Biomedical subjects

M Nakazato

Publications and source records attributed to M Nakazato.

At least 91 records · Page 5Linked to original sources

PubMed1998

Topological isomers of human uroguanylin: interconversion between biologically active and inactive isomers.

The solution structures of the two compounds of human uroguanylin (I and II), which were generated during disulfide bond forming reaction, were found to be topological isomers by 1H-nuclear magnetic resonance spectroscopy. These isomers are interconvertible in aqueous media at rates which vary with the pH and temperature of the solution. Because compound I is active in the cGMP producing assay, but compound II is not, this interconversion may be useful for evaluating the activity of human uroguanylin both in vivo and in vitro.

Amino Acid Sequence↗

PubMed1998

Identification of a novel splicing mutation and 1-bp deletion in the 17alpha-hydroxylase gene of Japanese patients with 17alpha-hydroxylase deficiency.

We report studies of two unrelated Japanese patients with 17alpha-hydroxylase deficiency caused by mutations of the 17alpha-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17alpha-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient's mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17alpha-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation.

Adolescent↗

PubMed1998

Bulbar palsy in senile onset familial amyloid polyneuropathy (30Val-->Met): transthyretin-amyloid deposits in the hypoglossal nerve root.

Japanese familial amyloid polyneuropathy (FAP type I) is characterized clinically by autonomic and sensorimotor polyneuropathy. Bulbar palsy or lower cranial amyloid neuropathy has not been reported in this disease. We report a 72-year-old man with senile onset FAP type I, who developed bulbar palsy and sensorimotor polyneuropathy without autonomic failures. He noticed dysesthesia and muscle weakness in the feet at the age of 67 years. Neurological examination showed bulbar signs and sensorimotor polyneuropathy, but not autonomic neuropathy. Electromyography showed denervation in the tongue and extremities. Sural nerve biopsy revealed a profound reduction of myelinated fibers. Neuropathological studies indicated axonal degeneration and transthyretin-amyloid deposits in the peripheral nerves and hypoglossal nerves or roots. DNA analysis demonstrated transthyretinMet30 mutation. We provide the novel possibility that bulbar palsy, due to amyloid neuropathy, can occur in FAP type I. Copyright Rapid Science Ltd

Journal Article↗

PubMed1998

Elevated concentrations of defensins in bronchoalveolar lavage fluid in diffuse panbronchiolitis.

Human neutrophils contain three isoforms of antimicrobial and cytotoxic peptides in the azurophil granules, which belong to a family of mammalian neutrophil peptides named defensins. Here we investigate the role of these peptides in diffuse panbronchiolitis (DPB). Defensins (human neutrophil peptide-1, -2 and -3) were measured by radioimmunoassay in bronchoalveolar lavage fluid (BALF) of 30 patients with DPB, 16 patients with idiopathic pulmonary fibrosis (IPF) and 15 healthy adults. The concentration of defensins was higher in BALF of patients with DPB than in patients with IPF and healthy subjects. DPB and IPF patients also had significantly higher plasma concentrations of defensins than controls. In patients with DPB, BALF concentration of defensins correlated significantly with neutrophil count or BALF concentration of interleukin (IL)-8. Immunohistochemistry of open-lung biopsy specimens from four DPB patients showed localization of defensins in neutrophils and mucinous exudate in the airways, and on the surface of bronchiolar epithelial cells. In vitro studies showed an enhanced extracellular release of defensins following stimulation of neutrophils with phorbol myristate acetate, N-formyl-methionyl-leucyl-phenyalamine, and human recombinant IL-8. Treatment of DPB with macrolides for 6 months significantly reduced neutrophil count and concentrations of defensins and IL-8 in BALF. Our results indicate accumulation of neutrophil-derived defensins in the airway in diffuse panbronchiolitis, and suggest that defensins may be a marker of neutrophil activity in this disease.

