scholarly journals A novel prespore-cell-inducing factor in Dictyostelium discoideum induces cell division of prespore cells

Development ◽  
1997 ◽  
Vol 124 (14) ◽  
pp. 2781-2787 ◽  
Author(s):  
A.A. Oohata ◽  
M. Nakagawa ◽  
M. Tasaka ◽  
S. Fujii
Keyword(s):  
Cell Division ◽  
Cell Differentiation ◽  
Dictyostelium Discoideum ◽  
Gel Filtration ◽  
Relative Molecular Mass ◽  
Heat Stable ◽  
Prespore Cell ◽  
Sds Page ◽  
Cell Induction ◽  
Low Cell Density

In Dictyostelium discoideum strain V12M2, at a very low cell density (approximately 10(2) cells/cm2), most amoebae differentiate into prespore cells in a salt solution containing cAMP if an adequately diluted conditioned medium (CM) is provided (Oohata, A. A. (1995) Differentiation 59, 283–288). This finding suggests the presence of factor(s) released into the medium that are involved in inducing prespore cell differentiation. In the present study, we report the presence of two types of factors that function synergistically in prespore cell induction; one is a heat-stable and dialysable factor(s) and the other is a heat-labile and non-dialysable factor termed psi (psi) factor (prespore-inducing factor). We purified and characterized the psi factor. Its relative molecular mass was determined to be 106x10(3) Mr by SDS-PAGE and 180x10(3) Mr by gel filtration HPLC, respectively. These results indicate that psi factor exists as a dimer under native conditions. In addition to inducing prespore cell differentiation, psi factor induced cell division of prespore cells in submerged culture. Our results suggest that psi factor plays important roles not only in prespore cell differentiation but also in the progress of the cell cycle in the prespore pathway in normal development.

scholarly journals A prespore-cell-inducing factor in Dictyostelium discoideum: its purification and characterization

1999 ◽  
Vol 343 (1) ◽  
pp. 265-271 ◽  
Author(s):  
Manabu NAKAGAWA ◽  
Akiko A. OOHATA ◽  
Hiromasa TOJO ◽  
Shigeru FUJII
Keyword(s):  
Cell Division ◽  
Cell Differentiation ◽  
Amino Acid ◽  
Conditioned Medium ◽  
Dictyostelium Discoideum ◽  
Periodic Acid ◽  
Limited Proteolysis ◽  
Amino Acid Sequences ◽  
Homology Search ◽  
Prespore Cell

Under starvation conditions, amoebae of Dictyostelium discoideum aggregate to form multicellular masses; the aggregates are then initiated to differentiate. We have reported previously that a signal substance exists in conditioned medium of D. discoideum, and we named it prespore-cell-inducing factor (psi, Ψ factor) [Oohata, Nakagawa, Tasaka, and Fujii (1997) Development 124, 2781-2787]. The factor can induce isolated amoebae to differentiate into prespore cells. Moreover, we suggested that it caused not only cell differentiation but also cell division. In the present study, we have purified Ψ factor from the conditioned medium and characterized it. The purified Ψ factor induced both prespore cell differentiation and cell division of prespore cells. Its apparent molecular mass was 180 kDa by gel filtration and 106 kDa by SDS/PAGE. Based on these results, Ψ factor exists as a dimer in normal conditions. Periodic acid/Schiff staining showed that Ψ factor was a glycoprotein. It was ascertained by Edman degradation that Ψ factor is blocked at the N-terminal. Treatment with pyroglutamate aminopeptidase removed the N-terminal block and allowed determination of the amino-acid sequence of Ψ factor. Moreover, three internal amino-acid sequences were determined in limited proteolysis experiments using trypsin and endoproteinase Lys-C. The homology search for these sequences supports the fact that Ψ factor is a novel differentiation factor.

