Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rapid, semiquantitative reverse transcriptase-polymerase chain reaction assay was developed to investigate signal transduction events involved in the induction of Crassulacean acid metabolism (CAM) in detached common ice plant (Mesembryanthemum crystallinum) leaves. Transcript abundance of Ppc1, a gene encoding the CAM-specific isoform of phosphoenolpyruvate carboxylase, increased rapidly in response to osmotic stress (dehydration and mannitol), ionic stress (NaCl), and exogenous abscisic acid treatment, but failed to accumulate in response to exogenous cytokinin or methyl jasmonate. Stress-induced accumulation of Ppc1, GapC1, and Mdh1 transcripts was inhibited by pretreating leaves with the calcium chelator ethyleneglycol-bis(aminoethyl ether)-N,N'-tetraacetic acid, suggesting that extracellular calcium participates in signaling events leading to CAM induction. Treatment of unstressed detached leaves with ionomycin, a Ca(2+) ionophore, and thapsigargin, a Ca(2+)-ATPase inhibitor, enhanced Ppc1 transcript accumulation, indicating that elevations in cytosolic [Ca(2+)] are likely to participate in signaling CAM induction. Inhibitors of Ca(2+)- or calmodulin-dependent protein kinases (N-[6-aminohexyl]-5-chloro-1-napthalenesulfonamide, Lavendustin C) and protein phosphatase 1 and 2A (okadaic acid) activity suppressed Ppc1 transcript accumulation in response to ionic and osmotic stresses, as well as abscisic acid treatment. These results suggest that both protein phosphorylation and dephosphorylation events participate in signaling during CAM induction. In contrast, pretreatment with cyclosporin A or ascomycin, inhibitors of protein phosphatase 2B activity, stimulated Ppc1 gene expression either directly or indirectly through promoting water loss.
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PMID:Signaling events leading to crassulacean acid metabolism induction in the common ice plant 1051 46

L-type voltage-sensitive Ca(2+) channels (L-VSCCs) play an important role in developmental and aging processes, as well as during normal function of brain neurons. Here, we tested a prediction of the hypothesis that membrane density of functional L-VSCCs is regulated by the level of gene expression for its alpha(1D) pore-forming subunit. If so, alpha(1D) mRNA and L-VSCC activity should be positively correlated within individual neurons. Conventional methods of aspiration and/or acute cell dissociation used in prior single-cell studies have generally yielded variable and incomplete recovery of intracellular mRNA. Thus, quantitative relationships between channel function and expression have been difficult to define. In this study, we used the partially dissociated ("zipper") hippocampal slice preparation as a method for collecting a single neuron's mRNA complement. This preparation, developed to expose neuronal somata for recording, also enables the extraction of a neuron with major processes largely intact. Thus, single-cell measures of gene/mRNA expression can be based on approximately the cell's full set of mRNA transcripts. In adult and aged rat hippocampal zipper slices, L-VSCC activity was first recorded in CA1 neurons in cell-attached patch mode. The same neurons were then extracted and collected for semiquantitative reverse transcriptase-PCR analysis of alpha(1D) and calmodulin A (CaM) mRNA content. Across multiple single neurons, a significant, positive correlation was found between the rank orders of L-VSCC activity and of alpha(1D), but not CaM, mRNA expression. Thus, these studies support the possibility that the level of alpha(1D) gene expression regulates the density of functional L-VSCCs.
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PMID:Expression of alpha 1D subunit mRNA is correlated with L-type Ca2+ channel activity in single neurons of hippocampal "zipper" slices. 1075 53

After separating by two-dimensional gel electrophoresis an extract of total proteins from human stratum corneum, two spots were extracted and analyzed for their peptide sequence. The resulting internal protein sequences provided evidence for the identification of a new calcium-binding protein. Cloning of the corresponding full-length cDNA was achieved by reverse transcriptase-polymerase chain reaction using two keratinocyte libraries, one from proliferating cultured keratinocytes and one from differentiated keratinocytes of reconstructed human epidermis. The cDNA had an open reading frame encoding a new calcium-binding protein of 146 amino acids, a member of the calmodulin family. We named this new protein calmodulin-like skin protein (CLSP), since reverse transcriptase-polymerase chain reaction studies of CLSP expression in 10 different human tissues revealed that this protein was particularly abundant in the epidermis where its expression is directly related to keratinocyte differentiation. Expression of the cloned cDNA in Escherichia coli yielded a recombinant protein which allowed its further characterization. rCLSP is able to bind calcium, and similarly to calmodulin, exposes thereafter hydrophobic parts which most likely interact with target proteins. Epidermal proteins retained by CaM affinity column are quantitatively and qualitatively distinct from those of the rCLSP column. Sequencing of a rCLSP affinity purified protein revealed 100% identity with transglutaminase 3, a key enzyme in terminal differentiation, indicating an important role of CLSP in this process.
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PMID:Identification and cloning of a new calmodulin-like protein from human epidermis. 1077 82

