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At least 721 records · Page 40Linked to original sources
PubMed2005

Assessment of bovine sperm viability by MTT reduction assay.

The MTT reduction assay depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate a simple and less costly MTT test to determine bovine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from eight bulls were included in this study. Semen sample was diluted to 30x10(6) sperms/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1h at 37 degrees C using spectrophotometer (MS2 Reader) at wave length 550nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for sperm viability, mitochondrial activity, and acrosomal integrity using SYBR-14, Rhodamine 123 and LysoTracker Green DNA-26, respectively. The correlation between the results of these tests was calculated using the Pearson correlation coefficients. The results revealed a strong correlation (P<0.001) between the results of MTT reduction rate and the results that simultaneously determined by flow cytometer, yielding correlation coefficients of r=0.950 for sperm viability, of r=0.926 for mitochondrial activity and of r=0.959 for acrosomal integrity. The same correlation coefficient was observed between the values of sperm viability calculated on the basis of MTT reduction rates and the results of flow cytometer. In conclusion, the MTT reduction test was found to be a reliable method in evaluating bovine semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.

Acrosome↗
PubMed2004

Expansion of chondrocytes in a three-dimensional matrix for tracheal tissue engineering.

BACKGROUND: The generation of autologous tracheal implants by tissue-engineering techniques is a promising concept for otherwise untreatable patients. A functional cartilaginous backbone represents a prerequisite for any bioartificial tracheal graft. The aim of this study was to define suitable cell types and culture conditions for the generation of tracheal cartilage. METHODS: We obtained tracheal, costal, and auricular cartilage from porcine donor animals (n = 10). The chondrocytes were cultured two-dimensionally in cell flasks or mixed with a liquid collagen solution forming a three-dimensional culture system. Labeling with carboxy fluorescein diacetate succinimidyl ester (CFDA SE) and biochemical reduction of formazan served to determine cell viability and proliferation. The extracellular matrix produced by the chondrocytes was characterized by Western blot. RESULTS: The CFDA SE labeling proved viability and the MTT assays documented a proliferation of the chondrocytes over time in vitro. While the chondrocytes in the three-dimensional cell culture system produced hyaline cartilage composed of collagen II, the two-dimensional culture conditions resulted in nonspecific collagen synthesis. CONCLUSIONS: Chondrocytes grown in a three-dimensional matrix can effectively proliferate and produce cartilage and are viable for more than 2 weeks. Costal chondrocytes are suitable for tracheal cartilage tissue engineering.

Animals↗
PubMed2004

Antiviral activity against human immunodeficiency virus-1 in vitro by myristoylated-peptide from Heliothis virescens.

An insect antiviral compound was purified from Heliothis virescens larval hemolymph by gel-filtration high pressure liquid chromatography (HPLC) and C-18 reverse-phase HPLC and its structure was determined by mass spectrometry. The antiviral compound is an N-myristoylated-peptide containing six amino acids with calculated molecular weight of 916 Da. The N-terminus contains the fatty acid myristoyl, and the C-terminus contains histidine with two methyl groups giving the histidine a permanent positive charge. The remainder of the compound is essentially non-polar. The structure of the compound corresponds with the 'myristate plus basic' motif expressed by certain viral proteins in their binding to the cytoplasmic side of the plasma membrane to initiate viral assembly and budding from a host cell. The insect antiviral compound may inhibit viral assembly and/or budding of viruses from host cells that could include the human immunodeficiency virus-1 (HIV-1) and herpes simplex virus-1 that use this motif for exit from a host cell. Using the formazan assay, the myristoylated-peptide was effective against HIV-1, with a nine times increase in the viability and protection in vitro of treated CEM-SS cells when compared with infected but untreated control cells.

Animals↗
PubMed2006

Mitochondria are a primary target of hypericin phototoxicity: synergy of intracellular calcium mobilisation in cell killing.

