Binding of 63Ni(II) to Ultrafiltrable Constituents of Rabbit Serum In Vivo and In Vitro

1975 ◽  
Vol 21 (4) ◽  
pp. 521-527 ◽  
Author(s):  
Noritake Asato ◽  
Maria van Soestbergen ◽  
F William Sunderman
Keyword(s):  
Rabbit Serum ◽  
Total Serum ◽  
In Vivo Experiments ◽  
The Mean ◽  
Layer Chromatography ◽  
In Vivo Administration ◽  
Mean Concentration

Abstract Binding of 63Ni(Il) to ultrafiltrable constituents of rabbit serum was studied (a) after in vitro incubation (2 h, 37 °C) of rabbit serum with 63NiCl2 (10-100 µmol/liter), and (b) at intervals (0.25-2 h) after in vivo administration of 63NiCl2 (40-160 µmol/kg body wt, i.v.). Serum ultrafiltrates were fractionated by thin-layer chromatography, and the separated compounds made visible by autoradiography and by ninhydrin staining. Several (≃5) ultrafiltrable 63Ni-complexes were demonstrable as distinct radiodense 63Ni-bands with chromatographic mobilities corresponding to those of ninhydrin-positive bands. Unbound 63Ni(II) was not detected in serum ultrafiltrates in either the in vitro or in vivo experiments. In sera (n = 10) incubated in vitro with 63Ni(II) (10 µmol/ liter), the mean percentage of ultrafiltrable 63Ni was 36% (range = 33-38) of total serum 63Ni. In contrast, in sera (n = 10) obtained 2 h after i.v. injection of 63Ni(II) (40 µmol/kg), the mean concentration of total serum 63Ni was 10.8 µmol/liter (range = 6-14), and the mean percentage of ultrafiltrable 63Ni was 15% (range = 9-21) of total serum 63Ni. The disparity between the percentages of ultrafiltrable 63Ni obtained in vitro and in vivo was obviated when the in vivo experiments were performed in rabbits bilaterally nephrectomized, with ligated common bile ducts. This investigation confirms the existence of several nickel receptors in serum ultrafiltrates and substantiates the role of ultrafiltrable complexes in the excretion of nickel.

scholarly journals The Extracellular Small Leucine-Rich Proteoglycan Biglycan Is a Key Player in Gastric Cancer Aggressiveness

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1330
Author(s):  
Filipe Pinto ◽  
Liliana Santos-Ferreira ◽  
Marta T. Pinto ◽  
Catarina Gomes ◽  
Celso A. Reis
Keyword(s):  
Clinical Value ◽  
Knock Out ◽  
Angiogenic Potential ◽  
In Vivo Experiments ◽  
Cancer Aggressiveness ◽  
Advanced Stages ◽  
Oncogenic Gene

Biglycan (BGN gene), an extracellular proteoglycan, has been described to be associated with cancer aggressiveness. The purpose of this study was to clarify the clinical value of biglycan as a biomarker in multiple independent GC cohorts and determine the in vitro and in vivo role of biglycan in GC malignant features. We found that BGN is commonly over-expressed in all analyzed cohorts, being associated with disease relapse and poor prognosis in patients with advanced stages of disease. In vitro and in vivo experiments demonstrated that biglycan knock-out GC cells display major phenotypic changes with a lower cell survival, migration, and angiogenic potential when compared with biglycan expressing cells. Biglycan KO GC cells present increased levels of PARP1 and caspase-3 cleavage and a decreased expression of mesenchymal markers. Importantly, biglycan deficient GC cells that were supplemented with exogenous biglycan were able to restore biological features, such as survival, clonogenic and migratory capacities. Our in vitro and in vivo findings were validated in human GC samples, where BGN expression was associated with several oncogenic gene signatures that were associated with apoptosis, cell migration, invasion, and angiogenesis. This study provided new insights on biglycan role in GC that should be taken in consideration as a key cellular regulator with major impact in tumor progression and patients’ clinical outcome.

Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

2007 ◽  
Vol 292 (4) ◽  
pp. L915-L923 ◽  
Author(s):  
Jaime Chávez ◽  
Patricia Segura ◽  
Mario H. Vargas ◽  
José Luis Arreola ◽  
Edgar Flores-Soto ◽  
...  
Keyword(s):  
Substance P ◽  
Guinea Pigs ◽  
Smooth Muscle Contraction ◽  
In Vivo Experiments ◽  
Intracellular Ca ◽  
Neurokinin 1 ◽  
Substance P Release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+ measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, ω-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of ∼50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.

