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1.
Five single-point aquaporin-2 (AQP2) mutations that cause non-X-linked nephrogenic diabetes insipidus (NDI) were characterized to establish the cellular defect and to develop therapeutic strategies. In Xenopus oocytes expressing AQP2 cRNAs, single-channel water permeabilities of mutants L22V, T126M, and A147T were similar to that of wild-type AQP2, whereas R187C and C181W were nonfunctional. In [35S]methionine pulse-chase experiments in transiently transfected CHO cells, half-times for AQP2 degradation were approximately 4 h for wild-type AQP2 and L22V, and mildly decreased for T126M (2.7 h), C181W (2.4 h), R187C (2.0 h), and A147T (1.8 h). Immunofluorescence showed three distinct AQP2-staining patterns: plasma membrane and endosomal staining (wild-type, L22V), endoplasmic reticulum (ER) staining (T126M > A147T approximately R187C), or a mixed pattern of reticular and perinuclear vesicular staining. Immunoblot of fractionated vesicles confirmed primary ER localization of T126M, R187C, and A147T. To determine if the AQP2-trafficking defect is correctable, cells were incubated with the "chemical chaperone" glycerol for 48 h. Immunoblot showed that glycerol produced a nearly complete redistribution of AQP2 (T126M, A147T, and R187C) from ER to membrane/endosome fractions. Immunofluorescence confirmed the cellular redistribution. Redistribution of AQP2 mutants was also demonstrated in transfected MDCK cells, and using the chaperones TMAO and DMSO in place of glycerol in CHO cells. Water permeability measurements indicated that functional correction was achieved. These results indicate defective mammalian cell processing of mutant AQP2 water channels in NDI, and provide evidence for pharmacological correction of the processing defect by chemical chaperones. 相似文献
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T Sch?neberg A Schulz H Biebermann A Grüters T Grimm K Hübschmann G Filler T Gudermann G Schultz 《Canadian Metallurgical Quarterly》1998,12(3):196-205
B cells and macrophages both activate NF-kappaB/Rel in response to lipopolysaccharide (LPS), but differ in sensitivity to LPS and in downstream genes that are activated. CD14 is a high-affinity receptor for LPS found on macrophages, but not B cells. We expressed human CD14 (hCD14) in the mouse B lymphoma, 70Z/3, and a mutant, 1B8, which responds slowly to LPS, to test whether expression of hCD14 could correct or bypass the defect in 1B8 cells. We compared the timing and extent of known responses to LPS in 70Z/3 cells and the 1B8 mutants. The hCD14+ 1B8 and 70Z/3 cells responded more rapidly and were sensitive to 100-fold lower levels of LPS than their untransfected counterparts. Degradation of the IkappaB-alpha and -beta molecules and translocation of the NF-kappaB/Rel complexes into the nucleus were more rapid and the steady-state levels of Igk mRNA and mIgM on the cell surface were markedly increased in cells that expressed hCD14. The LPS response of the hCD14+ 1B8 and 70Z/3 cells showed subtle differences. In the 1B8 hCD14 cells, the p50/p50 complexes were never abundant in nuclear extracts, and degradation of IkappaB-beta was slower than in hCD14 70Z/3 cells. This partial correction of the 1B8 phenotype suggests that the defective component in 1B8 participates in the CD14 signaling pathway and could include the B-cell LPS receptor itself. 相似文献
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The etiology, diagnosis and management of lithium-induced diabetes insipidus (LIDI) is discussed, including the presentation of a case report. It is suggested that lithium provokes LIDI by decreasing the responsive ness of the kidney to the antidiuretic hormone. Lithium-induced polyuria may be managed by concomitant treatment with a thiazide diuretic or discontinuation of the lithium. Caution must be employed, however, when using thiazides with lithium as these diuretics decrease renal clearance of lithium. 相似文献
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R Vargas-Poussou L Forestier MD Dautzenberg P Niaudet M Déchaux C Antignac 《Canadian Metallurgical Quarterly》1997,8(12):1855-1862
Congenital nephrogenic diabetes insipidus (CNDI) is a rare inherited disorder characterized by renal tubular insensitivity to the antidiuretic effect of arginine vasopressin (AVP). In a large majority of the cases, nephrogenic diabetes insipidus is an X-linked recessive disorder caused by mutations in the AVP V2 receptor gene (AVPR2). In the remaining cases, the disease is autosomal recessive or dominant and, for these patients, mutations in the aquaporin 2 gene (AQP2) have been reported. Fourteen probands belonging to 12 families were analyzed by single-strand conformational polymorphism and direct sequencing of the AVPR2 and AQP2 genes. Ten mutations of the AVPR2 gene (six previously reported mutations and four novel mutations: G107E, W193X, L43P, and 15delC) were identified. Three mutations of the AQP2 gene were also identified in two patients: the first patient is homozygous for the R85X mutation and the second is a compound heterozygote for V168 M and S216P mutations. Extrarenal responses to infusion of the strong V2 agonist 1-desamino-8-D-arginine vasopressin allowed AVPR2- and AQP2-associated forms of CNDI to be distinguished in three patients. This test also identified an unexpectedly high urinary osmolality (614 mosmol/kg) in a patient with a P322S mutation of AVPR2 gene and a mild form of CNDI. 相似文献
