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  • 1
    ISSN: 1617-4623
    Keywords: Key words Cassava (Manihot esculenta) ; Chemical stress ; Environmental stress ; Oxidative stress ; Superoxide dismutase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA, mSOD1, encoding cytosolic copper/zinc superoxide dismutase (CuZnSOD) was cloned and characterized from cell cultures of cassava (Manihot esculenta Crantz) which produce a high yield of SOD. mSOD1 encodes a 152-amino acid polypeptide with a pI value of 5.84. Southern analysis using an mSOD1-specific probe indicated that a single copy of the mSOD1 gene is present in the cassava genome. The mSOD1 gene is highly expressed in cultured cells, as well as in intact stems and tuberous roots. It is expressed at a low level in leaves and petioles. Transcripts of mSOD1 were not detected in nontuberous roots. Transcriptional level of mSOD1 reaches a high level at stationary phase, and then sharply decreases during further culture. In excised cassava leaves, the mSOD1 gene responded to various stresses in different ways. The stresses tested included changes in temperature and exposure to stress-inducing chemicals. Levels of mSOD1 transcript increased dramatically a few hours after heat stress at 37° C and showed a synergistic effect with wounding stress. Levels decreased in response to chilling stress at 4° C and showed an antagonistic effect with wounding stress. The gene was induced by abscisic acid, ethephon, NaCl, sucrose, and methyl viologen. These results indicate that the mSOD1 gene is involved in the response to oxidative stress induced by environmental change.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1617-4623
    Keywords: Key words Peroxidase cDNAs ; Suspension cultures ; Environmental stress ; Sweet potato (Ipomoea batatas)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two cDNAs for anionic peroxidase (PODs), swpa2 and swpa3, were isolated from suspension cultures of sweet potato (Ipomoea batatas), and their expression was investigated with a view to understanding the physiological function of PODs in relation to environmental stresses. Swpa2 (whose putative mature protein product would have a pI value of 4.1) and swpa3 (4.3) encode polypeptides of 358 and 349 amino acids, respectively. The genes from which they were derived are predominantly expressed in cultured cells of sweet potato; transcripts of swpa2 were not detected in any tissues of the intact plant, and transcripts of swpa3 were detected at a low level only in the stem tissue. During cell culture, the expression patterns of the two genes differed; the level of swpa2 RNA progressively increased during cell growth, whereas that of swpa3 reached a maximum at the stationary phase and decreased on further culture. The two genes responded differently to stresses such as wounding or chilling of leaves. Swpa2 was strongly induced 48 h after wounding, but swpa3 was not affected by this treatment. The two genes were also highly expressed upon chilling (4° C), but expression was reduced by prior acclimation at 15° C. In addition, both genes were strongly induced immediately after treatment with ozone, and expression had decreased to the basal level 12 h after treatment. The response of these two genes to stresses such as aging, wounding, and chilling are different from those of the POD genes (swpa1 encoding an anionic product and swpn1 a neutral peroxidase) that we described previously. The responses of the two genes were also different from each other. These results suggest that the two new POD genes are involved in overcoming oxidative environmental stress, and each POD gene may be regulated by cell growth and environmental stress in different ways.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-2013
    Keywords: Key words Smooth muscle ; Nonselective cationic current ; Carbachol (CCh) ; Protein kinase C (PKC) ; Desensitization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The possibility of the protein kinase C (PKC) pathway being a mechanism underlying the desensitization of carbachol- (CCh-)activated nonselective cationic current (I CCh) was investigated in a study of guinea-pig gastric myocytes. Using the conventional whole-cell patch-clamp technique with symmetrical CsCl-rich solution in pipette and bath, I CCh was induced by bath application of 50 µM CCh. With 0.5 mM EGTA [ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid] in the pipette solution (0.5 mM [EGTA]i), I CCh decayed spontaneously (desensitization of I CCh) to around 20% within 10 min. Desensitization of I CCh was significantly attenuated with 2 mM [EGTA]i. At a concentration of 20 µM OAG (1-oleoyl-2-acetyl-sn-glycerol), a PKC