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  • Nicotiana plumbaginifolia  (6)
  • Calcium  (5)
  • 1990-1994  (11)
  • 1925-1929
  • 1
    ISSN: 1432-2013
    Keywords: Renal proximal tubule ; Intracellular calcium ; Calcium influx ; Primary culture ; Calcium store depletion ; 86Rb efflux ; Calcium ; sensitive potassium channels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Cytoplasmic calcium changes and calcium influx evoked by adenosine triphosphate (ATP) were investigated in primary cultures of rabbit proximal convoluted tubule cells. Extracellular ATP (50 μM) induced a biphasic increase of [Ca2+]i measured with the calcium probe fura-2. In the early phase, the mobilization of intracellular pools resulted in a transient increase of [Ca2+]i from 106±11 nM (n=36) to 1059±115% (n=29) of the resting level within 10 s. In the presence of external calcium, [Ca2+]i then decreased within 3 min to a sustained level (398±38%,n=8). Measurements of fura-2 quenching by external manganese revealed that this phase was the result of an increased Ca2+ uptake, blocked by lanthanum (10 μM) and verapamil (100 μM) but not by the nifedipin (25 μM). Internal calcium store depletion by ATP induced an increased calcium influx through lanthanum- and verapamil-sensitive, nifedipininsensitive calcium channels, located on the apical membrane of the cells. As indicated by86Rb+ efflux measurements, ATP activated a potassium efflux that was blocked by barium andLeiurus quinquestriatus hebraeus (LQH) venom (containing charybdotoxin) indicating the involvement of Ca2+-sensitive K+ channels. Moreover, in the presence of the LQH venom, the internal calcium stores were not replenished after being depleted by ATP. Our results indicate that an ATPevoked hyperpolarization of the plasma membrane leads to increased Ca2+ influx, which facilitates the replenishment of the internal stores.
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  • 2
    ISSN: 1617-4623
    Keywords: Mutagenized seeds ; Nicotiana plumbaginifolia ; Ethanol selection ; Alcohol dehydrogenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Six independent mutant lines ofNicotiana plumbaginifolia resistant to ethanol, designated E3, E8, E101, E112, E144 and E251, were isolated as germinating seedlings on selective medium. In all cases, resistance to ethanol was conferred by a single recessive nuclear mutation at the same locus. Mutant seeds and pollen lacked detectable ADH activity, with the exception of E251 where a residual activity was detected. An antiserum directed againstArabidopsis thaliana ADH detected an ADH-related polypeptide of 44 kDa present in wild-type seeds and, to a lesser extent, in the seeds of the leaky mutant E251. No ADH-related polypeptide could be detected in seeds of the other mutants. However, all of them had a nearly normal level of ADH mRNA except one which did not synthesize any mRNA. These results suggest that these ethanol-resistant mutants are impaired in one of the structural genes coding for alcohol dehydrogenase. The corresponding locus has been designatedAdh1.
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  • 3
    ISSN: 1617-4623
    Keywords: Tobacco ; Nicotiana plumbaginifolia ; Nitrate reductase deficient mutants ; Functional complementation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The homeologous nitrate reductase (NR) structural genes from tobacco were used to complement nitrate reductase deficient mutants from tobacco and Nicotiana plumbaginifolia. A plasmid conferring kanamycin resistance and lambda genomic clones carrying the tobacco wild-type alleles of the genes were co-electroporated in protoplasts of the tobacco mutant. Among 266 plants regenerated from kanamycin resistant colonies, 3 were able to grow permanently on a medium containing nitrate as sole nitrogen source. One of these three plants was further characterized. The ability to grow on nitrate was transmitted as a new single Mendelian dominant marker linked to kanamycin resistance. Molecular analysis of this clone confirmed the integration of a copy of the wild-type allele, and the synthesis at a low level of an active NR. This NR activity is sufficient to regulate both exogenous wild-type and endogenous mutated alleles of the genes at the transcriptional level. A N. plumbaginifolia mutant carrying a mutation impairing NR mRNA production was transformed by Agrobacterium mediated transfer of the wild-type tobacco nia-2 gene cloned into a binary vector. Similarly, kanamycin resistant calli were tested for their ability to grow on nitrate. Among 70 kanamycin resistant transformants, 7 were restored for nitrate assimilation. Molecular analysis revealed the integration of the tobacco gene, and the synthesis at a low level of the NR mRNA and of a nitrate inducible active NR.