Hypericin, a naturally occurring anthraquinone synthesised by hypericum, upon light activation exhibits photodynamic cytotoxicity attributed mainly to the production of reactive oxygen species. This study aimed to elucidate the primary subcellular targets and mechanistic aspects of hypericin photosensitization in human prostate carcinoma cells. Depletion of intracellular glutathione (>85%) via inhibition of gamma-glutamyl-cysteine synthase had no effect on hypericin (5 microM) phototoxicity, thus precluding any direct oxidative involvement of H2O2. There was no change in intracellular SOD activity immediately after hypericin irradiation (1.5-5 J cm(-2)). Evaluation of the lysosomal enzyme hexosaminidase activity showed: (a) 60% cell loss 22 h following irradiation (1.5 J cm(-2)) and (b) a steady rate of lysosomal leakage to the cytosol (25%), at the same time and irradiation. However, lysosomal damage appears to be a slower process compared to the rapid loss of mitochondrial function, as reflected from parallel tetrazolium to formazan assays. The activity of cytosolic and mitochondrial aconitase, an enzyme exquisitely sensitive to oxidation, revealed a dose correlated loss of activity in the mitochondria immediately following hypericin photoactivation. The use of ionomycin, which modulates both internal Ca2+ stores and external Ca2+ transport during hypericin photosensitization, profoundly enhanced photocytotoxicity. Our data supports a direct mitochondrial hypericin phototoxicity that does not involve glutathione/H2O2 homeostasis. Further a potential synergistic treatment combining mitochondrial targeting of photosensitisers and Ca2+ mobilisation was identified.

Aconitate Hydratase↗
PubMed2006

Three-dimensional cell colonization in a sulfate rich environment.

Glycosaminoglycans (GAGs) have been explored for regenerating various tissues due to their involvement in diverse bioregulatory activity. However, understanding their influence on cell colonization in three-dimension (3-D) has been difficult due to variation in their molecular weight, degree of sulfation, and lack of in vitro models. This research focused on developing an in vitro model and evaluating the influence of MW (5, 10, and 500 kDa) of negatively charged dextran sulfate (DS), a semisynthetic GAG analog, on cell colonization. DS was combined with chitosan, a positively charged polymer in solution and porous 3-D matrices were formed inside 24-well plates using controlled rate freezing and lyophilization technique by two schemes: (i) chitosan structures were formed and then allowed to react with DS; (ii) DS was reacted with chitosan in solution and then matrices were formed. Scanning electron microscopy analysis showed that forming matrices after reacting DS with chitosan was more suitable for tissue regeneration. Analysis for the quantity and stability of DS by toluidine blue assay indicated significant presence of DS in the 3-D matrices even after seven days of incubation in phosphate buffered saline solution. Matrices formed by reacting 4% 5 kDa, 2% 10 kDa and 1% 500 kDa DS solution with chitosan had optimum porosity and mechanical stability. Next, 25,000 fibroblasts per matrix were seeded onto 3-D matrices and analyzed for proliferation by MTT-formazan assay, cytoskeletal organization by actin staining, and histological analysis by H/E staining. These results showed that cell growth was better on low MW containing 2-D membranes but high MW DS containing 3-D matrix supported cell growth similar to chitosan. Also, cells showed peripheral actin distribution in 3-D matrices. Analysis of fibronectin binding by ELISA showed negligible binding to all the DS-containing matrices, unlike chitosan. In summary, results show cell colonization on negatively charged matrices, similar to chitosan.

