Deoxyribonucleotide Synthesis in Phycovirus-Infected Green Algae. A New Virus-Induced Ribonucleotide Reductase

1993 ◽  
Vol 48 (1-2) ◽  
pp. 113-118 ◽  
Author(s):  
Claus Bornemann ◽  
Hartmut Follmann
Keyword(s):  
Green Algae ◽  
Ribonucleotide Reductase ◽  
Fold Increase ◽  
Optimum Concentration ◽  
Characteristic Parameters ◽  
Temperature Optimum ◽  
Allosteric Effector ◽  
Cell Extracts ◽  
Key Enzyme

Infection of Chlorella-like green algae with freshwater phycoviruses is associated with a large and rapid demand for DNA precursors which cannot be met by the algal deoxyribonucleotide-synthesizing enzymes. We have demonstrated in these cells an up to ten-fold increase of the key enzyme, ribonucleotide reductase, 1-2 h post infection. The enzyme activity has been partially enriched from cell extracts. In vitro, it differs from that of uninfected algae in three characteristic parameters, viz. eight-fold higher resistance to millimolar hydroxyurea concentrations, much higher optimum concentration of an allosteric effector nucleotide, thymidine triphosphate, and an unusually low temperature optimum at 20 °C. We conclude that the large DNA phycoviruses, like Herpes and pox viruses, code for their own specific ribonucleotide reductase.

Bovine granulosa cells express extracellular matrix proteins and their regulators during luteinization in culture

1996 ◽  
Vol 8 (2) ◽  
pp. 259 ◽  
Author(s):  
Y Zhao ◽  
MR Luck
Keyword(s):  
Extracellular Matrix ◽  
Granulosa Cells ◽  
Culture Media ◽  
Gelatin Zymography ◽  
Fold Increase ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Subunit Gene ◽  
Gelatinase Activity ◽  
Cell Extracts

This study investigated the ability of bovine granulosa cells to express and secrete collagen, metalloproteinase (MMP) activity and a tissue inhibitor of metalloproteinase (TIMP-1) during luteinization in vitro. Cells from mature (1-2 mL fluid volume) bovine follicles were cultured over 4 days in serum-free medium. Their luteinization during culture was confirmed by a 10-fold increase in progesterone secretion. Samples of cell extracts, culture media and follicular fluid were subjected to Western blotting to identify secreted proteins and to gelatin zymography to detect enzyme activity. Poly A+ RNA, isolated from cells before and after culture, was probed to detect expression of collagen alpha 1(I), collagen alpha 3(IV) and TIMP-1. The results revealed that: (1) the collagen alpha 1(I) subunit gene was expressed in cells before culture but with greater intensity by Day 4 culture; collagen I protein, on the other hand, was not detectable in culture medium; (2) the collagen alpha 3(IV) subunit gene was expressed at a low level in uncultured cells and could be detected on Day 4 of culture; low amounts of the protein were detected in medium; (3) a 92-kDa band of gelatinase activity (presumed MMP-9) was present in all medium samples, together with bands of unidentified activity; and (5) the TIMP-1 gene was expressed in uncultured cells but its expression increased markedly up to Day 4 of culture. These results show that granulosa luteinization is associated with an increase in the expression of collagen, collagen-degrading enzymes and TIMP-1. Collagen protein, however, may be only poorly synthesized in this culture model. The results suggest that granulosa-derived cells are a likely source of components of the extracellular matrix during post-ovulatory remodelling of early luteal tissue.

scholarly journals Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

1993 ◽  
Vol 4 (1) ◽  
pp. 79-92 ◽  
Author(s):  
L Connell-Crowley ◽  
M J Solomon ◽  
N Wei ◽  
J W Harper
Keyword(s):  
Mammalian Cells ◽  
Specific Activity ◽  
Fold Increase ◽  
Cyclin A ◽  
Cyclin B ◽  
Activation Process ◽  
Cyclin Dependent Kinase ◽  
Cell Extracts

