Anoxia-induced transcriptional upregulation of sarp-19: cloning and characterization of a novel EF-hand containing gene expressed in hepatopancreas of Littorina littorea

2004 ◽  
Vol 82 (2) ◽  
pp. 285-293 ◽  
Author(s):  
Kevin Larade ◽  
Kenneth B Storey
Keyword(s):  
Time Course ◽  
Signal Sequence ◽  
Calcium Ionophore ◽  
Fold Increase ◽  
Feedback Mechanism ◽  
Littorina Littorea ◽  
Transcript Levels ◽  
Reading Frame ◽  
Ef Hand

Many marine molluscs have well-developed biochemical adaptations that allow them to live without oxygen for long periods of time, but very little is currently known about the molecular biology underlying these processes. Differential screening of a cDNA library derived from the hepatopancreas of the marine snail Littorina littorea revealed a novel anoxia-induced gene, sarp-19 (snail anoxia-responsive protein, 19 kDa). Examination of the sarp-19 transcript revealed an open reading frame that encoded a protein of 168 amino acids containing an N-terminal signal sequence and two putative EF-hand domains. Expression analysis of transcript levels established that sarp-19 accumulated over a time course of anoxia exposure, reaching a maximum 5.6-fold increase after 96 h compared with aerobic controls. However, transcript levels were reduced by 50% within 1 h when aerobic conditions were reestablished. Nuclear runoff assays confirmed transcriptional upregulation of sarp-19 during anoxia exposure, and organ explant experiments showed that the gene was also responsive to anoxia exposure in vitro. sarp-19 transcripts were also elevated in response to freezing, suggesting that the protein may have a role in the physiological responses of this intertidal snail to both aerial exposure and winter freezing. Hepatopancreas explants treated with a calcium ionophore showed increased levels of the sarp-19 transcript, suggesting a possible feedback mechanism regulated by levels of intracellular calcium. Expression was also responsive to tissue incubation with cyclic GMP and phorbol 12-myristate 13-acetate but was not affected by cyclic AMP, implicating involvement of protein kinases G and C but not protein kinase A in the expression of sarp-19. The SARP-19 protein may play a role in calcium-activated signaling during anoxia exposure in L. littorea.Key words: anoxia, mollusc, gastropod, calcium, EF hand.

Developmental regulation of a novel repetitive protein of Trypanosoma brucei

1987 ◽  
Vol 7 (8) ◽  
pp. 2838-2844
Author(s):  
M R Mowatt ◽  
C E Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Tandem Repeats ◽  
Signal Sequence ◽  
Developmental Regulation ◽  
Developmentally Regulated ◽  
Hydrophobic Domain ◽  
Reading Frame ◽  
Amino Terminal ◽  
Membrane Bound

Trypanosoma brucei undergoes many morphological and biochemical changes during transformation from the bloodstream trypomastigote to the insect procyclic trypomastigote form. We cloned and determined the complete nucleotide sequence of a developmentally regulated cDNA. The corresponding mRNA was abundant in in vitro-cultivated procyclics but absent in bloodstream forms. The trypanosome genome contains eight genes homologous to this cDNA, arranged as four unlinked pairs of tandem repeats. The longest open reading frame of the cDNA predicts a protein of 15 kilodaltons, the central portion of which consists of 29 tandem glutamate-proline dipeptides. The repetitive region is preceded by an amino-terminal signal sequence and followed by a hydrophobic domain that could serve as a membrane anchor; the mRNA was found on membrane-bound polyribosomes. These results suggest that the protein is membrane associated.

A preprogalanin cDNA from the turtle pituitary and regulation of its gene expression

2007 ◽  
Vol 292 (4) ◽  
pp. R1649-R1656 ◽  
Author(s):  
John Yuh-Lin Yu ◽  
Chin-Hon Pon ◽  
Hui-Chen Ku ◽  
Chih-Ting Wang ◽  
Yung-Hsi Kao
Keyword(s):  
Mrna Expression ◽  
Untranslated Region ◽  
Signal Sequence ◽  
Biological Effects ◽  
In Vitro Study ◽  
The Body ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame

Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle ( Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5′-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3′-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH2 terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly1 and Thr29 substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48–81% and 76–96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17β-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.

