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Biomedical subjects

Y Natori

Publications and source records attributed to Y Natori.

At least 55 records · Page 3Linked to original sources

Effect of varying amino acid levels on protein metabolism in nephrotic rats during total parenteral nutrition.

In an attempt to determine appropriate diet in nephrotic syndrome, nephrotic rats, induced by puromycin aminonucleoside, were nourished by total parenteral nutrition fluid containing the same energy, but three different levels (1.65, 3.3, and 6.6%) of amino acids for 7 d. The fractional rate of total protein synthesis in the liver was determined by injecting a flooding dose of [3H]phenylalanine. The proportion of newly synthesized proteins retained and exported by the liver was estimated by injecting a tracer dose of [14C]leucine and then measuring the protein radioactivity remaining in the liver and present in the plasma after secretion was completed. Nephrotic animals synthesized more protein than control animals. Although the absolute synthesis rates of total protein in liver were increased with increasing amino acid administration, the absolute rates of synthesis of albumin were higher in the 3.3% group than in the other groups in nephrotic rats. However, kidney protein synthesis in nephrotic rats was higher in the 1.65% group than in the 3.3% group. Interestingly, the 3.3% group revealed the smallest urinary excretion of total protein and albumin. In addition, in the 3.3% group, plasma concentrations of total protein and albumin were higher, and plasma concentrations of total cholesterol and triglyceride were lower than in other groups. It was concluded that the 3.3% group, corresponding to a normal protein diet, has the greatest salutary effect on urinary protein excretion, followed by protein and lipid metabolism, in nephrotic rats. Not only protein intake but also the energy:protein ratio are important for diet therapy in nephrotic animals. The technique of total parenteral nutrition may be useful in defining the factors involved in glomerular permeability or permselectivity and intracellular protein metabolism.

Amino Acids↗

Endocytosis of serum albumin in regenerating rat liver.

Lysosomes, isolated from rat liver after 70% partial hepatectomy (PHX), were found, by Western blotting, to contain a considerable amount of serum albumin. The level of intralysosomal serum albumin after PHX showed biphasic patterns: it increased immediately after PHX, peaked at 30 min, rapidly declined within a few hours, rose again with a peak at 15 hr, and gradually declined thereafter. At 15 hr after PHX, the content of lysosomal proteins in the liver increased to twice the level of unoperated control, and the electron-microscopic observation of the isolated lysosomes revealed numerous large membrane-delimited structures with ground substances of variable electron opacities. The increase in the intralysosomal serum albumin at 30 min and 15 hr was accompanied by changes in the buoyant densities of endosomes in Percoll density gradients. At both time points, the density profiles of endosomes isolated from hepatectomized rats shifted to the denser direction, suggesting that PHX activates fusion and/or maturation of endosomes. Formaldehyde-treated bovine serum albumin is known to be taken up by the liver by receptor-mediated endocytosis. The uptake of the modified heterologous albumin was shown to be activated as early as 30 min after PHX. Both the uptake of serum albumin into lysosomes and the shift of buoyant density profile of endosomes after PHX were inhibited by the administration of adrenergic receptor antagonists, particularly by the alpha r-antagonist prazosin. Further, the concentration of catecholamines in rat serum, particularly that of norepinephrine, was found to increase immediately after PHX, relative to that in serum from sham-operated rats. These results suggest that the elevation of serum norepinephrine levels after PHX activates endocytosis and facilitates delivery of endocytosed serum albumin to lysosomes, where albumin is digested to yield amino acids for possible use in protein synthesis during liver regeneration.

Adrenergic alpha-Antagonists↗

Cloning and sequencing of cDNA encoding 4-aminobenzoate hydroxylase from Agaricus bisporus.

A cDNA clone encoding 4-aminobenzoate hydroxylase (EC 1.14.13.27) has been isolated using a probe prepared by PCR on the basis of partially determined amino acid sequences of the enzyme. The cDNA contained 1380-base pair open reading frame encoding 460 amino acid residues (M(r) 50974), 14-base pair 5'-untranslated region and 123-base pair 3'-untranslated region including a poly(A) tail of 20 nucleotides. All of the partially determined amino acid sequences were shown to be included in the deduced amino acid sequence. Homology analyses showed that the two regions on the enzyme share other flavoproteins such as salicylate hydroxylase and p-hydroxybenzoate hydroxylase.