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.

scholarly journals Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
Chaoyu Tian ◽  
Jiangang Yang ◽  
Yan Zeng ◽  
Tong Zhang ◽  
Yingbiao Zhou ◽  
...  
Keyword(s):  
Reaction System ◽  
Fold Increase ◽  
Cascade Reactions ◽  
Cascade Reaction ◽  
Galactinol Synthase ◽  
Plant Extraction ◽  
Cell Extracts ◽  
Multienzyme System ◽  
Raffinose Synthase

ABSTRACT Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained. IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

scholarly journals Phosphorylation of CK1δ: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo

2007 ◽  
Vol 406 (3) ◽  
pp. 389-398 ◽  
Author(s):  
Georgios Giamas ◽  
Heidrun Hirner ◽  
Levani Shoshiashvili ◽  
Arnhild Grothey ◽  
Susanne Gessert ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Fold Increase ◽  
Regulatory Subunit ◽  
Kinetic Properties ◽  
Casein Kinase 1 ◽  
Cell Extracts ◽  
Anchoring Protein

The involvement of CK1 (casein kinase 1) δ in the regulation of multiple cellular processes implies a tight regulation of its activity on many different levels. At the protein level, reversible phosphorylation plays an important role in modulating the activity of CK1δ. In the present study, we show that PKA (cAMP-dependent protein kinase), Akt (protein kinase B), CLK2 (CDC-like kinase 2) and PKC (protein kinase C) α all phosphorylate CK1δ. PKA was identified as the major cellular CK1δCK (CK1δ C-terminal-targeted protein kinase) for the phosphorylation of CK1δ in vitro and in vivo. This was implied by the following evidence: PKA was detectable in the CK1δCK peak fraction of fractionated MiaPaCa-2 cell extracts, PKA shared nearly identical kinetic properties with those of CK1δCK, and both PKA and CK1δCK phosphorylated CK1δ at Ser370in vitro. Furthermore, phosphorylation of CK1δ by PKA decreased substrate phosphorylation of CK1δ in vitro. Mutation of Ser370 to alanine increased the phosphorylation affinity of CK1δ for β-casein and the GST (gluthatione S-transferase)–p53 1–64 fusion protein in vitro and enhanced the formation of an ectopic dorsal axis during Xenopus laevis development. Anchoring of PKA and CK1δ to centrosomes was mediated by AKAP (A-kinase-anchoring protein) 450. Interestingly, pre-incubation of MiaPaCa-2 cells with the synthetic peptide St-Ht31, which prevents binding between AKAP450 and the regulatory subunit RII of PKA, resulted in a 6-fold increase in the activity of CK1δ. In summary, we conclude that PKA phosphorylates CK1δ, predominantly at Ser370in vitro and in vivo, and that site-specific phosphorylation of CK1δ by PKA plays an important role in modulating CK1δ-dependent processes.

Abstract 483: Clot Degradation Assay for the Analysis of Vascular Bed Fibrinolytic Reactivity

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Amanda L Clark ◽  
Neil Kumar ◽  
Elisa Roztocil ◽  
David Gillespie ◽  
John P Cullen
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Fold Increase ◽  
Clot Formation ◽  
Pulmonary Vasculature ◽  
Pulmonary Emboli ◽  
Cell Extracts ◽  
Vascular Beds ◽  
Fibrin Clots