Animals↗
PubMed2006

Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: 2,3,5-Triphenyltetrazolium chloride (TTC) staining measures tissue viability used to evaluate infarct size. The goal of this study was to compare viability of neuronal tissue during the early phases of ischemia-reperfusion assessed either by perfusion of the brain with TTC solution transcardially, in vivo, or by staining brain slices, in vitro. METHODS: The middle cerebral artery was occluded for 1 h in male SPRD rats and then reperfused for 0, 1, 4, 8, 16 and 24 h. Ischemic damage was evaluated by TTC staining, in vivo and in vitro, and by histology (Luxol Fast Blue and Fluoro-Jade staining, electron microscopy). RESULTS: Core volume of tissue injury measured in vivo was large at 0 h and steadily decreased by 50% (p<0.001) up to 16 h, but substantially increased from 16 to 24 h of reperfusion. In contrast, a significant core volume appeared at 4 h only, in vitro, and gradually increased up to 24 h. Core volume was larger in vivo than in vitro at all times except at 16 h when the opposite was observed. Evans blue administered intracardially stained TTC-negative areas at 1 and 24 h. Histology covered the evolution of serious tissue injury but also demonstrated some morphologically preserved neurons in the infracted area at 24 h. CONCLUSIONS: Formation of formazan from TTC can depend on both the staining method and the metabolic burden of the brain tissue causing uncertainties in the volume of ischemia-induced brain injury measured by TTC staining.

Animals↗
PubMed2004

Role of NADPH oxidase-mediated superoxide production in the regulation of E-selectin expression by endothelial cells subjected to anoxia/reoxygenation.

OBJECTIVE: Anoxia followed by reoxygenation (A/R) increases endothelial cell superoxide (O2-) generation which is implicated in E-selectin overexpression. The mechanisms which govern these processes are not fully understood and therefore the goal of our study was to determine the functional importance of NADPH oxidase in the regulation of E-selectin expression in human umbilical veins endothelial cells (HUVECs) submitted to A/R. METHODS: O2- production was estimated using lucigenin chemiluminescence and formazan accumulation. NADPH oxidase expression in HUVECs was studied by RT-PCR and Western blot and E-selectin by Northern blot analysis. NFkappaB activation was assessed by electrophoretic mobility shift assay. RESULTS: A/R caused an increased O2- production which was inhibited by the superoxide dismutase mimetic M40403 (50 micromol/l), the protein kinase C inhibitor chelerythrine (10 micromol/l), the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 micromol/l) and the NADPH oxidase assembly blocker apocynin (600 micromol/l). At the end of the anoxic period, the mRNA expression and the protein p47phox was increased as compared to normoxic HUVECs. NFkappaB activation of anoxic HUVECs was maximal after 1 h of reoxygenation and returned to basal normoxic levels after 2 h of reoxygenation. Apocynin reduced the NFkappaB activation at 1 h of reoxygenation. E-selectin mRNA expression was increased after 3 h of reoxygenation of anoxic HUVECs and the SOD mimetic M40403 as well as apocynin prevented this overexpression. CONCLUSIONS: Activated NADPH oxidase is a critical enzyme in E-selectin overexpression after A/R of HUVECs. Moreover, A/R increased expression of membranous and cytosolic NADPH oxidase subunits as well as the protein p47phox. Strategies aimed at preventing endothelial NADPH oxidase activation and/or activity may be useful in controlling leukocyte adhesion during ischemia/reperfusion.

Blotting, Southern↗
PubMed2004

Modification of intracellular free calcium in cultured F2408 embryo fibroblasts by 3-substituted-2-thiohydantoin derivatives.

3-substituted-2-thiohydantoin derivatives were synthesized and their structures elucidated by IR, 1H-NMR spectroscopy and elemental analysis. The cytotoxicity of the 2-thiohydantoin derivatives to rat embryo fibroblasts (F2408) in vitro was determined, and the effects of these compounds on intracellular free Ca2+, [Ca2+]i, were measured by spectrofluorophotometry. Cytotoxicity was determined by metabolic reduction of a tetrazolium salt to a formazan dye (MTT assay). Compounds 4 and 7 showed cytotoxic activity, with IC50 values in the range of 1-1.2 microM. Introduction of either chlorophenyl, metoxyphenyl, nitrophenyl or benzyl groups at C-3 resulted in concentration-dependent cytotoxic effects. Compounds 1-6 at 1 microM or more significantly increased [Ca2+]i in a dose-dependent manner in the cultured fibroblasts. This action may have been mediated through intracellular calcium stores.