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  • 4
    ISSN: 1617-4623
    Keywords: Nitrate reductase ; Reporter gene ; Nicotiana tabacum ; Nicotiana plumbaginifolia ; Transgenic plant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Reporter gene techniques and mutant analysis were used to identify the molecular basis of the regulation of the expression of nitrate reductase (NR) by nitrate and nitrate-, or ammonium-derived metabolites (N-metabolites), in the true diploïd species Nicotiana plumbaginifolia and in the amphidiploïd species Nicotiana tabacum. The N. plumbaginifolia mutant E23 results from the insertion of a Tnt1-like retrotransposon (Tnp2) in the first exon of the single-copy nia gene, which encodes nitrate reductase. One of the resulting transcripts ends in the 5′ LTR (long terminal repeat) sequence of this retrotransposon, and another one in the 3′ LTR. Nitrate and N-metabolites modulate the expression of these truncated transcripts, indicating that intron splicing and termination processes are not essential to these regulatory events. A GUS reporter sequence was transcriptionally linked to the promoter of the nia-1 gene of N. tabacum. This fusion was functional in transient expression assays done with protoplasts derived from mesophyll cells of N. tabacum. However none of the regulatory mechanisms known to affect steady-state levels of the nia-1 transcript were operative under these experimental conditions. Transgenic plants carrying either this fusion or translational fusions of GUS linked to the promoter of either the nia-1 or nia-2 gene of N. tabacum were obtained by Agrobacterium-mediated transfer. A low proportion of the transgenic plants (22 out of 105 independent transformants) expressed GUS activity although at a low level. Only 4 plants exhibited a detectable level of GUS mRNA. The concentration of this mRNA increased significantly in an NR-deficient background, indicating regulation by N-metabolites. Only 2 plants, however, showed regulation (induction) by nitrate. Attempts to use aux2 or nptII reporter sequences linked to either the nia-1 or nia-2 promoter as marker genes for the selection of regulatory mutants of the nitrate assimilation pathway were unsuccessful because of our inability to isolate transgenic plants in which these reporter genes were properly regulated by nitrate. The implications of these results are discussed.
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  • 5
    ISSN: 1617-4623
    Keywords: Nicotiana plumbaginifolia ; Nicotiana tabacum ; GATA-binding factor ; Nitrate reductase ; PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In higher plants, the expression of the nitrate assimilation pathway is highly regulated. Although the molecular mechanisms involved in this regulation are currently being elucidated, very little is known about the trans-acting factors that allow expression of the nitrate and nitrite reductase genes which code for the first enzymes in the pathway. In the fungus Neurospora crassa, nit-2, the major nitrogen regulatory gene, activates the expression of unlinked structural genes that specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein containing a single zinc finger motif defined by the C-X2-CX17-C-X2-C sequence. This DNA-binding domain recognizes the promoter region of N. crassa nitrogen-related genes and fragments derived from the tomato nia gene promoter. The observed specificity of the binding suggests the existence of a NIT2-like homolog in higher plants. PCR and cross-hybridization techniques were used to isolate, respectively, a partial cDNA from Nicotiana plumbaginifolia and a full-length cDNA from Nicotiana tabacum. These clones encode a NIT2-like protein (named NTL1 for nit-2-like), characterized by a single zinc finger domain, defined by the C-X2-C-X18-C-X2-C amino acids, and associated with a basic region. The amino acid sequence of NTL1 is 60% homologous to the NIT2 sequence in the zinc finger domain. The Ntl1 gene is present as a unique copy in the diploid N. plumbaginifolia species. The characteristics of Ntl1 gene expression are compatible with those of a regulator of the nitrate assimilation pathway, namely weak nitrate inducibility and regulation by light.
