scholarly journals Characterization and Expression Analysis of Genes Encoding Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxylase Kinase of Lotus japonicus, a Model Legume

2003 ◽  
Vol 16 (4) ◽  
pp. 281-288 ◽  
Author(s):  
Tomomi Nakagawa ◽  
Tomoko Izumi ◽  
Mari Banba ◽  
Yosuke Umehara ◽  
Hiroshi Kouchi ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Root Nodules ◽  
Expression Patterns ◽  
Phosphorylation Site ◽  
Lotus Japonicus ◽  
Specific Protein ◽  
Mrna Levels ◽  
Model Legume ◽  
Putative Phosphorylation Site

Phosphoenolpyruvate carboxylases (PEPCs), one form of which in each legume species plays a central role in the carbon metabolism in symbiotic root nodules, are activated through phosphorylation of a conserved residue by a specific protein kinase (PEPC-PK). We characterized the cDNAs for two PEPC isoforms of Lotus japonicus, an amide-translocating legume that forms determinate nodules. One gene encodes a nodule-enhanced form, which is more closely related to the PEPCs in amide-type indeterminate nodules than those in ureide-type determinate nodules. The other gene is expressed in shoots and roots at a low level. Both forms have the putative phosphorylation site, Ser11. We also isolated a cDNA and the corresponding genomic DNA for PEPC-PK of L. japonicus. The recombinant PEPC-PK protein expressed in Escherichia coli phosphorylated recombinant maize C4-form PEPC efficiently in vitro. The level of mRNA for PEPC-PK was high in root nodules, and those in shoots and roots were also significant. In situ hybridization revealed that the expression patterns of the transcripts for PEPC and PEPC-PK were similar in mature root nodules, but were different in emerging nodules. When L. japonicus seedlings were subjected to prolonged darkness and subsequent illumination, the activity of PEPC-PK and the mRNA levels of both PEPC and PEPC-PK in nodules decreased and then recovered, suggesting that they are regulated according to the amounts of photosynthates transported from shoots.

Phosphoenolpyruvate carboxylase during grape berry development: protein level, enzyme activity and regulation

2000 ◽  
Vol 27 (3) ◽  
pp. 221 ◽  
Author(s):  
Paraskevi Diakou ◽  
Laurence Svanella ◽  
Philippe Raymond ◽  
Jean-Pierre Gaudillère ◽  
Annick Moing
Keyword(s):  
Malic Acid ◽  
Protein Level ◽  
Phosphoenolpyruvate Carboxylase ◽  
Phosphorylation Site ◽  
Acid Synthesis ◽  
Grape Berry ◽  
Vitis Vinifera L ◽  
Normal Acid

The protein level and regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, involved in malic acid synthesis) was studied during the fruit development of two grape (Vitis vinifera L.) varieties, ‘Cabernet Sauvignon’ and ‘Gora Chirine’, with berries of normal and low organic acid content, respectively. The protein level and in vitro activity were higher in the low-acid variety than in the normal-acid variety for most stages. In vivo PEPC activity, measured using 14 CO2 labelling, was significantly higher in the low-acid variety than in the normal-acid variety about 1 week before and 1 week after veraison (the day which corresponds to the onset of ripening). However, partitioning into malate was the same for both varieties. Antibodies raised against the N-terminal part of SorghumPEPC recognised the grape berry PEPC, indicating the presence of the consensus phosphorylation site involved in PEPC regulation. PEPC phosphorylation status was estimated by studying sensitivity to pH and malate. Grape berry PEPC appeared more sensitive to low pH and malate during ripening (IC50 malate, 0.2–0.7 mM) compared to during the earlier stages of development (IC50 malate, 1.2–2 mM) for both varieties. Therefore, in the normal-acid variety, PEPC seems to participate in controlling malic acid accumulation but does not seem to control the differences in malic acid concentration observed between the two varieties.

scholarly journals GDF-9 and BMP-15 mRNA Levels in Canine Cumulus Cells Related to Cumulus Expansion and the Maturation Process

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 462 ◽  
Author(s):  
George Ramirez ◽  
Jaime Palomino ◽  
Karla Aspee ◽  
Monica De los Reyes
Keyword(s):  
Gene Expression ◽  
Estrous Cycle ◽  
Expression Patterns ◽  
Cumulus Cells ◽  
Mrna Levels ◽  
Cumulus Expansion ◽  
Polymerase Chain ◽  
Specific Regulation ◽  
Reproductive Phases

