Factors Involved in Crystal Formation in Cystinuria. In Vivo and in Vitro Crystallization Dynamics and a Simple, Quantitative Colorimetric Assay for Cystine

1971 ◽  
Vol 106 (1) ◽  
pp. 106-110 ◽  
Author(s):  
Bruce Ettinger ◽  
Felix O. Kolb
Keyword(s):  
Colorimetric Assay ◽  
Crystal Formation ◽  
Crystallization Dynamics

scholarly journals In vivo hypotensive effect and in vitro inhibitory activity of some Cyperaceae species

2013 ◽  
Vol 49 (4) ◽  
pp. 803-809
Author(s):  
Monica Lacerda Lopes Martins ◽  
Henrique Poltronieri Pacheco ◽  
Iara Giuberti Perini ◽  
Dominik Lenz ◽  
Tadeu Uggere de Andrade ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Colorimetric Assay ◽  
Hypotensive Effect ◽  
Ace Inhibition ◽  
Inhibitory Effect ◽  
Positive Control ◽  
Total Phenolic ◽  
Before And After

In 1820, French naturalist August Saint Hillaire, during a visit in Espírito Santo (ES), a state in southeastern Brazil, reported a popular use of Cyperaceae species as antidote to snake bites. The plant may even have a hypotensive effect, though it was never properly researched. The in vitro inhibitory of the angiotensin converting enzyme (ACE) activity of eigth ethanolic extracts of Cyperaceae was evaluated by colorimetric assay. Total phenolic and flavonoids were determined using colorimetric assay. The hypotensive effect of the active specie (Rhychonospora exaltata, ERE) and the in vivo ACE assay was measured in vivo using male Wistar Kyoto (ERE, 0.01-100mg/kg), with acetylcholine (ACh) as positive control (5 µg/kg, i.v.). The evaluation of ACE in vivo inhibitory effect was performed comparing the mean arterial pressure before and after ERE (10 mg/kg) in animals which received injection of angiotensin I (ANG I; 0,03, 03 and 300 µg/kg, i.v.). Captopril (30 mg/kg) was used as positive control. Bulbostylis capillaris (86.89 ± 15.20%) and ERE (74.89 ± 11.95%, ERE) were considered active in the in vitro ACE inhibition assay, at 100 µg/mL concentration. ACh lead to a hypotensive effect before and after ERE's curve (-40±5% and -41±3%). ERE showed a dose-dependent hypotensive effect and a in vivo ACE inhibitory effect. Cyperaceae species showed an inhibitory activity of ACE, in vitro, as well as high content of total phenolic and flavonoids. ERE exhibited an inhibitory effect on both in vitro and in vivo ACE. The selection of species used in popular medicine as antidotes, along with the in vitro assay of ACE inhibition, might be a biomonitoring method for the screening of new medicinal plants with hypotensive properties.

scholarly journals FTIR and UV‒vis study of chemically engineered biomaterial surfaces for protein immobilization

2002 ◽  
Vol 16 (3-4) ◽  
pp. 351-360 ◽  
Author(s):  
Herman Mansur ◽  
Rodrigo Oréfice ◽  
Marivalda Pereira ◽  
Zélia Lobato ◽  
Wander Vasconcelos ◽  
...  
Keyword(s):  
Colorimetric Assay ◽  
Protein Immobilization ◽  
Research Field ◽  
Male Rats ◽  
Visible Spectroscopy ◽  
Functional Abnormality ◽  
Specific Chemical ◽  
Uv Visible