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  • 6
    ISSN: 1617-4623
    Keywords: Mutagenized seeds ; Nicotiana plumbaginifolia ; Auxin-resistant mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Mutant lines of Nicotiana plumbaginifolia resistant to the synthetic auxins 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) were isolated as germinating seedlings on selective medium. In each case, resistance was conferred by a single recessive nuclear mutation at one of 3 loci designated iba1, iba2 and iba3. Labelling studies with 14C NAA suggest that resistance was not due to changes in the uptake or metabolism of NAA. Plants homozygous for the iba1 mutation exhibit a syndrome of atypical germination and growth suggestive of a defect in the biosynthesis, metabolism or localization of abscisic acid. Wild-type seeds treated with gibberellin exhibit the same syndrome, including resistance to NAA and IBA. On the basis of these observations, we propose that auxin toxicity in seeds may be mediated by a block in gibberellin biosynthesis.
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  • 7
    ISSN: 1617-4623
    Keywords: Transposable element ; Nitrate reductase ; Nicotiana plumbaginifolia ; γ-Ray mutagenesis ; Nucleotide sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after γ-ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 by insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 by terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5′ and 3′ ends of dTnp1 together with a perfect palindrome located after the 5′ inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.
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  • 8
    ISSN: 1433-2965
    Keywords: Bone mineral density ; Calcium ; Elderly ; Femoral neck ; Fracture ; Osteoporosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The efficacy of calcium (Ca) in reducing bone loss is debated. In a randomized placebo-controlled double-masked study, we investigated the effects of oral Ca supplements on femoral shaft (FS), femoral neck (FN) and lumbar spine (LS) bone mineral density (BMD), and on the incidence of vertebral fracture in vitamin-D-replete elderly. Ninety-three healthy subjects (72.1±0.6 years) were randomly allocated to three groups receiving 800 mg/day Ca in two different forms or a placebo for 18 months. Sixty-three patients (78.4±1.0 years) with a recent hip fracture were allocated to two groups receiving the two forms of Ca without placebo. FS BMD changes in Ca-supplemented non-fractured women were significantly different from those in the placebo group (+0.6±0.5% v −1.2±0.7%,p〈0.05). There was no difference in effect between the two forms of Ca. The changes of +0.7±0.8% v −1.7±1.6% in FN BMD of Ca-supplemented women and the placebo group did not reach statistical significance. In fractured patients, FS, FN and LS BMD changes were −1.3±0.8, +0.3±1.6 and +3.1±1.2% (p〈0.05 for the last). The rate of new vertebral fractures was 74.3 and 106.2 fractures per 1000 patient-years in Ca-supplemented non-fractured subjects and in the placebo group, respectively, and 144.0 in Ca-supplemented fractured patients. Thus, oral Ca supplements prevented a femoral BMD decrease and lowered vertebral fracture rate in the elderly.
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  • 9
    ISSN: 1432-1912
    Keywords: α1A-adrenoceptors ; α1B-adrenoceptors ; Rat kidney ; Inositol phosphates ; G protein ; Calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have compared the coupling mechanisms of rat renal α1A- and α1B-like adrenoceptors to inositol phosphate formation. The experiments were performed in parallel in native renal tissue preparations and in those where α1B-adrenoceptors had been inactivated by treatment with 10 μmol/l chloroethylclonidine for 30 min at 37°C; renal slices were used in most experiments but isolated renal cells were also used in some cases. The Ca2+ chelating agent, EGTA (5 mmol/l), reduced noradrenaline-stimulated inositol phosphate formation in native but enhanced it in chloroethylclonidine-treated renal slices. The inhibitory effect of EGTA was not mimicked by 100 nmol/l nifedipine. Inactivation of 87% of cellular Gi by 16–20 h treatment with 500 ng/ml pertussis toxin did not significantly affect noradrenaline-stimulated inositol phosphate formation in isolated renal cells but abolished the inhibitory effect of chloroethylclonidine. The adenylate cyclase activator, forskolin (20 μmol/l), inhibited noradrenaline-stimulated inositol phosphate formation in native and chloroethylclonidine-treated slices, and the inhibitory effects of chloroethylclonidine treatment and forskolin were additive. We conclude that in rat kidney inositol phosphate formation via α1B-like adrenoceptors may involve the influx of extracellular Ca2+ and a pertussis toxin-sensitive G-protein but is insensitive to inhibition by forskolin. In contrast α1A-like adrenoceptor-mediated inositol phosphate formation does not require the presence of extracellular Ca2+ or of Gi and is sensitive to inhibition by forskolin. In comparison to published data from other model systems we further conclude that the signaling mechanisms of α1-adrenoceptor subtypes may depend on their cellular environment.
