Up-regulation by IGF-I of proliferating cell nuclear antigen and Bcl-2 protein expression in human uterine leiomyoma cells.

IGF-I has been reported to play a role in regulating proliferation of human leiomyoma cells. There is, however, little evidence to suggest that IGF-I inhibits apoptosis in the leiomyoma cells. The present study was conducted to elucidate whether IGF-I affects apoptosis and Bcl-2 protein expression, an apoptosis-inhibiting gene product, in cultured leiomyoma cells. In addition, we examined the effect of IGF-I on proliferating cell nuclear antigen (PCNA) expression in cultured leiomyoma cells. Isolated human leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% FBS for 120 h and then stepped down to serum-free conditions for an additional 72 h in the absence or presence of graded concentrations of IGF-I (1.0, 10, and 100 ng/ml). The effects of IGF-I on Bcl-2 protein and PCNA expression in cultured leiomyoma cells were assessed by Western immunoblot analysis and immunocytochemical staining, whereas the effects of IGF-I on the cell viability and apoptosis of the cultured cells were determined by 3-(4,5-dimethylatriazol-2-yl)-2,5diphenyltetrasodium bromide (MTT) assay and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay, respectively. Immunocytochemical staining demonstrated that IGF-I treatment resulted in the increase in PCNA labeling index in cultured leiomyoma cells in a dose-dependent manner. Immunoblot analysis of proteins extracted from the cultured leiomyoma cells revealed that the addition of IGF-I (10 and 100 ng/ml) significantly increased the expression of 35-kDa immunoreactive PCNA and 26-kDa Bcl-2 protein, compared with those in control cultures. Cell survival and proliferation of cultured leiomyoma cells, assessed by MTT assay, was significantly augmented by IGF-I treatment, compared with those of control cultures. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay showed that the apoptosis-positive rate of leiomyoma cells treated with IGF-I was significantly decreased, compared with that in control cultures. The present results suggest that IGF-I plays crucial roles in leiomyoma cell growth, not only in promoting the proliferative potential by up-regulation of PCNA expression but also in down-regulating apoptosis by up-regulation of Bcl-2 protein expression in leiomyoma cells.

Human cystinuria-related transporter: localization and functional characterization.

BACKGROUND: Cystinuria has been proposed to be an inherited defect of apical membrane transport systems for cystine and basic amino acids in renal proximal tubules. Although the mutations of the recently identified transporter BAT1/b(0,+)AT have been related to nontype I cystinuria, the function and localization of human BAT1 (hBAT1)/b(0,+)AT have not been well characterized. METHODS: The cDNA encoding hBAT1 was isolated from human kidney. Fluorescence in situ hybridization was performed to map the hBAT1 gene on human chromosomes. Tissue distribution and localization of expression were examined by Northern blot and immunohistochemical analyses. hBAT1 cDNA was transfected to COS-7 cells with rBAT cDNA, and the uptake and efflux of 14C-labeled amino acids were measured to determine the functional properties. The roles of protein kinase-dependent phosphorylation were investigated using inhibitors or activators of protein kinases. RESULTS: The hBAT1 gene was mapped to 19q12-13.1 on the human chromosome, which is the locus of nontype I cystinuria. hBAT1 message was expressed predominantly in kidney. hBAT1 protein was localized in the apical membrane of proximal tubules in human kidney. When expressed in COS-7 cells with a type II membrane glycoprotein rBAT (related to b(0,+)-amino acid transporter), hBAT1 exhibited the transport activity with the properties of amino acid transport system b(0,+), which transported cystine as well as basic and neutral amino acids presumably via a substrate exchange mechanism. BAT1-mediated transport was reduced by the protein kinase A activator and enhanced by the tyrosine kinase inhibitor. CONCLUSIONS: hBAT1 exhibited the properties expected for a transporter subserving the high-affinity cystine transport system in renal proximal tubules. The hBAT1 gene was mapped to the locus of nontype I cystinuria, confirming the involvement of hBAT1 in cystinuria.

Isolation and characterization of the genes up-regulated in isolated neurons by aged garlic extract (AGE).