A role for the EphA family in the topographic targeting of vomeronasal axons

Development ◽  
2001 ◽  
Vol 128 (6) ◽  
pp. 895-906
Author(s):  
B. Knoll ◽  
K. Zarbalis ◽  
W. Wurst ◽  
U. Drescher
Keyword(s):  
Tyrosine Kinases ◽  
Expression Patterns ◽  
Eph Receptors ◽  
Accessory Olfactory Bulb ◽  
In Vivo Experiments ◽  
Receptor Concentration ◽  
High Ligand

We have investigated the role of the Eph family of receptor tyrosine kinases and their ligands in the establishment of the vomeronasal projection in the mouse. Our data show intriguing differential expression patterns of ephrin-A5 on vomeronasal axons and of EphA6 in the accessory olfactory bulb (AOB), such that axons with high ligand concentration project onto regions of the AOB with high receptor concentration and vice versa. These data suggest a mechanism for development of this projection that is the opposite of the repellent interaction between Eph receptors and ligands observed in other systems. In support of this idea, when given the choice of whether to grow on lanes containing EphA-F(c)/laminin or F(c)/laminin protein (in the stripe assay), vomeronasal axons prefer to grow on EphA-F(c)/laminin. Analysis of ephrin-A5 mutant mice revealed a disturbance of the topographic targeting of vomeronasal axons to the AOB. In summary, these data, which are derived from in vitro and in vivo experiments, indicate an important role of the EphA family in setting up the vomeronasal projection.

Role of Activated Platelets in the Homing, Maturation, and Differentiation of Endotheleal Progenitor Cells (EPCs) to Vascular Subendotheleal Matrix.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3938-3938
Author(s):  
Eli I. Lev ◽  
Jing-fei Dong ◽  
Marcin Bujak ◽  
Khatira Aboulfatova ◽  
Neal S. Kleiman ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Vascular Injury ◽  
Human Platelets ◽  
In Vitro Experiments ◽  
Cell Markers ◽  
Femoral Arteries ◽  
In Vivo Experiments

Abstract We and others have found that platelets play an important role in the recruitment of endothelial progenitor cells to sights of vascular injury. However, it is not clear whether the EPCs mature and differentiate to endothelial cells following recruitment to the vascular injury sites. In addition, there is limited in vivo data to support the role of EPCs in re-endothlialization following vascular injury. We conducted in vitro experiments to investigate the maturation of EPCs on platelet based-media and in vivo experiments to evaluate the recruitment of EPCs following vascular injury. In in vitro experiments human EPCs were isolated from donated buffy coats by magnetic microbeads and flow cytometry cell sorting using CD133 and VEGFR-2, respectively, as cell markers. Isolated viable EPCs (CD133+, VEGFR-2+ cells) were plated on human fibronectin or a monolayer of washed human platelets. Cell colonies were counted 7 days after plating and stained for the endothelial cell markers CD31 (PECAM-1) and CD144 (VE-cadherin). The mean number of colony-forming cells was 35±2.6 colonies/106 cells on platelets, which was significantly higher than 18±4.2 colonies/106 cells on fibronectin (n = 4, P<0.01). Apart from the difference in colony numbers, the EPC colonies grew faster on the platelet substrate, were larger, and had more spindle-shaped cells (Figure 1 - staining of EPC colonies for CD31 and CD144). In the in vivo experiments a model of transluminal injury to mouse femoral arteries was used. Femoral artery denudation was performed by 0.25-mm-diameter angioplasty guidewire. Injured femoral arteries were compared to the contra-lateral controls (uninjured), and were harvested 1.5 hours following the injury and immunostaining performed with an anti-VEGFR-2 antibody. Four experiments showed a markedly higher number of VEGFR-2+ cells in the artery that has undergone denudation. These experiments indicate that a media composed of platelets promotes the maturation and differentiation of EPCs. Furthermore, in vivo, EPCs are recruited early following vascular injury. Thus, homing, maturation, and differentiation of EPCs are mediated by platelets.

scholarly journals Role of transferrin in determining internal iron distribution

Blood ◽  
1977 ◽  
Vol 49 (6) ◽  
pp. 957-966
Author(s):  
P Pootrakul ◽  
A Christensen ◽  
B Josephson ◽  
CA Finch
Keyword(s):  
Isotope Ratio ◽  
Hepatic Uptake ◽  
The Other ◽  
Iron Salts ◽  
Iron Deficient ◽  
Loading And Unloading ◽  
The Mean