Introduction Pulmonary emboli cause significant morbidities and risk of mortality to affected patients, yet the mechanism behind their formation is still elusive. A greater understanding of clot formation and fibrinolysis in the pulmonary artery (PA) and iliac vein (IV) vascular beds has become an important area of research in order to find better therapeutics for the treatment of deep vein thrombi and pulmonary emboli. The aim of this study was to develop a novel in vitro clot degradation assay for the comparison of fibrinolytic reactivity of different vascular beds. Methods Thrombin was added to fibrinogen at 37 ° C for 24 hours and the resulting fibrin clot was centrifuged, dried and weighed. Human PA and IV endothelial cells were incubated for 24 hours and cellular extract and media samples were collected and evaluated for their ability to degrade fibrin clots in the presence of plasminogen. Fibrinolysis, stimulated by plasmin formed by endothelial cell derived plasminogen activator, was measured as percent clot degradation by weight. Results Fibrinogen and thrombin dose responses were completed to find the appropriate concentrations of each to use for clot formation: fibrinogen (6.04x10 -6 M) and thrombin (0.0005 NIH units). To validate the clot degradation assay, a uPA dose response was conducted from 150fM to 15nM uPA and demonstrated that each ten-fold increase in uPA concentration corresponded to an average of 14% degradation (p<0.05, n=3) up to 72.5% degradation in the presence of 15 nM uPA. While PA cell extracts showed no effect on clot degradation, IV cell extracts increased degradation by 30%. However, when clots were exposed to PA media there was a 14% increase in clot degradation (p<0.05, n=3), whereas the corresponding IV media samples had no effect on clot weight. Conclusions This study demonstrates the development of a novel and reproducible methodology to examine fibrinolytic reactivity in cell culture samples. Endothelial cells from the pulmonary artery circulation exhibit greater ability to extrinsically degrade fibrin clots when compared to endothelial cells from the iliac venous bed which may explain, in part, why more clots are found in the deep veins and not the pulmonary vasculature.

scholarly journals Characterization of translation systems in vitro from three developmental stages of Strongylocentrotus purpuratus

1989 ◽  
Vol 258 (2) ◽  
pp. 553-561 ◽  
Author(s):  
A C Lopo ◽  
C C Lashbrook ◽  
J W B Hershey
Keyword(s):  
Developmental Stages ◽  
Strongylocentrotus Purpuratus ◽  
Fold Increase ◽  
Translation Control ◽  
Ribonuclease Inhibitor ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Transit Times ◽  
Translation Systems

We have developed and characterized cell-free systems active in translation from unfertilized eggs, 30-min zygotes and hatched blastulae of the sea urchin Strongylocentrotus purpuratus. The ion concentrations selected for preparation of the lysates were 150 mM-K+, 40 mM-Na+, 40 mM-Cl-, 5 x 10(-7) M free Ca2+ and 1 mM free Mg2+. It was necessary to include the ribonuclease inhibitor RNas in the preparations to obtain full activity consistently. The pH optimum was 7.2 and was extremely sharp for the three S. purpuratus lysates. The temperature optima of the three lysates were remarkably similar to those of the intact unfertilized egg and embryos. Lysates from unfertilized egg and 30-min zygotes showed a temperature optimum at 15 degrees C. The hatched blastula lysate showed a broader temperature optimum with a shift to about 20 degrees C. The optimized lysates incorporated radiolabelled amino acids into polypeptides for up to 90 min. The polypeptides synthesized ranged in Mr from 200,000 to 20,000, suggesting that the mRNA in the lysates was intact and capable of directing the synthesis of complete polypeptides. Furthermore, the three lysates were capable of initiation, as demonstrated by inhibition of initiation using the inhibitors edeine and 7-methylguanosine 5′-triphosphate (m7GTP). At 15 degrees C, the transit times for the three lysates were: unfertilized egg, 40 min; 30-min zygotes and hatched blastula lysates, 20 min. These transit times are similar to those of intact eggs and embryos, and significantly, reflect the two-fold increase in elongation rate seen following fertilization in intact embryos. Thus, these lysates display many features and characteristic responses typical of intact eggs and embryos, indicating that the lysates should be useful tools for the analysis of translation control in early embryogenesis.