Animals↗
PubMed2005

Reduction of MTT by flavonoids in the absence of cells.

The tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) is used to determine cell viability in assays of cell proliferation and cytotoxicity. MTT is reduced in metabolically active cells to yield an insoluble purple formazan product. However, in this study, using a colorimetric method with MTT, we demonstrated that luteolin and quercetin (both are flavonoids) can reduce MTT in the absence of living cells. We also examined effects of some experimental conditions on the reaction, such as concentrations of flavonoids, incubation time, the structure and the existing form in solvents of flavonoids. The maximal absorbance values, which were 0.355 (luteolin) and 0.491 (quercetin), were observed when the concentrations of flavonoids were 200 microg/ml. Incubating MTT with flavonoids for 8 h, the absorbance values got the maximum, which were 0.320 (luteolin) and 0.398 (quercetin). The ability of flavonoids reducing MTT in RPMI-1640 with 10% fetal calf serum was higher than that in anhydrous ethanol. And the ability of quercetin reducing MTT was higher than that of luteolin both in RPMI-1640 with 10% fetal calf serum and anhydrous ethanol. Based on the results, this undescribed reaction can significantly influence the results of experiments using the MTT assay to measure the effects of flavonoids on cell growth.

Cell Survival↗
PubMed2004

Protective effect of taurine against free radicals damage in the rat myocardium.

Free radicals are highly cytotoxic to the heart and are involved in ischemia/reperfusion injury. In this study, we tested the ability of taurine to neutralize the deleterious effects of free radicals generated ex vivo and in vitro. Taurine was added at a concentration of 0.1 mM to the drinking water of experimental rats during 6 months. The animal hearts were then isolated and submitted to regional ischemia and reperfusion; ventricular fibrillation was significantly reduced as compared to a control group of non-treated animals. Moreover, at a concentration of 1 mM, taurine provided significant cardio-protection against the deleterious effect of free radicals generated by the electrolysis of Krebs-Henseleit buffer. When isolated hearts were perfused with electrolysed buffer, extensive fiber necrosis occurred, as observed by staining with nitro blue tertrazolium, a soluble dye which yields a dark blue formazan stain in the presence of reducing agents This stain was barely detectable when taurine was added to the perfusing electrolysed buffer. To further understand the protecting mechanism of taurine, we used xanthine-xanthine-oxidase as a superoxide (O2-) generating system and monitored the O2- through yield O2--dependent cytochrome c reduction. We demonstrated that taurine did not affect this system, which indicated that it did not scavenge O2- directly. On the other hand, taurine inhibited the auto-oxidation of adrenaline to adrenochrome at pH 7.8 where this auto-oxidation is O2--independent and superoxide dismutase insensitive. We thus conclude that taurine acts as a potent, but non-specific, scavenger of free radicals that cause heart damage and protects against reperfusion-induced ventricular

Administration, Oral↗
PubMed2004

APC0576 decreases production of pro-inflammatory chemokine and extracellular matrix by human Tenon's capsule fibroblasts.