In Vitro Analyses of Umbilical Cord Blood (UCB) Derived Monocytes Support Adjunct Topical Application to Augment Platelet Rich Plasma (PRP) in Wound Healing.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1263-1263 ◽  
Author(s):  
Graham C. Chapman ◽  
Nicholas Greco ◽  
Richard Patrick Weitzel ◽  
Phil Paul ◽  
Peter Haviernik ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cell ◽  
Topical Application ◽  
Time Course ◽  
Basal Medium ◽  
Fold Increase ◽  
Conditioned Media ◽  
Tubule Formation ◽  
Cell Networks

Abstract Currently, PRP is used clinically as a topical application to augment healing of both surgical and chronic, non-healing wounds. PRP exerts its efficacy by releasing growth factors that enhance clot formation and vasculogenesis. We conducted in vitro functional analyses comparing PRP and/or UCB-derived monocytes including cytokine production, cell migration, and HUVEC tubule formation in standard matrigel assays to test the hypothesis whether topical concurrent application of PRP and UCB-derived monocytes may serve to augment wound healing beyond the ability of topical PRP alone. UCB was obtained according to institutional guidelines and collected into bags with citrate dextrose (Allegiance). MNC were separated on a Histopaque-1077 (Sigma) density gradient. UCB CD14+ monocytes were isolated using AutoMACS magnetic cell sorter (Miltenyi), and cultured in RPMI with 1% HSA. PRP was isolated from adult peripheral blood by centrifugation. To determine if the addition of UCB monocytes may improve the wound healing effects of PRP alone, VEGF, bFGF, and PDGF secreted by monocytes alone, PRP alone, and monocytes supplemented with 3% PRP, were measured by ELISA (RayBiotech) daily over 4 days. PRP alone elicited no measurable secretion of VEGF. UCB-derived monocytes alone showed a low, constant production of VEGF over the four days of 0.868ng/ml. PRP supplemented with UCB-derived monocytes secreted VEGF at a 7.6-fold increase over either PRP or UCB monocytes alone, with a peak production at day three of 6.638ng/ml. PRP alone produced no measurable secretion of bFGF over the four day time course. UCB monocytes alone secreted bFGF in an increasing manner during the same time course. During days one to four, bFGF secreted by UCB monocytes was 33.8, 27.9, 115.4, and 452.1pg/ml, respectively. The presence of PRP suppressed this secretion, as PRP combined with UCB monocytes constantly secreted bFGF at an average of 39.9pg/ml throughout days one to four. Finally, secretion of PDGF was highest in conditions including PRP combined with UCB monocytes. PRP alone constantly produced PDGF at an average of 3,144pg/ml over a 4 day time course. Monocytes alone secreted PDGF constantly at a lower average of 597pg/ml. PRP supplemented with UCB monocytes secreted PDGF at a concentration 5.9-fold higher than PRP alone, producing an average of 18,534pg/ml over four days. To determine whether UCB-derived monocytes respond to cytokines elicited by injured vascular endothelial cells, we measured UCB-derived monocyte chemotaxis to HUVEC conditioned media in hypoxic conditions (5% O2). Migration experiments were conducted using Transwell plates with 8.0 μm pores. Monocytes were cultured in RPMI with 5% FBS at a concentration of 5×106/ml and were allowed to migrate for four hours to either: media alone, PRP, HUVEC-conditioned media, or HUVEC-conditioned media supplemented with PRP. We observed a 3.3 fold increase in the migration of the monocytes to HUVECconditioned media over that of basal media. Experiments with PRP alone showed no significant difference in monocyte migration compared to basal medium. To determine whether UCB-derived monocytes may serve to augment endothelial cell function beyond that elicited by PRP alone, matrigel experiments were conducted by adding HUVEC in endothelial cell basal medium. HUVEC tubule formation in matrigel in basal media was compared in three conditions including media conditioned with: 1) PRP alone, 2) UCB monocytes alone, or 3) a combination of PRP + UCB monocytes. We compared the kinetics and stability of enclosed endothelial cell networks formed by HUVEC. No significant benefit was seen with addition of PRP conditioned media. The number of enclosed endothelial cell networks reached a higher maximum with the addition of monocyte conditioned media (137 networks) as well as PRP + monocyte conditioned media (142 networks), compared to non-conditioned media (80 networks). UCB monocyte and PRP + UCB monocyte conditioned media also improved the stability of the enclosed cell networks in culture as structures persisted beyond 24h, while none were present in the PRP-conditioned or non-conditioned media matrigel cultures. Figure Figure In summary, these in vitro analyses support the hypothesis that UCB-derived monocytes significantly improve efficacy of PRP alone in augmentation of vasculogenesis and cell migration to vascular endothelial injury, thereby supporting potential concurrent topical application of UCB-derived monocytes to PRP in wound healing.

