Does the parathyroid 'see' phosphate?
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Animals↗
Biomedical subjects
S Tatsumi
Publications and source records attributed to S Tatsumi.
Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands.
The extracellular concentration of inorganic phosphate (Pi) is an important determinant of parathyroid cell function. The effects of Pi may be mediated through specific molecules in the parathyroid cell membrane, one candidate molecule for which would be a Na+-dependent Pi cotransporter. A complementary DNA encoding a Na+-Pi cotransporter, termed rat PiT-1, has now been isolated from rat parathyroid. The 2890-bp complementary DNA encodes a protein of 681 amino acids that shows sequence identities of 97% and 93% with the type III Na+-Pi cotransporters mouse PiT-1 and human PiT-1, respectively. Expression of rat PiT-1 in Xenopus oocytes revealed that it possesses Na+-dependent Pi cotransport activity. PiT-1 messenger RNA (mRNA) is widely distributed in rat tissues and is most abundant in brain, bone, and small intestine. The amount of PiT-1 mRNA in the parathyroid of vitamin D-deficient rats was reduced compared with that in normal animals and increased markedly after administration of 1,25-dihydroxyvitamin D3. Furthermore, the abundance of PiT-1 mRNA in the parathyroid was much greater in rats fed a low-Pi diet than in those fed a high-Pi diet. Thus, rat PiT-1 may contribute to the effects of Pi and vitamin D on parathyroid function.
Amino Acid Sequence↗
Cloning, functional expression and dietary regulation of the mouse neutral and basic amino acid transporter (NBAT).
The Na+-independent dibasic and neutral amino acid transporter NBAT is among the least hydrophobic of mammalian amino acid transporters. The transporter contains one to four transmembrane domains and induces amino acid transport activity via a b0,+-like system when expressed in Xenopus oocytes. However, the physiological role of NBAT remains unclear. Complementary DNA clones encoding mouse NBAT have now been isolated. The expression of mouse NBAT in Xenopus oocytes also induced an obligatory amino acid exchange activity similar to that of the b0,+-like system. The amount of NBAT mRNA in mouse kidney increased during postnatal development, consistent with the increase in renal cystine and dibasic transport activity. Dietary aspartate induced a marked increase in cystine transport via the b0,+ system in mouse ileum. A high-aspartate diet also increased the amount of NBAT mRNA in mouse ileum. In the ileum of mice fed on the aspartate diet, the extent of cystine transport was further increased by preloading brush border membrane vesicles with lysine. Hybrid depletion of NBAT mRNA from ileal polyadenylated RNA revealed that the increase in cystine transport activity induced by the high-aspartate diet, as measured in Xenopus oocytes, was attributable to NBAT. These results demonstrate that mouse NBAT has an important role in cystine transport.
Amino Acid Sequence↗
Relative contributions of Na+-dependent phosphate co-transporters to phosphate transport in mouse kidney: RNase H-mediated hybrid depletion analysis.
Reabsorption of Pi in the proximal tubule of the kidney is an important determinant of Pi homoeostasis. At least three types (types I-III) of high-affinity Na+-dependent Pi co-transporters have been identified in mammalian kidneys. The relative roles of these three types of Na+/Pi co-transporters in Pi transport in mouse kidney cortex have now been investigated by RNase H-mediated hybrid depletion. Whereas isolated brush-border membrane vesicles showed the presence of two kinetically distinct Na+/Pi co-transport systems (high Km-low Vmax and low Km-high Vmax), Xenopus oocytes, microinjected with polyadenylated [poly(A)+] RNA from mouse kidney cortex, showed only the high-affinity Pi uptake system. Kidney poly(A)+ RNA was incubated in vitro with antisense oligonucleotides corresponding to Npt-1 (type I), NaPi -7 (type II) or Glvr-1 (type III) Na+/Pi co-transporter mRNAs, and then with RNase H. Injection of such treated RNA preparations into Xenopus oocytes revealed that an NaPi-7 antisense oligonucleotide that resulted in complete degradation of NaPi-7 mRNA (as revealed by Northern blot analysis), also induced complete inhibition of Pi uptake. Degradation of Npt-1 or Glvr-1 mRNAs induced by corresponding antisense oligonucleotides had no effect on Pi transport, which was subsequently measured in oocytes. These results indicate that the type II Na+/Pi co-transporter NaPi-7 mediated most Na+-dependent Pi transport in mouse kidney cortex.
