scholarly journals Effect of heparin administration to sheep on the release profiles of circulating activin A and follistatin

2004 ◽  
Vol 181 (2) ◽  
pp. 307-314 ◽  
Author(s):  
KL Jones ◽  
DM De Kretser ◽  
DJ Phillips
Keyword(s):  
Area Under The Curve ◽  
Activin A ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Large Pool ◽  
Heparin Administration ◽  
Repeat Administration ◽  
Clearance Mechanism ◽  
Exclusion Chromatography ◽  
Dose Dependent

Activin A and follistatin are normally present in relatively low amounts in the circulation. Heparin administration elicits a rapid and robust release of these proteins, although this phenomenon is poorly defined. In the present studies, the response to heparin administration was evaluated in the plasma of adult ewes in terms of whether it was dose-dependent, could be neutralized, was responsive to multiple stimulation, and the nature of the activin A and follistatin released. Activin A and follistatin were rapidly released by heparin in a dose-dependent manner (25, 100 or 250 IU/kg), with differences in the response as adjudged by peak concentration, timing of the peak and area under the curve. The heparin response could be blocked by pretreatment with protamine; conversely protamine injection alone (2 mg/kg) elicited release of follistatin but not activin A. Repeat administration of heparin at three-hourly intervals resulted in activin and follistatin responses to each injection, but each subsequent stimulation increased and extended the responses, consistent with saturation of the heparin clearance mechanism. Size exclusion chromatography of plasma samples confirmed that the majority of activin and follistatin released by heparin was a complex, whereas follistatin released by protamine was unbound. These data are consistent with a large pool of activin A and follistatin resident on extracellular matrices, with the rapid response implicating the vascular endothelium as the prime site of release following administration of these commonly used anticoagulant therapies.

scholarly journals Serum Extracellular Vesicle-Derived circHIPK3 and circSMARCA5 Are Two Novel Diagnostic Biomarkers for Glioblastoma Multiforme

2021 ◽  
Vol 14 (7) ◽  
pp. 618
Author(s):  
Michele Stella ◽  
Luca Falzone ◽  
Angela Caponnetto ◽  
Giuseppe Gattuso ◽  
Cristina Barbagallo ◽  
...  
Keyword(s):  
Glioblastoma Multiforme ◽  
Roc Analysis ◽  
Characteristic Curve ◽  
Area Under The Curve ◽  
Digital Pcr ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Circular Rnas ◽  
Diagnostic Biomarkers ◽  
Exclusion Chromatography

Glioblastoma multiforme (GBM) is the most frequent and deadly human brain cancer. Early diagnosis through non-invasive biomarkers may render GBM more easily treatable, improving the prognosis of this currently incurable disease. We suggest the use of serum extracellular vesicle (sEV)-derived circular RNAs (circRNAs) as highly stable minimally invasive diagnostic biomarkers for GBM diagnosis. EVs were isolated by size exclusion chromatography from sera of 23 GBM and 5 grade 3 glioma (GIII) patients, and 10 unaffected controls (UC). The expression of two candidate circRNAs (circSMARCA5 and circHIPK3) was assayed by droplet digital PCR. CircSMARCA5 and circHIPK3 were significantly less abundant in sEVs from GBM patients with respect to UC (fold-change (FC) of −2.15 and −1.92, respectively) and GIII (FC of −1.75 and −1.4, respectively). Receiver operating characteristic curve (ROC) analysis, based on the expression of sEV-derived circSMARCA5 and circHIPK3, allowed us to distinguish GBM from UC (area under the curve (AUC) 0.823 (0.667–0.979) and 0.855 (0.704 to 1.000), with a 95% confidence interval (CI), respectively). Multivariable ROC analysis, performed by combining the expression of sEV-derived circSMARCA5 and circHIPK3 with preoperative neutrophil to lymphocyte (NLR), platelet to lymphocyte (PLR) and lymphocyte to monocyte (LMR) ratios, three known diagnostic and prognostic GBM markers, allowed an improvement in the GBM diagnostic accuracy (AUC 0.901 (0.7912 to 1.000), 95% CI). Our data suggest sEV-derived circSMARCA5 and circHIPK3 as good diagnostic biomarkers for GBM, especially when associated with preoperative NLR, PLR and LMR.