The role of diffusible molecules in regulating the cellular differentiation of Dictyostelium discoideum

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 1-16 ◽  
Author(s):  
J.G. Williams
Keyword(s):  
Developmental Biology ◽  
Cell Differentiation ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Cellular Differentiation ◽  
Prespore Cell ◽  
Additional Role ◽  
Acting In Concert ◽  
Morphogenetic Fields

A central problem in developmental biology is to understand how morphogenetic fields are created and how they act to direct regionalized cellular differentiation. This goal is being pursued in organisms as diverse as moulds, worms, flies, frogs and mice. Each organism has evolved its own solution to the challenge of multicellularity but there appear to be common underlying principles and, once pattern formation is fully understood in any system, some general truths seem certain to be revealed. As a non-obligate metazoan, Dictyostelium discoideum has proven a particularly tractable system in which to identify and characterize cellular morphogens. Cyclic AMP and ammonia stimulate prespore cell differentiation and ammonia plays an additional role in repressing terminal cellular differentiation. Differentiation Inducing Factor (DIF) acts to direct prestalk cell differentiation and adenosine may play a synergistic role in repressing prespore cell differentiation. This review summarizes the evidence for these interactions and describes a number of models which show how this small repertoire of diffusible molecules, acting in concert, may direct the formation of a differentiated structure.

scholarly journals Purification and properties of porphobilinogen deaminase from Arabidopsis thaliana.

1994 ◽  
Vol 299 (3) ◽  
pp. 895-902 ◽  
Author(s):  
R M Jones ◽  
P M Jordan
Keyword(s):  
Arabidopsis Thaliana ◽  
Gel Filtration ◽  
Chain Termination ◽  
Porphobilinogen Deaminase ◽  
Ph Optimum ◽  
Heat Stable ◽  
Purification And Properties ◽  
Sds Page ◽  
Plant Arabidopsis Thaliana

Porphobilinogen deaminase (EC 4.3.1.8) has been purified to homogeneity (16,000-fold) from the plant Arabidopsis thaliana in yields of 8%. The deaminase is a monomer of M(r) 35,000, as shown by SDS/PAGE, and 31,000, using gel-filtration chromatography. The pure enzyme has a Vmax. of 4.5 mumol/h per mg and a Km of 17 +/- 4 microM. Determination of the pI and pH optimum revealed values of 5.2 and 8.0 respectively. The sequence of the N-terminus was found to be NH2-XVAVEQKTRTAI. The deaminase is heat-stable up to 70 degrees C and is inhibited by NH3 and hydroxylamine. The enzyme is inactivated by arginine-, histidine- and lysine-specific reagents. Incubation with the substrate analogue and suicide inhibitor, 2-bromoporphobilinogen, results in chain termination and in inactivation.

scholarly journals Molecular Properties of Three Soluble Invertase Forms from Lilium longiflorum Flower Buds

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 581e-581
Author(s):  
Anil P. Ranwala ◽  
William B. Miller
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Lilium Longiflorum ◽  
Acid Invertase ◽  
Single Band ◽  
Relative Molecular Mass ◽  
Wheat Coleoptile ◽  
Flower Buds ◽  
Con A ◽  
Sds Page

Three soluble invertase isoforms from Lilium longiflorum flower buds that had been separated by DEAE-Sephacel chromatography were purified to near homogeneity by further chromatography on hydroxylapetite, Con-A sepharose, phenyl agarose, and Sephacryl S-200 gel filtration. Nondenaturing polyacrylamide gel electrophoresis (PAGE) gave a single band in all three invertases that corresponded to a band of invertase activity in a duplicate gel. The SDS-PAGE of the purified invertase I resulted in a single band with apparent relative molecular mass of 78 kDa. Invertase II and III were resolved to a similar polypeptide pattern by SDS-PAGE with three bands of 54, 52, and 24 kDa. Antiserum of tomato acid invertase cross-reacted with all three invertase protein bands. Antiserum of wheat coleoptile acid invertase cross-reacted only with 54 and 52 kDa bands of invertase II and III but did not recognize invertase I protein. Con-A peroxidase was bound to invertase I protein and all three protein bands of invertase II and III, suggesting that all proteins were glycosylated. Invertase I protein could be completely deglycosylated by incubating with peptide-N-glycosidase F to result in a peptide of 75 kDa. Invertase II and III were partially deglycosylated by peptide-N-glycosidase F resulting proteins bands of 53, 51, 50, and 22 kDa.

scholarly journals Purification and Characterization of Carboxymethyl Cellulase from Bacillus sp. Isolated from a Paddy Field

2012 ◽  
Vol 61 (1) ◽  
pp. 51-55 ◽  
Author(s):  
PONNUSWAMY VIJAYARAGHAVAN ◽  
S.G. PRAKASH VINCENT
Keyword(s):  
Paddy Field ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Relative Molecular Mass ◽  
Bacillus Sp ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Ph Range

