Biomedical subjects

Y Someya

Publications and source records attributed to Y Someya.

At least 55 records · Page 3Linked to original sources

PubMed1994

[Organization of the human GLUT2 gene and regulation of the GLUT2 gene expression].

The high Km glucose transporter, termed GLUT2, is expressed in pancreatic beta-cells and hepatocytes in a tissue specific manner. Previous studies revealed that higher glucose levels cause increased GLUT2 gene expression in beta-cells and hepatocytes and that 11 exons of human GLUT2 gene are spanning around 30 kilobases in q26.1-->q26.3 region of was has been chromosome 3. While the putative transcriptional start site has been reported, we made a series of deletion analyses of the human GLUT2 gene promoter region and showed that the sequences located downstream of the putative transcriptional start site are essential for transcription not only in a hamster beta-cell line but also in primary cultured rat hepatocytes.

Animals↗

PubMed1994

[The detection of GLUT2 gene mutation by polymerase-chain reaction single stranded conformation polymorphism (PCR-SSCP) method].

The polymerase chain reaction-single stranded conformation Polymorphism (PCR-SSCP) procedure was applied to examine whether the mutation in the liver/islet glucose transporter (GLUT2) gene could be associated with non-insulin-dependent diabetes mellitus (NIDDM) in Japanese. Samples were processed through 30-40 cycles of 1 min denaturation at 94 degrees C, 1 min annealing at 55-68 degrees C for 1-2 min, extention at 72 degrees C for 1 min, and denaturation at 94 degrees C for 1 min. We identified a silent mutation in codon 479 for PheTTT/TTC.

Base Sequence↗

PubMed1993

Second-site mutation of Ala-220 to Glu or Asp suppresses the mutation of Asp-285 to Asn in the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli.

A carboxyl group of Asp-285 is essential for tetracycline/H+ antiport mediated by the transposon Tn10-encoded metal-tetracycline/H+ antiporter (TetA) of Escherichia coli (Yamaguchi, A., Akasaka, T., Ono, N., Someya, Y., Nakatani, M., and Sawai, T. (1992) J. Biol. Chem. 267, 7490-7498). Spontaneous tetracycline resistance revertants were isolated from E. coli cells carrying the Asn-285 mutant tetA gene. All of the revertants were due to the second-site mutation at codon 220 of GCG (Ala) to GAG (Glu). The Km value of the tetracycline transport mediated by the revertant TetA protein was about 4-fold higher than that of the wild-type, indicating that the revertant is a low affinity mutant. A Glu-220 and Asn-285 double mutant constructed by site-directed mutagenesis showed the same properties as the revertants, confirming that the mutation of Ala-220 is solely responsible for the suppression. The Asp-220 mutation of the Asn-285 mutant resulted in a lower level of restoration of the tetracycline resistance and the transport activity than in the case of the Glu-220 mutation. A single mutation replacing Ala-220 with Glu or Asp caused about a 2-4-fold decrease in the tetracycline resistance, but no crucial change in the transport activity. It is not likely that Glu-220 is required for a charge-neutralizing salt bridge because an unpaired negative charge in a Glu-220 or Asp-220 single mutant did not cause a serious change in the activity. An alternative explanation is reasonable; Asp-285 directly contributes to the binding of a cationic substrate, metal-tetracycline chelation complex, or proton, and an acidic residue at position 220 can take over the role of Asp-285.

Alanine↗

PubMed1993

Cloning, functional expression and pharmacological characterization of a fourth (hSSTR4) and a fifth (hSSTR5) human somatostatin receptor subtype.

Somatostatin exerts diverse effects in various tissues upon binding its specific membrane receptors. Recently, we have cloned three different somatostatin receptor subtypes. Here we report the sequence and functional expression of a fourth and a fifth human somatostatin receptor subtype, termed hSSTR4 and hSSTR5, respectively. The hSSTR4 encodes a protein of 388 amino acids and the hSSTR5 is a protein of 364 amino acids. There is 42-60% identity among the amino acid sequences of the five human somatostatin receptor subtypes identified to date. RNA blotting studies reveal that the hSSTR4 is expressed as a single transcript of 4.8 kb in MIA PaCa-2 cells, a cell line derived from human pancreatic cancer while the hSSTR5 is undetectable in the tissues examined. The hSSTR4 and hSSTR5 transiently expressed in COS1 cells exhibit specific binding to somatostatin-14 with IC50 values of 1.6 and 0.16 nM, respectively. We also have characterized the binding affinity of various somatostatin analogues to the hSSTR4 and hSSTR5. The rank of the potency of the analogues are: somatostatin-14 = somatostatin-28 >> RC-160 >> SMS201-995 for the hSSTR4 and somatostatin-28 > somatostatin-14 >> RC-160 > SMS201-995 for the hSSTR5. These results suggest that diverse actions of somatostatin are mediated by at least five somatostatin receptor subtypes with potentially different function.