Animals↗
Immunohistochemical and RT-PCR detection of Na+-dependent inorganic phosphate cotransporter (NaPi-2) in rat brain.
Expression of a renal Na+-dependent inorganic phosphate (Pi) cotransporter (NaPi-2) was studied in rat forebrain with reverse transcription and polymerase chain reaction (RT-PCR) and immunohistochemistry. RT-PCR analysis for total RNA from whole brain and sequencing of the PCR products showed expression of NaPi-2 mRNA in the brain. Immunohistochemical analysis revealed NaPi-2 staining in many nonpyramidal neurons of all six layers throughout neocortical areas and in neurons of proisocortical and periallocortical areas. NaPi-2-immunoreactive neurons were also detectable in the piriform cortex, hippocampal formation, caudate-putamen, amygdaloid nuclei and lateral geniculate nucleus. Furthermore, NaPi-2 staining was shown in ependymal cells and microvascular endothelial cells. The present results suggest that NaPi-2 is synthesized within the brain and involved in maintaining Pi homeostasis of certain neurons and/or the entire brain.
Animals↗
Gene structure and functional analysis of the human Na+/phosphate co-transporter.
Three lambda phage clones encompassing the Na+/phosphate co-transporter (NaPi-3) gene and its 5' flanking region were isolated from a human genomic DNA library. The gene comprises 13 exons and 12 introns and spans approx. 14 kb. All exon-intron junctions conform to the GT/AG rule. The major transcription-initiation site was determined by primer-extension analysis and is an adenosine residue 57 bp upstream of the 3' end of the first exon. There is a typical TATA box 28 bp upstream of the major transcription-initiation site and various cis-acting elements, including a cAMP-responsive element, AP-1, AP-2 and SP-1 sites in the 5' flanking region. This region also contains three direct-repeat-like sequences that resemble the consensus binding sequence for members of the steroid-thyroid hormone receptor superfamily, including vitamin D. Deletion analysis suggests that the region from nt-2409 to nt-1259 in the 5' flanking region may be involved in kidney-specific gene expression. Vitamin D responsiveness of the NaPi-3 promoter was also detected in COS-7 cells co-transfected with a human vitamin D receptor expression vector. The presence of the three vitamin D receptor- responsive elements in the NaPi-3 promoter may be important in mediating the enhanced expression of the gene by 1,25-dihydroxyvitamin D3.
Amino Acid Sequence↗
Blockade of voltage-sensitive sodium channels by NS-7, a novel neuroprotective compound, in the rat brain.
The effects of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7), a novel neuroprotective compound, on the voltage-sensitive sodium channels (VSSC) were examined in the rat brain and cardiac myocytes. NS-7 inhibited [3H]batrachotoxinin A 20 alpha-benzoate (BTX) binding (neurotoxin receptor site 2) in brain membranes with a Ki value of 1 microM, while the compound was less effective in the cardiac myocytes (Ki = 13 microM). Aconitine, on the other hand, inhibited [3H]BTX binding to brain membranes and cardiac myocytes with the same potency. In contrast. NS-7 had no affinity for [3H]saxitoxin binding in brain (neurotoxin receptor site 1). In superfused slices of the rat cerebral cortex, NS-7 inhibited the veratridine (5 microM)-evoked glutamate release in a concentration-dependent manner, the IC50 value of which was 7.7 microM, whereas the compound showed a weak and not significant suppression of KCl-evoked glutamate release. The tissue concentrations of NS-7 in the rat cerebral cortex and heart were 89 and 28 nmole/g tissue, respectively, 5 min after its intravenous injection (8 mg/kg). Furthermore, in the cerebral cortex, NS-7 distributed preferentially to the membrane-enriched synaptosomal fraction. Since neurotoxin receptor site 2 is located in the transmembrane region of the VSSC moiety, the channel function may be substantially inhibited by a peripheral administration of NS-7. These results suggest that the blockade of neurotoxin receptor site 2 of VSSC in the brain contributes to the neuroprotective action of NS-7.
Aconitine↗
A novel cognition enhancer NS-105 modulates adenylate cyclase activity through metabotropic glutamate receptors in primary neuronal culture.