In vivo and in vitro effects of dexamethasone on pituitary thyrotrophin-releasing hormone-like peptide concentrations in the rat

1995 ◽  
Vol 145 (2) ◽  
pp. 333-341 ◽  
Author(s):  
K O Akinsanya ◽  
H Jamal ◽  
M A Ghatei ◽  
S R Bloom
Keyword(s):  
Anterior Pituitary ◽  
Chromatographic Analysis ◽  
Fold Increase ◽  
Pituitary Hormones ◽  
Exchange Chromatography ◽  
Pituitary Cells ◽  
Thyrotrophin Releasing Hormone ◽  
Cell Extracts

Abstract The novel peptide, pyroglutamyl-glutamyl-proline amide (pGlu-Glu-ProNH2; EEP), which has structural and immunological similarities to TRH (pGlu-His-ProNH2) has recently been shown to contribute to total TRH-like immunoreactivity (t-TRH-LI) detected in the rabbit prostate and rat and porcine anterior pituitary. In this study, the effects of dexamethasone (DEX) on rat pituitary TRH-like peptide levels in the rat were determined. TRH-like immunoreactivity (TRH-LI) was separated by ion exchange chromatography and detected by TRH RIA. Anion exchange chromatographic analysis suggested that EEP-like immunoreactivity (EEP-LI) accounted for 15·0 ± 1·2 pmol t-TRH-LI/g (70·4 ± 3·9%) in the control anterior pituitary with the remaining t-TRH-LI being due to TRH-LI. Following DEX treatment pituitary EEP-LI and TRH-LI increased by 200% and 400% (P<0·001) respectively, constituting a 2·5-fold increase in t-TRH-LI in the pituitary. TRH-LI now accounted for 45·7±5·3% of t-TRH-LI compared with 29·6 ±4·1% in the controls. TRH-LI, but not EEP-LI, was detected in the hypothalamus and posterior pituitary, suggesting that EEP-LI is synthesised within the anterior pituitary. DEX also caused a 2·6-fold rise (P<0·001) in t-TRH-LI in dispersed, cultured anterior pituitary cells. Chromatographic analysis of cultured pituitary cell extracts revealed that the majority of t-TRH-LI (>98%) was due to TRH-LI. A possible explanation for the change in EEP-LI and TRH-LI levels in the in vivo and in vitro pituitary samples is that hypothalamic influences are necessary for the continued production of EEP-LI and are not present in vitro. Alternatively, the dissociation of the cell–cell interactions and/or the accumulation of cell products, particularly pituitary hormones in vitro, may result in a loss of the in vivo paracrine influences or the introduction of factors which inhibit EEP-LI and stimulate TRH-LI. Journal of Endocrinology (1995) 145, 333–341

Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

1976 ◽  
Vol 35 (02) ◽  
pp. 350-357 ◽  
Author(s):  
Hana Bessler ◽  
Galila Agam ◽  
Meir Djaldetti
Keyword(s):  
Protein Synthesis ◽  
Fold Increase ◽  
Human Platelets ◽  
Polystyrene Latex ◽  
Latex Particles ◽  
The Third ◽  
Platelet Protein ◽  
Dose Dependent ◽  
Phagocytotic Activity

SummaryA three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

Monitoring Anticoagulant Therapy by Activated Partial Thromboplastin Time: Hirudin Assessment

1994 ◽  
Vol 72 (05) ◽  
pp. 685-692 ◽  
Author(s):  
Michael T Nurmohamed ◽  
René J Berckmans ◽  
Willy M Morriën-Salomons ◽  
Fenny Berends ◽  
Daan W Hommes ◽  
...  
Keyword(s):  
Whole Blood ◽  
Partial Thromboplastin Time ◽  
Ex Vivo ◽  
Fold Increase ◽  
Heparin Therapy ◽  
Activated Partial Thromboplastin Time ◽  
Recombinant Hirudin ◽  
Interindividual Differences ◽  
The Relationship