The behavior in vivo of transferrin in loading and unloading iron from its two sites was examined in rats. Radioiron entering the plasma from the gastrointestinal tract in iron-deficient, normal and iron-loaded rats did not differ in its subsequent tissue distribution between erythroid marrow and liver of normal recipients from a second isotope added to the same plasma in vitro. Loading studies in vitro were then carried out employing a reticulocyte incubation model designed to place one isotope predominantly on one site of transferrin, more available to the erythron, and the second isotope on the other site, more available to the liver. In 15 groups of animals in which 3 different iron salts were employed to load transferrin with iron, the mean isotope ratio in the erythron was 1.03 (+/-0.06 SD) and the mean liver ratio was 0.75 (+/-0.21 SD). It was found that the incubation of plasma with reticulocytes resulted in contamination of the plasma by radioactive hemoglobin. After allowance was made for hepatic uptake of radiohemoglobin in the 13 groups in which proper correction could be made, the isotope ratio in the liver became 0.97 (+/-0.17 SD). It is concluded that iron atoms from the two sites of transferrin have similar tissue distributions in vivo in the experimental situations examined.

scholarly journals Blocking Jak/STAT signalling using tofacitinib inhibits angiogenesis in experimental arthritis

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Paola Di Benedetto ◽  
Piero Ruscitti ◽  
Onorina Berardicurti ◽  
Noemi Panzera ◽  
Nicolò Grazia ◽  
...  
Keyword(s):  
Serum Levels ◽  
Tube Formation ◽  
Boyden Chamber ◽  
Arthritis Score ◽  
Ethical Approval ◽  
Synovial Tissues ◽  
In Vivo Administration

Abstract Objective During rheumatoid arthritis (RA), the angiogenic processes, occurring with pannus-formation, may be a therapeutic target. JAK/STAT-pathway may play a role and the aim of this work was to investigate the inhibiting role of a JAK-inhibitor, tofacitinib, on the angiogenic mechanisms occurring during RA. Methods After ethical approval, JAK-1, JAK-3, STAT-1, STAT-3 and VEGF expression was evaluated on RA-synovial-tissues. In vitro, endothelial cells (ECs), stimulated with 20 ng/ml of VEGF and/or 1 μM of tofacitinib, were assessed for tube formation, migration and proliferation, by Matrigel, Boyden chamber assay and ki67 gene-expression. In vivo, 32 mice received collagen (collagen-induced arthritis (CIA)) and 32 mice PBS (control). At day 19, CIA and controls mice were divided: 16 mice receiving vehicle and 16 mice receiving tofacitinib. At day 35, the arthritis score, the thickness of paw joints and the serum levels of VEGF and Ang-2 were evaluated. Results The expression of JAK-1, JAK-3, STAT-1, STAT-3 and VEGF in synovial tissue of RA-patients were significantly higher than healthy controls. In vitro, tofacitinib inhibited the ECs ability to form vessels, to proliferate and to migrate. In vivo, administration of tofacitinib prevented the increase of the arthritis score, the paw thickness, the synovial vessels and VEGF and Ang-2 serum-accumulation, when compared to CIA without tofacitinib. Conclusions We explored the anti-angiogenic role of tofacitinib, reporting its ability to inhibit in vitro the angiogenic mechanisms of ECs and in vivo the formation of new synovial vessels, occurring in CIA model. These findings suggest that the therapeutic effect of tofacitinib during RA may be also related to its anti-angiogenic activity.

scholarly journals Downregulation of Microparticle Release and Pro-Inflammatory Properties of Activated Human Polymorphonuclear Neutrophils by LMW Fucoidan

2018 ◽  
Vol 11 (4) ◽  
pp. 330-346 ◽  
Author(s):  
João Alfredo Moraes ◽  
Ana Clara Frony ◽  
Pedro Barcellos-de-Souza ◽  
Marcel Menezes da Cunha ◽  
Thayanne Brasil Barbosa Calcia ◽  
...  
Keyword(s):  
Polymorphonuclear Neutrophils ◽  
In Vivo Experiments ◽  
Formyl Peptide ◽  
Human Pmns ◽  
Oxidative Effects ◽  
Species Production ◽  
Bacterial Products