The competence to undergo expansion is a characteristic of cumulus cells (CCs). The aim was to investigate the expression of GDF-9 and BMP-15 mRNA in canine cumulus cells in relation to cumulus expansion and meiotic development over the estrous cycle. CCs were recovered from nonmatured and in vitro-matured (IVM) dog cumulus oocyte complexes (COCs), which were obtained from antral follicles at different phases of the estrous cycle. Quantitative real-time polymerase chain reaction (q-PCR) was used to evaluate the relative abundance of GDF-9 and BMP-15 transcripts from the CCs with or without signs of expansion. The results were evaluated by ANOVA and logistic regression. The maturity of the oocyte and the expansion process affected the mRNA levels in CCs. There were differences (p < 0.05) in GDF-9 and BMP-15 gene expression in CCs isolated from nonmatured COCs when comparing the reproductive phases. Lower mRNA levels (p < 0.05) were observed in anestrus and proestrus in comparison to those in estrus and diestrus. In contrast, when comparing GDF-9 mRNA levels in IVM COCs, no differences were found among the phases of the estrous cycle in expanded and nonexpanded CCs (p < 0.05). However, the highest (p < 0.05) BMP-15 gene expression in CCs that did not undergo expansion was exhibited in anestrus and the lowest (p < 0.05) expression was observed in estrus in expanded CCs. Although the stage of the estrous cycle did not affect the second metaphase (MII )rates, the expanded CCs obtained at estrus coexisted with higher percentages of MII (p < 0.05). In conclusion, the differential expression patterns of GDF-9 and BMP-15 mRNA transcripts might be related to cumulus expansion and maturation processes, suggesting specific regulation and temporal changes in their expression.

Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2328-2334 ◽  
Author(s):  
Mı́riam Molina-Arcas ◽  
Beatriz Bellosillo ◽  
F. Javier Casado ◽  
Emili Montserrat ◽  
Joan Gil ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chronic Lymphocytic Leukemia ◽  
Expression Patterns ◽  
Individual Variability ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Nucleoside Transporter ◽  
Drug Bioavailability

Nucleoside derivatives are currently used in the treatment of hematologic malignancies. Although intracellular events involved in the pharmacologic action of these compounds have been extensively studied, the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in leukemia cells has not been comprehensively addressed. We have monitored the amounts of mRNA for the 5 nucleoside transporter isoforms cloned so far (CNT1, CNT2, CNT3, ENT1, and ENT2) in several human cell types and in normal human leukocytes. We then examined the expression patterns of these plasma membrane proteins in patients with chronic lymphocytic leukemia (CLL) and correlated them with in vitro fludarabine cytotoxicity. Despite a huge individual variability in the mRNA amounts for every transporter gene expressed in CLL cells (CNT2, CNT3, ENT1, and ENT2), no relationship between mRNA levels and in vitro fludarabine cytotoxicity was observed. Fludarabine accumulation in CLL cells was mostly, if not exclusively, mediated by ENT-type transporters whose biologic activity was clearly correlated with fludarabine cytotoxicity, which reveals a role of ENT-mediated uptake in drug responsiveness in patients with CLL.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

Expression of a KCC1 Splice Variant Is Suppressed by NF-ΚB in Erythroid Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1669-1669
Author(s):  
Kathleen P. Anderson ◽  
Scott C. Crable ◽  
Suzan M. Hammond ◽  
Patrick G. Gallagher ◽  
Clinton H. Joiner
Keyword(s):  
Sickle Cell ◽  
Splice Variant ◽  
Expression Patterns ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Mrna Levels ◽  
Red Cell ◽  
Rt Pcr ◽  
Erythroid Precursor

