GbOse4Cer, and GgOse3Cer) and the p-bromophenacyl and 2,4-dinitrophenyl hydrazide derivatives of the gangliosides (GM4, GM3, GM2, GM1, GD1a) into individual molecular species on a C18 reversed-phase column is described. Peaks were identified by comparing their relative retention times to the relative retention time of the corresponding glycosphingolipid of known molecular species composition. As little as 5 to 10 pmol of each molecular species of neutral glycosphingolipids and 3 to 5 pmol of the gangliosides can be detected. The effects of changes in the proportion of acetonitrile, methanol, and water in the mobile phase and of column temperature on the molecular species separation are described. A procedure for the tentative identification of glycosphingolipid molecular species based on their relative retention times is presented.

Chromatography, High Pressure Liquid↗
PubMed1988

Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance.

Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.

Alleles↗
PubMed1988

Effects of mannitol in the prevention of evolving myocardial infarction.

The ability of hyperosmolar mannitol to salvage evolving myocardial infarction was studied in 50 open-chest dogs subjected to 3 hours occlusion of the left anterior descending coronary artery. In 28 dogs, 20% hyperosmolar mannitol was infused into the distal half of the infarcted portion after commencement of reperfusion, leaving the proximal half mannitol free. In the other 22 dogs, physiological saline was infused instead of mannitol. The wall thickness at the distal half of the infarcted portion was increased 2 hours after reperfusion in both the saline and the mannitol groups. After 2 hours reperfusion the chest was closed. Thirteen out of 28 dogs in the mannitol group and 12 out of 22 dogs in the saline group died over one week. Seven days later, myocardial samples were measured for myocardial water content and creatine phosphokinase activity (CPK). Myocardial water content was increased in the infarcted portion in both the saline and the mannitol groups. In the saline group, myocardial CPK was reduced markedly in the proximal half (54.8 +/- 7.8% at the inner layer and 53.1 +/- 4.7% at the outer layer). These depletions were even more marked in the distal half (47.9 +/- 5.8 and 48.3 +/- 3.3%). In the mannitol groups, although the CPK was similarly depressed in the proximal half (44.8 +/- 5.8 and 41.1 +/- 5.4%), the CPK depletion in the distal half was less (48.1 +/- 6.1 and 58.1 +/- 6.7%) than the proximal half, suggesting mannitol preserved CPK depletion in the infarcted portion. It was concluded that intracoronary infusion of mannitol after reperfusion may salvage the infarcted myocardium.

Animals↗
PubMed1988

Reduced early diastolic extension in the infarcted portion in patients with old myocardial infarction.

To study relaxation characteristics of the infarcted myocardium, cyclic changes in the global left ventricular (LV) volume were measured in 20 patients with old myocardial infarction (OMI) and 17 normals (Normal) and those in the regional segment length were measured in 9 patients with anterior old myocardial infarction (anterior OMI) and 11 normals. The LV volume was calculated by using biplane LV cineangiograms. The regional segment length was calculated by measuring the spatial length between the 2 points of the ramifying branches on the left coronary arteries by using biplane coronary cineangiograms. The LV filling volume before atrial contraction (VR) was significantly less in the OMI compared with that in the normals (Normal 38 +/- 6 (mean +/- SD) ml/m2 vs 30 +/- 7 ml/m2: p less than 0.01), while filling volume by atrial contraction (Va) did not significantly differ (Normal 15 +/- 4 ml/m2 vs OMI 17 +/- 5 ml/m2). The lengthening of the segmental wall during diastole before atrial contraction (%LR) in the infarcted portion was 5.0 +/- 2.9% which was also significantly less than that in the non-infarcted portion (9.6 +/- 4.2%). The extent of lengthening by atrial contraction (%La) did not differ between the 2 portions (non-infarcted portion 3.8 +/- 1.1% vs infarcted portion 3.5 +/- 1.2%). Reduction of %LR was speculated to be caused by the incomplete relaxation in the myocardium adjacent to the infarcted portion and stiff myocardium in the infarcted portion. This study suggests that the infarcted myocardium may lead to a reduction of diastolic expansion before atrial contraction.

Angina Pectoris↗
PubMed1988

Nonspecific lipid transfer protein in the assay of a membrane-bound enzyme CMP-N-acetyl-neuraminate:lactosylceramide sialyltransferase.

CMP-N-acetylneuraminate:lactosylceramide alpha-2,3-sialyltransferase is tightly associated with the luminal side of the Golgi membrane as is its lipid substrate, lactosylceramide. In order to understand the kinetics, properties, and regulation of this enzyme, it is necessary to alter the amount and type of substrate in the membrane while minimizing changes in the membrane environment or in the conformation of the enzyme. Therefore, nonspecific lipid transfer protein, which accelerates the transfer of phospholipids, cholesterol, and glycosphingolipids between membranes was used to study the properties and kinetics of rat liver CMP-N-acetylneuraminate:lactosylceramide sialyltransferase. These results are compared to those obtained in parallel experiments using detergent-solubilized substrate. Enzyme activity was increased four- to fivefold by transfer protein and was consistently higher than the activity measured in the presence of detergents. In contrast to the results obtained with detergents, the enzyme activity increased linearly with both Golgi protein and with incubation time for up to 60 min. The Km values for the water-soluble substrate, CMP-neuraminic acid, were virtually identical when determined in the presence of transfer protein (0.23 mM) or detergents (0.27 mM). On the other hand, the apparent Km values for the lipophilic substrate, lactosylceramide, were markedly different when determined in the presence of transfer protein (47.9 microM) or in the presence of detergents (1.2 microM). These observations suggest that transfer protein is a useful tool to study the properties and kinetics of membrane-bound enzymes when both the enzyme and substrate are components of the same membrane.