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  • 10
    ISSN: 1432-1912
    Keywords: Px purinoceptors ; Calcium ; Magnesium ; Zinc ; Rat vas deferens ; α,β-methyleneATP binding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In this study we have examined the effect of metal cations (as their chloride salts) on the binding of [3H]α,β-methylene ATP ([3H]αβmeATP) to rat vas deferens membranes using a vacuum filtration receptor binding assay. Whereas NaCl and KCl (0.01 and 30 mM) did not affect total binding of 1 nM [3H]αβmeATP, several divalent and trivalent cation salts markedly increased binding. The trivalent cation salts, FeCl3 and AlCl3 (0.1 to 100 μM), produced the greatest increases in total binding of [3H]αβmeATP, however, their effects were most probably due to precipitation of the radioligand. In contrast, several divalent cations, at concentrations between 1 μM and 1–10 mM, increased total binding of [3H]αβmeATP to rat vas deferens by between 87% and 215% while having no effect on either filter binding or non specific binding. The following pEC50 values for potentiating binding of the radioligand were obtained: ZnCl2 (5.44), MnCl2 (4.52), CaCl2 (4.17), CoCl2 (4.06), MgCl2 (3.67) and BaCl2 (3.10). Both EDTA and EGTA (0.01–1 mM) inhibited the binding of the radioligand. The effects of ZnCl2, CaCl2 and MgCl2 were examined in saturation studies. In the absence of added divalent cations, [3H]αβmeATP labelled both high (pKd = 9.15) and low (pKd = 7.06) affinity binding sites. The affinity of the radioligand for its high affinity sites was increased by 3 mM CaCl2 (pKd = 9.56) and by 30 μM ZnCl2 (pKd = 9.46) but not by 3 mM MgCl2. The Bmax of the low affinity site for [3H]αβmeATP was increased (approximately 4 fold) by both 3 mM MgCl2 and 30 μM ZnCl2 but not by 3 mM CaCl2. The selective effect of CaCl2 on the high affinity binding sites enabled these sites to be labelled in the presence of 3 mM CaCl2 using a low concentration of [3H]αβmeATP (1 nM); the sites exhibited the binding characteristics expected of the P2x purinoceptor. The selective effect of MgCl2 on the low affinity binding sites enabled these sites to be labelled in the presence of 3 mM MgCl2 and using a high concentration of [3H]αβmeATP (100 nM). A comparison of the binding characteristics of the high and low affinity sites for [3H]αβmeATP revealed several other differences, in addition to their cation selectivity. First, the adenine analogues ADP, αβmeATP and adenosine tetraphosphate possessed between 13 and 62 fold higher affinity for the high affinity [3H]αβmeATP binding sites than for the low affinity binding sites. Secondly, GTP-γ-S and pyrophosphate were selective ligands for the low affinity [3H]αβmeATP binding sites possessing approximately 43 and 1995 fold, respectively, higher pIC50 values at the low affinity sites than at the high affinity sites. Finally, treatment of the membranes with 0.01–1 mM N-ethyl maleimide increased low affinity binding of the radioligand while not affecting binding to the high affinity sites. The binding characteristics of the low affinity sites suggest that they do not equate with functional P2x purinoceptors; their identity remains to be determined. There was evidence for heterogeneity of both the high and low affinity sites for [3H]αβmeATP since competition curves to several nucleotide and polyphosphate compounds displayed Hill slopes less than unity. In conclusion the present study has demonstrated that cations have a marked effect on the binding of [3H]αβmeATP in rat vas deferens. Of particular interest was the ability of CaCl2 to increase the affinity of the radioligand for its high affinity sites enabling these sites to be selectively labelled, while the ability of MgCl2 to increase the Bmax of the low affinity binding sites enabled these sites to be selectively labelled.
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