Amino Acid Sequence↗

PubMed1993

The in vivo assembly and function of the N- and C-terminal halves of the Tn10-encoded TetA protein in Escherichia coli.

The tetA gene was cut into its N- and C-terminal halves at the central EcoRI site and the two halves were subcloned individually or together under a separate lac promoter/operator. The expression of the C-terminal half was detected with a C-terminal-specific antibody. The amount of the N-terminal half in the cytoplasmic membrane was not affected by the presence of the C-terminal half. In contrast, the amount of the C-terminal half in the membrane was increased in the presence of the N-terminal half, indicating that the N-terminal half helps the stable folding of the C-terminal half in the membrane. Each half individually showed no tetracycline transport activity, however, when both halves were expressed together, the resultant complex showed about 40% of the tetracycline transport activity of the wild-type per number of the C-terminals of TetA protein in the membrane.

Amino Acid Sequence↗

PubMed1993

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The structural resemblance and functional difference in the role of the duplicated sequence motif between hydrophobic segments 2 and 3 and segments 8 and 9.

The properties of site-directed mutants as to the putative hydrophilic loop region between hydrophobic segments 2 and 3 in the transposon Tn10-encoded metal-tetracycline/H+ antiporter (TET) were reported in our previous paper (Yamaguchi, A., Someya, Y., and Sawai, T. (1992) J. Biol. Chem. 267, 19155-19162). The loop between hydrophobic segments 8 and 9 contains a conserved sequence motif, GXXXXKXGEK, which is a derivative of the sequence motif, GXXXXRXGRR, in loop2-3. Site-directed mutagenesis studies on loop8-9 revealed that the two loops exhibit significant structural resemblance, that is, 1) when the Gly residue at the eighth position in each loop was replaced by various amino acid residues, the residual activity of the resultant mutants corresponded well to the beta-turn propensity of the substituent, 2) the Cys mutant as to the fourth position in each loop was most profoundly inactivated by N-ethylmaleimide among 10 Cys mutants as to each loop, and 3) the reactivity of a Cys residue introduced at the third position in loop8-9 with N-ethylmaleimide was lower than that in the case of the other Cys mutants, probably due to the residue being partially cryptic as to the attack of the reagent, similar to in the case of the corresponding residue in loop2-3, the latter being entirely cryptic. The Gly at the first position in loop8-9 is less important than the corresponding Gly in loop2-3, however, since the TET protein suffered a loss of activity when a bulky side chain was introduced at the first position in loop8-9 as well as in loop2-3, the structural roles of the 2 glycines are likely to be similar. These findings suggested that loop2-3 and loop8-9 may occupy similar positions in the three-dimensional structure of the TET protein. On the other hand, the two loops showed a significant functional difference; the negative charge of Asp66 and the positive charge of Arg70 in loop2-3 were essential for the transport function, but, in contrast, there was no functionally essential residue in loop8-9, indicating that loop2-3 may form an "active" leaflet in the TET protein, while loop8-9 may be a "silent" counterpart.

Amino Acid Sequence↗

PubMed1993

Two 3',5'-cyclic-adenosine monophosphate response elements in the promoter region of the human gastric inhibitory polypeptide gene.

Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions of the human gastric inhibitory polypeptide (GIP) promoter into hamster insulinoma (HIT T15) cells indicated that the region between -180 and +14 is sufficient for basal promoter activity. Two CRE-BP1 binding sites were identified in this promoter region by DNase I footprinting with the bacterially expressed cAMP response element (CRE) binding protein, CRE-BP1. Mutation analyses showed that these two CREs are required for the basal promoter activity, and furthermore that one site, at nucleotide-158, contributed mainly to the cAMP inducibility of the GIP promoter in HIT T15 cells. Interestingly, the GIP promoter activity was repressed by the c-jun proto-oncogene product, possibly through the CREs.

Animals↗

PubMed1993

Possible compensatory role of parathyroid hormone-related peptide on maintenance of calcium homeostasis in patients with non-insulin-dependent diabetes mellitus.