Leishmania major biotin protein ligase forms a unique cross-handshake dimer

2021 ◽  
Vol 77 (4) ◽  
pp. 510-521
Author(s):  
Manoj Kumar Rajak ◽  
Sonika Bhatnagar ◽  
Shubhant Pandey ◽  
Sunil Kumar ◽  
Shalini Verma ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Size Exclusion ◽  
Covalent Attachment ◽  
Mitochondrial Enzymes ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Terminal Domain ◽  
Exclusion Chromatography ◽  
Partial Unfolding ◽  
Biotin Protein Ligase

Biotin protein ligase catalyses the post-translational modification of biotin carboxyl carrier protein (BCCP) domains, a modification that is crucial for the function of several carboxylases. It is a two-step process that results in the covalent attachment of biotin to the ɛ-amino group of a conserved lysine of the BCCP domain of a carboxylase in an ATP-dependent manner. In Leishmania, three mitochondrial enzymes, acetyl-CoA carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, depend on biotinylation for activity. In view of the indispensable role of the biotinylating enzyme in the activation of these carboxylases, crystal structures of L. major biotin protein ligase complexed with biotin and with biotinyl-5′-AMP have been solved. L. major biotin protein ligase crystallizes as a unique dimer formed by cross-handshake interactions of the hinge region of the two monomers formed by partial unfolding of the C-terminal domain. Interestingly, the substrate (BCCP domain)-binding site of each monomer is occupied by its own C-terminal domain in the dimer structure. This was observed in all of the crystals that were obtained, suggesting a closed/inactive conformation of the enzyme. Size-exclusion chromatography studies carried out using high protein concentrations (0.5 mM) suggest the formation of a concentration-dependent dimer that exists in equilibrium with the monomer.

Epithelial permeability factor: a serum protein that condenses actin and opens tight junctions

1992 ◽  
Vol 262 (6) ◽  
pp. C1403-C1410 ◽  
Author(s):  
A. D. Marmorstein ◽  
K. H. Mortell ◽  
D. R. Ratcliffe ◽  
E. B. Cramer
Keyword(s):  
Tight Junctions ◽  
Total Activity ◽  
Size Exclusion ◽  
Heat Inactivation ◽  
Epithelial Permeability ◽  
Lectin Affinity Chromatography ◽  
Permeability Factor ◽  
Exclusion Chromatography ◽  
Dose Dependent ◽  
Darby Canine Kidney

An epithelial permeability factor (EPF) in human serum lowered, within 1 h, the transepithelial electrical resistance and opened the tight junctions of a cultured kidney epithelium (Madin-Darby canine kidney) when it came in contact with the basolateral surface of the kidney epithelium. Size-exclusion chromatography of serum or heat-inactivated serum resolved seven peaks of EPF activity (approximately 15, approximately 30, approximately 45, approximately 60, approximately 120, and approximately 240 kDa and greater than 240 kDa) with 65% of the activity at approximately 45, approximately 60, and approximately 120 kDa. Heat inactivation, which had no effect on total activity, caused a significant decrease in the activity at 120 kDa and an equivalent rise in activity at 45 kDa. Although acid charcoal extraction or lectin affinity chromatography did not remove activity, EPF activity was eliminated by pepsin. Heat-inactivated serum or fractions containing EPF had no effect on ZO-1 localization but did cause a dose-dependent focal condensation of the perijunctional actin ring at sites where three or more cells were in contact. These data suggest that EPF is a protein that appears to form multimers that interact with the basolateral surface of the epithelium and cause constriction of the cytoskeleton and an increase in permeability at specific sites along the tight junction.

scholarly journals The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

2020 ◽  
Author(s):  
Jaehyoun Lee ◽  
Seunghee Oh ◽  
Saikat Bhattacharya ◽  
Ying Zhang ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Pyruvate Dehydrogenase ◽  
Biochemical Analysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Acetyl Coa ◽  
Cell Extracts ◽  
Exclusion Chromatography ◽  
Dehydrogenase Complex