Aged garlic extract (AGE) produces neurotrophic effects on cultured fetal rat hippocampal neurons. These studies examined the molecular events triggered by AGE that might account for a suppression of neuronal cell death. Genes differentially expressed by the addition of AGE in primary cultured hippocampal neurons isolated from fetal rat brain were screened using mRNA differential display. Four cDNA clones were significantly enhanced at their transcriptional level; they were designated as #24, #110, #153 and #155. Quantitative reverse transcription-polymerase chain reaction (RT-PCR), as well as dot-blot hybridization combined with RT-PCR, confirmed that the transcription from these four genes was elevated at least twofold, particularly the mRNA of #153, which was increased >20 times 72 h after the addition of AGE. A homology search of the respective cDNA sequences in the DNA database revealed that #153 is an alpha 2-microglobulin-related protein (alpha 2MRP) gene. The others genes were not identified. Induction of the alpha 2MRP gene expression occurred within 24 h after addition of AGE. These findings suggest a possible mechanism by which AGE may regulate gene expression and bring about a neurotrophic effect. Further, our results suggest that alpha 2MRP may function at the initial step of the molecular events triggered by AGE and play an important role in the survival of hippocampal neurons.

A role for ghrelin in the central regulation of feeding.

Ghrelin is an acylated peptide that stimulates the release of growth hormone from the pituitary. Ghrelin-producing neurons are located in the hypothalamus, whereas ghrelin receptors are expressed in various regions of the brain, which is indicative of central-and as yet undefined-physiological functions. Here we show that ghrelin is involved in the hypothalamic regulation of energy homeostasis. Intracerebroventricular injections of ghrelin strongly stimulated feeding in rats and increased body weight gain. Ghrelin also increased feeding in rats that are genetically deficient in growth hormone. Anti-ghrelin immunoglobulin G robustly suppressed feeding. After intracerebroventricular ghrelin administration, Fos protein, a marker of neuronal activation, was found in regions of primary importance in the regulation of feeding, including neuropeptide Y6 (NPY) neurons and agouti-related protein (AGRP) neurons. Antibodies and antagonists of NPY and AGRP abolished ghrelin-induced feeding. Ghrelin augmented NPY gene expression and blocked leptin-induced feeding reduction, implying that there is a competitive interaction between ghrelin and leptin in feeding regulation. We conclude that ghrelin is a physiological mediator of feeding, and probably has a function in growth regulation by stimulating feeding and release of growth hormone.

Effect of furanocoumarin derivatives in grapefruit juice on the uptake of vinblastine by Caco-2 cells and on the activity of cytochrome P450 3A4.

1. The presence of inhibitors of drug efflux transporters, such as P-glycoprotein (P-gp), in grapefruit juice (GFJ) was confirmed based on the uptake of [(3)H]-vinblastine (VBL) by Caco-2 cells. 2. The uptake of [(3)H]-VBL by Caco-2 cells was significantly increased by the ethyl acetate extract of GFJ as well as by cyclosporin A. The extract was separated on a Cosmosil column and the eluate with 60% methanol increased [(3)H]-VBL uptake, while the activity to inhibit CYP3A4 was greatest in the 70 and 80% eluates. 3. These results show that the major inhibitor of efflux transport of VBL is different from that of CYP3A4. 4. Further separation of the 60% methanol eluate afforded dihydroxybergamottin (DHBG). Both ethyl acetate extract of GFJ and DHBG increased steady-state [(3)H]-VBL uptake by LLC-GA5-COL300 cells. Besides DHBG, other furanocoumarins contained in GFJ, such as bergamottin, FC726, bergaptol and bergapten, increased the steady-state uptake of [(3)H]-VBL by Caco-2 cells. 5. The order of inhibitory potency of these compounds was FC726>DHBG>bergamottin>bergapten>bergaptol . While, the IC(50) values for inhibition of CYP3A4 were 0.075, 0.45, 1.0, 1.0 and >20 microM, respectively. Bergaptol specifically inhibited VBL efflux. 6. DHBG was thus identified as a candidate for inhibitors of VBL transport, together with other furanocoumarins. Moreover, partly involvement of the P-gp inhibition was suggested. 7. Therefore, the inhibition of efflux transport of drugs as well as of drug metabolism by CYP3A4 could be an important cause of drug-GFJ interaction.

Cloning and characterization of a human brain Na(+)-independent transporter for small neutral amino acids that transports D-serine with high affinity.

