Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We have isolated a cDNA encoding a calcium-dependent protein kinase (CDPK) in Nicotiana tabacum, which we designated NtCDPK1. The deduced amino acid sequence of NtCDPK1 suggests that this protein contains the kinase domain at the amino terminus and the autoregulatory and calmodulin-like domains at the carboxy terminus. NtCDPK1 is highly homologous to DcCPK1, a CDPK of carrot, showing 76.5% amino acid sequence identity. NtCDPK1 transcripts are present in roots, stems and flowers, but are almost undetectable in leaves. In leaves, NtCDPK1 mRNA accumulation is stimulated by phytohormones (ABA, GA and cytokinin), Ca2+, methyl jasmonate, wounding, fungal elicitors, chitosan, and NaCl. The recombinant full-length NtCDPK1 protein is catalytically active and highly stimulated by Ca2+. A truncated recombinant NtCDPK1 which lacks the C-terminal calmodulin-homologous domain also undergoes autophosphorylation, but the kinase activity is not stimulated by Ca2+. Phosphoamino acid analysis showed that NtCDPK1 phosphorylates serine and threonine residues. Finally, a 60 kDa protein which matches the expected size of NtCDPK1 was immunodetected in the membrane fraction by an antiserum reacting with NtCDPK1. Immunoprecipitation and in vitro phosphorylation using the antiserum also generated a 60 kDa phosphoprotein only in the membrane fraction. These results suggest that NtCDPK1 is associated with the membrane.
Plant Mol Biol 1999 Mar
PMID:Characterization of NtCDPK1, a calcium-dependent protein kinase gene in Nicotiana tabacum, and the activity of its encoded protein. 1034 4

Scrapie is a neurodegenerative disease in sheep and goats. Neuropathological examination shows astrocytosis. One issue is whether the astrocytosis seen in scrapie is a function of an increase in reactivity of individual cells, or whether there is actual replication of astrocytes. We used double-label immunohistochemistry for proliferating cell nuclear antigen (PCNA) and for glial fibrillary acidic protein (GFAP) to determine the mitotic state of cells and to confirm their identity as astrocytes. Brain sections from hamsters (strain LVG/LAK) infected with 139H or 263K scrapie isolates were examined. GFAP immunostaining was increased in astrocytes in most regions of the brains of scrapie-infected hamsters. These qualitative observations were confirmed by computerized image analysis quantification. A proportion of the hypertrophic astrocytes (0.5-10.8%, depending on specific location) were PCNA immunoreactive. The PCNA-immunopositive astrocytes were most frequently found in cerebral cortex, corpus callosum, subependymal areas, fimbria, caudate, thalamus, hypothalamus, hippocampus, and dentate gyrus. Our results suggest that the astrocytosis seen in scrapie-infected animals is, at least in part, owing to actual replication of astrocytes in these animals. We hypothesize that the astrocytes may be an important locus for the disease process.
J Mol Neurosci 1998 Dec
PMID:Astrocytosis and proliferating cell nuclear antigen expression in brains of scrapie-infected hamsters. 1034 95

We have used differential display to detect altering mRNA levels in response to desiccation and rehydration in leaves of the desiccation tolerant grass Sporobolus stapfianus. One of the RT-PCR products identified was used to isolate a cDNA of 999 bp which encodes a protein of 210 amino acids (predicted size 23 kDa). This protein displays considerable sequence similarity to mammalian and plant Rab2, a small GTP-binding protein, possessing several conserved motifs common to these regulatory proteins. Sporobolus Rab2 was expressed in Escherichia coli yielding a protein with an apparent molecular mass of ca. 30 kDa which was shown to have the ability to bind GTP. Rab2 transcript accumulated early in response to a decrease in relative water content (RWC) and remained high even in dried leaves. Rehydration of desiccated leaves resulted in a decrease in levels within 3 h of rewetting, with a brief increase at ca. 12 h. Accumulation of Rab2 transcript was also evident during drying and rehydration of the roots of S. stapfianus, as well as in leaves of the desiccation-sensitive grass Sporobolus pyramidalis. Earlier work on S. stapfianus concluded that the plant hormone ABA has little effect on inducing desiccation tolerance, however Rab2 transcript does exhibit a small increase in accumulation in response to exogenous ABA. A possible role for Rab2 with respect to desiccation tolerance and damage repair is discussed.
Plant Mol Biol 1999 Mar
PMID:Characterization of a desiccation-responsive small GTP-binding protein (Rab2) from the desiccation-tolerant grass Sporobolus stapfianus. 1035 94