Adenosine Diphosphate↗

Detection of N-acetylgalactosaminyltransferase mRNA which determines expression of Sda blood group carbohydrate structure in human gastrointestinal mucosa and cancer.

The Sda blood group carbohydrate structure, GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4GlcNAc-R, is expressed on glycolipid and glycoprotein in human gastrointestinal mucosa. The expression of the Sda determinant dramatically decreases in cancer tissue. The activity of the beta1,4N-acetylgalactosaminyltransferase (Sda-GalNAcT), which transfers GalNAc to NeuAcalpha2-3Galbeta1-4Glc(NAc)-R, correlates with the expression of the Sda immuno-epitope. From the total RNA fraction of human gastric mucosa, we have amplified a cDNA segment by reverse-transcription-polymerase-chain reaction (RT-PCR), using primers designed according to the cDNA sequence of a murine beta1,4GalNAcT which synthesizes the Sda determinant. An RT-PCR product of 390 bp shared 85% nucleotide identity with the murine Sda-related beta1,4GalNAcT. This RT-PCR product hybridized to a transcript in mRNA prepared from human gastric mucosa. In RT-PCR using specific primers to this PCR product, Sda-GalNAcT mRNA was detected in all samples of normal stomach and small intestine examined and the majority of normal colonic specimens. Six out of nine cases of gastric cancer, and 9 out of 13 cases of colonic cancer failed to produce the target DNA. These results correlate with the beta1,4GalNAcT activity measured in the same samples. In conclusion, a segment of the cDNA for betal,4GalNAcT which determines expression of the Sda carbohydrate structure was obtained, and reduced transcription of this beta 1,4GalNAcT resulted in the disappearance of the Sda epitope in gastrointestinal cancer.

Animals↗

Vitamin B6 deficiency accelerates metabolic turnover of cystathionase in rat liver.

Although most of cystathionase was found to exist as an inactive apoenzyme in the liver of vitamin B6-deficient rats, the concentrations of the immunoreactive enzyme protein were virtually the same for control and vitamin B6-deficient livers. Under vitamin B6 deficiency, however, the rate of synthesis of cystathionase, measured by incorporation of labeled amino acid into the immunoprecipitated enzyme, was increased severalfold due to an increased level of cystathionase mRNA. Western blot analysis of lysosomal proteins showed that the amount of cystathionase in the lysosomes from the liver of vitamin B6-deficient rats was also increased severalfold. This observation suggests that lysosomes specifically recognize the apocystathionase for sequestration in preference to the holoenzyme. The present study provides the molecular basis for dual roles of vitamin B6 in controlling the metabolic turnover of cystathionase; it regulates synthesis of the enzyme by modulating the expression of cystathionase gene, and it regulates degradation of the enzyme by different susceptibilities of apo- and holoenzymes to lysosomal proteolysis.

Animals↗

Jak3 expression in glomerular epithelia of IgA nephropathy (IgA-N) patients.