We isolated a cDNA for the human homologue of system asc transporter Asc-1 from human brain. The encoded protein designated as hAsc-1 (human Asc-1) exhibited 91 % sequence identity to mouse Asc-1. Consistent with mouse Asc-1, hAsc-1 required 4F2 heavy chain for its functional expression in Xenopus oocytes. hAsc-1 exhibited the properties of amino acid transport system asc which transports small neutral amino acids in a Na(+)-independent manner. hAsc-1 transported D-serine at high affinity with a K(m) value of 22.8 microM. In brain, 2.0 kb mRNA was highly expressed. hAsc-1 gene was mapped to human chromosome 19, region q12-q13.1. Because of the high-affinity transport with the K(m) value close to the physiological concentration of D-serine, together with the high levels of expression in brain, hAsc-1 is proposed to play significant roles in the D-serine mobilization in brain.

Purification and characterization of rat des-Gln14-Ghrelin, a second endogenous ligand for the growth hormone secretagogue receptor.

Ghrelin, a peptide purified from the stomach, is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and potently stimulates growth hormone release from the pituitary. Ghrelin is modified with an n-octanoyl group at Ser(3). This modification is essential for the activity of ghrelin. Previously, it was not known whether other ligands for GHS-R existed. Here, we report the purification of the second endogenous ligand for GHS-R from rat stomach. This ligand, named des-Gln(14)-ghrelin, is a 27-amino acid peptide, whose sequence is identical to ghrelin except for one glutamine. Southern blotting analysis under low hybridization conditions indicates that no homologue for ghrelin exists in rat genomic DNA. Furthermore, genomic sequencing and cDNA analysis indicate that des-Gln(14)-ghrelin is not encoded by a gene distinct from ghrelin but is encoded by an mRNA created by alternative splicing of the ghrelin gene. This is the first example of a novel mechanism that produces peptide multiplicity. Des-Gln(14)-ghrelin has an n-octanoyl modification at Ser(3) like ghrelin, which is also essential for its activity. Des-Gln(14)-ghrelin-stimulated growth hormone releases when injected into rats. Thus, growth hormone release is regulated by two gastric peptides, ghrelin and des-Gln(14)-ghrelin.

Identification and characterization of a Na(+)-independent neutral amino acid transporter that associates with the 4F2 heavy chain and exhibits substrate selectivity for small neutral D- and L-amino acids.

A cDNA was isolated from the mouse brain that encodes a novel Na(+)-independent neutral amino acid transporter. The encoded protein, designated as Asc-1 (asc-type amino acid transporter 1), was found to be structurally related to recently identified mammalian amino acid transporters for the transport systems L, y(+)L, x(C)(-), and b(0,+), which are linked, via a disulfide bond, to the type II membrane glycoproteins, 4F2 heavy chain (4F2hc), or rBAT (related to b(0,+) amino acid transporter). Asc-1 required 4F2hc for its functional expression. In Western blot analysis in the nonreducing condition, a 118-kDa band, which seems to correspond to the heterodimeric complex of Asc-1 and 4F2hc, was detected in the mouse brain. The band shifted to 33 kDa in the reducing condition, confirming that Asc-1 and 4F2hc are linked via a disulfide bond. Asc-1-mediated transport was not dependent on the presence of Na(+) or Cl(-). Although Asc-1 showed a high sequence homology (66% identity at the amino acid level) to the Na(+)-independent broad scope neutral amino acid transporter LAT2 (Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., and Kanai, Y. (1999) J. Biol. Chem. 274, 19745-19751), Asc-1 also exhibited distinctive substrate selectivity and transport properties. Asc-1 preferred small neutral amino acids such as Gly, L-Ala, L-Ser, L-Thr, and L-Cys, and alpha-aminoisobutyric acid as substrates. Asc-1 also transported D-isomers of the small neutral amino acids, in particular D-Ser, a putative endogenous modulator of N-methyl-D-aspartate-type glutamate receptors, with high affinity. Asc-1 operated preferentially, although not exclusively, in an exchange mode. Asc-1 mRNA was detected in the brain, lung, small intestine, and placenta. The functional properties of Asc-1 seem to be consistent with those of a transporter subserving the Na(+)-independent small neutral amino acid transport system asc.