PURPOSE: To control intraocular pressure in patients treated by trabeculectomy, progressive inflammation, fibroblast proliferation, and the enhanced expression of extracellular matrix (ECM), causes of scar formation at the bleb, must be prevented. Using human Tenon's capsule fibroblasts (TCFs), we examined the effect of APC0576, a suppressor of NF-kappaB-dependent gene activation of human vascular endothelial cells that does not adversely affect cell viability, on the production of pro-inflammatory chemokine and ECM. Its effect on TCF proliferation was also assessed. METHODS: Enzyme-linked immunosorbent assay was used to detect the pro-inflammatory cytokines IL-8 and MCP-1 in the supernatant of TCFs stimulated with IL-1alpha in the presence or absence of APCO576 and ECM proteins, COOH-terminal peptide of type I procollagen (PIP), fibronectin (FN), and laminin (LN) in supernatants and lysates of TCFs stimulated with IL-1alpha or TGF-beta. The proliferative response of IL-1alpha-stimulated TCFs was examined using the SF formazan solution reaction. RESULTS: APC0576 significantly suppressed the production by TCFs of IL-8 (p<0.0001), MCP-1(p<0.0001), PIP (supernatants: p<0.0001, cell lysates: p<0.0001), LN (supernatants: p<0.0001, lysates: p<0.0001), and FN (supernatants: p<0.0001, lysates: p<0.0001) as well as their proliferation (p<0.0001). CONCLUSIONS: APC0576 suppressed pro-inflammatory chemokine and ECM production in TCFs as well as their proliferation. It may represent a novel candidate for the postoperative management of patients treated by trabeculectomy.

Aged↗
PubMed2005

Separation of active and inactive fractions from starved culture of Vibrio parahaemolyticus by density dependent cell sorting.

The co-existence of physiologically different cells in bacterial cultures is a general phenomenon. We have examined the applicability of the density dependent cell sorting (DDCS) method to separate subpopulations from a long-term starvation culture of Vibrio parahaemolyticus. The cells were subjected to Percoll density gradient and separated into 12 fractions of different buoyant densities, followed by measuring the cell numbers, culturability, respiratory activity and leucine incorporation activity. While more than 78% of cells were in lighter fractions, about 95% of culturable cells were present in heavier fractions. The high-density subpopulations also had high proportion of cells capable of forming formazan granules. Although this was accompanied by the cell specific INT-reduction rate, both leucine incorporation rates and INT-reduction rates per cell had a peak at mid-density fraction. The present results indicated that DDCS could be used to separate subpopulations of different physiological conditions.

Cell Respiration↗
PubMed2006

An NADH-tetrazolium-coupled sensitive assay for malate dehydrogenase in mitochondria and crude tissue homogenates.

A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.

Animals↗
PubMed2004

Antiradical activity of different copper(II) Schiff base complexes and their effect on alloxan-induced diabetes.

Considering the important role of antioxidants in biological systems, the group of copper(II) complexes derived from salicylaldehyde and alpha- or beta-alanine and its thiourea derivative and copper(II) complexes derived from pyruvic acid and beta-alanine were studied. The antiradical activity of the tested compounds was studied by both in vitro and in vivo methods. The chemical methods based on inhibition of INT-formazane or 3-nitrotyrosine formation were used for the evaluation of SOD-mimic and antiperoxynitrite activity, respectively. In the case of in vivo activity evaluation, an alloxan-induced diabetes mellitus model in mice was used, the mechanism of action of alloxan being closely connected with the formation of free radicals selectively damaging the pancreatic beta-cells. Since all the substances studied showed different positive effects, it is obvious that they have not acted only as a source of copper(II) ions but their effect is related to their specific chelate structure. The obtained results are a contribution to the knowledge of copper(II) Schiff base complexes with ligands of aldimine or ketimine type and form the basis for further preclinical tests of these bioactive agents in biological models of oxidative stress.

Animals↗
PubMed2005

Comparison of three assays for the quantification of Candida biomass in suspension and CDC reactor grown biofilms.

A common assay to measure yeast metabolic activity in biofilms is based on the reduction of the tetrazolium salt XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} to a colored formazan. However, a recent report, also confirmed by our own findings about the shortcomings of the chromogenic XTT assay, has prompted us to investigate alternative methods for yeast biomass quantification. To this end, two fluorogenic assays using fluorescein diacetate (FDA) and SYTO 9 as well as the XTT assay were comparatively evaluated with regard to the linear range of Candida albicans and Candida parapsilosis cell number-response curves, precision and intra- and interspecies variability. Reading of fluorescence and absorbance was carried out in a multilabel microtiter plate reader. All three assays were adequate for the determination of planktonic yeast biomass, but the FDA and SYTO 9 assays present practical advantages. When applied to the quantification of yeast biofilm biomass obtained in the CDC biofilm reactor, the FDA assay proved superior.