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SM Mulders DG Bichet JP Rijss EJ Kamsteeg MF Arthus M Lonergan M Fujiwara K Morgan R Leijendekker P van der Sluijs CH van Os PM Deen 《Canadian Metallurgical Quarterly》1998,102(1):57-66
Calcyclin (CaCY) is a member of the S100 subfamily of helix-loop-helix (EF-hand) calcium-binding proteins. Human CaCY was overexpressed in Escherichia coli and purified with an overall yield of 40 mg/l culture. Ca2+ and Zn2+ binding properties of CaCY were examined with respect to the oxidation state of the single Cys residue at position 3. CaCY with the SH group either reduced, blocked or oxidized stays as a dimer as shown by analytical ultracentrifugation. Upon binding of Ca2+, CaCY exhibits 30% enhancement of the Tyr fluorescence, the apparent binding constant (Ka) being 2.8-5.8x10(4) M(-1). Oxidized CaCY binds Ca2+ approximately twice as weakly than its reduced form. The affinity for Ca2+ is increased in the presence of caldesmon, which could be a potential target molecule. Fully reduced CaCY binds Zn2+ with an affinity of at least 1.0x10(7) M(-1). As compared to Ca2+, Zn2+ binding results in a three times greater enhancement of the Tyr fluorescence. Saturation occurs at a Zn2+/CaCY ratio of 2:1. The reactivity of Cys3 is reduced by Zn2+ binding, although oxidized CaCY still binds Zn2+. On the basis of the effects of thiol-directed labels on the affinities for Ca2+ and Zn2+, the fluorescence changes accompanying the binding, and the CaCY reactivity with a hydrophobic probe, it was concluded that the two cations bind to CaCY at different sites: Ca2+ binds probably at the EF-hand type sites, whereas binding of at least one Zn2+ ion involves the Cys residue, and results in a different structural change. 相似文献
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A 45-year-old man, with a 10-year history of manic depression treated with lithium, was admitted with hyperosmolar, nonketotic coma. He gave a five-year history of polyuria and polydipsia, during which time urinalysis had been negative for glucose. After recovery from hyperglycaemia, he remained polyuric despite normal blood glucose concentrations; water deprivation testing indicated nephrogenic diabetes insipidus, likely to be lithium-induced. We hypothesize that when this man developed type 2 diabetes, chronic polyuria due to nephrogenic diabetes insipidus was sufficient to precipitate hyperosmolar dehydration. 相似文献
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Nephrogenic diabetes insipidus (NDI) is characterized by resistance of the kidney to the action of arginine-vasopressin (AVP); it may be due to genetic or acquired causes. Recent advances in molecular genetics have allowed the identification of the genes involved in congenital NDI. While inactivating mutations of the vasopressin V2 receptor are responsible for X-linked NDI, autosomal recessive NDI is caused by inactivating mutations of the vasopressin-regulated water channel aquaporin-2 (AQP-2). About 70 different mutations of the V2 receptor have been reported, most of them missense mutations. The functionally characterized mutants show a loss of function due to defects in their synthesis, processing, intracellular transport, AVP binding, or interaction with the G protein/adenylyl cyclase system. Thirteen different mutations of the AQP-2 gene have been reported. Functional studies of three AQP-2 mutations reveal impaired cellular routing as the main defect. The great number of different mutations with various functional defects hinders the development of a specific therapy. Gene therapy may, however, eventually become applicable to the congenital forms of NDI. At present all gene-therapeutic approaches lack safety and efficiency, which is of particular relevance in a disease that is treatable by an adequate water intake. The progress with regard to the molecular basis of antidiuresis contributes to the understanding of acquired forms of NDI on a molecular level. Recent data show that lithium dramatically reduces the expression of AQP-2. Likewise, hypokalemia reduces the expression of this water channel. The exact mechanisms leading to this reduced expression of AQP-2 remain to be determined. 相似文献
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K Goji M Kuwahara Y Gu M Matsuo F Marumo S Sasaki 《Canadian Metallurgical Quarterly》1998,83(9):3205-3209
Novel mutations of the aquaporin-2 (AQP2) gene have been detected in Japanese female siblings with autosomal-recessive nephrogenic diabetes insipidus. The patients were compound heterozygote for point mutations at nucleotide position 374 (C374T) and at position 523 (G523A) in exon 2 of the AQP2 gene, resulting in substitution of methionine for threonine at codon 125 (T125M) and arginine for glycine at codon 175 (G175R). The water permeability (Pf) of oocytes injected with wild-type complementary RNA increased 9.0-fold compared with the Pf of water-injected oocytes, whereas the increases in the Pf of oocytes injected with T125M and G175R complementary RNA were only 1.7-fold and 1.5-fold, respectively. Immunoblot and immunocytochemistry indicated that the plasma membrane expressions of T125M and G175R AQP2 proteins were comparable to that of the wild-type, suggesting that although neither the T125M nor G175R mutation had a significant effect on plasma membrane expression, they both distorted the structure and function of the aqueous pore of AQP2. These results provide evidence that the nephrogenic diabetes insipidus in patients with T125M and G175R mutations is attributable not to the misrouting of AQP2, but to the disrupted water channel function. 相似文献