Effect of histamine H2-receptor antagonist on the phosphorus-binding abilities of calcium carbonate and calcium lactate in hemodialysis patients.

The effect of histamine H2-receptor antagonist (famotidine) on the phosphorus-binding abilities of calcium carbonate and calcium lactate were examined in 13 chronic hemodialysis patients. In seven patients receiving calcium carbonate, famotidine (20 mg/d) was given because of gastroduodenal disorders, and calcium carbonate was replaced with calcium lactate as a phosphorus binder after 4 wk of treatment with famotidine. With the 4-wk administration of famotidine accompanied by calcium carbonate, the serum phosphorus level increased from 6.3+/-0.9 to 7.1+/-0.5 mg/dl (P<0.05). However, with the substitution of calcium lactate, the serum phosphorus level decreased significantly when compared to that before substitution (6.3+/-0.2 and 6.0+/-0.9 mg/dl after 4 and 8 wk of substitution, respectively), despite continued administration of famotidine. Serum calcium, creatinine, alkaline phosphatase, high sensitive parathyroid hormone, blood urea nitrogen, arterial blood pH, and bicarbonate were not significantly altered during the trial period. In six control patients treated with calcium carbonate alone, there were no statistical changes in serum calcium and phosphorus levels after substitution of calcium lactate for calcium carbonate. These results suggest that famotidine significantly affects the phosphorus-binding ability of calcium carbonate, but not that of calcium lactate. A careful observation of changes in the serum phosphorus level should be required in hemodialysis patients receiving calcium carbonate and histamine H2-receptor antagonists. Calcium lactate may be useful as a phosphorus binder in such hemodialysis patients.

The augmentation of pituitary adenylate cyclase-activating polypeptide (PACAP) in streptozotocin-induced diabetic rats.

Pituitary adenylate cyclase activating polypeptide (PACAP), which was isolated from ovine hypothalamic extract, has been shown to have a physiological role in the regulation of insulin or islet functions. In streptozotocin (STZ)-induced diabetic rats, we examined the content of PACAP immunoreactivity and gene expression of three specific receptors. Four weeks after administration of STZ (50 mg/kg), plasma glucose levels increased 3.3-fold, and plasma insulin levels decreased to one-tenth as compared with the control. The content of PACAP immunoreactivity in the pancreas potently increased by 30%, but the content of vasoactive intestinal polypeptide (VIP) immunoreactivity was not changed. In the other tissues, the content of PACAP immunoreactivity did not significantly change except in the hypothalamus, which showed a 10% increment. In the expression level of PACAP/VIP receptors, semi-quantitative RT-PCR analysis revealed that VIP1/PACAP receptor mRNA significantly increased as compared with the other two types of receptors in the pancreas of STZ-induced diabetic rats. These findings suggest that PACAP and VIP1/PACAP receptor might be involved in the pathophysiology of diabetes mellitus.

Inhibition of vinblastine efflux mediated by P-glycoprotein by grapefruit juice components in caco-2 cells.

We investigated the effect of components in grapefruit juice (GFJ) on the transport of vinblastine, a substrate of P-glycoprotein (P-gp), across Caco-2 cells. The apical to basolateral flux of [3H]vinblastine was increased in the presence of GFJ extracts. The steady-state uptake of [3H]vinblastine from the apical side was significantly increased in the presence of GFJ in a dose-dependent manner within the range of 2.5 to 50% (v/v) of GFJ. Although naringin and naringenin reduced apical efflux of [3H]vinblastine at the concentration present in GFJ and increased steady-state uptake from the apical side to 124 and 240%, respectively, the observed effect of naringin was not enough to account for the effect of GFJ and naringenin is not naturally present in GFJ. To investigate the effective components in GFJ, we examined the inhibitory effect of several organic solvent extracts of GFJ on the transport of [3H]vinblastine in Caco-2 cells. Organic solvent extracts of GFJ enhanced the apical to basolateral transcellular transport and inhibited the apical efflux. The permeability coefficient of apical to basolateral transport of [3H]vinblastine increased in the order of the ethyl acetate>diethyl ether>methylene chloride extracts of GFJ. Since the extracted amount of naringenin by ethyl acetate was less than that with the other organic solvents, the primary inhibitor in GFJ is suggested to be different from this flavonoid. The present study demonstrated the existence of inhibitory components in GFJ for the P-gp function in Caco-2 cells, which are distinct from known components such as naringin or naringenin.