Biofilms↗
PubMed2005

Development of a semi-automated colorimetric assay for screening anti-leishmanial agents.

MTS or {3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl}-2H-tetrazolium, inner salt) is converted into soluble formazan by mitochondrial dehydrogenase of viable cells, thus serving as an indicator of cell viability. Accordingly, a MTS-based assay was developed to evaluate anti-leishmanial activity in Leishmania promastigotes from strains responsible for visceral, cutaneous or mucocutaneous leishmaniasis. The assay was initially optimized for the appropriate wavelength (490 nm), culture medium (M-199), incubation time (3 h) and temperature (37 degrees C). Increasing absorbance with increasing cell density confirmed linearity of the assay that was maintained up to 2.5 x 10(6) cells/200 microl. The growth kinetics of six L. donovani strains and six non-L. donovani strains consistently indicated higher absorbances in the L. donovani strains highlighting the importance of strain-specific customization of the MTS assay. The IC(50) values (i.e., the concentration at which 50% of growth was inhibited) of amphotericin B, miltefosine and pentamidine isethionate obtained by the MTS assay corroborated with previously published data. Taken together, the MTS assay thus permits a simple, reproducible and reliable semi-automated method for evaluating cell viability, effective for drug-screening and growth kinetic studies.

Amphotericin B↗
PubMed2006

Response to copper stress in aposymbiotically grown lichen mycobiont Cladonia cristatella: uptake, viability, ergosterol and production of non-protein thiols.

The mycobiont of lichens usually determines the morphology of the symbiotic organism and is also dominates in terms of biomass. However, its role for sensitivity or tolerance of lichens to heavy metals is almost unknown. In the present study, the influence of copper (Cu) on the aposymbiotically-grown mycobiont of Cladonia cristatella was assessed. Intracellular Cu uptake was correlated with increasing Cu concentrations over a 24-h exposure time. Viability, measured as the degree of reduction of triphenyltetrazolium chloride to triphenyl formazan, as well as to ergosterol levels, decreased with growing Cu concentrations tested. Reduced glutathione (GSH) was found to be the most abundant low-molecular-weight thiol in the hyphae of C. cristatella and its intracellular content increased at concentrations of 10mum Cu. Higher Cu concentrations caused a significant decrease in GSH, possibly due to heavy metal-induced oxidation of GSH to glutathione disulphide (GSSG). Free cysteine levels were relatively constant. As expected, we did not observe the production of phytochelatins in the mycobiont, contrary to what is found in intact lichens and axenic cultures of their photobionts.

Ascomycota↗
PubMed2005

Development of a modified MTT assay for screening antimonial resistant field isolates of Indian visceral leishmaniasis.

The semi-automated MTT colorimetric assay has previously been applied on Leishmania promastigotes based on the ability of viable parasites to reduce the tetrazolium salt to an insoluble formazan product. As promastigotes are non-adherent, application of the MTT assay in its original form has a major drawback of a high and variable background absorbance due to incomplete removal of phenol red, a component of most media. We have accordingly optimised a modified MTT assay wherein the absorbance linearity was maintained for cells ranging from 1x10(4) to 1x10(7) being 0.04+/-0.003-2.38+/-0.04. In contrast, the original MTT assay had a narrower linearity range of 1x10(6)-1x10(7) cells, absorbances being 0.05+/-0.005-1.54+/-0.005. The modified MTT assay was effectively applied to study growth kinetics and identification of antimonial resistant field isolates. Considering the growing problem of antimonial unresponsiveness in the Indian subcontinent, this modified MTT assay is a useful tool for Leishmania research.

Amphotericin B↗