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BACKGROUND: The AVPR2 gene encodes the type 2 vasopressin receptor, a member of the vasopressin/oxytocin receptor subfamily of G protein-coupled receptors. Disruption of AVPR2 causes X-linked congenital nephrogenic diabetes insipidus (NDI), yet the functional significance of most gene sequence variations found in association with NDI has not been proven. The large number of naturally occurring AVPR2 mutations constitutes a model system for studying the structure-function relationship of G protein-coupled receptors. This analysis can be aided by examining amino acid sequence variation and conservation among evolutionarily disparate members of the subfamily. METHODS: Twenty-five new NDI patients were evaluated by DNA sequencing for mutations in AVPR2. Receptors encoded by eighteen NDI alleles were tested for physiologic signaling activity in response to varying concentrations of arginine vasopressin (AVP) in a sensitive cell culture assay. Seventeen amino acid sequences from the vasopressin/oxytocin receptor subfamily were aligned and conserved residues were identified and correlated with the locations of NDI associated variations. RESULTS: Twenty-four variant alleles were found among the 25 new patients. Thirteen had no prior family history of expressed NDI. All 18 of the NDI-associated AVPR2 alleles tested for function demonstrated diminished response to stimulation with AVP. Twelve failed to respond at all, whereas six signaled only at high AVP concentrations. Evolutionarily conserved residues clustered in the transmembrane domains and in the first and second extracellular loops, and NDI-associated missense mutations appeared mostly in the conserved domains. CONCLUSIONS: Sporadic cases are frequent and they usually represent the X-linked rather than the autosomal form of NDI. Genetic and functional testing can confirm this in individual cases. Mutations in this study affecting ligand binding domains tend to retain partial signaling in vitro, whereas those that introduce a charged residue in a transmembrane domain are inactive. The minimal partial signaling observed in cultured cells is unlikely to correlate with clinically significant urine concentrating ability. Other AVPR2 mutations with milder effects on receptor function probably exist, but may not be expressed clinically as typical NDI. 相似文献
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In five patients (a boy aged 10 years, a boy aged 3 months, his brother aged 1 week, the brother of the mother of the last-mentioned two boys who had died at the age of one, and a girl of kindergarten age) congenital nephrogenic diabetes insipidus was diagnosed. This rare syndrome (prevalence 1:500,000) is caused by renal insensitivity to the antidiuretic hormone arginine vasopressin. In infancy the symptoms of this disorder are aspecific, and the main symptoms of the disease, polyuria and polydipsia, often remain unnoticed at this young age. A simple anamnesis and a few laboratory tests should suggest the diagnosis. Early diagnosis and genetic counselling are possible as the molecular effects involved have been elucidated. 相似文献
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Nephrogenic diabetes insipidus is a rare, mostly X-linked recessive disorder characterised by renal tubular resistance to the antidiuretic effect of arginine vasopressin. The gene responsible for the X-linked nephrogenic diabetes insipidus, the G-protein-coupled vasopressin V2-receptor, has been localised on the Xq28 region. In this study four patients were investigated with molecular genetic methods. Diagnosis was based on clinical symptoms and lack of increase of urinary osmolality after administration of the arginine vasopressin, or the synthetic vasopressin analogue DDAVP. Three different mutations (C112R, N317K, W323S) were found in three patients, while no mutation was detected in the fourth patient. Since earlier histiocytosis X has been diagnosed in this patient, this patient has probably central diabetes insipidus. Although the main symptoms of the disease can be found in all patients, there are significant differences in the seriousness of the symptoms as well as in some other symptoms. The explanations might be the different mutations in the V2-receptor gene and the various other genetic and environmental factors; these findings provide further evidence that X-linked nephrogen diabetes insipidus results from defects in the V2-receptor gene. 相似文献