Abstract The K+Cl- cotransporter (KCC) plays a significant role in the maintenance of red cell volume. Activity of the cotransporter is higher in sickle (SS) compared to normal (AA) reticulocytes, and contributes to SS dehydration. Thus, KCC is considered a potential modifier gene for sickle cell disease (SCD). We have demonstrated the presence of transcripts for KCC1, KCC3, and KCC4 in human reticulocytes (Exp.Hem.2005;33:624–31) and shown that one splice variant of KCC1 (KCC1ex1b), which codes for a protein with a small (7aa) alternative N-terminal exon, is detected in AA, but not SS reticulocytes. Studies with murine KCC1 have demonstrated that proteins produced by N-terminal truncation are inactive for K+Cl- cotransport, and function as dominant negative regulators of full-length KCC1 and KCC3 proteins. Since high level expression of this variant in AA cells compared to SS cells might explain the relatively low KCC activity in AA reticulocytes, we have identified the promoter for the KCC1ex1b transcript and investigated the regulatory elements that control its expression. Here we report the involvement of TNFα and NF-ΚB in the transcriptional regulation of the KCC1ex1b variant. Although KCC1ex1b is not expressed in reticulocytes isolated from sickle cell patients, we found that SS erythroid precursor cells cultured in vitro express this variant. SS and AA peripheral blood mononuclear cells were cultured in semi-liquid media with stem cell factor and erythropoietin, and collected after 5, 10, and 14 days in culture. Cells harvested at 14 days and isolated by binding to micromagnetic beads coated with transferrin receptor antibody were 95–98% positive for glycophorin A. RNA was extracted and analyzed by semi-quantitative RT-PCR, using primers for KCC1ex1 and KCC1ex1b. In both AA and SS cells, the transcript level for KCC1ex1b rose over the time in culture, while the KCC1ex1 transcript was constant. This difference between the in vitro and in vivo expression patterns for the KCC1ex1b variant could be explained by regulation via an external factor, such as a cytokine present in the blood of sickle cell patients, but absent in the in vitro culture system. The levels of numerous cytokines, including TNF, VEGF, and various interleukins, are elevated in SCD. We therefore assayed the effect of TNFα on endogenous KCC1ex1b expression in K562 cells by RT-PCR analysis at 24 and 48 hours after the addition of TNFα to the tissue culture medium. The steady-state mRNA levels of the KCC1ex1b variant decreased approximately 40% in response to TNF treatment. The transcription factor NF-ΚB is activated by TNF signaling, and an NF-ΚB consensus site is present in the KCC1ex1b promoter region. We assayed the effect of co-expressing NF-ΚB and our KCC1ex1b promoter constructs in K562 cells. NF-ΚB expression produced an 8-fold decrease in luciferase activity from these promoter constructs indicating NF-ΚB transcriptionally represses this promoter, either directly or indirectly. Our current model proposes that induction or modulation of the expression of the KCC1ex1bvariant could be an important factor in the control of red cell hydration.

scholarly journals PP2A Regulates HDAC4 Nuclear Import

2008 ◽  
Vol 19 (2) ◽  
pp. 655-667 ◽  
Author(s):  
Gabriela Paroni ◽  
Nadia Cernotta ◽  
Claudio Dello Russo ◽  
Paola Gallinari ◽  
Michele Pallaoro ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Histone Deacetylases ◽  
Phosphorylation Site ◽  
Class Ii ◽  
Binding Studies ◽  
Nuclear Entry ◽  
Putative Phosphorylation Site

Different signal-regulated serine/threonine kinases phosphorylate class II histone deacetylases (HDACs) to promote nuclear export, cytosolic accumulation, and activation of gene transcription. However, little is known about mechanisms operating in the opposite direction, which, possibly through phosphatases, should promote class II HDACs nuclear entry and subsequent gene repression. Here we show that HDAC4 forms a complex with the PP2A holoenzyme Cα, Aα, B/PR55α. In vitro and in vivo binding studies demonstrate that the N-terminus of HDAC4 interacts with the catalytic subunit of PP2A. HDAC4 is dephosphorylated by PP2A and experiments using okadaic acid or RNA interference have revealed that PP2A controls HDAC4 nuclear import. Moreover, we identified serine 298 as a putative phosphorylation site important for HDAC4 nuclear import. The HDAC4 mutant mimicking phosphorylation of serine 298 is defective in nuclear import. Mutation of serine 298 to alanine partially rescues the defect in HDAC4 nuclear import observed in cells with down-regulated PP2A. These observations suggest that PP2A, via the dephosphorylation of multiple serines including the 14-3-3 binding sites and serine 298, controls HDAC4 nuclear import.