[Effect of histamine H2-receptor antagonists on the phosphorus-binding ability of phosphate binders in hemodialysis patients].

We examined the effects of histamine H2-receptor antagonists on the phosphorus binding ability of phosphate binders. Serum calcium, phosphorus, ALP, PTH and arterial blood pH and bicarbonate were measured during treatment with histamine H2-receptor antagonists accompanied by calcium carbonate in sixteen patients undergoing maintenance hemodialysis. Seven patients receiving histamine H2-receptor antagonists without calcium carbonate were selected as controls. In the sixteen patients receiving calcium carbonate, serum calcium, ALP, PTH and arterial blood pH and bicarbonate were not significantly altered during treatment with histamine H2-receptor antagonists, but serum phosphorus levels increased significantly after four (5.6 +/- 1.1 mg/dl) and eight weeks (5.9 +/- 0.8 mg/dl) of treatment as compared with that before treatment (4.8 +/- 1.2 mg/dl). Furthermore, serum phosphorus levels decreased significantly eight weeks after the discontinuation of treatment with histamine H2-receptor antagonists. In the seven control patients there were no statistical differences in serum calcium and phosphorus levels measured before and after treatment with histamine H2-receptor antagonists. In seven other patients receiving histamine H2-receptor antagonists with calcium carbonate, calcium carbonate was replaced with calcium lactate as the phosphate binder after four weeks of treatment with histamine H2-receptor antagonists. With the 4-week administration of histamine H2-receptor antagonists accompanied by calcium carbonate, the serum phosphorus level increased, similarly to that of the first study (from 6.3 +/- 0.9 to 7.1 +/- 0.5 mg/dl). However, with the substitution of calcium lactate, the serum phosphorus level decreased significantly (6.3 +/- 0.2 and 6.0 +/- 0.9 mg/dl after four and eight weeks, respectively, despite continued administration of histamine H2-receptor antagonists). These results suggest that histamine H2-receptor antagonists significantly affect the phosphorus binding ability of calcium carbonate, but not of calcium lactate. Although the exact mechanism remains obscure, one possible explanation may be related to the rise in pH of the gastric juice. Careful observation of changes in the serum phosphorus level is required in hemodialysis patients receiving calcium carbonate and histamine H2-receptor antagonists. Calcium lactate may be useful as a phosphate binder in such hemodialysis patients.

Nimodipine improves brain energy metabolism and blood rheology during ischemia and reperfusion in the gerbil brain.

Whether nimodipine improves cerebral blood flow (CBF) and metabolism in cerebral ischemia remains a controversial issue. We investigated the effect of nimodipine on CBF, brain energy metabolism, using a laser-Doppler flowmeter and in vivo 31phosphorus nuclear magnetic resonance (31P NMR) spectroscopy, and blood rheology during forebrain ischemia and reperfusion in gerbils. Eighty-three adult gerbils received nimodipine (1 micrograms/kg/min), or an equal volume of the vehicle, or saline, over 60 min prior to a transient forebrain ischemia for 60 min. We measured sequential changes in phosphocreatine (PCr) / inorganic phosphate (Pi) ratio, beta-ATP/Pi ratio, and intracellular pH (pHi) during ischemia and reperfusion by 31P NMR spectroscopy, and the measurement of whole blood viscosity (WBV) at 60 min after reperfusion. CBF was measured continuously throughout the study by a laser-Doppler flowmeter. During forebrain ischemia, PCr/Pi and beta-ATP/Pi ratios were higher significantly in the nimodipine-treated group (p < 0.05 and 0.01) than in the vehicle- or saline-treated groups. During reperfusion, PCr/Pi and beta-ATP/Pi ratios recovered significantly only in the nimodipine-treated group (p < 0.05 and 0.01). The WBV at high shear rate (562.5 s-1) lowered significantly in the nimodipine-treated group (p < 0.05) compared with the vehicle- or saline-treated group. CBF was higher significantly only during administration of nimodipine in the nimodipine-treated group (p < 0.01) than other groups. Nimodipine improved brain energy metabolism and blood rheology during forebrain ischemia and reperfusion in the gerbil brain.