Adult↗

PubMed1998

Tissue distribution, cellular source, and structural analysis of rat immunoreactive uroguanylin.

Uroguanylin, a member of the guanylin peptide family, acts on guanylyl cyclase C (GC-C) to regulate intestinal and renal fluid and electrolyte transport through the second messenger, cGMP. Using an antiserum raised against synthetic rat uroguanylin, we established an RIA and identified three endogenous molecular forms in the intestine and kidney: a 15-amino acid uroguanylin, an 18-amino acid uroguanylin that is a monobasic processing product, and a 9.4-kDa prouroguanylin. Prouroguanylin is the major molecular form in these two tissues, whereas only 15-amino-acid uroguanylin is present in the urine. Rat uroguanylin is most abundant in the proximal small intestine, its content decreasing toward the colon. Uroguanylin is present immunohistochemically in the endocrine cells in the intestine and stomach, B cells in the pancreatic islets, and tubular epithelial cells in the kidney. Uroguanylin has a widespread tissue distribution and is located in cells that function in an endocrine, paracrine, and/or luminocrine (luminal secretion) fashion. Uroguanylin may have physiological functions other than the regulation of fluid and electrolyte transport in the intestine and kidney.

Amino Acid Sequence↗

PubMed1998

Elevated pleural fluid levels of defensins in patients with empyema.

BACKGROUND: Defensins, also known as human neutrophil peptides, are antimicrobial peptides present in the azurophil granules of neutrophils. We measured their level in pleural effusion in various pulmonary diseases to investigate whether they could be used as a diagnostic marker in the differential diagnosis of specific pleural diseases. PATIENTS AND PARTICIPANTS: We analyzed pleural effusion samples collected from 61 patients, including 50 exudates (11 with empyema, 3 parapneumonic, 15 tuberculous, 18 neoplastic, 3 miscellaneous) and 11 transudates as controls. MEASUREMENTS: Defensins were measured by radioimmunoassay and also analyzed by reverse-phase high-performance liquid chromatography. The concentrations of interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF) in pleural effusion fluid were measured by enzyme-linked immunosorbent assay to examine the correlation between these cytokines and defensins. RESULTS: The concentration of defensins in all samples of empyema was >5,100 ng/mL and the mean concentration (13,265.8+/-1,895.2 ng/mL) in these samples was the highest among other groups. The concentration in the other 50 pleural effusion samples tested was <2,800 ng/mL. Defensins were mostly of the mature type in empyema. Pleural effusion levels of IL-8 and G-CSF in patients with empyema were also significantly higher than those in other samples. There was a significant correlation between defensins and IL-8 or G-CSF in pleural effusion fluid (r=0.762, and 0.827, respectively). CONCLUSIONS: Our results suggest that the high effusion concentrations of defensins in pleural effusion may constitute an important component of the host defense system or may have a cytotoxic role in empyema. Our results also indicate that the high levels of IL-8 and G-CSF in empyema may indirectly explain the elevated levels of defensins by increasing the number of neutrophils in the pleural space.

Biomarkers↗

PubMed1998

[Guanylin family: new intestinal peptides regulating salt and water homeostasis].

Guanylin and uroguanylin are novel peptides that are first isolated from rat jejunum and opossum urine, respectively. They bind to and activate guanylyl cyclase-C (GC-C) to regulate intestinal and renal fluid and electrolyte transport through the second messenger, cyclic GMP. Heat-stable enterotoxins produced by pathogenic bacteria have close structural similarities to guanylin and uroguanylin, and they use this mimicry to act on GC-C, causing life-threatening secretory diarrhea. Guanylin primarily is restricted to the intestine, whereas uroguanylin is present in the stomach kidney, lung and pancreas in addition to intestine. Guanylin and uroguanylin in the intestine are secreted into the lumen and blood in response to sodium chloride administration. These peptides will function in salt and water transport in the intestine and kidney by luminocrine and/or endocrine actions. Guanylin peptide family links the intestine with the kidney and could play the physiological roles in the control of water and electrolyte balance.