The biomaterials research field has broadened in the last 3 decades, including replacement of diseased or damaged parts, assist in healing, correct and improve functional abnormality, drug delivery systems, immunological kits and biosensors. Proteins play crucial role in almost every biological system. They are involved in enzymatic catalysis, transport and storage, coordinated motion, mechanical support, immune protection, control of growth and cell differentiation among many others. The immobilization of proteins onto surface functionalized substrates has been one of the most promising areas in bioengineering field. It is important to note that the term immobilization can refer either to a temporary or to a permanent localization of the biomolecule on or within a support. Proteins have very particular chain configurations and conformations that promote high levels of specificity during chemical interactions. In the present work, we aimed to study the phenomenon of protein immobilization onto biomaterial with chemically engineered surface. We have tailored the surface of the porous gels of SiO2with 5 different silane surface modifying agents: tetraethoxysilane (TEOS), 3‒mercaptopropyltrimethoxysilane (MPTMS) and 3‒aminopropyltriethoxysilane (APTES), 3‒glycidoxypropyltrimethoxysilane (GPTMS) and 3‒isocyanatopropyltriethoxysilane (ICPES). Fourier Transform Infrared Spectroscopy (FTIR) was used to characterize the presence of all specific chemical groups in the materials. The surface functionalized gels were then immersed in porcine insulin (PI) solutions for protein immobilization. The incorporation of protein within the gels was also monitored by FTIR spectroscopy. The kinetics of protein adsorption and desorption from the gel matrixin vitrotests were monitored by UV‒visible spectroscopy. We could not observe any evidence of denaturation of insulin after its desorption from gel matrices using UV‒visible spectroscopy technique.In vivotests with adult male rats were used to verify the immobilized insulin bioactivity after implantation of different biomaterial with functionalized surfaces. Plasma glucose levels were obtained by using the Glucose GOD‒ANA Colorimetric Assay. All surface modified materials have presented acute hypoglycemic peak response associated with the insulin bioactivity.

Triphenyltin acetate toxicity : a biochemical and ultrastructural study on mouse thymocytes

1996 ◽  
Vol 15 (3) ◽  
pp. 219-225 ◽  
Author(s):  
E. Bollo ◽  
L. Ceppa ◽  
E. Cornaglia ◽  
C. Nebbia ◽  
B. Biolatti ◽  
...  
Keyword(s):  
Colorimetric Assay ◽  
Immunosuppressive Agents ◽  
Primary Cultures ◽  
Lactic Dehydrogenase ◽  
Neutral Red ◽  
Transmission Electron ◽  
Dose Dependent ◽  
Triphenyltin Acetate

1 Triphenyltin acetate (TPTA) has been shown to exert in vivo a selective toxic effect on the immune system. To assess in vitro possible alterations induced by TPTA exposure, primary cultures of mouse thymocytes were incubated up to 24 h with graded amounts (1-12 μM) ofthe organotin. 2 The cytotoxic activity has been evaluated with the MTT colorimetric assay, the neutral red (NR) assay and the lactic dehydrogenase (LDH) cellular release. Cell pellets were fixed with 2.5% glutaraldehyde, resin-embedded and ultrathin sections were observed through transmission electron microscopy. 3 After 2 h of incubation, dose-dependent increases of cytotoxicity were observed in thymocytes submitted to MTT and NR tests (up to 41.43% and 18.9%, respectively), while 22 h later this overt effect on cell viability was noticed merely in cells exposed to 12 μM TPTA. Dose- dependent increases of LDH leakage in the culture medium were observed all throughout the study. 4 Morphological investigations revealed features (chro matin condensation, cell membranes fragmentation and formation of membrane bound apoptotic bodies) sugges tive of apoptosis. 5 This study indicates that TPTA is cytotoxic to mouse thymocytes: morphologically, the rising of apoptosis is likely to be recognized, as previously reported in different in vitro studies with other immunosuppressive agents as dioxin and corticosteroids.

In vitro activity of ABT-888 (A), 5-FU (F), and dacarbazine (D) in neuroendocrine tumors (NET).

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 236-236
Author(s):  
Sam Joseph Lubner ◽  
Yash Somnay ◽  
Dustin A. Deming ◽  
Kyle D. Holen ◽  
Herbert Chen
Keyword(s):  
Neuroendocrine Tumors ◽  
Antitumor Effect ◽  
Colorimetric Assay ◽  
Phase 1 ◽  
Parp Inhibitor ◽  
Neuroendocrine Cells ◽  
Low Grade ◽  
Bon Cells