SummaryBackground. Recombinant hirudin (RH) is a new anticoagulant for prophylaxis and treatment of venous and arterial thrombosis. To which extent the activated partial thromboplastin time (APTT) is suitable for monitoring of RH has not been properly evaluated. Recently, a capillary whole blood device was developed for bed-side monitoring of the APTT and it was demonstrated that this device was suitable to monitor heparin therapy. However, monitoring of RH was not evaluated.Study Objectives. To evaluate in vitro and ex vivo the responsiveness and reproducibility for hirudin monitoring of the whole blood monitor and of plasma APTT assays, which were performed with several reagents and two conventional coagulometers.Results. Large interindividual differences in hirudin responsiveness were noted in both the in vitro and the ex vivo experiments. The relationship between the APTT, expressed as clotting time or ratio of initial and prolonged APTT, and the hirudin concentration was nonlinear. A 1.5-fold increase of the clotting times was obtained at 150-200 ng/ml plasma. However, only a 2-fold increase was obtained at hirudin levels varying from 300 ng to more than 750 ng RH/ml plasma regardless of the assays. The relationship linearized upon logarithmic conversion of the ratio and the hirudin concentration. Disregarding the interindividual differences, and presuming full linearity of the relationship, all combinations were equally responsive to hirudin.Conclusions. All assays were equally responsive to hirudin. Levels up to 300 ng/ml plasma can be reliably estimated with each assay. The manual device may be preferable in situations where rapid availability of test results is necessary.

Gene transfer of endothelial NO-synthase restores migratory capacity of endothelial progenitor cells from patients with coronary artery diseases

2007 ◽  
Vol 30 (4) ◽  
pp. 96
Author(s):  
Michael R. Ward ◽  
Qiuwang Zhang ◽  
Duncan J. Stewart ◽  
Michael J.B. Kutryk
Keyword(s):  
Risk Factors ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Fold Increase ◽  
No Synthase ◽  
Endothelial Progenitor ◽  
Migratory Capacity ◽  
Acute Mi ◽  
Cad Risk

Autologous endothelial progenitor cells (EPCs) have been used extensively in the development of cell-based therapy for acute MI. However, EPCs isolated from patients with CAD and/or CAD risk factors have reduced regenerative activity compared to cells from healthy subjects. As in endothelial cells, endothelial NO synthase (eNOS) expression and subsequent NO production are believed to be critical determinants of EPC function. Recently, the ability of EPCs to migrate in vitro in response to chemotactic stimuli has been shown to predict their regenerative capacity in clinical studies. Therefore, we hypothesized that the regenerative function of EPCs from patients with or at high risk for CAD will be enhanced by overexpression of eNOS, as assessed by migratory capacity. Methods: EPCs were isolated from the blood of human subjects with CAD risk factors (>15% Framingham risk score; FRS) (± CAD) by Ficoll gradient separation and differential culture. Following 3 days in culture, cells were transduced using lentivirus vectors containing either eNOS or GFP (sham) at an MOI of 3. The cells were cultured for an additional 5 days before being used in functional assays. Cell migration and chemotaxis in response to VEGF (50 ng/mL) and SDF-1 (100 ng/mL) were assessed using a modified Boyden Chamber assay. Results: Transduction at an MOI of 3 led to a ~90-100-fold increase in eNOS mRNA expression and a 5-6 fold increase in eNOS protein expression, as assessed by qRT-PCR and Western Blotting. Moreover, there was a significant improvement in the migration of EPCs following eNOS transduction compared to sham-transduced EPCs in response to both VEGF (44.3 ± 8.4 vs. 31.1 ± 4.6 cells/high power field; n=10, p < 0.05) and SDF-1 (51.9 ± 11.1 vs. 34.5 ± 3.3 cells/HPF; n=10, p < 0.05). Conclusions: These data show that the reduced migration capacity of EPCs isolated from patients with CAD and/or CAD risk factors can be significantly improved through eNOS overexpression in these cells. Thus, eNOS transduction of autologous EPCs may enhance their ability to restore myocardial perfusion and function following acute MI. We intend to further explore the regenerative potential of eNOS-transduced EPCs using various in vitro and in vivo models.

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