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

AB0144 STRATIFICATION OF PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME BY MEASURING B-CELL HEALTH

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1372-1373
Author(s):  
G. M. Verstappen ◽  
J. C. Tempany ◽  
H. Cheon ◽  
A. Farchione ◽  
S. Downie-Doyle ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Cell Differentiation ◽  
Research Support ◽  
Differentiation Capacity ◽  
Cell Responses ◽  
Healthy Donors

Background:Primary Sjögren’s syndrome (pSS) is a heterogeneous immune disorder with broad clinical phenotypes that can arise from a large number of genetic, hormonal, and environmental causes. B-cell hyperactivity is considered to be a pathogenic hallmark of pSS. However, whether B-cell hyperactivity in pSS patients is a result of polygenic, B cell-intrinsic factors, extrinsic factors, or both, is unclear. Despite controversies about the efficacy of rituximab, new B-cell targeting therapies are under investigation with promising early results. However, for such therapies to be successful, the etiology of B-cell hyperactivity in pSS needs to be clarified at the individual patient level.Objectives:To measure naïve B-cell function in pSS patients and healthy donors using quantitative immunology.Methods:We have developed standardised, quantitative functional assays of B-cell responses that measure division, death, differentiation and isotype switching, to reveal the innate programming of B cells in response to T-independent and dependent stimuli. This novel pipeline to measure B-cell health was developed to reveal the sum total of polygenic defects and underlying B-cell dysfunction at an individual level. For the current study, 25 pSS patients, fulfilling 2016 ACR-EULAR criteria, and 15 age-and gender-matched healthy donors were recruited. Standardized quantitative assays were used to directly measure B cell division, death and differentiation in response to T cell-independent (anti-Ig + CpG) and T-cell dependent (CD40L + IL-21) stimuli. Naïve B cells (IgD+CD27-) were sorted from peripheral blood mononuclear cells and were labeled with Cell Trace Violet at day 0 to track cell division until day 6. B cell differentiation was measured at day 5.Results:Application of our standardized assays, and accompanying parametric models, allowed us to study B cell-intrinsic defects in pSS patients to a range of stimuli. Strikingly, we demonstrated a hyperresponse of naïve B cells to combined B cell receptor (BCR) and Toll-like receptor (TLR)-9 stimulation in pSS patients. This hyperresponse was revealed by an increased mean division number (MDN) at day 5 in pSS patients compared with healthy donors (p=0.021). A higher MDN in pSS patients was observed at the cohort level and was likely attributed to an increased division burst (division destiny) time. The MDN upon BCR/TLR-9 stimulation correlated with serum IgG levels (rs=0.52; p=0.011). No difference in MDN of naïve B cells after T cell-dependent stimulation was observed between pSS patients and healthy donors. B cell differentiation capacity (e.g., plasmablast formation and isotype switching) after T cell-dependent stimulation was also assessed. At the cohort level, no difference in differentiation capacity between groups was observed, although some pSS patients showed higher plasmablast frequencies than healthy donors.Conclusion:Here, we demonstrate defects in B-cell responses both at the cohort level, as well as individual signatures of defective responses. Personalized profiles of B cell health in pSS patients reveal a group of hyperresponsive patients, specifically to combined BCR/TLR stimulation. These patients may benefit most from B-cell targeted therapies. Future studies will address whether profiles of B cell health might serve additional roles, such as prediction of disease trajectories, and thus accelerate early intervention and access to precision therapies.Disclosure of Interests:Gwenny M. Verstappen: None declared, Jessica Catherine Tempany: None declared, HoChan Cheon: None declared, Anthony Farchione: None declared, Sarah Downie-Doyle: None declared, Maureen Rischmueller Consultant of: Abbvie, Bristol-Meyer-Squibb, Celgene, Glaxo Smith Kline, Hospira, Janssen Cilag, MSD, Novartis, Pfizer, Roche, Sanofi, UCB, Ken R. Duffy: None declared, Frans G.M. Kroese Grant/research support from: Unrestricted grant from Bristol-Myers Squibb, Consultant of: Consultant for Bristol-Myers Squibb, Speakers bureau: Speaker for Bristol-Myers Squibb, Roche and Janssen-Cilag, Hendrika Bootsma Grant/research support from: Unrestricted grants from Bristol-Myers Squibb and Roche, Consultant of: Consultant for Bristol-Myers Squibb, Roche, Novartis, Medimmune, Union Chimique Belge, Speakers bureau: Speaker for Bristol-Myers Squibb and Novartis., Philip D. Hodgkin Grant/research support from: Medimmune, Vanessa L. Bryant Grant/research support from: CSL

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