Amino Acid Sequence↗

PubMed1997

Transcription of the thyroid transcription factor-1 (TTF-1) gene from a newly defined start site: positive regulation by TTF-1 in the thyroid.

Regulation of the thyroid transcription factor-1 (TTF-1) gene expression in the thyroid was investigated. We identified a new transcription start site as nucleotide (nt) -1917, 1700 bp upstream of previously described site, and the region encompassing nt -1242 to -14 as the first intron. Although a probe targeting exon 2 hybridized to both 3.7 and 2.7 kp transcripts, a probe targeting newly identified exon 1 mainly reacted with 3.7 kb transcript, indicating that there exsits a transcript from -1917. Chloramphenicol acetyltransferase (CAT) reporter gene assays demonstrated that 5'-flanking region of the start site exhibited promoter activity in FRTL-5 cells but not in rat liver cells, suggesting that this region confers the thyrocyte-selective expression of the gene. Two consensus TTF-1 binding motifs were detected in this promoter region, and electrophoretic mobility-shift assays showed that oligonucleotide probes, each containing one of these motifs, formed a complex with the recombinant TTF-1 homeodomain. Moreover, recombinant TTF-1 increased the transcriptional activity in FRT cells which do not express TTF-1. These results suggest that transcription from the newly identified start site in the TTF-1 gene is positively regulated by TTF-1 in the thyroid.

Animals↗

PubMed1997

Detection of three transthyretin gene mutations in familial amyloidotic polyneuropathy by analysis of DNA extracted from formalin-fixed and paraffin-embedded tissues.

We identified three different missense mutations of the transthyretin (TTR) gene in three Japanese patients with familial amyloidotic polyneuropathy by analysis of their DNAs extracted from formalin-fixed and paraffin-embedded tissues. Patient 1 carried the TTR methionine-30 (Met) mutation (G to A transition at position 1679). DNA sequencing analysis of the TTR gene from patient 2 showed a G to T transversion at position 3830 in exon 3, resulting in an amino acid replacement of serine-50 (Ser) with isoleucine (Ile). Patient 3 had the novel mutation (G to T transversion at position 7314) in exon 4, resulting in an amino acid replacement of alanine-109 (Ala) with Ser. We established DNA diagnostic methods for detecting TTR Ile50 by polymerase chain reaction (PCR)-induced mutation restriction analysis and for TTR Ser109 by PCR-restriction fragment length polymorphism. Gene analysis of archival paraffin-embedded tissues is useful for the precise diagnosis of FAP and for clarifying its molecular pathogenesis in patients for whom fresh genomic DNA is not available.

Aged↗

PubMed1997

Genomic structure and chromosomal localization of human uroguanylin.

Uroguanylin, a member of the guanylin peptide family, is a novel peptide regulator in intestinal salt and water transport. We isolated the gene for uroguanylin from a human genomic library and determined its structure. This gene consists of three exons and two introns within an overall length of 2.5 kb. The 5' flanking region has TATA and CAAT boxes. The gene also has multiple binding sites for promoter-specific transcription factor, activator protein-1, and activator protein-2, and a cAMP-regulated enhancer element. Fluorescence in situ hybridization showed the uroguanylin gene at human chromosome 1p33-p34. RNA blot analysis showed that human uroguanylin mRNA is expressed in the gastric fundus and pylorus as well as in the intestine.

Amino Acid Sequence↗

PubMed1997

Urine and plasma levels of uroguanylin and its molecular forms in renal diseases.