Significance of glutathione depletion and oxidative stress in early embryogenesis in glucose-induced rat embryo culture.

Recent studies have demonstrated the protective effects of supplementing free oxygen radical scavenging enzymes against hyperglycemia-induced embryonic malformations. In this study, the glutathione (GSH)-dependent protection system in hyperglycemia-induced embryopathy was investigated. Rat embryos at the early head-fold stage (day 9.5) cultured in 66.7 mmol/l glucose for 48 h showed significant growth retardation and an increase in the frequency of malformations. The concentration of GSH and activity of the rate-limiting GSH-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), significantly decreased in embryos exposed to hyperglycemia compared with controls (7.9 +/- 0.6 vs. 12.5 +/- 0.9 nmol/mg protein, P < 0.01 and 13.3 +/- 1.9 vs. 22.6 +/- 1.1 microU/mg protein, P < 0.01, respectively). Decreased activity of gamma-GCS in embryos exposed to hyperglycemia was associated with decreased expression of gamma-GCS mRNA levels. However, the activities of superoxide dismutase and glutathione peroxidase did not significantly change in these embryos. Extracellular and intracellular free oxygen radical formations estimated by Lucigenin-dependent chemoluminescence and flow cytometric analysis using 2',7'-dichlorofluorescein diacetate increased in isolated embryonic cells taken from embryos cultured under hyperglycemia. Supplementation of 2 mmol/l GSH ester into the hyperglycemic culture nearly restored GSH concentration in these embryos (11.9 +/- 0.5 vs. 12.5 +/- 0.9 nmol/mg protein) and reduced the formation of free oxygen radical species leading to almost complete normalization of growth retardation and embryonic dysmorphogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of immunoreactive human C-type natriuretic peptide in brain and heart.

Amino acid sequence of human C-type natriuretic peptide (CNP) has recently been deduced to be identical to those of porcine and rat CNPs in the bioactive unit of C-terminal 22 residues (CNP-22) (1). Thus, tissue concentrations and molecular forms of immunoreactive (ir-) CNP in human brain and heart were determined or characterized using a radioimmunoassay established for porcine CNP. In human brain (hypothalamus and medullapons), ir-CNP was detected at a concentration of 1.04 pmol/g, being about 25 times or 70 times higher than ir-atrial (A-type) natriuretic peptide (ANP) or ir-brain (B-type) natriuretic peptide (BNP). CNP was present mainly as CNP-53, with CNP-22 as well as 13K CNP (presumed to be pro-CNP) as minor components. In heart, 1 approximately 5 pmol/g of ir-CNP was detected in both atrium and ventricle, but this ir-CNP was shown to be derived from crossreactivity of ANP. These results demonstrated that human CNP functions exclusively in the central nervous system in contrast to ANP and BNP which mainly function in the circulation system.

[Evaluation of the changes in intracranial water, sodium, phosphorus metabolites and intracellular cerebral pH in rats with acute dilutional hyponatremia].

Although most prominent among the clinical manifestations associated with hyponatremia are central nervous system (CNS) symptoms, the alterations in brain function remain poorly understood. In the present study, the alterations in intracellular cerebral pH and intracranial water, sodium and phosphorus metabolites content in rats with acute dilutional hyponatremia were examined by using an in vivo nuclear magnetic resonance (NMR) technique which noninvasively provides continuous informations on intracellular phenomena. Acute dilutional hyponatremia was induced on anesthetized male Sprague-Dawley rats by intraperitoneal injection of distilled water with an initial dose of 10 ml/100 g bw, followed by an additional dose of 5 ml/100 g bw 40 min later. Arterial blood sampling and NMR measurements were made before and every 60 min after the initial injection of distilled water. The treatment with distilled water resulted in dramatic falls in serum Na, Cl and osmolality at 60 min after water loading (Na; from 143.4 +/- 2.6 to 112.3 +/- 1.3 mmol/l, Cl; from 101.02 +/- 2.2 to 78.4 +/- 5.4 mmol/l, Osm; from 306.3 +/- 5.8 to 247.3 +/- 7.3 mOsm/kg H20). 1H-NMR imaging showed the accumulation of brain water as dilutional hyponatremia developed. Intracranial Na content measured by 23Na-NMR spectroscopy decreased significantly at 60 min after of water loading to about 70% of that observed under control condition. Since it has been demonstrated that solute extrusion from the brain with resultant reduction of brain swelling occurs within 60 min after the dilution, this result may be, at least in part, explained by this protective mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor.