ABSTRACTThe pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

scholarly journals Activin A suppresses proliferation of interleukin-3-responsive granulocyte-macrophage colony-forming progenitors and stimulates proliferation and differentiation of interleukin-3-responsive erythroid burst-forming progenitors in the peripheral blood

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 2891-2897 ◽  
Author(s):  
T Mizuguchi ◽  
M Kosaka ◽  
S Saito
Keyword(s):  
Activin A ◽  
Dependent Manner ◽  
Hematopoietic Progenitors ◽  
Erythroid Progenitors ◽  
Interleukin 3 ◽  
Liquid Suspension ◽  
Proliferation And Differentiation ◽  
Macrophage Colony ◽  
Dose Dependent ◽  
Stimulating Factor

Abstract We examined the effects of activin A on the proliferation and differentiation of immature hematopoietic progenitors prepared from peripheral blood (PB) using methylcellulose and liquid-suspension culture. In a kinetic analysis, colony formation by PB granulocyte- macrophage colony-forming unit (CFU-GM) was delayed in a dose-dependent manner by the addition of activin A only when stimulated with interleukin-3 (IL-3), but not when stimulated with granulocyte colony- stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or stem cell factor (SCF) plus G-CSF. DNA-synthesizing CFU-GM was increased by IL-3, but this effect was abolished by activin A. In contrast, PB erythroid burst-forming unit (BFU-E) was accelerated by the addition of activin A only when exposed to IL-3 plus erythropoietin (Epo), but not when exposed to Epo or Epo plus SCF. DNA- synthesizing BFU-E was increased by IL-3 and activin A, alone and additively in combination. In a mixed culture of myeloid and erythroid progenitors, activin A increased the numbers of BFU-E and CFU-Mix colonies at concentrations of 1 and 10 ng/mL and decreased the number of CFU-GM colonies in a dose-dependent manner. However, in a liquid- suspension culture of erythroid progenitors, activin A decreased total cell count and the percentage of hemoglobin-containing cells only when cells were exposed to IL-3 plus Epo. These results indicate that activin A suppresses the proliferation of IL-3-responsive CFU-GM progenitors and stimulates the proliferation and differentiation of IL- 3-responsive BFU-E progenitors, and suggest that activin A acts as a commitment factor of immature hematopoietic progenitors for erythroid differentiation.

scholarly journals Prebiotic fibres dose-dependently increase satiety hormones and alter Bacteroidetes and Firmicutes in lean and obese JCR:LA-cp rats

2011 ◽  
Vol 107 (4) ◽  
pp. 601-613 ◽  
Author(s):  
Jill A. Parnell ◽  
Raylene A. Reimer
Keyword(s):  
Gastric Emptying ◽  
Energy Expenditure ◽  
Gut Microbiota ◽  
Peptide Yy ◽  
Therapeutic Potential ◽  
Area Under The Curve ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Total Bacteria

There is a growing interest in modulating gut microbiota with diet in the context of obesity. The purpose of the present study was to evaluate the dose-dependent effects of prebiotics (inulin and oligofructose) on gut satiety hormones, energy expenditure, gastric emptying and gut microbiota. Male lean and obese JCR:LA-cp rats were randomised to either of the following: lean 0 % fibre (LC), lean 10 % fibre (LF), lean 20 % fibre (LHF), obese 0 % fibre (OC), obese 10 % fibre (OF) or obese 20 % fibre (OHF). Body composition, gastric emptying, energy expenditure, plasma satiety hormone concentrations and gut microbiota (using quantitative PCR) were measured. Caecal proglucagon and peptide YY mRNA levels were up-regulated 2-fold in the LF, OF and OHF groups and 3-fold in the LHF group. GhrelinO-acyltransferase mRNA levels were higher in obesev.lean rats and decreased in the OHF group. Plasma ghrelin response was attenuated in the LHF group. Microbial species measured in the Bacteroidetes division decreased, whereas those in the Firmicutes increased in obesev.lean rats and improved with prebiotic intake.BifidobacteriumandLactobacillusincreased in the OHFv.OC group.Bacteroidesand total bacteria negatively correlated with percentage of body fat and body weight. Enterobacteriaceae increased in conjunction with glucose area under the curve (AUC) and glucagon-like peptide-1 AUC.Bacteroidesand total bacteria correlated positively with ghrelin AUC yet negatively with insulin AUC and energy intake (P < 0·05). Several of the mechanisms through which prebiotics act (food intake, satiety hormones and alterations in gut microbiota) are regulated in a dose-dependent manner. The combined effects of prebiotics may have therapeutic potential for obesity.

