Masamiti Tatibana

Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor

The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the probands lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes.

Metabolic fate of pyrimidines and purines in dietary nucleic acids ingested by mice.

1. In order to study the metabolism and tissue utilization of pyrimidines or purines ingested as dietary nucleic acid components, [14C]uracil, [14C]cytosine-labeled RNA, [14C]thymine-labeled DNA, or [14C]adenine-labeled RNA was fed to mice. 2. Absorption and catabolism of each ingested radioactive material were rapid; 80% or more of the ingested radioactivity was excreted as catabolic products over an 8-h period. 3. Utilization of the ingested radioactive materials for tissue synthesis of nucleic acids was limited under the usual conditions, the extent being 2--5%, 4 h after feeding. Such acid-insoluble radioactivity was localized principally in gastrointestinal tissue, and much lesser amounts, albeit significant, were found in the liver. 4. With increase in the dose of dietary nucleic acids, the amounts of utilized (nucleic acids and nucleotides) and utilizable (nucleosides and free bases) forms of uracil and cytosine and of adenine were increased in all tissues examined. Relationship between the dose and utilization together with additional findings support the view that gastrointestinal tissue and the liver utilize and degrade a greater part of the exogenous nucleic acid bases before their entry into the systemic circulation. 5. The metabolism of DNA thymine was unique in that it was significantly utilized for DNA synthesis in tissues other than the gastrointestinal tissue and liver to a comparative extent. The spleen was particularly active in this respect, and the hyperplastic, hematopoietic spleen was three times more active than the normal spleen. 6. Principal components of partially digested products in the intestinal lumen 1 h after the ingestion were uridine (33%) and cytidine (22%) in the case of [14C]uracil, [14C]cytosine-labeled RNA and inosine (53%) in the case of [14C]adenine-labeled RNA, in accordance with the view that purines and pyrimidines in nucleic acids are absorbed mainly in the form of nucleosides.

Expression of CDK5 (PSSALRE kinase), a neural cdc2-related protein kinase, in the mature and developing mouse central and peripheral nervous systems

CDK5 is a cdc2-related protein kinase that is known to be highly expressed in mature brain. In this study, we obtained a mouse CDK5 cDNA by screening an adult mouse cDNA library. Northern blot analysis demonstrated that the mouse CDK5 mRNA was expressed especially highly in brain, and moderately in kidney, testis and ovary. In brain the expression of CDK5 is already seen at embryonal 12.5 days (E12.5), and it gradually increases through the embryonal stage. After birth, the expression is maintained at a high level to adulthood. In situ hybridization demonstrated that the expression of CDK5 mRNA was distributed in neurons throughout the brain, spinal cord and peripheral ganglia, especially in the hippocampal pyramidal cells, cerebellar Purkinje cells, cortical neurons, olfactory mitral cells, mesencephalic and motor trigeminal nuclei and trigeminal ganglion. In any portion, no apparent expression was observed in glia. During development, the expression of CDK5 was already seen at E12.5 intensely in trigeminal and dorsal root ganglia, and moderately and diffusely in the central nervous system. The expression pattern of CDK5 is quite in contrast with that of CDC2. The fact that CDK5 is expressed in terminally differentiated non-dividing neurons predicts an alternative function(s) in addition to controlling the cell cycle.

Two carbamyl phosphate synthetases of mammals: Specific roles in control of pyrimidine and urea biosynthesis

Abstract Livers of ureotelic animals are known to contain a high activity of ammonia- and acetylglutamate-dependent carbamyl-P synthetase, which is present in the mitochondria and is considered to provide carbamyl-P principally for arginine and urea biosynthesis. The glutamine-dependent synthetase, the occurrence of which in mammals was only recently demonstrated, is considered to function specifically for pyrimidine biosynthesis. The latter activity is present in the cytosol of growing tissues, either normal or malignant, as well as of liver. The mechanisms for control of their activities are quite different; each fits its postulated respective roles. The glutamine enzyme from mouse spleen is subject to inhibition by UTP (negative allosteric effector) and to activation by PP-ribose-P (positive allosteric effector). Through the activating effect of PP-ribose-P the initial step of the pyrimidine pathway may be intimately related to the important reactions in nucleotide metabolism that utilize PP-ribose-P. The sensitivity is not found for the ammonia enzyme. For control of the ammonia enzyme we propose a hypothesis that acetylglutamate, the essential allosteric activator for the enzyme, may work as a physiological regulator for the entry of ammonia nitrogen into the urea cycle. Experimental support for the hypothesis is provided. A highly specific enzyme for acetylglutamate synthesis is present in the liver mitochondria and its activity is controlled specifically by arginine. The known and newly revealed distinctions between the localization and properties of two synthetases may assure the complete or almost complete segregation as well as independent control of the arginine and pyrimidine pathways in mammals. The carbamyl-P metabolism in various organisms is discussed for comparative purposes.