scholarly journals Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study

2000 ◽  
Vol 350 (3) ◽  
pp. 723-734 ◽  
Author(s):  
Christine LACABARATZ-PORRET ◽  
Sophie LAUNAY ◽  
Elisabeth CORVAZIER ◽  
Raymonde BREDOUX ◽  
Béla PAPP ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Western Blotting ◽  
Time Course ◽  
In Vitro Study ◽  
Fold Increase ◽  
Distinct Pattern ◽  
Platelet Glycoprotein ◽  
Protein Patterns ◽  
Fold Induction

The endoplasmic reticulum (ER) plays a key role in Ca2+ signalling through Ca2+ release via inositol 1,4,5-trisphosphate receptors (InsP3-Rs) and Ca2+ uptake by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs). Here, we investigated the organization of platelet ER and its biogenesis during megakaryocytopoiesis. First, erythro/megakaryoblastic MEG 01, UT7, M-O7e and CHRF 288-11 cell lines, platelets and thrombopoietin-induced UT7-Mpl cells were selected for the study of SERCA2b and SERCA3 proteins by Western blotting using the antibodies IID8 and PL/IM430, respectively. As judged by platelet glycoprotein IIIa (GPIIIa) expression, an increase in SERCA3 proteins was observed while that of SERCA2b remained unchanged throughout maturation. Second, these studies were extended to the newly described alternatively spliced SERCA3a–c RNAs and InsP3-Rs using the in vitro model of PMA-induced differentiation of MEG 01 cells. Time-course and dose–response studies showed a maximal approx. 4-fold up-regulation of SERCA3 proteins using 10-8 M PMA for 3 days, which paralleled induction of GPIIIa expression. SERCA3 induction was found to occur at the level of mRNA. The modulation of the different SERCA3 species (i.e. 3a, 3b and 3c) was isoform-specific: while SERCA3a was slightly increased, an approx. 3-fold induction of SERCA3b, and a 4-fold induction of SERCA3c, was observed after 24h of PMA treatment. Isoform-specific Western blotting and/or reverse transcriptase PCR studies showed that InsP3-R types I, II and III are expressed in MEG 01 cells, as well as in platelets. Study of the expression of these InsP3-R types in PMA-induced MEG 01 cells revealed that: (i) InsP3-RI protein and mRNA showed no changes; (ii) InsP3-RII mRNA was up-regulated and peaked at hour 48 and (iii) InsP3-RIII mRNA and protein showed a transitory maximal 3- and 2.3-fold increase at hours 6 and 30, respectively. Upon PMA treatment of CHRF 288-11 cells, in which GPIIIa is not induced upon treatment, a similar pattern of regulation of InsP3-R types II and III was seen, but a distinct pattern of SERCA3 regulation was observed. These results suggest a profound reorganization of ER-protein patterns during megakaryocytopoiesis and underline the role of SERCA3 gene regulation in the control of Ca2+-dependent platelet functions.

A human intracellular apyrase-like protein, LALP70, localizes to lysosomal/autophagic vacuoles

1999 ◽  
Vol 112 (15) ◽  
pp. 2473-2484 ◽  
Author(s):  
A. Biederbick ◽  
S. Rose ◽  
H.P. Elsasser
Keyword(s):  
Fluorescent Protein ◽  
Signal Sequence ◽  
In Vitro Transcription ◽  
Autophagic Vacuole ◽  
Reading Frame ◽  
Vacuole Membrane ◽  
Conserved Regions ◽  
Punctate Pattern ◽  
Green Fluorescent

Using antibodies against autophagic vacuole membrane proteins we identified a human cDNA with an open reading frame of 1848 bp, encoding a protein of 70 kDa, which we named lysosomal apyrase-like protein of 70 kDa (LALP70). Sequence analysis revealed that LALP70 belongs to the apyrase or GDA1/CD39 family and is almost identical to a human uridine diphosphatase, with the exception of nine extra amino acids in LALP70. Members of this family were originally described as ectoenzymes, with some intracellular exceptions. Transfected LALP70 fused to the green fluorescent protein localized in the cytoplasm with a punctate pattern in the perinuclear space. These structures colocalized with the autophagic marker monodansylcadaverine and the lysosomal protein lamp1. Hydrophobicity analysis of the encoded protein revealed a transmembrane region at the N and C termini. Most of the sequence is arranged between these transmembrane domains, and contains four apyrase conserved regions. In vitro transcription/translation in the presence of microsomes showed that no signal sequence is cleaved off and that the translation product is protected from trypsin treatment. Our data indicate that LALP70 is a type III lysosomal/autophagic vacuole membrane protein with the apyrase conserved regions facing the luminal space of the vacuoles.