Exposition of neutrophils (polymorphonuclear neutrophils, PMNs) to bacterial products triggers exacerbated activation of these cells, increasing their harmful effects on host tissues. We evaluated the possibility of interfering with the classic immune innate responses of human PMNs exposed to bacterial endotoxin (lipopolysaccharide, LPS), and further stimulated with bacterial formyl peptide (N-formyl-methionine-leucine-phenylalanine, fMLP). We showed that the low- molecular-weight fucoidan (LMW-Fuc), a polysaccharide extracted from brown algae, attenuated the exacerbated activation induced by fMLP on LPS-primed PMNs, in vitro, impairing chemotaxis, NET formation, and the pro-survival and pro-oxidative effects. LMW-Fuc also inhibited the activation of canonical signaling pathways, AKT, bad, p47phox and MLC, activated by the exposition of PMN to bacterial products. The activation of PMN by sequential exposure to LPS and fMLP induced the release of L-selectin+ microparticles, which were able to trigger extracellular reactive oxygen species production by fresh PMNs and macrophages. Furthermore, we observed that LMW-Fuc inhibited microparticle release from activated PMN. In vivo experiments showed that circulating PMN-derived microparticles could be detected in mice exposed to bacterial products (LPS/fMLP), being downregulated in animals treated with LMW-Fuc. The data highlight the autocrine and paracrine role of pro-inflammatory microparticles derived from activated PMN and demonstrate the anti-inflammatory effects of LMW-Fuc on these cells.

Optoelectronic transducer for in vivo recording of oviductal contractile activity

1980 ◽  
Vol 239 (3) ◽  
pp. R326-R331
Author(s):  
S. A. Halbert ◽  
R. J. Bourdage ◽  
J. L. Boling ◽  
J. A. Ringo ◽  
R. J. Blandau
Keyword(s):  
Contractile Activity ◽  
Red Light ◽  
Muscular Activity ◽  
Optical Signal ◽  
In Vivo Experiments ◽  
Optoelectronic Transducer ◽  
Optical Analog

An optoelectronic instrument to record oviductal muscular activity in chronically instrumented animals was evaluated in in vitro and in vivo experiments. The intensity of red light transmitted through the oviduct was modulated by contractions of the oviductal wall producing an optical analog of the mechanical events. Accuracy of the analog was tested by Fourier analysis of signals from mechanical and optoelectronic transducers placed at the same site on the oviduct; the results validated the use of the optical device as a contraction event sensor. Contractions of the tubal mesenteries had less effect on the optical signal than on signals from extraluminal mechanical transducers. Optical and photographic recordings of luminal transport in exposed oviducts showed a correspondence of intraluminal movements to events in the optical contraction signal. This instrument does not alter tubal function, and thus it is an especially useful experimental tool to investigate the role of oviductal muscular activity in fertility.

Quantification of the Active Decitabine-Triphosphate (DAC-TP) Metabolite: A Novel Pharmacoanalytical Endpoint for Optimization of Hypomethylating Therapy in Acute Myeloid Leukemia (AML)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3578-3578
Author(s):  
Hongyan Wang ◽  
Ping Chen ◽  
Jiang Wang ◽  
Ramasamy Santhanam ◽  
Josephine Aimiuwu ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Dna Methyltransferases ◽  
Analytical Methodology ◽  
In Vivo Experiments ◽  
Accuracy And Precision ◽  
Coefficients Of Variation ◽  
The Mean