scholarly journals The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243489
Author(s):  
Jaehyoun Lee ◽  
Seunghee Oh ◽  
Saikat Bhattacharya ◽  
Ying Zhang ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Pyruvate Dehydrogenase ◽  
Biochemical Analysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Acetyl Coa ◽  
Cell Extracts ◽  
Exclusion Chromatography ◽  
Dehydrogenase Complex

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

scholarly journals Breath Hydrogen Produced by Ingestion of Commercial Hydrogen Water and Milk

2009 ◽  
Vol 4 ◽  
pp. BMI.S2209 ◽  
Author(s):  
Akito Shimouchi ◽  
Kazutoshi Nose ◽  
Makoto Yamaguchi ◽  
Hiroshi Ishiguro ◽  
Takaharu Kondo
Keyword(s):  
Baseline Level ◽  
Area Under The Curve ◽  
Distilled Water ◽  
Dependent Manner ◽  
Breath Hydrogen ◽  
Milk Intolerance ◽  
Maximal Level ◽  
Hydrogen Water ◽  
Dose Dependent ◽  
Sustained Increase

Objective To compare how and to what extent ingestion of hydrogen water and milk increase breath hydrogen in adults. Methods Five subjects without specific diseases, ingested distilled or hydrogen water and milk as a reference material that could increase breath hydrogen. Their end-alveolar breath hydrogen was measured. Results Ingestion of hydrogen water rapidly increased breath hydrogen to the maximal level of approximately 40 ppm 10–15 min after ingestion and thereafter rapidly decreased to the baseline level, whereas ingestion of the same amount of distilled water did not change breath hydrogen (p < 0.001). Ingestion of hydrogen water increased both hydrogen peaks and the area under the curve (AUC) of breath hydrogen in a dose-dependent manner. Ingestion of milk showed a delayed and sustained increase of breath hydrogen in subjects with milk intolerance for up to 540 min. Ingestion of hydrogen water produced breath hydrogen at AUC levels of 2 to 9 ppm hour, whereas milk increased breath hydrogen to AUC levels of 164 ppm hour for 540 min after drinking. Conclusion Hydrogen water caused a rapid increase in breath hydrogen in a dose-dependent manner; however, the rise in breath hydrogen was not sustained compared with milk.

Anti-Angiogenic Activity of Bortezomib Is blocked by a 50–60 KDa Protein Secreted by Various tumor Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5448-5448
Author(s):  
Johann Kern ◽  
Gerold Untergasser ◽  
Heinz Zoller ◽  
Gunther Gastl ◽  
Eberhard Gunsilius ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Lines ◽  
Chorioallantoic Membrane ◽  
Carcinoma Cells ◽  
Size Exclusion ◽  
Cam Assay ◽  
Cycle Arrest ◽  
Exclusion Chromatography ◽  
Dose Dependent