By using reverse transcriptase-polymerase chain reaction, two cDNAs were isolated that encode major intrinsic membrane proteins (MIPs) that are expressed in nitrogen-fixing root nodules of Lotus japonicus. Lotus intrinsic membrane protein 1 (LIMP 1) is expressed at high levels in both nodule and root tissues and shows highest sequence similarity to members of the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Functional analysis of LIMP 1 by expression in Xenopus laevis oocytes show that it is a water-specific aquaporin. In contrast, LIMP 2 shows the highest sequence similarity to soybean nodulin 26 (67.8% amino acid sequence identity). LIMP 2 is also a nodulin, showing expression only in mature nitrogen fixing nodules of L. japonicus. LIMP 2 is a multifunctional aquaglyceroporin, and displays the ability to flux both water as well as glycerol upon expression in Xenopus oocytes. Additionally, the carboxyl terminal region of LIMP 2 has a conserved phosphorylation motif that is phosphorylated by a calmodulin-like domain protein kinase. Overall, the data show that L. japonicus nodules contain two structurally and functionally distinct MIP proteins: one (LIMP 2) which appears to be the nodulin 26 ortholog of L. japonicus and another (LIMP 1) which appears to be a member of the TIP subfamily.
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PMID:Water-selective and multifunctional aquaporins from Lotus japonicus nodules. 1080 45

Plant phosphoenolpyruvate carboxylase (PEPc) activity and allosteric properties are regulated by PEPc kinase (PPcK) through reversible phosphorylation of a specific serine (Ser) residue near the N terminus. We report the molecular cloning of PPcK from the facultative Crassulacean acid metabolism (CAM) common ice plant (Mesembryanthemum crystallinum), using a protein-kinase-targeted differential display reverse transcriptase-polymerase chain reaction approach. M. crystallinum PPcK encodes a minimal, Ca(2+)-independent Ser/threonine protein kinase that is most closely related to calcium-dependent protein kinases, yet lacks both the calmodulin-like and auto-inhibitory domains typical of plant calcium-dependent protein kinase. In the common ice plant PPcK belongs to a small gene family containing two members. McPPcK transcript accumulation is controlled by a circadian oscillator in a light-dependent manner. McPPcK encodes a 31.8-kD polypeptide (279 amino acids), making it among the smallest protein kinases characterized to date. Initial biochemical analysis of the purified, recombinant McPPcK gene product documented that this protein kinase specifically phosphorylates PEPc from CAM and C(4) species at a single, N-terminal Ser (threonine) residue but fails to phosphorylate mutated forms of C(4) PEPc in which this specific site has been changed to tyrosine or aspartate. McPPcK activity was specific for PEPc, Ca(2+)-insensitive, and displayed an alkaline pH optimum. Furthermore, recombinant McPPcK was shown to reverse the sensitivity of PEPc activity to L-malate inhibition in CAM-leaf extracts prepared during the day, but not at night, documenting that PPcK contributes to the circadian regulation of photosynthetic carbon flux in CAM plants.
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PMID:A minimal serine/threonine protein kinase circadianly regulates phosphoenolpyruvate carboxylase activity in crassulacean acid metabolism-induced leaves of the common ice plant. 1093 63

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.
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PMID:A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats. 1095 42

Because amphetamine releases two to three times more dopamine in schizophrenia patients than in control subjects, and because calcium-calmodulin-dependent protein kinase II has a key role in the enhanced action of amphetamine-induced dopamine release in rats, the synaptic content of calcium-calmodulin-dependent protein kinase IIbeta mRNA was measured (by quantitative competitive RT-PCR; reverse transcriptase-polymerase chain reaction) in seven frontal cerebral cortices of post-mortem brains from patients who had schizophrenia and in seven control tissues. The results indicate that the mRNA of this kinase is elevated in the schizophrenia frontal cortex.
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PMID:Schizophrenia: elevated mRNA for calcium-calmodulin-dependent protein kinase IIbeta in frontal cortex. 1104 61