Jak3 is a member of the Janus kinase family which plays an important role in cytokine signal transduction. Jak3 associates the gamma(c) chain of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, and is essential for the signal transduction of these cytokines. We have isolated Jak3 kinase from renal mesangial cells and demonstrated the constitutive expression of Jak3 in glomeruli in vivo. To investigate the physiological and pathological role of Jak3 in glomeruli, we prepared anti-Jak3 antibody and analysed the localization of Jak3 in glomeruli of renal biopsy samples from various nephritis patients and normal subjects. Among 61 nephritis patients and four normal subjects investigated in the present study, Jak3 was selectively localized to glomerular epithelia of IgA-N patients (14/34 cases) and focal glomerulosclerosis patients (1/5 cases), but not detected in minimal changes (n = 6), membranous glomerulonephropathy (n = 7), crescentic glomerulonephritis (n = 4), lupus nephritis patients (n = 5), and normal subjects (n = 4). The intense immunoreactivity for Jak3 is significantly associated with the decrease in creatinine clearance (81.5 +/- 10.4 ml/min versus 104.3 +/- 29.6 ml/min; P < 0.05, Student's t-test) and the increase in level of serum creatinine (1.13 +/- 0.33 mg/dl versus 0.75 +/- 0.23 mg/dl; P < 0.01, Student's t-test) in IgA-N patients. Furthermore, gamma(c) chain was concomitantly expressed with Jak3 in glomerular epithelia in vivo and in vitro, suggesting that signal transduction via gamma(c)-Jak3 cascade may be involved in the pathogenesis of glomerular injury of IgA-N. Taken together with the recent findings that IL-4-secreting T lymphocytes in affected glomeruli injure glomerular epithelium, the responsiveness of glomerular epithelium for IL-4 may be pathologically enhanced in IgA-N.

Animals↗

Cytotoxicity of sera from rats with puromycin aminonucleoside nephrosis.

Administration of puromycin aminonucleoside (PAN) to rats induces acute nephrosis with hyperlipidemia, and, in some experimental conditions, it results in chronic focal glomerulosclerosis. In this study, we examined the cytotoxicity of serum from rats with PAN-induced nephrosis, since hypercholesterolemia is considered to cause injury to vascular walls in atherosclerosis, the mechanism of which is analogous to that of glomerulosclerosis. About half of the tested sera from nephrotic rats (9 out of 17) were cytotoxic to cultured aortic endothelial cells. The toxic substance(s) was heat-stable and was extracted in the lipid fraction. Serum levels of triglyceride and cholesterol were markedly higher in the group of rats with cytotoxic serum than in the group with noncytotoxic serum. No cytotoxicity was associated with sera from control rats or the corresponding lipid fractions. Cytotoxic sera were also effective against cultured glomerular epithelial and mesangial cells. These results indicate that cytotoxic lipid is produced in rats with PAN nephrosis and the results raise the possibility that the cytotoxic lipid in nephrotic serum might contribute to lipid-mediated glomerular injury which may induce glomerulosclerosis at a subsequent stage.

Animals↗

Isolation and characterization of a novel perchloric acid-soluble protein inhibiting cell-free protein synthesis.

We found a novel protein in the postmitochondria supernatant fraction of rat liver, which is soluble in 5% perchloric acid and strongly inhibits protein synthesis in a rabbit reticulocyte lysate system. The protein extracted from the supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The protein was shown to consist of two identical subunits with a molecular mass of 14 kDa. By immunoscreening with the rabbit antisera against the protein, a cDNA encoding the protein was cloned and sequenced. The cDNA contained an open reading frame of 411 base pairs encoding a 136-amino acid protein with a molecular mass of 14,149 Da. The deduced amino acid sequence was completely identical with that constructed from all of the above peptides. Interestingly, the perchloric acid-soluble protein inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner from RNase A. The protein is likely to inhibit an initiation stage of cell-free protein synthesis. Among the rat tissues tested, the protein was located only in liver and kidney. These findings are the first report on a new inhibitor that may be involved in the regulation of protein synthesis in those tissues.

Amino Acid Sequence↗

Interaction of rat liver lysosomes with basic polypeptides.

In order to gain knowledge on the interaction of lysosomes with proteins, we have assessed the equilibrium densities of the lysosomal membrane and matrix markers after in vitro incubation of rat liver lysosomes with various polypeptides. The addition of basic polypeptides, polylysine or protamine, to the suspension of lysosomes brought about a profound alteration of lysosomal membrane, causing extensive leakage of lysosomal matrix enzymes. Electron microscopic observation revealed a remarkable aggregation of lysosomes by the basic polypeptides. Polyglutamic acid, an acidic polypeptide, did not produce such effect. ATP was found to stabilize lysosomes during incubation, particularly with basic polypeptides.

Animals↗

Vitamin B6 modulates expression of albumin gene by inactivating tissue-specific DNA-binding protein in rat liver.