scholarly journals Gene Expression Analysis of the Streptococcus pneumoniae Competence Regulons by Use of DNA Microarrays

2000 ◽  
Vol 182 (21) ◽  
pp. 6192-6202 ◽  
Author(s):  
Scott Peterson ◽  
Robin T. Cline ◽  
Hervé Tettelin ◽  
Vasily Sharov ◽  
Donald A. Morrison
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Microarrays ◽  
Target Genes ◽  
Expression Patterns ◽  
In Vitro Models ◽  
Regulatory Genes ◽  
Mrna Levels ◽  
Similar Expression Pattern ◽  
Genes Encoding

ABSTRACT Competence for genetic transformation in Streptococcus pneumoniae is coordinated by the competence-stimulating peptide (CSP), which induces a sudden and transient appearance of competence during exponential growth in vitro. Models of this quorum-sensing mechanism have proposed sequential expression of several regulatory genes followed by induction of target genes encoding DNA-processing-pathway proteins. Although many genes required for transformation are known to be expressed only in response to CSP, the relative timing of their expression has not been established. Overlapping expression patterns for the genes cinA andcomD (G. Alloing, B. Martin, C. Granadel, and J. P. Claverys, Mol. Microbiol. 29:75–83, 1998) suggest that at least two distinct regulatory mechanisms may underlie the competence cycle. DNA microarrays were used to estimate mRNA levels for all known competence operons during induction of competence by CSP. The known competence regulatory operons, comAB, comCDE, andcomX, exhibited a low or zero initial (uninduced) signal, strongly increased expression during the period between 5 and 12 min after CSP addition, and a decrease nearly to original values by 15 min after initiation of exposure to CSP. The remaining competence genes displayed a similar expression pattern, but with an additional delay of approximately 5 min. In a mutant defective in ComX, which may act as an alternate sigma factor to allow expression of the target competence genes, the same regulatory genes were induced, but the other competence genes were not. Finally, examination of the expression of 60 candidate sites not previously associated with competence identified eight additional loci that could be induced by CSP.

scholarly journals Thyroid hormone receptor β2 is strongly up-regulated at all levels of the hypothalamo–pituitary–thyroidal axis during late embryogenesis in chicken

2007 ◽  
Vol 196 (3) ◽  
pp. 519-528 ◽  
Author(s):  
Sylvia V H Grommen ◽  
Lutgarde Arckens ◽  
Tim Theuwissen ◽  
Veerle M Darras ◽  
Bert De Groef
Keyword(s):  
Thyroid Hormone ◽  
Mrna Expression ◽  
Thyroid Hormone Receptor ◽  
Expression Patterns ◽  
Perinatal Period ◽  
Mrna Levels ◽  
Late Embryogenesis ◽  
Mrna Content ◽  
Hpt Axis

In this study, we tried to elucidate the changes in thyroid hormone (TH) receptor β2 (TRβ2) expression at the different levels of the hypothalamo–pituitary–thyroidal (HPT) axis during the last week of chicken embryonic development and hatching, a period characterized by an augmented activity of the HPT axis. We quantified TRβ2 mRNA in retina, pineal gland, and the major control levels of the HPT axis – brain, pituitary, and thyroid gland – at day 18 of incubation, and found the most abundant mRNA content in retina and pituitary. Thyroidal TRβ2 mRNA content increased dramatically between embryonic day 14 and 1 day post-hatch. In pituitary and hypothalamus, TRβ2 mRNA expression rose gradually, in parallel with increases in plasma thyroxine concentrations. Using in situ hybridization, we have demonstrated the presence of TRβ2 mRNA throughout the diencephalon and confirmed the elevation in TRβ2 mRNA expression in the hypophyseal thyrotropes. In vitro incubation with THs caused a down-regulation of TRβ2 mRNA levels in embryonic but not in post-hatch pituitaries. The observed expression patterns in pituitary and diencephalon may point to substantial changes in TRβ2-mediated TH feedback active during the perinatal period. The strong rise in thyroidal TRβ2 mRNA content could be indicative of an augmented modulation of thyroid development and/or function by THs toward and after hatching. Finally, THs proved to exert an age-dependent effect on pituitary TRβ2 mRNA expression.

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