236 Background: Low-grade neuroendocrine tumors (NET) have few cytotoxic chemotherapy options. Data suggests that a combination of temozolomide and a fluoropyrimidine has clinical efficacy. ABT-888 is a novel poly-ADP ribose polymerase (PARP) inhibitor that has been tested in a phase 1 setting with temozolomide, with synergy demonstrated in preclinical models of other tumors. We proposed an in-vitro study of ABT-888 with varying concentrations of dacarbazine (D), and 5-FU (F) on human neuroendocrine cells (BON). Methods: BON cells were incubated with varying concentrations and combinations of ABT-888 (2.5, 5 and 10uM), F (25-100uM), and D (25-100uM) for 96 hours. After incubation, cell growth was measured by MTT rapid colorimetric assay with absorbance reported as mean % control. Western blot analysis was performed for chromogranin A, PARP, γH2AXand XIAP to assess for cell death and on-target effects for the population treated with 5-FU. Combination indices (CI) were calculated using the Chou-Talalay method using CompuSyn. CI’s below 1 signified synergy. Results: ABT-888 alone did not demonstrate any antitumor effect (103%). D alone had antitumor effect (74% at 50uM, 67% at 100uM) which was improved by adding ABT-888 (70% at 50uM D; CI 0.73; p=0.06, 60% at 100 uM D; CI 0.88; p=0.0003). F alone had antitumor effect (82% at 50uM and 71% at 100uM) which was improved by adding 2.5uM ABT-888 (71% at 50uM of F; CI=0.59, and 58% at 100uM of F; CI=0.74), and further enhanced with 5uM of concomitant ABT-888 (56% at 50uM of F; CI=0.68, and 49% at 100uM of F; CI=0.76). Western analysis of lysates showed markers of increased apoptosis, decreased PARP, and decreased expression of CgA. The combination of F+D did not demonstrate increased cytotoxicity with the addition of ABT-888 (58% with/without ABT-888 p=0.61). Conclusions: ABT-888 demonstrated in vitro synergy against BON cells with F or D. The combination of all three compounds (A+F+D) did not demonstrate synergy above F+D. Synergy was statistically significant with increasing doses of cytotoxic compounds which are achievable in vivo with current doses of A, F, and D. The combination of ABT-888 with temozolomide or a fluoropyrimidine merits further study in human clinical trials.

scholarly journals A Simple in vivo Assay Using Amphipods for the Evaluation of Potential Biocompatible Metal-Organic Frameworks

2021 ◽  
Vol 9 ◽  
Author(s):  
Ines Sifaoui ◽  
Idaira Pacheco-Fernández ◽  
José E. Piñero ◽  
Verónica Pino ◽  
Jacob Lorenzo-Morales
Keyword(s):  
Ex Vivo ◽  
Colorimetric Assay ◽  
Metal Organic Frameworks ◽  
Screening Method ◽  
Toxicity Tests ◽  
Toxicity Screening ◽  
Metal Organic ◽  
Potential Use

In this study, the application of amphipods in vivo assays was evaluated. The main aim of this work was to check the potential use of this model in biocompatibility assessments of metal-organic frameworks (MOFs). Hence, six different MOFs were synthesized and the in vitro and ex vivo cytotoxicity was first assessed using a colorimetric assay and a macrophage cell line. Obtained results were compared to validate the in vivo toxicity tests carried out using amphipods and increasing concentrations of the different MOFs. Amphipods do not require the need of ethics approval and also are less expensive to keep than conventional in vivo models, showing its potential as a fast and reliable platform in toxicity studies. The obtained results showed that the amphipods based-assay was simple, easy to replicate and yielded toxicity data corresponding to the type of MOFs tested. In addition, it was observed that only CIM-80(Al) and CIM-84(Zr) did not show any toxicity to the animals at the different tested concentrations. Therefore, the developed in vivo model could be applied as a high-throughput toxicity screening method to evaluate the toxicity of numerous materials, chemicals and therapeutic agents among others.

scholarly journals Vitamin K1 Inhibition of Renal Crystal Formation through Matrix Gla Protein in the Kidney

2019 ◽  
Vol 44 (6) ◽  
pp. 1392-1403 ◽  
Author(s):  
Yang Li ◽  
Xiuli Lu ◽  
Baoyu Yang ◽  
Jing Mao ◽  
Shan Jiang ◽  
...  
Keyword(s):  
Cell Viability ◽  
Matrix Gla Protein ◽  
Collagen I ◽  
Crystal Formation ◽  
Vitamin K1 ◽  
Crystal Deposition ◽  
Treatment And Prevention ◽  
Hk2 Cells