The effect of (+)-5-oxo-D-prolinepiperidinamide monohydrate (NS-105), a novel cognition enhancer, on adenylate cyclase activity was investigated in cultured neurons of the mouse cerebral cortex. NS-105 (10(-7) and 10(-6) M) inhibited forskolin-stimulated cyclic AMP formation, an action that was dependent on pertussis toxin-sensitive G proteins. Conversely, in pertussis toxin-pretreated neurons, NS-105 (10(-7)-10(-5) M) significantly enhanced the forskolin-stimulated cyclic AMP formation, and this action was completely reversed by cholera toxin. A metabotropic glutamate receptor agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S, 3R-ACPD) produced similar bi-directional actions on the cyclic AMP formation. Both of these inhibitory and facilitatory actions of NS-105 and 1S, 3R-ACPD were blocked by L(+)-2-amino-3-phosphopropinoic acid (L-AP3). NS-105 (10(-6) M) and 1S, 3R-ACPD (10(-4) M) significantly enhanced isoproterenol- and adenosine-stimulated cyclic AMP formation. The enhancement of such Gs-coupled receptor agonists-stimulated cyclic AMP formation was also produced by quisqualate but not by L(+)-2-amino-4-phosphonobutanoate (L-AP4). The phosphoinositides hydrolysis was enhanced by 1S, 3R-ACPD (10(-4) M) but not by NS-105 (10(-6) M), however, 1S, 3R-ACPD-induced increase in phosphoinositides turnover was attenuated by NS-105. These findings suggest that NS-105 stimulates metabotropic glutamate receptor subclasses that are coupled both negatively and positively to adenylate cyclase, but it acts as an antagonist at the receptor subclasses that are linked to phosphoinositides hydrolysis.
Adenylate Cyclase Toxin↗
Acute regulation by dietary phosphate of the sodium-dependent phosphate transporter (NaP(i)-2) in rat kidney.
Alteration of the dietary intake of phosphate (P(i)) leads to rapid changes in renal P(i) transport activity. The present study, examined the underlying cellular mechanisms of the rapid regulation, with special reference to renal P(i) cotransporter. Rats were fed either a low-P(i) (0.02%) diet (CLP rats), the low-P(i) diet followed by a high-P(i) (1.2%) diet (AHP rats), or a normal (0.6%) diet (control rats). Na(+)-dependent P(i) transport activity in the brush border membrane was significantly increased in CLP rats compared with control rats, and this activity decreased rapidly within 2 h after the change of diet in AHP rats. Kinetic analysis of P(i) transport in the AHP rats indicated that the reduction was accompanied by a decrease in the apparent Vmax for Na(+)-dependent P(i) uptake. Northern blot analysis showed no difference in the abundance of NaP(i)-2 mRNA of the kidney between AHP and CLP rats. In contrast, Western blot analysis of renal brush border membrane proteins of AHP rats indicated a significant decrease in the abundance of NaP(i)-2 protein as compared with CLP rats. Immunoreactive signals for NaP(i)-2 were detected in lysosomal fractions of AHP and CLP rats. Immunohistochemical analysis showed that, NaP(i)-2 immunoreactivity in AHP rats was largely reduced in the apical membrane of the proximal tubular epithelial cells. Neither cycloheximide nor actinomycin D affected high-P(i)-induced reduction of NaP(i)-2 protein in the brush border membrane of AHP rats, indicating that de novo protein synthesis of an unidentified regulator protein was not involved in the mechanism of this reduction. In contrast, treatment with colchicine, which disrupts microtubulers, abolished the effect of high-P(i) diet on NaP(i)-2 expression. These results suggested that rapid endocytotic internalization of NaP(i)-2 may occur specifically in the brush border membrane following an acute increase in dietary P(i) intake.
Animals↗
Structural organization of the human vitamin D receptor chromosomal gene and its promoter.