We report the characterisation of two cytochrome b5 genes and their spatial and temporal patterns of expression during development in olive, Olea europaea. A PCR-generated probe, based on a tobacco cytochrome b5 sequence, was used to isolate two full-length cDNA clones (cytochrome b5-15 and cytochrome b5-38) from a library derived from 13 WAF olive fruits. The cDNAs encoded proteins of 17.0 and 17.7 kDa, which contained all the characteristic motifs of cytochromes b5 from other organisms and exhibited 63% identity and 85% similarity with each other. The olive cytochrome b5-15 cDNA was then used as a probe for more detailed analysis. Southern blotting revealed a gene family of at least 4-6 members while northern blotting and in situ hybridisation showed a highly specific pattern of gene expression. Very low levels of cytochrome b5 mRNA were detected in tissues characterised by high rates of lipid accumulation, such as young expanding leaves, maturing seeds and ripening mesocarp. The cytochrome b5 genes were not induced at 6 degrees C and their response to ABA was relatively slow compared with fatty acid desaturase genes. In contrast, high levels of cytochrome b5 gene expression were found in young fruits at the pattern formation (globular/heart) stage of embryogenesis and in vascular and transmitting tissues of male and female reproductive organs. The data are consistent with a major role for cytochrome b5 in developmental processes related to plant reproduction in addition to being an electron donor to microsomal desaturases.
Plant Mol Biol 1999 May
PMID:Temporal and spatial gene expression of cytochrome B5 during flower and fruit development in olives. 1039 47

We have isolated and sequenced a 9.5 kb genomic region from A. thaliana, located on chromosome 2, which contains two tandemly arranged closely related genes (AtM10 and AtM17) coding for a new family of LEA proteins. The deduced proteins have a molecular mass of 11 and 29 kDa, respectively, are extremely hydrophilic except at their N-termini and share 70% amino acid (aa) identity. A 47 aa motif containing a 6-cysteine domain is present once in AtM10 and four times in AtM17. The short intergenic region, the identical position of the intron and the overall sequence homology suggest that these two genes evolved through a duplication event. This conclusion is supported by the presence of two homologous strictosidine synthase-like (pseudo)genes downstream from AtM17 and AtM10. Expression studies, using AtM10 and AtM17 cDNAs, revealed that both transcripts accumulate exclusively in seeds from late embryogenesis until two days after imbibition. Expression of both genes in young seedlings is repressed during ABA, salt or drought treatment, whereas a cold stress induces the expression of AtM17 only. In situ hybridization revealed that AtM10 transcripts are detected throughout the embryo while those of AtM17 are more localized to cotyledon cells.
Plant Mol Biol 1999 May
PMID:Structure, organization and expression of two closely related novel Lea (late-embryogenesis-abundant) genes in Arabidopsis thaliana. 1039 54

Two aquaporin genes were isolated from a cDNA library of canola (Brassica napus L.). The first aquaporin, BnPIP1 of 1094 bp, encoding a putative polypeptide of 287 amino acids with a predicted molecular mass of 30.4 kDa and a pI of 7.8, belongs to the family of plasma membrane intrinsic protein (PIPs) aquaporins. The B. napus aquaporin showed 85-94% identity to the Arabidopsis thaliana PIPs. ABA priming of seed induced high levels of BnPIP1 transcript which remained after subsequent re-drying of the seed. The second aquaporin, Bny-TIP2 of 1020 bp, encoded a putative polypeptide of 253 amino acids with a predicted molecular mass of 25.8 kDa and a pI of 5.8. Bngamma-TIP2 showed 83-90% identity to gamma-TIP genes from a variety of plant species. Bngamma-TIP2 was expressed only when radicle protrusion occurred in either untreated or primed seeds. Seeds primed with PEG or ABA germinated earlier and showed a higher final percentage of germination than unprimed seed, particularly under salt and osmotic stresses at low temperature. Transcripts of both BnPIP1 and Bngamma-TIP2 genes were present earlier during germination of primed seeds than non-primed seed. From these results, we conclude that BnPIP1 is related to the water transportation required for enzymatic metabolism of storage nutrients at the early stages of canola seed germination whereas Bngamma-TIP2 expression is related to cell growth associated with radicle protrusion. Priming induced the expression of BnPIP1 but had no effect on Bngamma-TIP2.
Plant Mol Biol 1999 Jul
PMID:Characterization and expression of plasma and tonoplast membrane aquaporins in primed seed of Brassica napus during germination under stress conditions. 1048 Mar 87