Uroguanylin activates the intestinal and possibly the renal guanylate cyclase C receptor, and stimulates Cl- secretion. We developed a sensitive radioimmunoassay (RIA) for human uroguanylin and measured its concentration in the urine and plasma. Twenty-four-hour urinary excretion of immunoreactive (ir-) uroguanylin for persons with a high-salt diet (10 g/day) was 137.8 +/- 14.4 pmol/day, significantly higher than that for persons with a low-salt diet (7 g/day, 95.1 +/- 16.3 pmol/day, P < 0.05). There were significantly positive correlations between the urinary excretion of ir-uroguanylin and Na+, Cl-, K+ or cyclic GMP (cGMP). We demonstrated the presence of messenger RNA of guanylate cyclase C in the medulla of human kidney. The concentration of plasma ir-uroguanylin significantly correlated with that of serum creatinine (r = 0.71, P < 0.001). Biologically active uroguanylin-16 accounted for 99% of the endogenous uroguanylin molecules in normal urine and 60% in plasma, the remainder being the 10 kDa precursor. The precursor content increased in the urine and plasma as the severity of renal impairment increased. These findings suggest that bioactive uroguanylin-16 is involved in the regulation of electrolyte homeostasis and that the kidney participates in the metabolism and excretion of uroguanylin.

Circadian Rhythm↗

PubMed1997

Elevated concentrations of human neutrophil peptides in plasma, blood, and body fluids from patients with infections.

Neutrophil peptides, also called defensins, are antimicrobial molecules localized in the azurophil granules of neutrophils. We used a sensitive radioimmunoassay to measure the concentrations of human neutrophil peptides (HNPs) 1-3 in the plasma and blood of 86 healthy volunteers who served as controls and 54 patients with various infections. The respective mean plasma concentrations of HNPs 1-3 in the patients at the onset of bacterial infection, nonbacterial infection, and pulmonary tuberculosis were 4.2, 3.2, and 1.8 times the mean value (+/- SE) for the controls (254.8 +/- 7.1 pg/microL). Plasma concentrations of HNPs 1-3 for the patients were correlated with the peripheral blood neutrophil counts (correlation coefficient = 0.629; P = .0001). The mean concentration (+/- SE) of blood HNPs 1-3 in patients with bacterial infections was higher than the mean value (+/- SE) for controls (10.4 +/- 0.6 ng/microL), whereas the mean concentration (+/- SE) in patients with nonbacterial infections or tuberculosis did not differ from that for controls. Reverse-phase high performance liquid chromatography of plasma samples from patients with bacterial infections showed high concentrations of two precursors of HNPs 1-3, indicating that the biosynthesis of HNPs in neutrophil precursor cells is stimulated in response to infection and/or that the release of pro-HNPs is enhanced. HNPs 1-3 concentrations in the pleural fluid, bronchoalveolar lavage fluid, urine, and cerebrospinal fluid were also elevated in patients with infections. Our results suggest that HNPs have physiological significance in infection and that this family of peptides may be useful in assessing neutrophil function during infection.

Adolescent↗

PubMed1997

Thyroid-stimulating hormone-induced down-regulation of thyroid transcription factor 1 in rat thyroid FRTL-5 cells.

Thyroid transcription factor 1 (TTF-1) is thought to play an important role in the expression of genes that encode thyroid-specific proteins such as thyroglobulin, thyroid peroxidase, and TSH-receptor. The role of TSH in the regulation of TTF-1 messenger RNA (mRNA) and protein abundance was investigated in rat thyroid FRTL-5 cells. Northern blot analysis revealed that TSH reduced TTF-1 mRNA abundance in a dose- and time-dependent manner. Immunoblot analysis with rabbit antibodies prepared against a recombinant fragment of TTF-1 expressed in bacteria showed that TSH also reduced the amount of TTF-1 protein in FRTL-5 cells. Whereas the effect of TSH on TTF-1 mRNA was apparent after 3 h, the effect on TTF-1 protein was not apparent until 12 h after TSH addition to the cells. Both TTF-1 mRNA and protein were significantly decreased after the addition of (Bu)2 cAMP or forskolin for 24 h, whereas they were not decreased by 12-O-tetradecanoyl-phorbol-13-acetate. These results indicate that TSH down-regulates TTF-1 expression in FRTL-5 cells via the cAMP pathway.