Abstract Abstract 3578 Decitabine (DAC) is successfully used for treatment of patients (pts) with myelodysplastic syndromes and AML. Following cellular uptake, DAC is thought to be activated to DAC-TP and incorporated into DNA. The DAC-TP/DNA complex binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately anti-leukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated pts has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a sensitive and specific LC-MS/MS method for quantification of DAC-TP. The assay exhibited excellent accuracy and precision. The accuracy values were 83.7–109.4%, as determined by calculating the percentage of measured DAC-TP relative to the respective nominal concentrations (50, 500 and 5,000 nM) of the quality control samples. The within-day coefficients of variation (CVs) were 19.9 % (n=6) at 50 nM and 4.7–7.0 % between 500–5,000 nM; the between-day CVs (n=3) were 15.2 % at 50 nM and 7.5–10.2 % between 500–5,000 nM. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant MV4–11, and in THP-1 and FDC-P1/Kitmut cells (in vitro); and in bone marrow (BM) and spleen of normal and FDC-P1/Kitmut-driven AML mice (in vivo). DAC-TP reached peak levels (0.8, 1.4 and 0.5 pmol/106 cells) in 1–4 hours and declined to 20 % of its peak concentration after 24 hours incubation with 2.5 μM DAC in MV4–11, THP-1 and FDC-P1/Kitmut cells, respectively. Inhibition of hENT1 that mediates DAC transport into the cells and dCK that phosphorylates DAC into DAC-TP by NBTI and 2-thio-2′-deoxycytidine, respectively, significantly inhibited DAC-TP accumulation in AML cells. DAC-TP decay was instead blocked by tetrahydrouridine (THU)-induced inhibition of CDA, the catabolizing enzyme for cytidine and deoxycytidine and analogs. Consistent with these results, low dCK and hENTs but not CDA expression were detected in DAC-resistant MV4–11 cells, which showed 60 % decrease in DAC-TP levels as compared to their parental counterparts. DAC/DAC-TP-mediated downregulation of DNMT proteins (preferentially DNMT1 and DNMT3a) was also demonstrated in the AML cells even at DAC-TP concentrations as low as 0.1–1.3 pmol/106 cells in vitro after 4 hours DAC incubation. In the in vivo experiments, DAC-TP levels in leukemic mice were comparable to that in normal C57BL/6 mice, 0.3 pmol/106 cells in BM and 199.2 pmol/g tissue in spleen at 4-hours and 0.2 pmol/106 cells in BM and 165.3 pmol/g tissue in spleen at 24-hours following an i.v. bolus of 6.5 mg/kg DAC. In BM of leukemic mice, not only DNMT1 and DNMT3a but also DNMT3b protein expression reduced 80 % (DNMT3a) or diminished (DNMT1 and DNMT3b). The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells (PBMC) from AML pts treated with a 10-day regimen of DAC given 20 mg/m2/day i.v. over 1 hour. In BM samples, the mean DAC-TP levels were 0.8 ± 0.6 (Day 1) and 0.9 ± 0.5 pmol/106 cells (Day∼5) in complete responsive (CR) pts (n=4); and 0.4 ± 0.3 (Day 1) and 0.12 ± 0.02 pmol/106 cells (Day∼5) in non-responsive (NR) pts (n=3). In PBMC samples, the mean DAC-TP levels were 0.5 ± 0.2 (Day 1) and 1.2 ± 0.4 pmol/106 cells (Day∼5) in CR pts (n=3); and 0.02 ± 0.02 (Day 1) and 0.21 ± 0.04 pmol/106 cells (Day∼5) in NR pts (n=3). These data suggested that higher levels are seemingly associated with clinical response, but a larger number of pts need to be tested. In conclusion, monitoring the intracellular concentration of DAC-TP is feasible, and DAC-TP levels correlate with DNMT downregulation and may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Decreased Fast Ripples in the Hippocampus of Rats with Spontaneous Recurrent Seizures Treated with Carbenoxolone and Quinine

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Consuelo Ventura-Mejía ◽  
Laura Medina-Ceja
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
High Frequency Oscillations ◽  
The Mean ◽  
Eeg Recordings ◽  
The Right ◽  
Spontaneous Recurrent Seizures

Background. In models of temporal lobe epilepsy and in patients with this pathology, high frequency oscillations called fast ripples (FRs, 250–600 Hz) can be observed. FRs are considered potential biomarkers for epilepsy and, in the light of manyin vitroandin silicostudies, we thought that electrical synapses mediated by gap junctions might possibly modulate FRsin vivo.Methods. Animals with spontaneous recurrent seizures induced by pilocarpine administration were implanted with movable microelectrodes in the right anterior and posterior hippocampus to evaluate the effects of gap junction blockers administered in the entorhinal cortex. The effects of carbenoxolone (50 nmoles) and quinine (35 pmoles) on the mean number of spontaneous FR events (occurrence of FRs), as well as on the mean number of oscillation cycles per FR event and their frequency, were assessed using a specific algorithm to analyze FRs in intracranial EEG recordings.Results. We found that these gap junction blockers decreased the mean number of FRs and the mean number of oscillation cycles per FR event in the hippocampus, both during and at different times after carbenoxolone and quinine administration.Conclusion. These data suggest that FRs may be modulated by gap junctions, although additional experimentsin vivowill be necessary to determine the precise role of gap junctions in this pathological activity associated with epileptogenesis.

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