activator, inhibited I CCh at 0.5 mM [EGTA]i but far less at 2 mM [EGTA]i (18% and 81% of control, respectively). The same cationic current induced by intracellular guanosine-5′-O-(3-thiotriphosphate) (GTP[γ-S]) was not inhibited by OAG with 0.5 mM [EGTA]i. The pretreatment of gastric myocytes with PKC inhibitors, either 1 µM chelerythrine or 1 µM peptide inhibitor, attenuated the desensitization of I CCh. [Ca2+]i was also measured by single cell microfluorometry using fura-2. Under CCh stimulation with 2 mM [EGTA]i, [Ca2+]i did not increase above 100 nM while it increased to around 260 nM with 0.5 mM [EGTA]i. These results suggest that the desensitization of I CCh is partly due to the Ca2+-dependent PKC pathway in guinea-pig gastric myocytes.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-2013
    Keywords: Key words Carbachol ; Nonselective cationic current ; Protein kinase C ; smooth muscle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The effect of protein kinase C (PKC) on carbachol (CCh)-activated nonselective cationic current (I CCh) was investigated in guinea-pig gastric myocytes using a PKC activator, phorbol 12, 13 dibutyrate (PDBu). Pretreatment with 1 μ M PDBu suppressed I CCh by 96.5 ± 2.9% (n = 14) in a reversible manner in nystatin-perforated mode. In the presence of 1 μM chelerythrine , a PKC inhibitor, inhibition of I CChby PDBu was not seen. In whole-cell mode, the inhibition of I CCh by PDBu was dependent on intracellular MgATP. In the presence of MgATP in the pipette, PDBu decreased I CCh by 98.8 ± 1.2% (n = 5) as was observed in nystatin-perforated mode. However, PDBu had little effect on I CCh in the absence of MgATP in the pipette; the extent of inhibition was 12.7 ± 4.3% (n = 8). PDBu also suppressed the generation of cationic current induced by intracellularly perfused GTP[γS]. In the PDBu-pretreated group (n = 9) and PDBu-untreated control group (n = 6), GTP[γS]-induced currents were 6.7 ± 2.4 pA and 236 ± 23 pA, respectively. These results suggest that PKC modulates I CCh at postreceptor sites via protein phosphorylation.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-2013
    Keywords: Key words Smooth muscle ; Nonselective cationic current ; Carbachol ; Myosin light chain kinase ; ML-7
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (I CCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. I CCh was induced by carbachol (CCh, 50 μM) at a holding potential of –30 mV or –60 mV. ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited I CCh concentration dependently in a reversible manner (53 ± 8.6% at 1 μM, mean ± SE, n = 11). In addition, amplitudes of I CCh were only 37 ± 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca2+ channel current. The peak value of Ba2+ current at 0 mV was reduced to 35 ± 7.4% (n = 9) by 3 μM of ML-7. As I CCh is known to have an intracellular Ca2+ dependence, we tried to exclude the possibility that ML-7 inhibited I CCh indirectly via suppression of Ca2+ current and the similar inhibitory effects of ML-7 on I CChwere confirmed under the following conditions: (1) clamp of membrane potential at –60 mV; (2) clamp of intracellular [Ca2+] to 1 μM by 10 mM BAPTA; (3) pre-inhibition of Ca2+ channel by verapamil. Different from the effects on I CCh, ML-7 barely inhibited the same cationic current induced by guanosine 5’-O-(3-thiotriphosphate) (GTP[γS], 0.2 mM) in the pipette solution. These results suggest that a Ca2+/calmodulin-MLCK-dependent pathway can modulate the activation of I CCh in guinea-pig gastric antral myocytes.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-203X
    Keywords: Key wordsArabidopsis thaliana ; Environmental stress ; Glutathione S-transferase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A glutathione S-transferase (GST) gene was cloned in Arabidopsis thaliana. The gene, designated ATGST 1, contained the entire transcription unit in three exons interrupted by two introns. The combined sequence of three exons had an open reading frame which predicted a GST protein of 208 amino acids. Gene transcription has been reported to be induced by pathogen attack and dehydration. In the present study northern blot analysis using a gene-specific DNA probe in the 3′ untranslated region revealed that expression of the gene was also rapidly induced by other environmental stresses such as wounding, low temperature, high salt and DPE herbicide treatment. The promoter region of the gene contained the sequence motif ATTTCAAA that is known to be present in ethylene-responsive elements and other motifs that are highly conserved amongst stress-inducible gene promoters.
    Type of Medium: Electronic Resource
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