Animals↗

PubMed1997

Increased expression of the Na+/I- symporter in cultured human thyroid cells exposed to thyrotropin and in Graves' thyroid tissue.

The Na+/I- symporter (NIS) is important in hormone synthesis in the thyroid gland. NIS activity, as reflected by I- uptake, was increased by TSH (1 mU/mL) or forskolin (10 mumol/L) in primary cultured human thyroid cells. Northern blot analysis revealed that incubation of these cells with TSH or forskolin for 24 h increased the abundance of NIS messenger ribonucleic acid (mRNA) 2.3- and 2.5-fold, respectively. Immunoblot analysis revealed 2.7- and 2.4-fold increases, respectively, in the amount of NIS protein after 48 h, suggesting that elevated levels of intracellular cAMP induced the expression of NIS in human thyrocytes. We then studied the levels of NIS mRNA and protein in Graves' thyroid tissue and found that the amount of NIS mRNA in thyroid tissue from individuals with Graves' disease (n = 5) was 3.8 times that in normal thyroid tissue (n = 5). The abundance of NIS mRNA was significantly correlated with that of thyroid peroxidase or thyroglobulin mRNAs, but not with that of TSH receptor mRNA, in the Graves' and normal thyroid tissue specimens. The amount of NIS protein was also increased 3.1-fold in Graves' thyroid tissue compared with that in normal thyroid tissue. The increased expression of NIS may thus contribute to the development of Graves' disease.

Blotting, Northern↗

PubMed1997

A 5'-splice site mutation in the cytochrome P450 steroid 17alpha-hydroxylase gene in 17alpha-hydroxylase deficiency.

17alpha-Hydroxylase deficiency (17OHD) is an autosomal recessive disorder that produces an excess of mineralocorticoids and sexual differentiation abnormalities. Using DNA sequencing analysis of the 17alpha-hydroxylase (CYP17) gene from a Japanese patient with 17OHD, we identified a new type of genetic abnormality in this disease, a G to A transition at position +5 in the splice donor site of intron 7 of the CYP17 gene. In vitro expression analysis of an allelic minigene that consists of exons 6-8 of the patient's CYP17 gene showed that the transition causes the skipping of exon 7. This exon skipping alters the translational reading frame of exon 8 and introduces a premature stop codon (TAA) at amino acid position 410 proximal to the heme iron-binding site essential for the enzymatic activity of CYP17. Restriction enzyme analysis showed that the patient is homozygous for the mutated CYP17 gene, and the parents are heterozygotes. This is the first reported patient with 17OHD caused by the splice site mutation in the CYP17 gene.

Adrenal Hyperplasia, Congenital↗

PubMed1997

Thyroid transcription factor-1 activates the promoter activity of rat thyroid Na+/I- symporter gene.