Enzymatic synthesis of acetylglutamate by mammalian liver preparations and its stimulation by arginine

Summary An enzymatic activity to synthesize N-acetyl-L-glutamate, known as an essential allosteric activator of the ammonia-dependent carbamyl phosphate synthetase, from L-glutamate and acetyl-CoA was demonstrated in the extracts of rat and mouse liver mitochondria. A partially purified enzyme had a strict substrate specificity: various amino acids and acyl-CoA analogues could not substitute for the both substrates. A notable finding is that the enzyme activity was markedly stimulated by L-arginine. The effect of arginine was specific, and all other amino acids tested, including usual components of proteins and intermediates of urea cycle as well as several guanidino compounds were totally ineffective. Arginine also stimulated the acetylglutamate synthesis in intact mitochondria. The occurrence of an enzyme specific for acetylglutamate synthesis and control of its activity by arginine may provide an important regulatory mechanism for the production of carbamyl phosphate as the first step of urea biosynthesis.

Insulin-resistant diabetes associated with partial deletion of insulin-receptor gene

The insulin-receptor genes from a 16-year-old girl with type A insulin resistance, who presented with fasting hyperinsulinaemia, acanthosis nigricans, and reduced insulin binding, and from her family were examined. One allele of her insulin-receptor gene inherited from her mother contained a 1.2 kb deletion arising from a recombination between two Alu elements. The deletion removed the 14th exon in the beta subunit and altered the reading frame, to produce a stop codon after aminoacid 867. Pedigree analysis indicated that this mutation alone will not cause diabetes, and the proband is possibly a compound heterozygote. 4 other members of her family were heterozygous for the same mutation; all 4 had a decrease in insulin binding and slight impairment of glucose tolerance. Perhaps the same mutation is an underlying feature of some cases of non-insulin-dependent diabetes mellitus.

Expression of c‐myc oncogene in colorectal polyps as a biological marker for monitoring malignant potential

The expression of oncogenes (c‐myc, c‐fos, c‐Ki‐ras, c‐Ha‐ras, and p53) was examined by Northern blot analysis using freshly isolated human colorectal and gastric cancers and noncancerous portions as the controls. Remarkably high levels of c‐myc expression were found in colorectal cancers (eight of 11), but not in gastric cancers. High levels of c‐myc expression were also detected in colorectal polyps and in metastatic liver tumors. In colorectal polyps, the transcript levels significantly correlated with the histologic malignancy and the size. In contrast, neither c‐fos nor c‐Ki‐ras was overexpressed in colorectal and gastric cancers, and transcripts of c‐Ha‐ras and p53 were not evident in any tissue examined. In light of these observations the c‐myc expression may be specifically associated with the evolution of colorectal cancer as well as progression and maintenance stages, hence may prove to be a useful marker to evaluate the malignant potential of colorectal polyps.

Activation by 5-phosphoribosyl 1-pyrophosphate of glutamine-dependent carbamyl phosphate synthetase from mouse spleen

Summary Glutamine-dependent carbamyl phosphate synthetase from hematopoietic mouse spleen, the first enzyme of the pyrimidine biosynthesis, is subject to a remarkable activation by PP-ribose-P. The effect is specific; ribose 5-phosphate, inorganic pyrophosphate, or a mixture of both can not substitute for it. The activation is reversible and there is no measurable consumption of PP-ribose-P during the reaction. PP-ribose-P stimulates the reaction by increasing the affinity of the enzyme for MgATP2-, a substrate, without changing the maximal velocity. These results support an allosteric mechanism for this effect. A low value of the activation constant for PP-ribose-P (less than 10 μ M ) suggests a physiological importance of this activation in regulation of general nucleotide metabolism.

Cell-free synthesis of the enzymes of peroxisomal β-oxidation

Three enzymes of peroxisomal β-oxidation of rat liver were synthesized in a cell-free protein-synthesizing system derived from rabbit reticulocyte lysate. The invitro products of acyl-CoA oxidase and enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase multifunctional protein were similar in size to or slightly larger than the subunit of the respective mature enzymes. The invitro product of peroxisomal 3-ketoacyl-CoA thiolase was about 3,000 daltons larger than the mature subunit. The hepatic levels of translatable mRNAs coding for these three enzymes were about 10 times higher in rats fed a di(2-ethylhexyl)phthalate-containing diet than in control animals.

Tissue-differential expression of two distinct genes for phosphoribosyl pyrophosphate synthetase and existence of the testis-specific transcript

Cloning of cDNA coding for rat phosphoribosyl pyrophosphate (PPRibP) synthetase (EC 2.7.6.1) revealed two distinct types of subunit, referred to as PRS I and PRS II (Taira et al. (1987) J. Biol. Chem. 262, 14867-14870). Tissue-specific expression of PRS I and PRS II genes (designated PRPS1 and PRPS2, respectively), was shown for 16 rat organs, using Northern blot analysis. The 2.3 kb PRPS1 mRNA level was high in the brain and adrenal gland, whereas the 3.7 kb PRPS2 mRNA level prevailed in the lung and spleen. Both genes were highly expressed in the thymus, adipose tissue and testis. In other mammals (mouse, calf and human), these two types of mRNA were also detected in various tissues and cell lines. Thus, the expression of each gene is regulated in a tissue-specific manner and there may be functional differences between catalytic and/or regulatory properties of subunits PRS I and II of this enzyme. In the testis, an additional PRPS1-related transcript of 1.4 kb was noted in rats, mice and humans. This transcript may belong to a group of testis-specific gene expressions or functions.