A cDNA (C2C-Prx) corresponding to a 2Cys-peroxiredoxin (2Cys-Prx) was isolated from a leaf cDNA library of Chinese cabbage. The predicted amino acid sequence of C2C-Prx has 2 conserved cysteines and several peptide domains present in most of the 2Cys-Prx subfamily members. It shows the highest sequence homology to the 2Cys-Prx enzymes of spinach (88%) and Arabidopsis (86%). Southern analysis using the cDNA insert of C2C-Prx revealed that it consists of a small multigene family in Chinese cabbage genome. RNA blot analysis showed that the gene was predominantly expressed in the leaf tissue of Chinese cabbage seedlings, but the mRNA was generally expressed in most tissues of mature plant, except roots. The expression of C2C-Prx was slightly induced by treatment with H2O2 (100 microM) or Fe3+/O2/DTT oxidation system, but not by ABA (50 microM) or GA3 (10 microM). The C2C-Prx is encoded as a preprotein of 273 amino acids containing a putative chloroplast-targeting signal of 65 amino acids at its N-terminus. The N-terminally truncated recombinant protein (deltaC2C-Prx) migrates as a dimer in a non-reducing SDS-polyacrylamide gel and as a monomer in a reducing condition. The deltaC2C-Prx shows no immuno cross-reactivity to antiserum of the yeast thiol-specific antioxidant protein, and vice versa. The deltaC2C-Prx prevents the inactivation of glutamine synthetase and the DNA cleavage in the metal-catalyzed oxidation system. In the yeast thioredoxin system containing thioredoxin reductase, thioredoxin, and NADPH, the deltaC2C-Prx exhibits peroxidase activity on H2O2.
Plant Mol Biol 1999 Jul
PMID:Molecular cloning, expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage. 1048 17

Membrane intrinsic proteins facilitate movement of small molecules often times functioning as water channels. We have identified two genes from rice which encode proteins with characteristic features of plasma membrane intrinsic proteins (PIP). They possess six membrane-spanning domains, an NPA repeat, overall high sequence homologies and characteristic C- and N-terminal hallmark motifs which allowed assignment of OsPIP1a to the PIP1 subfamily and of OsPIP2a to the PIP2 subfamily. OsPIP1a and OsPIP2a showed similar but not identical expression patterns. The two genes were expressed at higher levels in seedlings than in adult plants and expression in the primary root was regulated by light. In internodes of deepwater rice plants which were induced to grow rapidly by submergence, transcript levels were slightly induced in the intercalary meristem (IM) and slightly reduced in the elongation zone (EZ) after 18 h. In internodes of GA-induced excised stem sections transcript levels transiently declined in the IM and EZ after 1 h and subsequently recovered to elevated levels after 18 h. GA also induced OsPIP expression in non-growing tissue after 18 h. In the IM of submergence-induced stem sections transcript levels remained constitutive. The different growth-promoting treatments showed no direct correlation between growth rate and OsPIP gene expression in dividing or expanding cells. In fact, treatment of excised stem sections with ABA or drought stress induced similar changes in OsPIP expression in the growing zone during the first 6 h as GA did. We conclude that regulation of OsPIP1a and OsPIP2a expression is not primarily controlled by growth. GA-induced growth may however change the water status of cells which in turn results in altered PIP abundance.
Plant Mol Biol 1999 Aug
PMID:Expression of two PIP genes in rapidly growing internodes of rice is not primarily controlled by meristem activity or cell expansion. 1052 23

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5'-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The beta-glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.
Plant Mol Biol 2000 Mar
PMID:Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression. 1080 11

The therapeutic potential of synthetic inhibitors to the major cysteine-proteinase from Trypanosoma cruzi (cruzain or cruzipain) was recently demonstrated in animal models of Chagas' disease. A possible limitation of this strategy would be the emergence of parasite populations developing resistance to cysteine-proteinase inhibitors. Here, we describe the properties of a phenotypically stable T. cruzi cell line (R-Dm28) that displays increased resistance to Z-(SBz)Cys-Phe-CHN2, an irreversible cysteine-proteinase inhibitor which preferentially inactivates cathepsin L-like enzymes. Isolated from axenic cultures of the parental cells (IC50 1.5 microM), R-Dm28 epimastigotes exhibited 13-fold (IC50) 20 microM) higher resistance to this inhibitor and did not display cross-resistance to unrelated trypanocidal drugs, such as benznidazol and nifurtimox. Western blotting (with mAb), affinity labeling (with biotin-LVG-CHN2) and FACS analysis of R-Dm28 log-phase epimastigotes revealed that the cruzipain target was expressed at lower levels, as compared with Dm28c. Interestingly, this deficit was paralleled by increased expression of an unrelated Mr 30 000 cysteine-proteinase whose activity was somewhat refractory to inhibition by Z-(SBz)Cys-Phe-CHN,. N-terminal sequencing of the affinity-purified biotin-LVG-proteinase complex allowed its identification as a cathepsin B-like enzyme. Increased antigenic deposits of this proteinase were found in the grossly enlarged and electron dense reservosomes from R-Dm28 epimastigotes. Our data suggest that R-Dm28 resistance to toxic effects induced by the synthetic inhibitor may result from decreased availability of the most sensitive cysteine-proteinase target, cruzipain. The deficit in metabolic functions otherwise mediated by this cathepsin L-like proteinase is likely compensated by increased expression/accumulation of a cathepsin B-like target.
Mol Biochem Parasitol 2000 Jun
PMID:Altered expression of cruzipain and a cathepsin B-like target in a Trypanosoma cruzi cell line displaying resistance to synthetic inhibitors of cysteine-proteinases. 1092 56


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