The vitamin D receptor (VDR) is known to mediate the pleiotropic biological actions of 1,25-dihydroxyvitamin D3 through its ability to modulate the expression of target genes. The regulation of this ligand-activated cellular transcription factor is reported to occur at both transcriptional and posttranslational levels. To begin to address the molecular basis by which the VDR gene is regulated transcriptionally, we report here an initial characterization of the human VDR gene and its promoter. We isolated several overlapping A-phage and cosmid clones that cover more than 100 kb of human DNA and contained the entire VDR gene. The gene is comprised of 11 exons that, together with intervening introns, span approximately 75 kb. The noncoding 5'-end of the gene includes exons 1A, 1B, and 1C. Eight additional exons (exons 2-9) encode the structural portion of the VDR gene product. While primer extension and S1 nuclease-mapping studies reveal several common transcriptional start sites, three unique mRNA species are produced as a result of the differential splicing of exons 1B and 1C. The DNA sequence lying upstream of exon 1A is GC rich and does not contain an apparent TATA box. Several potential binding sites for the transcription factor SP1 and other activators are evident. Fusion of DNA fragments containing putative promoter sequences upstream of the luciferase structural gene followed by transient transfection of these plasmids into several mammalian cell lines resulted in significant reporter activity. Due to the size and complexity of the 5'-end of the VDR gene, we examined the activity of a DNA fragment surrounding exon 1C. An intron fragment 3' of exon 1C conferred retinoic acid responsivity when fused to a reporter gene plasmid, suggesting a molecular mechanism for the previously observed ability of retinoic acid to induce the VDR. The recovery of the gene for the human VDR will enable further studies on the transcriptional regulation of this gene.
Bacteriophage lambda↗
[Primary hypophosphatemic rickets].
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Adult↗
Effects of truncation of the COOH-terminal region of a Na+-independent neutral and basic amino acid transporter on amino acid transport in Xenopus oocytes.
To determine the role of a neutral and basic amino acid transporter (NBAT) in amino acid transport, we microinjected several COOH-terminal deletion mutants of NBAT cRNA into Xenopus oocytes and measured transport activity for arginine, leucine, and cystine in the presence and absence of sodium. Wild-type NBAT significantly stimulated the uptake of all three amino acids 10-20-fold compared with controls. On the other hand, no mutant, except a Delta511-685 mutant, stimulated the uptake of these amino acids. The Delta511-685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Delta511-685 mutant also increased the uptake of leucine. The Delta511-685 mutant did not stimulate cystine uptake in the presence or absence of sodium. The stimulation of arginine uptake by the Delta511-685 mutant was inhibited by a 100-fold excess of unlabeled leucine in the presence of sodium. Inhibition of L-arginine uptake by L-homoserine was seen only in the presence of sodium, and an increase in the inhibition of L-arginine uptake by L-histidine was seen when the extracellular pH was decreased. Furthermore, an inward current in oocytes injected with the Delta511-685 mutant was recorded electrophysiologically when basic amino acids were applied. Homoserine was also taken up, but sodium was necessary for their transport. These properties of the Delta511-685 mutant correspond to those of the y+ amino acid transporter. If NBAT is a component of the b0,+-like amino acid transport system, it is unlikely that a mutant protein (Delta511-685) is able to stimulate an endogenous y+-like transport system. These results suggest that NBAT functions as a activator of the amino acid transport system in Xenopus oocytes.
Amino Acid Sequence↗
Production and characterization of a monoclonal antibody against medulloblastoma-derived cell line.
A murine monoclonal antibody of the immunoglobulin M subclass, termed M1C, was prepared by fusion of P3X63Ag8U.1 mouse myeloma cells with spleen cells from BALB/c mice immunized with the recently established human medulloblastoma cell line MED-3. MED-3 cells are unique in expressing both neuronal and glial phenotypes. The antigen recognized by M1C was expressed predominantly in the cytoplasm of antigen-bearing cells, but was also present in lower concentration on the cell surface. Flow cytometry showed M1C binding to MED-3 cells increased during the G2/M phase, and decreased after induction of differentiation with N6, O2-dibutyryl cyclic adenosine monophosphate. Biochemical analysis indicated that M1C recognized a 25-kd protein. Immunoperoxidase staining of cultured cell lines showed that although M1C recognized a large panel of malignant tumor-derived cell lines, the staining of malignant glioma-derived cell lines was strongest. Immunohistochemical examination of various human tissues revealed that M1C reacted strongly with malignant gliomas but did not recognize low grade astrocytomas, neuroblastomas, or tumors from other organs. Cross-reactivity with normal tissues was restricted to peripheral blood lymphocytes. The immunoreactivity of M1C was quite different from the two previously described monoclonal antibodies against medulloblastoma. M1C is the first reported MoAb against medulloblastoma, which recognizes a differentiation- and proliferation-dependent antigen expressed in malignant gliomas in particular, and may be useful in determining the differentiation and proliferation features of medulloblastomas and gliomas.
Antibodies, Monoclonal↗
Comparison of heavy metal concentrations in human umbilical cord in 1980 and 1990.