The level of albumin mRNA in the liver of vitamin B6-deficient rats was found to be 7-fold higher than that of control rats. Since the transcriptional activity of the albumin gene, as measured by a nuclear run-on assay, was increased 5-fold in vitamin B6 deficiency, the higher concentration of albumin mRNA in the liver of vitamin-deficient rats could be attributed to the enhanced rate of transcription. The promoter proximal sequences of the albumin gene interact with a number of tissue-specific transcription factors including HNF-1 and C/EBP. We determined the binding activities of liver nuclear extracts to the HNF-1- and C/EBP-binding sites by gel mobility-shift assay and found that the activities of the extract prepared from liver of vitamin B6-deficient rats were greater than those of controls. As the concentrations of C/EBP in nuclear extracts from control and vitamin-deficient rats, estimated by Western-blot analysis, were essentially the same, the lower binding activity of the extract from control liver is probably due to inactivation of tissue-specific factors by pyridoxal phosphate and/or its analogues. We therefore examined the effect of pyridoxal phosphate and its analogues on the binding activity of nuclear extract in vitro and found that only pyridoxal phosphate effectively inhibited the binding. These observations indicate that vitamin B6 modulates albumin gene expression through a novel mechanism that involves inactivation of tissue-specific transcription factors by direct interaction with pyridoxal phosphate.

Albumins↗

Detection and purification of two 14 kDa phospholipase A2 isoforms in rat kidney: their role in eicosanoid synthesis.

Phospholipase A2 (PLA2) activity in the soluble fraction of rat kidney yielded three peaks on DEAE cellulose column chromatography. From these three, we purified two PLA2 isoforms to near-homogeneity. Both had a molecular weight of approx. 14,000 on SDS-PAGE, and immunochemical and enzymological studies indicated that one is a 14 kDa type I PLA2 and the other a 14 kDa type II PLA2. RNA blot analysis confirmed that rat kidney contains both types of PLA2 and that administration of lipopolysaccharides and mercury chloride into rats increased type II PLA2 mRNA levels in kidney. When cultured rat mesangial cells were incubated with purified type I or type II PLA2 in combination with the calcium ionophore A23187 at suboptimal condition, augmentation of prostaglandin E2 production was observed. Type I and type II forms of PLA2 may play a role in arachidonate metabolism in rat kidney.

Animals↗

Production of functional chick liver HMG 2a protein in Escherichia coli.

An efficient Escherichia coli system for the production of a variant form of high-mobility group-2a protein (HMG 2a), having the additional 5 amino acid residues (Ala-Pro-Thr-Leu-Glu) at the NH2-terminal, has been constructed. cDNA encoding HMG 2a was ligated with the Omp A signal peptide sequence and was inserted into an inducible bacterial expression vector pSH-L. After the plasmid introduced into E. coli was expressed by temperature shift, the recombinant product was purified by trichloacetic acid precipitation followed by Bio-Rex 70 column chromatography. The purified product showed the expected NH2-terminal sequence and the superhelical activity of circular DNA similar to the authentic HMG 2a isolated from chick liver.

Amino Acid Sequence↗

Microsurgical anatomy of the superior orbital fissure.

The microsurgical anatomy of the superior orbital fissure was examined in cadaver specimens. The cavernous sinus fills the posterior margin and the orbital contents fill the anterior margin of the fissure. All of the nerves coursing in the walls of the cavernous sinus pass through the superior orbital fissure to reach the orbit. The fissure has a narrow lateral part and a larger medial part. The annular tendon from which the rectus muscles arise is situated in front of the upper half of the medial part of the fissure and is attached to the lateral margin near the junction of the lateral and medial parts. The fissure is divided into three sectors: lateral, central, and inferior. The lateral sector, which corresponds to the narrow lateral part, transmits the trochlear, frontal, and lacrimal nerves and the superior ophthalmic vein, all of which course outside the annular tendon. The central sector, which is situated behind and is aligned with the lateral part of the annular tendon, transmits the superior and inferior divisions of the oculomotor nerve, the abducens and nasociliary nerves, and the sensory and sympathetic roots of the ciliary ganglion, all of which pass through the annular tendon. The inferior sector, which is located below the annular tendon and origin of the inferior rectus muscle, is filled with a posterior extension of the orbital fat and transmits the inferior ophthalmic vein. The relationship and course of the nerves in each sector and the incisions that may be used to open and expose the contents of the fissure are reviewed.