We have cloned 15 kbp of rat thyroid Na+/I- symporter gene from liver genomic library, which contains 6 kbp upstream sequence from the translation initiation site. Southern blot analysis of the genomic DNA from the liver has revealed that thyroid Na+/I- symporter gene is the single gene in the rat. To study the tissue-selective expression mechanism of the gene, we at first determined the transcriptional start site of the gene. Results of a rapid amplification of cDNA end procedure as well as that of primer extension analysis indicated that the transcriptional start sites clustered between -96, -95, and -93 bp of the gene (A in ATG is designated as +1). Chimeras containing 1.9 kbp (-1967 to -46 bp) of the 5'-flanking sequence of the Na+/I- symporter gene and luciferase gene expressed significant enzyme activity when transfected into a rat thyroid cell line, FRTL-5, but little activity was observed in BRL-3A rat liver cells. Deletion analysis of the constructs indicated that a minimal region, exhibiting promoter activity and cell specificity, is located between -291 and -134 bp of the gene. Deoxyribonuclease I footprinting shows that nuclear extracts from FRTL-5, but not BRL-3A, cells protect a region between -245 and -230 bp. Electrophoretic mobility shift assays have demonstrated that nuclear extracts from FRTL-5 cells formed a specific DNA-protein complex with an oligonucleotide probe corresponding to -250 to -211 bp of the gene, but that from BRL-3A cells did not, suggesting that thyrocyte-selective nuclear factors bind to the region. When the nuclear extracts from FRTL-5 cells were preincubated with antibody against thyroid transcription factor-1 (TTF-1), homeodomain containing nuclear protein, formation of the complex was abolished and the band was supershifted. We also found that the probe formed a DNA-protein complex with the recombinant TTF-1 homeodomain, and mutations of the binding site eliminated factor binding. When pRc/CMV-TTF-1 was cotransfected with the minimal promoter fragment of thyroid Na+/I- symporter gene into FRT cells, which express no TTF-1, it caused a significant increase in the transcriptional activity of the reporter construct, but not of the construct having mutated TTF-1-binding element. These results suggest that TTF-1 confers the cell-selective expression of Na+/I- symporter gene in thyrocytes.

Animals↗

PubMed1997

Increased plasma guanylin levels in patients with impaired renal function.

Guanylin, a 15-amino acid peptide, activates intestinal guanylate cyclase C receptor, thereby regulating intestinal fluid and electrolyte transport through the second messenger, cyclic GMP. To examine the role of the kidney in guanylin metabolism, we used a radioimmunoassay (RIA) to measure plasma concentrations of guanylin in 3 groups; normal individuals, patients who had renal disorders with normal or elevated serum creatinine levels (0.4 < Cre < 11.9 mg/dl), and patients who received hemodialysis (HD). The plasma concentration of immunoreactive guanylin in the normal individuals was 32.3 +/- 4.8 fmol/ml. The concentrations in 32 non-HD patients were correlated with their serum creatinine concentrations (r = 0.81, p < 0.0001). In 16 HD patients the plasma concentrations of immunoreactive guanylin before the start of HD were correlated with their dialysis duration (r = 0.63, p < 0.01). The plasma levels of immunoreactive guanylin in HD patients for whom EVAL membranes were used decreased one hour after the start of HD as compared with the prior levels. The plasma levels in HD patients for whom PC membranes were used showed no change. Ten kilodalton guanylin is the main component of guanylin molecules in the plasma and hemofiltrates of HD patients. These findings suggest that the kidney has a major role in the elimination and/or metabolism of guanylin. Uroguanylin, a member of the guanylin family that was recently isolated from human urine, also acts on the guanylate cyclase C receptor. Further studies of guanylin family peptides should provide a better understanding of the physiological roles of the kidney in the control of water and electrolyte balance.

Binding Sites↗

PubMed1997

[Measurement of plaque volume using three-dimensional intravascular ultrasound: in vitro study].

The usefulness of three-dimensional echocardiography using intravascular ultrasound (3D-IVUS) for the measurement of plaque volume was evaluated by comparing plaque volume derived from 3D-IVUS with that directly measured in 10 autopsied iliac or femoral plaque models (5-15 mm long). Using IVUS (3.5 F, 30 MHz), sequential cross-sectional images for three-dimensional datasets were acquired with a motorized catheter pullback device connected to the three-dimensional reconstruction system. Three-dimensional reconstruction was performed from the sum of the two-dimensional cross-sectional views. Plaque volumes were calculated using a summation of disks algorithm based on the reconstructed multiple short-axis cross-sections from the three-dimensional data. Three-dimensional IVUS demonstrated a good correlation with direct measurement of plaque volume (y = 0.71x + 0.001, r = 0.80, SEE = 0.003 ml), so is useful for the measurement of plaque volumes in the experimental models.

Arteriosclerosis↗