Recent studies have revealed that altered mineral and vitamin D metabolism is observed in diabetic patients with the complication of osteopenia. In order to elucidate the role of parathyroid hormone-related peptide (PTHrP) on calcium homeostasis in diabetes, we have measured the serum level and urinary excretion of PTHrP as well as other serum calcium-regulating hormones in 106 patients with non-insulin-dependent diabetes mellitus (NIDDM) and 43 control subjects. The serum concentration of intact PTH was 2.34 +/- 0.13 (mean +/- SEM) pmol/l in NIDDM patients, which is significantly lower than the value of 3.11 +/- 0.14 pmol/l in the controls (p < 0.01). Both serum calcium and calcitonin, however, were not statistically different from controls. On the other hand, circulating PTHrP in NIDDM was 40.1 +/- 1.4 pmol/l, which is significantly elevated when compared to 27.3 +/- 1.3 pmol/l in the controls (p < 0.01). Moreover, urinary excretion of PTHrP also was significantly higher in NIDDM (p < 0.01). In the present study, the circulating calcium level was well preserved in NIDDM patients, although the PTH levels were shown to be decreased. The elevated serum PTHrP might, therefore, have a physiologically compensatory role on the calcium regulatory systems in NIDDM. Furthermore, this elevation is most likely due to the excess production of this peptide and not to the decrease in urinary excretion.

Adaptation, Physiological↗

PubMed1992

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3.

The region including the conserved Ser65-Asp66 dipeptide in the tetracycline/H+ antiporter (TET) encoded by transposon Tn10 is thought to play a gating role (Yamaguchi, A., Ono, N., Akasaka, T., Noumi, T., and Sawai, T. (1990) J. Biol. Chem. 265, 15525-15530). The dipeptide is in putative interhelix loop2-3, which also includes the conserved sequence motif, GXXXXRXGRR, found in all TET proteins and sugar/H+ symporters. Through the combination of localized random and site-directed mutagenesis, each residue in loop2-3 was replaced. Among 10 residues in putative loop2-3, the important residues, of which substitution resulted in significant reduction or complete loss of the transport activity, were Gly62, Asp66, Gly69, and Arg70. The defect in the transport activity of the Gly62 and Gly69 substitution mutants corresponded to the steric hindrance by the substituents as to the putative beta-turn structure of the peptide backbone containing these glycines. Of 3 conserved Arg residues, the replacement of only Arg70 caused complete loss of the activity except for replacement with Lys, indicating the importance of a positive charge at this position, which is similar to the essentiality of a negative charge at Asp66. A "charge-neutralizing" intra-loop salt bridge between Asp66 and Arg70 was not likely because the double mutant in which Asp66 and Arg70 were replaced with asparagine and leucine, respectively, showed no transport activity. A triple mutant with only one positive charge at Arg70 in this loop showed about half the wild-type activity, indicating that the polycationic nature of the loop was not critical for the activity. Cys mutants as to the unessential residues in the loop were modifiable with N-ethylmaleimide, except for the Met64----Cys and Arg71----Cys mutants; however, the modification of only the Ser65----Cys mutant caused significant inhibition of the transport activity, indicating that position 65 is a unique position in the structure of loop2-3.

Amino Acid Sequence↗

PubMed1992

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. Roles of the aspartyl residues located in the putative transmembrane helices.

Three conserved aspartyl residues located in the putative transmembrane helices in the Tn10-encoded metal-tetracycline/H+ antiporter were replaced by Asn, Lys, or Glu with oligonucleotide-directed site-specific mutagenesis. Replacement of Asp84 or Asp15 by Asn or Lys caused a severe defect in tetracycline transport activity, however, the Glu84 and Glu15 mutants retained 150 and 40% of the wild type activity, respectively, indicating the critical role of the negative charge. The increase in the activity of the Glu84 mutant was due to an increase in the affinity for the substrate. H+/tetracycline coupling was intact in these mutants, including Asn and Lys mutants. On the other hand, all of the Asp285-substitution mutants showed a severe defect in tetracycline transport activity and a complete lack of tetracycline-coupled H+ transport. However, since in vivo tests showed the tetracycline resistance for the Glu285 mutant, a negative charge in position 285 plays some role in maintaining the possible down-hill and/or low affinity efflux of accumulated tetracycline from intact cells. Similar work was done for Asp365, and here the Asn and Glu mutants showed decreased but high activity, while the Lys mutant was only marginally active (5%), indicating that a negative charge is not so demanding in position 365, possibly because it is not in the membrane.

Amino Acid Sequence↗

PubMed1992

Effect of pancreas transplantation on decreased levels of circulating bone gamma-carboxyglutamic acid-containing protein and osteopenia in rats with streptozotocin-induced diabetes.