Animals↗
PubMed1987

Paradoxical fall of tachycardia- and hypoxia-induced coronary flow under conditions of severe coronary stenosis in dogs.

It has been stated that the coronary flow paradoxically falls in response to tachycardia if the coronary artery is stenotic and "compliant". To clarify this, we measured coronary vascular resistance by cannulating the left anterior descending coronary artery in open-chest dogs. In constant flow perfusion of 41 +/- 5 ml/min/100 gm, coronary perfusion pressure was decreased by pacing, while at lower flow of 14 +/- 3 ml, it was increased by pacing, indicating that coronary vascular response was reversed. In constant pressure perfusion, coronary vascular resistance was reduced by pacing at high perfusion pressure, while it was paradoxically increased by pacing at low perfusion pressure. In the third experiment at constant flow perfusion, perfusing blood was changed from arterial to venous blood to induce myocardial hypoxia. At high flow, venous blood perfusion reduced coronary vascular resistance, while at low flow it increased coronary vascular resistance. All three experiments indicated that at high perfusion, tachycardia and hypoxia caused a reduction in coronary vascular resistance to meet the increased myocardial oxygen demand; however, at low perfusion, those stimuli increased coronary vascular resistance. The present study showed that the coronary vascular response is reversed at low flow and suggested that those stimuli might reduce flow further in patients with stenotic coronary artery and could be one of the mechanisms causing the development of myocardial infarction in those patients.

Animals↗
PubMed1986

Forme fruste of von Recklinghausen's disease: unilateral association of an orbital neurofibroma, a trigeminal neurinoma, and an acoustic neurinoma. Case Report.

A patient who presented with a tumor of the left ambient cistern, a left cerebellopontine angle tumor, and a left orbital tumor causing left hearing loss and left exophthalmos without café au lait spots or cutaneous neurofibromas is described. There was no family history of von Recklinghausen's disease. A cerebellopontine angle tumor removed by a suboccipital craniectomy was an acoustic neurinoma. An ambient cistern tumor was approached through a subtemporal route. A tumor arising from the trigeminal nerve was also a neurinoma. An orbital neurofibroma was excised by a frontal craniotomy with removal of the orbital roof. This rare unilateral association of neurinomas and a neurofibroma on the left side was thought to be a forme fruste of von Recklinghausen's disease, and it could be considered a presentation of a mosaic of von Recklinghausen's disease.

Adult↗
PubMed1986

[Effects of nitroglycerin on left ventricular geometry and compliance in man].

The effects of nitroglycerin (NTG) on relaxation characteristics of the infarcted and non-infarcted myocardium were investigated by calculating a segment length on the epicardium of the left ventricle for 16 patients with old myocardial infarction. The spatial segment length was measured between two points which were identified as a junction of ramifying branches of the left coronary arteries using biplane coronary cineangiography. Regional myocardial stiffness was expressed as delta P/delta L, where delta P was an increment of left ventricular (LV) diastolic pressure from the lowest LV diastolic pressure to the pressure at the maximal segment length, and delta L was the difference of two segment lengths corresponding to those pressures. Myocardial stiffness decreased from 0.0402 +/- 0.0293 mmHg/mm to 0.0212 +/- 0.0157 with intracoronary NTG (p less than 0.01) and from 0.0220 +/- 0.0090 to 0.0136 +/- 0.0124 with sublingual NTG (p less than 0.001) in the non-infarcted portions. However, it was unchanged with both intracoronary and sublingual NTG in the infarcted portions. NTG may cause venous pooling and may decrease diastolic wall tension of the left ventricle as its indirect effect on the non-infarcted myocardium. Also, the non-infarcted myocardium may be influenced by dilatation of the epicardial coronary artery. Muscle stiffness of the infarcted myocardium was unchanged, probably due to the rigidity of myocardial fibrosis. It was concluded that in myocardial infarction diastolic distensibility of the non-infarcted portion can be improved by NTG both through indirect and direct effects.

Compliance↗
PubMed1986

[Reduction of myocardial segment shortening during angina-free period in patients with angina pectoris].

In 16 patients with angina pectoris who had no histories of myocardial infarction, myocardial segment shortening was studied during angina-free periods. Myocardial segment length in the anterior wall of the left ventricle was calculated by measuring the spatial length between two points identified as junctions of ramifying branches of the left coronary arteries using biplane coronary cineangiography. Segment shortening was classified according to the severity of coronary arterial stenosis. The patients were categorized according to the severity of coronary arterial stenosis: as 1) the 0% stenosis (normal); 2) the 50% stenosis group; and 3) the 75-90% stenosis group. Total segment shortening in the normal group was the same as that in the 50% stenosis group (10.4 +/- 2.5%). However, in the 75-90% stenosis group, segment shortening was reduced to 7.3 +/- 2.5%. Effective segment shortening during the ejection period was reduced (5.0 +/- 1.8%) in the 75-90% stenosis group, as compared with the normal group (8.4 +/- 2.4%) and the 50% stenosis group (7.2 +/- 3.6%). This study demonstrated that segment shortening was reduced at rest in patients with angina pectoris who had had no previous infarction. A possible mechanism of this reduced segment shortening during angina-free periods may be irreversible myocardial alteration from recurrent ischemic attacks.

Angina Pectoris↗