scholarly journals Arachidonic acid metabolism in TNS-induced chronic and immunologic enteritis in rats, and the effect of 5-ASA

1993 ◽  
Vol 2 (5) ◽  
pp. 391-395 ◽  
Author(s):  
F. J. Zijlstra ◽  
A. P. M. van Dijk ◽  
N. Selve ◽  
J. H. P. Wilson
Keyword(s):  
Arachidonic Acid ◽  
Time Course ◽  
Fold Increase ◽  
Arachidonic Acid Metabolism ◽  
Initial Increase ◽  
Aminosalicylic Acid ◽  
Metabolite Formation ◽  
Distal Intestine ◽  
Eicosanoid Formation

Inflammation of the rat distal intestine was induced by intradermal sensitization and subsequent multiple intrajejunal challenge with the hapten 2,4,6-trinitrobenzenesulphonic acid (TNBS) via an implanted catheter. The time course of the inflammatory reaction was followed by determination of the enteritis score and measurement ofin vitroeicosanoid formation of homogenates of the gut after 0, 1, 2, 4, 7, 14 and 21 days of local daily challenge with 0.08% TNBS. There was a small initial increase of eicosanoid formation, reached at days 1 and 2, followed by a significant increase in metabolism of arachidonic acid on day 21. Although at day 1 a four-fold increase in inflammation score was reached, no further significant changes were observed during the following 3 weeks. The greatest increase in metabolite formation was observed in prostanoids TxB2, PGE2. and PGD2and the 5-lipoxygenase product LTC4, whereas minor changes were found for LTB4and other lipoxygenase products such as 12- and 15-HETE. The formation of these metabolites was already inhibited by low-dose 5-aminosalicylic acid (5-ASA), given orally twice daily during the 3 weeks challenge period, while the enteritis score was affected dosedependently.

scholarly journals Characterisation and preliminary functional analysis of N-acetyltransferase 13 from Schistosoma japonicum

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yalan Tang ◽  
Kerou Zhou ◽  
Qingqing Guo ◽  
Cheng Chen ◽  
Jing Jia ◽  
...  
Keyword(s):  
Schistosoma Japonicum ◽  
Egg Production ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Female Adult ◽  
Male Adult ◽  
Transcript Levels ◽  
Reading Frame ◽  
Interfering Rna

Abstract Background N-acetyltransferase 13 (NAT13) is a probable catalytic component of the ARD1A-NARG1 complex possessing alpha (N-terminal) acetyltransferase activity. Results In this study, a full-length complementary DNA (cDNA) encoding Schistosoma japonicum NAT13 (SjNAT13) was isolated from schistosome cDNAs. The 621 bp open reading frame of SjNAT13 encodes a polypeptide of 206 amino acids. Real-time PCR analysis revealed SjNAT13 expression in all tested developmental stages. Transcript levels were highest in cercariae and 21-day-old worms, and higher in male adult worms than female adult worms. The rSjNAT13 protein induced high levels of anti-rSjNAT13 IgG antibodies. In two independent immunoprotection trials, rSjNAT13 induced 24.23% and 24.47% reductions in the numbers of eggs in liver. RNA interference (RNAi) results showed that small interfering RNA (siRNA) Sj-514 significantly reduced SjNAT13 transcript levels in worms and decreased egg production in vitro. Conclusions Thus, rSjNAT13 might play an important role in the development and reproduction of schistosomes.

scholarly journals Developmental regulation of a novel repetitive protein of Trypanosoma brucei.