The transcription factor NFAT (nuclear factor of activated T-cells) plays a central role in mediating Ca(2+)-dependent gene transcription in a variety of cell types. Sustained increases in intracellular calcium concentration ([Ca(2+)]i) are presumed to be required for NFAT dephosphorylation by the Ca(2+)/calmodulin-dependent protein calcineurin and its subsequent nuclear translocation. Here, we provide the first identification and characterization of NFAT in native smooth muscle, showing that NFAT4 is the predominant isoform detected by reverse transcriptase-polymerase chain reaction and Western blot analysis. PDGF induces NFAT4 translocation in smooth muscle, leading to an increase in NFAT transcriptional activity. NFAT4 activation by PDGF depends on Ca(2+) entry through voltage-dependent Ca(2+) channels, because its nuclear accumulation is prevented by the Ca(2+) channel blocker nisoldipine and the K(+) channel opener pinacidil. Interestingly, elevation of [Ca(2+)]i by membrane depolarization or ionomycin treatment are not effective stimuli for NFAT4 nuclear accumulation, indicating that Ca(2+) influx is necessary but not sufficient for NFAT4 activation. In contrast, membrane depolarization readily activates the Ca(2+)-dependent transcription factor CREB (cAMP-responsive element-binding protein). The calcineurin blockers CsA and FK506 also prevented the PDGF-induced NFAT4 nuclear localization. These results indicate that both the nature of the calcium signal and PDGF-induced modulation of nuclear import-export of NFAT are critical for NFAT4 activation in this tissue.
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PMID:NFAT4 movement in native smooth muscle. A role for differential Ca(2+) signaling. 1127 65

Brain-derived neurotrophic factor (BDNF), which supports spiral ganglion neuron (SGN) survival in vivo and in vitro, is synthesized by SGNs. The BDNF gene generates multiple different transcripts, each from its own promoter region. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we find that SGNs express only the downstream transcripts III and IV in vivo and in vitro. Using RT-PCR assays of BDNF transcripts and transfection of BDNF promoter-reporter constructs, we tested the hypothesis, originally derived from studies of cortical neurons, that depolarization induces BDNF expression via a signaling pathway that includes Ca2+/calmodulin-dependent kinases (CaMKs) and the transcription factor, Ca2+/cyclic AMP response element binding protein (CREB). In contrast, we found that in SGNs in vivo BDNF expression is constitutive and is not increased by electrical activation. Similarly, BDNF expression in vitro is not increased by stimuli that activate CREB, including depolarization, cAMP, or transfection of activated CaMK mutants. However, transfection of dominant-negative CREB mutants did abrogate gene expression driven by BDNF promoters III and IV, indicating that CREB is necessary for constitutive BDNF expression. Thus, BDNF synthesis within SGNs makes possible an autocrine or paracrine mechanism that can contribute to support SGN survival but SGNs are distinctive in that this mechanism is constitutive and not activity-regulated.
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PMID:BDNF synthesis in spiral ganglion neurons is constitutive and CREB-dependent. 1137 82

Fumonisins are a family of mycotoxins produced by Fusarium moniliforme, which is the most common mold found on corn throughout the world. These compounds are both toxic and carcinogenic for animals, and perhaps humans, with the kidney being the most sensitive organ to fumonisin toxicity. The molecular mechanism of fumonisin toxicity appears to involve disruption of de novo biosynthesis of sphingolipids and accumulation of sphinganine. The goals of this study were to determine whether fumonisin B(1) kills LLC-PK(1) renal kidney epithelial cells by inducing apoptosis and to identify genes affected by sphinganine that mediate fumonisin B(1)-induced cell death. Fumonisin B(1) produced morphological changes (i.e., cell shrinkage, membrane blebbing) and time-dependent increases in DNA fragmentation demonstrating that the toxin induces apoptosis. Simultaneously, fumonisin B(1) blocked sphingolipid biosynthesis and caused accumulation of sphinganine. To further investigate the role of sphinganine in fumonisin B(1)-induced apoptosis, beta-fluoroalanine (betaFA) was used to inhibit serine palmitoyltransferase, which catalyzes an earlier step in the sphingolipid biosynthetic pathway. betaFA blocked sphinganine accumulation and prevented fumonisin B(1)-induced DNA fragmentation, confirming that apoptosis induced by fumonisin B(1) is dependent upon accumulation of sphinganine. To examine gene expression, differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was applied to RNA isolated after 16 h of exposure to fumonisin B(1). Differential expression in response to fumonisin B(1) of a gene identified as calmodulin has been verified by Northern analysis. Sphinganine appears to mediate the effect because betaFA reduces induction of calmodulin mRNA by fumonisin B(1). Fumonisin B(1) also increases calmodulin protein in a concentration-dependent manner and the calmodulin antagonist W7 blocks fumonisin B(1)-induced DNA fragmentation, supporting a role for calmodulin in fumonisin B(1)-induced apoptosis. In contrast, fumonisin B(1) had no effect on expression of bcl-2 family genes (bax, bcl-2, and bcl-x). These findings demonstrate that fumonisin B(1) kills LLC-PK(1) kidney cells by inducing apoptosis. Further, the results establish a sequence of events for fumonisin B(1)-induced apoptosis involving initial disruption of sphingolipid metabolism and accumulation of sphinganine (or a metabolite), which, in turn, induces expression of calmodulin.
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PMID:Fumonisin B(1) induces apoptosis in LLC-PK(1) renal epithelial cells via a sphinganine- and calmodulin-dependent pathway. 1160 88


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