Background and Objectives: Vitamin K (VK) plays a major role in modifying the binding of calcium in bones and blood vessels. Understanding the effect of VK on crystal formation in the kidney would contribute to advancing the treatment and prevention of kidney stones. Methods: Rats were treated with vitamin K1 (VK1) for 8 weeks. VK1 levels were detected and crystal formation were observed. HK2 cells were exposed to calcium oxalate monohydrate crystals. Apoptosis and cell viability were detected. Crystal deposition was analyzed using atomic absorption assay. The adenovirus vectors expressing matrix Gla protein (MGP) and siMGP were constructed to elucidate the effect and mechanism of VK1 on crystal formation. MGP expression in vivo and in vitro was analyzed by Western blot. The mRNA levels of monocyte chemoattractant protein-1 (MCP-1) and collagen I was measured by semiquantitative RT-PCR. Results: The concentrations of VK1 in whole blood and kidney tissues rose under treatment with VK1. Crystal formation was inhibited from the second to the 6th week, the frequency and quality of crystal formation decreased significantly, and the location of crystal formation was limited to a greater extent in the rats treated by VK1 compared to the control group. Warfarin treatment in the crystals-exposed HK2 cells significantly increased the number of crystals adhering to cells and the number of apoptotic cells and reduced cell viability. VK1 treatment reversed warfarin’s above influence. VK1 inhibited the upregulations of MCP-1 and collagen I in kidney tissues under crystal load. VK1 treatment increased MGP expression in vivo and in vitro, and MGP is necessary for VK1 to play a role in crystal deposition in cells. Conclusions: VK1 treatment can inhibit the formation of renal crystals in vivo. VK1 increases MGP expression and functions through MGP to reduce crystal deposition in cells and provide cell protection. Our findings suggest that VK1 treatment could be a potential strategy for the treatment and prevention of nephrolithiasis.

scholarly journals Stimulation of precipitation of calcium phosphate by matrix vesicles

1978 ◽  
Vol 170 (3) ◽  
pp. 681-691 ◽  
Author(s):  
R Felix ◽  
W Herrmann ◽  
H Fleisch
Keyword(s):  
Calcium Phosphate ◽  
Matrix Vesicles ◽  
Crystal Formation ◽  
Freezing And Thawing ◽  
Minimum Concentration ◽  
Vesicle Preparation ◽  
Before And After ◽  
Stimulation Of

The ability of matrix vesicles isolated from the epiphysial growth plate of 6-week-old chicks to facilitate the precipitation of calcium phosphate was studied in vitro. The vesicles lowered the minimum concentration product [ca2+]X[p1] needed to induce crystal formation, thereby showing the vesicles are nucleators of crystallization. After freezing and thawing the vesicles at pH6.0, part but not all of this ability to nucleate disappeared. Freezing and thawing markedly decreased the Ca and Pi content of the vesicles, suggesting that part of the nucleating activity may have been due to mineral already present. After removal of the mineral the residual nucleating activity could be destroyed by extracting the vesicles with lipid solvents or by treatment with enzymes such as phosphoilipase C, neuraminidase or proteinase. Matrix vesicles obtained from chicks treated with 1-hydroxyethane-1, 1-diphosphonate, a compound that inhibits calcification in vivo, showed impaired nucleating activity, both before and after treatment at pH6.0. The vesicle preparation bound some diphosphonate in vitro, probably to the mineral present in the preparation, since no binding could be detected in vesicles preincubated at pH6.0. No difference was found in the nucleating activity of vesicles isolated from rachitic chicks which had or had not received cholacalciferol 48 h before death. These results suggest that matrix vesicles possess intrinsic nucleating activity that may be important in biological calcification.

scholarly journals A Cysteine-Rich Motif in Poliovirus Protein 2CATPaseIs Involved in RNA Replication and Binds Zinc In Vitro

2000 ◽  
Vol 74 (1) ◽  
pp. 334-343 ◽  
Author(s):  
Thomas Pfister ◽  
Keith W. Jones ◽  
Eckard Wimmer
Keyword(s):  
Molecular Mechanisms ◽  
Colorimetric Assay ◽  
Rna Replication ◽  
Cysteine Residue ◽  
Reduced Form ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Cell Organelles

ABSTRACT Protein 2CATPase of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2CATPase engages in these processes are not known. To characterize functional domains of 2CATPase, we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2CATPase. This region, which is well conserved among enteroviruses and rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that replaced the first cysteine residue of the motif with a serine was lethal. A mutant virus which lacked the second of four potential coordination sites for zinc was temperature sensitive. At the restrictive temperature, RNA replication was inhibited whereas translation and polyprotein processing, assayed in vitro and in vivo, appeared to be normal. An intragenomic second-site revertant which reinserted the missing coordination site for zinc and recovered RNA replication at the restrictive temperature was isolated. The cysteine-rich motif was sufficient to bind zinc in vitro, as assessed in the presence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding, however, was not required for hydrolysis of ATP. 2CATPase as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells.

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