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Chronic diarrhea commonly causes hypokalemia. However, life-threatening hypokalemia due to chronic diarrhea has not been reported in the adult caused by chronic ingestion of a fructose beverage. We report an adult patient who had severe hypokalemia from chronic osmotic diarrhea as a result of drinking 4 to 6 liters of Big Red daily for several months. This resulted in sustained hypokalemia, complicated by hypokalemic nephropathy and subsequent nephrogenic diabetes insipidus. All abnormalities cleared with potassium replacement, salt restriction, modest thiazide treatment, and abstinence from Big Red. 相似文献
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Twenty-one families were selected from the published reports in which the propositus had the triad of juvenile diabetes mellitus, diabetes insipidus, and optic atrophy. The data were consistent with the hypothesis of an autosomal gene which, in the homozygote, causes juvenile diabetes mellitus and one or more of diabetes insipidus, optic atrophy, and nerve deafness. Heterozygotes appear to have an increased probability of developing juvenile diabetes mellitus. 相似文献
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A 61-year-old man who had a 10-year history of anhidrosis was found to have idiopathic diabetes insipidus. He showed no spontaneous sweating or pilocarpine-induced sweat response. Skin pathology showed a normal eccrine gland. Microneurography detected no skin sympathetic nerve activity. Within a month of desmopressin treatment for diabetes insipidus, sweating and skin sympathetic nerve activity returned. 相似文献
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Y Ala D Morin B Mouillac N Sabatier R Vargas N Cotte M Déchaux C Antignac MF Arthus M Lonergan MS Turner MN Balestre G Alonso M Hibert C Barberis GN Hendy DG Bichet S Jard 《Canadian Metallurgical Quarterly》1998,9(10):1861-1872
X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine vasopressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. To study the cause of loss of function of mutant V2 receptors, we expressed 12 mutations (N55H, L59P, L83Q, V88M, 497CC-->GG, deltaR202, I209F, 700delC, 908insT, A294P, P322H, P322S) in COS-7 cells. Eleven of these, including P322H, were characterized by a complete loss of function, but the mutation P322S demonstrated a mild clinical and in vitro phenotype. This was characterized by a late diagnosis without any growth or developmental delay and a significant increase in urine osmolality after intravenous 1-deamino[D-Arg8]AVP administration. In vitro, the P322S mutant was able to partially activate the Gs/adenylyl cyclase system in contrast to the other V2R mutants including P322H, which were completely inactive in this regard. This showed not only that Pro 322 is important for proper V2R coupling, but also that the degree of impairment is strongly dependent on the identity of the substituting amino acid. Three-dimensional modeling of the P322H and P322S mutant receptors suggested that the complete loss of function of the P322H receptor could be due, in part, to hydrogen bond formation between the His 322 side chain and the carboxyl group of Asp 85, which does not occur in the P322S receptor. 相似文献
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In Drosophila bipectinata, flies with bilateral outgrowths on thorax were detected in sepia eye colour mutant stock. Such thoracic outgrowths have not been reported earlier in any species of Drosophila and appear to be unique phenotypic change in D. bipectinata caused due to autosomal recessive mutation. By using a double recessive mutant stock (se og) and a wild type stock, crosses were made and on the basis of recombination data of F1 females, the og mutation was mapped on the second chromosome of D.bipectinata. Further, D. bipectinata exhibits spontaneous male recombination in low frequency. 相似文献
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OBJECTIVE: To characterize the biochemical mechanisms of expression of the pyruvate dehydrogenase (PDH) E1alpha subunit exon 10 R302C missense mutation. BACKGROUND: Mutations in the X-linked E1alpha subunit gene are responsible for most cases of PDH deficiency, an important cause of neurodevelopmental defects and neurodegeneration with primary lactic acidemia. Although the disease shows extreme allelic heterogeneity, the R302C mutation has been defined in several unrelated cases. METHODS: Cell lines expressing selectively either the mutant or wild-type E1alpha alleles against identical genetic backgrounds were generated from the fibroblasts of a female heterozygous for the R302C mutation. Enzyme activity, mRNA, polypeptide expression, and turnover were studied in each. RESULTS: The residual PDH activity was below measurable levels in the cell line (B5) expressing only the mutant allele and normal in the wild-type polypeptide expressing (A10) cell line, confirming that the R302C mutation alone is sufficient to cause a severe PDH deficiency. The mutant polypeptide was less stable than the wild-type polypeptide, but the steady-state level of the mutant E1alpha protein was reduced only two- to threefold. CONCLUSIONS: The primary mechanism of expression of the R302C mutation must be limitation of catalytic efficiency. We speculate that catalysis may be inhibited in the mutant polypeptide because conformational changes are induced near serine 300, a residue that is particularly important as a regulatory phosphorylation site in the wild-type polypeptide. 相似文献