Diabetic osteopenia has been known as one of the chronic complications of diabetes mellitus, and a decrease in bone turnover has been thought to be one of the pathophysiological characteristics of this complication. In order to investigate the effect of long-term insulin therapy on low bone turnover in diabetes, pancreas transplantation was performed on streptozotocin-induced diabetic rats. Plasma levels of bone gamma-carboxyglutamic acid-containing protein(osteocalcin) in untreated diabetic rats were 0.9 +/- 0.1 (mean +/- SEM) nmol/l, significantly lower than the value of 4.2 +/- 0.6 nmol/l in control rats (p less than 0.01). Pancreas transplantation reversed this decrease to 6.3 +/- 1.1 nmol/l, which was not significantly different from the value in control rats. The circulating levels of calcitriol were significantly decreased in the untreated diabetic group (p less than 0.01), and the decrease was fully reversed by pancreas transplantation. In addition, the decreases in bone length, strength and weight were also improved by the transplantation. This evidence clearly shows that the improvement of metabolic derangements in diabetes by insulin is essential for the prevention of deterioration in diabetic osteopenia. It is possible, therefore, that insulin exerts an indirect beneficial influence through the metabolic amelioration on the decreases in bone turnover and circulating osteocalcin in diabetes mellitus, or has a direct stimulatory effect on the osteoblasts via the insulin receptor since its presence has been shown recently in osteoblastic cells.

1-Carboxyglutamic Acid↗

PubMed1992

Glucose as regulator of glucose transport activity and glucose-transporter mRNA in hamster beta-cell line.

To investigate the role of glucose in regulating glucose transporters in pancreatic beta-cells, we studied the hamster clonal beta-cell line HIT-T15, which retains responsiveness to glucose. Northern blot analysis demonstrates that GLUT2 and GLUT1 mRNA are abundant in HIT cells. After a 24-h culture with various concentrations of glucose (0-22.2 mM [0-400 mg/dl]), the GLUT2 mRNA level in HIT cells increased by 40% at 22.2 mM (400 mg/dl) glucose compared with 11.1 mM (200 mg/dl) without a change in mRNA stability. It also decreased proportionally to the reduction of glucose concentration. Glucose deprivation resulted in a decrease of GLUT2 mRNA to an almost undetectable level, with a marked increase in the degradation rate of mRNA. In contrast, the GLUT1 mRNA was not affected by glucose. We show that glucose uptake is highest in HIT cells incubated at 2.8-5.5 mM (50-99 mg/dl) glucose for 24 h, and that levels in cells cultured at 0 mM (0 mg/dl) and 22.2 mM (400 mg/dl) glucose decrease to approximately 20% of the maximum level. This decrease is consistent with the effects of glucose on glucose-stimulated insulin secretion in HIT cells. Our results indicate that glucose is involved in regulating GLUT2 mRNA and glucose uptake activity and that the glucose responsiveness of the insulin secretion correlates with the glucose-induced change in glucose uptake activity in HIT cells.

Animals↗

PubMed1991

Tissue distribution and species difference of the brain type glucose transporter (GLUT3).

The complementary DNA for the human brain type glucose transporter (GLUT3) was used to determine its tissue specific expression in human, monkey, rabbit, rat, and mouse. Under high stringent conditions, 4.1 and 3.2 kilobase (kb) GLUT3 transcripts in monkey and a single 4.1 kb GLUT3 mRNA in rabbit, rat, and mouse were detected by RNA blot analysis. Although the GLUT3 transcripts were widely distributed, as are the erythrocyte type glucose transporter (GLUT1) transcripts, this mRNA is most abundant in the brain. However, the relative abundance of GLUT3 mRNA in the various regions of the monkey brain shows a different pattern from that of GLUT1 mRNA: GLUT3 is most highly expressed in the frontal lobe of the cerebrum, whereas GLUT1 is most abundant in the basal ganglia and the thalamus. Moderately higher GLUT3 mRNA levels were detected in the parietal lobe of the cerebrum, hippocampus, and cerebellum than the levels of GLUT1 transcripts. We also detected GLUT3 mRNA in adult human psoas major muscle, although it has been reported that the GLUT3 gene is scarcely expressed in adult human skeletal muscle of the thigh. In addition, in the rat and the mouse, no transcripts of the GLUT3 gene were detected in liver, kidney, small intestine, skeletal muscle, or fat besides in brain. Thus, the expression of the GLUT3 gene seems to be restricted to the brain in rodents. These results suggest that the expression of GLUT1 and GLUT3 genes might be regulated by different mechanisms.

Animals↗

PubMed1991

Antigen-specific directional target cell lysis by perforin-negative T lymphocyte clones.

Despite a number of reports indicating that perforin, a pore-forming protein, is the primary effector molecule mediating specific target cell lysis by cytotoxic T lymphocytes (CTL), several lines of evidence suggest the existence of perforin-independent mechanisms. We established class II-restricted, soluble protein-specific CD4+ T cell clones with killing function which do not express a detectable amount of perforin and perforin mRNA. Nevertheless, these clones induced cytolysis and DNA fragmentation of target cells in a specific and highly directional manner which was not inhibitable by antibody against TNF/lymphotoxin. These data not only indicate the existence of cytotoxic T cell subsets which do not utilize perforin, but also suggest that perforin is not mandatory for specific target lysis by T cells.

Animals↗

PubMed1987