Adult↗

How the developing septo-preoptic medical basal hypothalamus stimulates the development of placode-derived LHRH neurons.

We examined the effects of the developing cerebral cortex (CC) and septo-preoptic medial basal hypothalamus (S-MBH) on the development of LHRH neurons in vitro. The serum-free basal culture medium (BCM) was supplemented with CC or S-MBH extracts prepared from 18.5-day-old embryos or from 2-day-old newborns, and the olfactory placode (NAP) of 12-day-old embryos was cultured. The migration of LHRH neurons was found on Day 3 in the cultures supplemented with the embryonic S-MBH extract (Group 3), where the cell development proceeded showing a numerical increase of the cells and the elongation of neurites. In cultures supplemented with the newborn S-MBH extract (Group 5), the cell development was less intensive in comparison with that of Group 3, while in cultures which had no brain extracts (Group 1), the neurons failed to survive a long term culture. The effects of the CC were less than of S-MBH extracts. Analysis of the protein composition of the extracts by electrophoretic and immunoblotting examinations demonstrated a protein spot of 70-kD in the embryonic S-MBH extract. Because the protein spot was identified to be alpha-fetoprotein (AFP), we further examined the effects of AFP. When the anti-AFP immunoglobulin was added to the Group 3 culture, the stimulative effects of the embryonal extract were inhibited, and the addition of AFP to Group 1 cultures did not show stimulative effects. We conclude that the developing S-MBH, the migrating target of LHRH neurons, contains some essential factors for the development of LHRH neurons, but further analysis is needed to determine the chemical natures of these factors.

Animals↗

Pyridoxal 5'-phosphate modulates expression of cytosolic aspartate aminotransferase gene by inactivation of glucocorticoid receptor.

The level of mRNA for cytosolic aspartate aminotransferase (cAST) in the liver of vitamin B6-deficient rats was found to be 7-fold higher than that of the control rats. The administration of hydrocortisone to adrenalectomized vitamin B6-deficient rats induced expression of hepatic cAST mRNA and the induction was suppressed by the simultaneous administration of pyridoxine. Since the 5' regulatory region of the rat cAST gene contains several sequences showing homology to glucocorticoid-responsive elements, we synthesized an oligonucleotide probe of glucocorticoid-responsive element sequence and assayed the binding activity of liver nuclear extract to the oligonucleotide by gel mobility shift analysis. We found that the binding activity of nuclear extract prepared from the liver of vitamin B6-deficient rats was far greater than that of the control rats, indicating that the DNA-binding activity of glucocorticoid receptor was enhanced by vitamin B6 deficiency. We further found that preincubation of the nuclear extract from the vitamin-deficient liver with pyridoxal 5'-phosphate brought about a rapid and extensive decrease in the binding of the extract to the glucocorticoid-responsive element. Congeners of pyridoxal phosphate, such as pyridoxamine 5'-phosphate, pyridoxal, pyridoxamine and pyridoxine, did not show an inhibitory effect. These observations suggest that pyridoxal 5'-phosphate modulates cAST gene expression by inactivating the binding activity of glucocorticoid receptor to glucocorticoid-responsive elements.

Adrenalectomy↗

Effect of vitamin B6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle.

The effect of vitamin B6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle was investigated. The level of phosphorylase mRNA in the muscle of vitamin B6-deficient rats was reduced to 40% of that in the control rats. By contrast, the phosphorylase mRNA level was increased 5-fold in the liver of the deficient animals. It was also found that the expression of the beta-actin gene, generally regarded as a 'housekeeping' gene, was unaffected by B6 deficiency in the muscle but was enhanced in the liver of the deficient animals. These observations suggest that vitamin B6 may modulate the transcriptional activation of the phosphorylase gene in a tissue-specific manner.

Actins↗