1987 ◽  
Vol 7 (8) ◽  
pp. 2838-2844 ◽  
Author(s):  
M R Mowatt ◽  
C E Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Tandem Repeats ◽  
Signal Sequence ◽  
Developmental Regulation ◽  
Developmentally Regulated ◽  
Hydrophobic Domain ◽  
Reading Frame ◽  
Amino Terminal ◽  
Membrane Bound

Trypanosoma brucei undergoes many morphological and biochemical changes during transformation from the bloodstream trypomastigote to the insect procyclic trypomastigote form. We cloned and determined the complete nucleotide sequence of a developmentally regulated cDNA. The corresponding mRNA was abundant in in vitro-cultivated procyclics but absent in bloodstream forms. The trypanosome genome contains eight genes homologous to this cDNA, arranged as four unlinked pairs of tandem repeats. The longest open reading frame of the cDNA predicts a protein of 15 kilodaltons, the central portion of which consists of 29 tandem glutamate-proline dipeptides. The repetitive region is preceded by an amino-terminal signal sequence and followed by a hydrophobic domain that could serve as a membrane anchor; the mRNA was found on membrane-bound polyribosomes. These results suggest that the protein is membrane associated.

scholarly journals Cloning of an intracellular protein that binds selectively to mitogenic acidic fibroblast growth factor

1998 ◽  
Vol 336 (1) ◽  
pp. 213-222 ◽  
Author(s):  
Elona KOLPAKOVA ◽  
Antoni WIĘDŁOCHA ◽  
Harald STENMARK ◽  
Olav KLINGENBERG ◽  
Pål Ø. FALNES ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
High Efficiency ◽  
Signal Sequence ◽  
Binding Protein ◽  
Fibroblast Growth ◽  
Acidic Fibroblast Growth Factor ◽  
Reading Frame ◽  
Intracellular Binding

In addition to its extracellular action, there is evidence that acidic fibroblast growth factor (aFGF) acts inside cells. To identify intracellular proteins interacting with aFGF, we screened a HeLa cell library in the yeast two-hybrid system using pLex-aFGF as a bait. A clone binding to aFGF, but not to the non-mitogenic mutant aFGF-K132E, was isolated and characterized. The insert contained an open reading frame corresponding to a novel protein of 42 kDa. The protein, termed aFGF intracellular binding protein (FIBP), is mainly hydrophilic and does not contain an N-terminal signal sequence. In vitro-translated FIBP bound specifically to a fusion protein of maltose-binding protein and aFGF. FIBP became post-translationally associated with microsomes added to the cell-free protein synthesizing system, and the membrane-associated protein bound aFGF with high efficiency. Immunoblots and fluorescence microscopy demonstrated that the protein is present in nuclei and, to a lesser extent, associated with mitochondria and other cytoplasmic membranes. The possibility is discussed that FIBP may be involved in the mitogenic action of aFGF. The nucleotide sequences described in this paper have been submitted to GeneBank database under accession numbers AF010187 (human FIBP) and AF010188 (simian FIBP).

Regulation of guinea pig heart phosphorylase kinase by cAMP, protein kinase, and calcium

1981 ◽  
Vol 240 (3) ◽  
pp. E340-E349 ◽  
Author(s):  
J. S. Hayes ◽  
S. E. Mayer
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Time Course ◽  
Activity Ratio ◽  
Fold Increase ◽  
Maximal Activity ◽  
Phosphorylase Kinase ◽  
Adrenergic Stimulation ◽  
Guinea Pig Heart

In skeletal muscle the activation of phosphorylase kinase (PK) associated with phosphorylation of the enzyme can be measured as an increase in the pH 6.8:8.2 activity ratio. Phosphorylation leads to a 20- to 30-fold increase in PK activity (PKA) at pH 6.8 and a large decrease in the Km for phosphorylase. Perfused guinea pig hearts exposed to isoproterenol (0.3 microM) showed an increase in PKA, but without an increase in the pH 6.8:8.2 activity ratio. In a 10-fold dilution of guinea pig heart cytosol exposed to cAMP + methylisobutylxanthine, PKA was stimulated twofold at pH 7.5. Addition of exogenous protein kinase stimulated PKA fourfold. Both methods of activation were reversible and were blocked by the heat-stable inhibitor of protein kinase. Guinea pig heart PK was Ca2+-dependent requiring 0.6 microM Ca2+ for half-maximal activity. Kinetic studies indicate that the Km of guinea pig heart PK for phosphorylase b at pH 6.8 is not markedly reduced after in vitro activation (35%). This could explain the observed lack of increase in the pH 6.8:8.2 activity ratio after exposure of hearts to isoproterenol. The time course for the activation of inotropic state and the glycogenolytic pathway in perfused guinea pig hearts by isoproterenol showed that these processes were maximally activated within 25 s. However, PK remained activated for 2 min, long after the other biochemical and physiological parameters had returned to control values. These data suggest that Ca2+, not phosphorylation state, is important in regulating the return of dP/dt to control levels after beta-adrenergic stimulation.

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