Abstract Background: Bortezomib is a reversible proteasome inhibitor displaying significant clinical activity in the treatment of multiple myeloma. Apart from direct antitumor cell activity via inhibition of the nuclear factor kappa B pathway in myeloma cells additional anti-angiogenic effects have been reported. Aims: This study aimed to analyze the cellular effects and molecular targets of bortezomib in endothelial cells. In particular, we wanted to assess the its effects on proliferating as well as quiescent endothelial cells in vitro. Anti-angiogenic activity in vivo was studied in the chicken chorioallantoic membrane (CAM) model using tumor xenografts. Methods: Cell viablility of human umbilical vein endothelial cells (HUVEC) and endothelial colony forming cells (ECFC) was determined by flow cytometry; DNA synthesis was measured by BrDU-incorporation. All experiments were performed on mitotic and growth-arrested cells, respectively. Proteins involved in cell cycle arrest and apoptosis were analysed by Western Blots and antibody microarrays. Anti-angiogenic effects in vivo were studied in the chorioallantoic membrane (CAM) assay and the B16F10 xenograft model. Secretome analysis of bortezomib-resistant cells was performed by size exclusion chromatography (SEC), 2D gel electrophoresis (2D-PAGE) and mass spectroscopy (MS, Maldi-TOF). Results: In proliferating endothelial cells, bortezomib significantly reduced cell viability and proliferation in a dose-dependent fashion with an EC50 of 5 ng/mL. This is about ten times lower than the EC50 determined for established multiple myeloma cell lines (OPM-2, LP1 and RPMI 8226). A dose-dependent increase of cell cycle arrest was accompanied by upregulation of the cycline-dependent kinase inhibitors p21CIP1, p27 KIP1 and the tumor suppressor p53. Bortezomib at higher concentrations induced apoptosis only in proliferating endothelial cells by induction of the pro-apoptotic Bok and Noxa proteins. Vessel formation in the CAM-assay was strongly inhibited by bortezomib. In contrast,, growth and vascularization of melanoma (B16F10) xenografts in the CAM assay was not inhibited by bortezomib. A protein secreted spontaneously by melanoma cells neutralized the antiangiogenic action of bortezomib. By size exclusion chromatography (SEC) and proteasome activity assays a 50–60 KDa protein could be isolated and identified to cause this activity. Besides melanoma cells a variety of other cell lines derived from soldi tumors, such as the colorectal carcinoma cells HRT18 and prostate carcinoma cells PC3 were found to release this protein constitutively at high concentrations. Mass spectrometry data on this protein will be presented at the meeting. Conclusions: Besides its direct anti-myeloma activity by suppressing the nuclear factor kappa B pathway in myeloma cells bortezomib induces apoptosis in proliferating endothelial cells via the p53 pathway and its downstream targets Noxa and Bok, and inhibits angiogenesis in vivo.. Unexpetedly, we observed many solid tumor cell lines to release a soluble protein that can effectively block the antiangiogenic activity of bortezomib in vivo..

scholarly journals Effect of activin on production and secretion of prolactin and growth hormone in cultured rat GH3 cells

2000 ◽  
pp. 506-511 ◽  
Author(s):  
N Tamura ◽  
M Irahara ◽  
A Kuwahara ◽  
K Ushigoe ◽  
H Sugino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Activin A ◽  
Gh3 Cells ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Inhibitory Effects ◽  
Gh Secretion ◽  
Gh Gene ◽  
Dose Dependent ◽  
Cells Cultured

OBJECTIVE: To evaluate the effect of the growth factor activin A on the secretion of prolactin (PRL) and GH in cultured GH3 cells. METHODS: The concentrations of PRL and GH secreted from GH3 cells cultured in media with and without activin A were measured by RIA, and the expression of PRL mRNA and GH mRNA were analyzed using the Northern blot method. RESULTS: Activin A significantly inhibited PRL release from GH3 cells cultured for 48h in a dose-dependent manner (activin: 0.3-3nM). The inhibitory effects of 3nM activin A were observed in the culture from 12h to 48h (53.2% of control). Activin A (3nM) also significantly inhibited the expression of PRL mRNA at 24h (33.8% of control). In contrast, activin A significantly stimulated GH release from GH3 cells cultured for 48h in a dose-dependent manner (activin: 0.3-3nM). The stimulatory effect of 3nM activin A was observed in the culture for 48h (157.6% of control). Activin A (3nM) also significantly stimulated the expression of GH mRNA at 24h (183.6% of control). In spite of these significant changes in PRL and GH secretion, pit-1 mRNA levels were not significantly changed by activin A. CONCLUSIONS: These findings indicated that activin A modulates PRL and GH secretion through the regulation of PRL and GH gene transcription in GH3 cells, but that these effects are unrelated to pit-1 gene expression.

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