The well-known Minamata disease was caused by mercury; the Itai-Itai disease by cadmium; and lead poisoning by gasoline additives. Following our previous investigation on heavy metal concentrations in the umbilical cords in 1980, total mercury, cadmium and lead concentrations in the umbilical cords (from 20 males and 20 females) have been measured in 1990 for comparison in the present study. The changes in metal concentrations in 1980 and 1990 were: from 0.007 +/- 0.005 microgram/g to 0.011 +/- 0.008 microgram/g for total mercury; from 0.019 +/- 0.016 microgram/g to 0.006 +/- 0.005 microgram/g, from 0.151 +/- 0.123 microgram/g to 0.046 +/- 0.038 microgram/g for lead. Total mercury accumulated in the umbilical cords increased to approximately 1.6-fold, while cadmium and lead decreased to approximately 1/3-fold during these 10 years.
Adult↗
Mutations of the basic amino acid transporter gene associated with cystinuria.
To investigate the function of a basic and neutral amino acid transporter-like protein (rBAT) which is a candidate gene for cystinuria, we analysed the rBAT gene in cystinuric patients. Patient 1 is a compound heterozygote with mutations in the rBAT gene causing a glutamine-to-lysine transition at amino acid 268, and a threonine-to-alanine transition at amino acid 341, who inherited these alleles from his mother (E268K) and father (T341A), respectively. Injection of T341A and E268K mutant cRNAs into oocytes decreased transport activity to 53.9% and 62.5% of control (L-cystine transport activity in oocytes injected with wild-type rBAT cRNA), respectively. Co-injection of E268K and T341A into oocytes strongly decreased amino acid transport activity to 28% of control. On the other hand, co-injection of wild-type and mutant rBAT did not decrease transport activity. Furthermore, immunological studies have demonstrated that the reduction of amino acid transport is not due to a decrease in the amount of rBAT protein expressed in oocyte membranes. These results indicate that mutations in the rBAT gene are crucial disease-causing lesions in cystinuria. In addition, co-injection experiments suggest that rBAT may function as a transport activator or regulatory subunit by homo- or hetero-multimer complex formation.
Adolescent↗
Cloning and functional expression of a Na(+)-dependent phosphate co-transporter from human kidney: cDNA cloning and functional expression.
A cDNA clone encoding a protein 69% identical in amino acid sequence with that of the Na/P(i) co-transporter NaP(i)-1 was isolated from a human kidney cDNA library. The DNA sequence was identical with that of NPT-1 cDNA published by Chong, Kristjansson, Zoghbi and Hughe (1993) (Genomics, 18, 355-359). In the present study, we have characterized the function of the encoded protein and the tissue distribution of its mRNA. Injection of RNA transcribed from NPT-1 into Xenopus oocytes resulted in expression of Na/P(i) co-transport activity showing a high affinity for P(i) transport (Km 0.29 mM). Kinetic characterization ([P(i)], [Na+]) demonstrated that the expressed transport activity has properties similar to those displayed by oocytes injected with human kidney poly(A)+ RNA. Northern blotting demonstrated that NPT-1 mRNA is expressed in renal cortex, liver and brain but not in other tissues. Hybrid depletion with antisense oligonucleotides to NaP(i)-3 and NPT-1 completely inhibited poly(A)+ RNA-induced Na(+)-dependent P(i) uptake in oocytes. These findings indicate that two high-affinity Na/P(i) cotransporters (NaP(i)-3 and NPT-1) are present in human kidney cortex.
Animals↗
Estimation of age from image processing of soft X-ray findings in Japanese male thyroid cartilages.
A method was developed for estimating the age at death based on image processing of soft X-ray pictures of thyroid cartilage from Japanese males. Data for 501 individuals were used to derive equations for this determination. Prior to image processing, gross morphological findings were used for estimation. Soft X-ray pictures indicated ossification with age and one index for image processing was introduced. The exponential function was applied to age estimation.
Adult↗
Estimation of age from soft X-ray findings of Japanese females thyroid cartilages.
A method for estimating the age at death using the image processing of soft X-ray pictures of the thyroid cartilages of Japanese females was examined. The soft X-ray pictures showed increasing ossification with age. One index for image processing was introduced. It is ossification rate (OSFR). Corresponding to this index, one age estimation equation was obtained. This age estimation equation showed accuracies of 60% or more for the age range of +/- 5 years and about 80% for the age range of +/- 8 years.
Adult↗