The sequence of 5,037 amino acids composing the ryanodine receptor from rabbit skeletal muscle sarcoplasmic reticulum has been deduced by cloning and sequencing the complementary DNA. The predicted structure suggests that the calcium release channel activity resides in the C-terminal region of the receptor molecule, whereas the remaining portion constitutes the 'foot' structure spanning the junctional gap between the sarcoplasmic reticulum and the transverse tubule.

Effect of supplementing low protein diets with the limiting amino acids on the excretion of N1-methylnicotinamide and its pyridones in rats.

We have hypothesized that the ratio of the excreted by-products of niacin metabolism, N1-methyl-2-pyridone-5-carboxamide (2-pyr) + N1-methyl-4-pyridone-3-carboxamide (4-pyr)/N1-methylnicotinamide (MNA), might be useful as an index to assess the adequacy of amino acid intake in rats. The experiment reported herein was performed to test this hypothesis. When a 10, 20 or 40% casein diet supplemented with 0.1, 0.2 or 0.4% L-methionine, respectively, was fed to rats, the urinary excretion of MNA decreased, and that of 4-pyr increased, as the level of dietary casein and methionine increased. Therefore, the ratio of (2-pyr + 4-pyr)/MNA increased with increasing dietary casein and methionine levels. When the limiting amino acids of casein or soy protein isolate were added to a low casein or low soy protein isolate diet, the urinary ratio of (2-pyr + 4-pyr)/MNA also increased. These results indicate that the increased urinary ratio of (2-pyr + 4-pyr)/MNA can serve as a biological marker for adequate amino acid intake.

The primary structure of human gamma-glutamyl transpeptidase.

A cDNA hybridizable to that of rat gamma-glutamyl transpeptidase (GGT) was cloned from a cDNA library of human fetal liver. The insert of the cDNA clone contained 1866 bp consisting of an open reading frame (ORF) of 1709 bp (569 amino acids (aa), N-terminal portion truncated) and a 135-bp 3'-untranslated region followed by a polyadenylated tail. In parallel, amino acid sequences of N-terminal portions of heavy and light chains of a purified human GGT were determined. Two stretches of amino acid sequences identical to the N-terminal sequences of heavy and light chains were found in the ORF. We therefore concluded that the clone is a cDNA for human GGT. From the amino acid sequence deduced from cDNA, the heavy and the light chains of the purified enzyme are estimated to be composed of 351 aa (Mr 38,336) and of 189 aa (Mr 20,000), respectively. The heavy chain is preceded by a signal peptide of at least 29 aa presumed to be cleaved by bromelain treatment. Six putative N-glycosylation sites are present in the heavy subunit region and one in the light subunit region. Primary structure and hydrophobicity profile are closely similar to those of rat GGT.

Immunocytochemical demonstration of dynorphin(PH-8P)-like immunoreactive elements in the human hypothalamus.

PH-8P (dynorphin[1-8])-like immunoreactive neuronal perikarya, processes, and terminals located within the human hypothalamus were investigated by the avidin-biotin peroxidase complex (ABC) immunocytochemical procedure. Immunopositive neurons were distributed throughout the hypothalamus. The distributional pattern was found to be similar to that in other mammalian species by the use of antisera against dynorphin. A large number of immunoreactive neuronal perikarya were detected in the supraoptic nucleus (SON) and the magnocellular portion of the paraventricular nucleus (PVN). Their processes appeared to project to the posterior pituitary via the internal layer of the median eminence and their distribution seemed to be less dense than in other mammalian species. PH-8P and vasopressin were colocalized in the neuronal perikarya in the human SON unlike the colocalization of these peptides in the rat SON and PVN. There were a few immunoreactive terminals in the external layer of the median eminence; their immunoreactive substances may be released into the portal veins to act on anterior pituitary cells. In addition, PH-8P-like immunoreactive neurons in the human hypothalamus may project to the extrahypothalamic area.