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PubMed2006

Silicone based polyurethane materials: a promising biocompatible elastomeric formulation for cardiovascular applications.

The biocompatibility of a new material for cardiovascular applications constituted by a poly(ether)urethane (PEtU) and a silicone [polydimethylsiloxane (PDMS)] was evaluated. The achieved material shows properties similar to both polyurethanes and silicones. The material was transformed into porous membranes by a spray-deposition technique. Since any material preparation and manufacturing procedure may introduce some toxicity, in vitro cytotoxicity screening tests were carried out. Human umbilical vein endothelial cells (HUVECs) and a mouse fibroblasts cell line (L929) were cultivated with extracts obtained from materials containing 10, 40 and 100% (w/w) of PDMS. The commercially available Estane 5714-F1 and Cardiothane 51 were used as controls. Extracts were incubated up to 72 hours with HUVECs and L929 cells. The cytotoxic effect was evaluated by light microscopy, cell viability (MTT reduction and neutral red uptake) and proliferation (5-bromo-2'-deoxyuridine incorporation) tests. In vivo studies were carried out using materials containing the same PDMS percentages as for in vitro experiments. The same commercial controls were used. Results obtained with cell culture studies agreed with those obtained in the in vivo experiments and showed that the material preparation and manufacturing procedure do not introduce any toxicity in the products at each PDMS concentration investigated.

Animals↗
PubMed2006

Promising in vitro performances of a new nickel-free stainless steel.

Stainless steel is a metallic alloy largely employed in orthopaedics. However, the presence in its composition of a high quantity of nickel, an agent known to trigger toxic and allergic responses, is cause for concern. In this study, we have investigated the in vitro biocompatibility of a new nickel-reduced stainless steel, namely Böhler P558, in comparison to the conventional stainless steel AISI 316L. The neutral red (NR) uptake and the amido black (AB) tests were performed on L929 fibroblasts and MG63 osteoblasts to assess the cytotoxicity, while cytogenetic effects were evaluated on CHOK1 cells by studying the frequency of Sister Chromatid Exchanges (SCE) and chromosomal aberrations. Ames test was used to detect the mutagenic activity. The expression of selected markers typical of differentiated osteoblasts, such as alkaline phosphatase activity (ALP), type I collagen (CICP) and osteocalcin (OC) production, were also monitored in MG63 cells cultured on the tested materials. Our results indicate the absence of significant cytotoxicity and genotoxicity for both test alloys. ALP, CICP and OC analyses confirmed that both materials support the expression of these phenotypic markers. Overall, these data show that this Ni-free alloy possesses good in vitro biocompatibility and could have a potential for orthopaedic applications.

Animals↗
PubMed1999

Toxicity of ifosfamide and its metabolite chloroacetaldehyde in cultured renal tubule cells.

Renal injury is a common side effect of the chemotherapeutic agent ifosfamide. Current evidence suggests that the ifosfamide metabolite chloroacetaldehyde may contribute to this nephrotoxicity. The present study examined the effects of ifosfamide and chloroacetaldehyde on rabbit proximal renal tubule cells in primary culture. The ability of the uroprotectant medication sodium 2-mercaptoethanesulfonate (mesna) to prevent chloroacetaldehyde-induced renal cell injury was also assessed. Chloroacetaldehyde (12.5-150 microM) produced dose-dependent declines in neutral red dye uptake, ATP levels, glutathione content, and cell growth. Coadministration of mesna prevented chloroacetaldehyde toxicity while pretreatment of cells with the glutathione-depleting agent buthionine sulfoximine enhanced the toxicity of chloroacetaldehyde. Ifosfamide (1000-10,000 microM) toxicity was detected only at concentrations of 4000 microM or greater. Analysis of media collected from ifosfamide-treated cell cultures revealed the presence of several ifosfamide metabolites, demonstrating that renal proximal tubule cells are capable of biotransforming this chemotherapeutic agent. This primary renal cell culture system should prove useful in studying the cause and prevention of ifosfamide nephrotoxicity.

Acetaldehyde↗
PubMed1990

Separation and culture of living adrenaline- and noradrenaline-containing cells from bovine adrenal medullae.

Separation of viable adrenaline-containing from noradrenaline-containing chromaffin cells in large amounts has been achieved. The procedure involves collagenase digestion of bovine adrenomedullary tissue, isolation of cells through gentle filtration, separation of chromaffin from nonchromaffin cells on discontinuous gradients of the radiopaque contrast Renografin, and separation of adrenaline-enriched from noradrenaline-enriched fractions after centrifugation on self-generated Percoll gradients. Collection of 1-ml Percoll fractions gave two clear-cut catecholamine peaks. The denser peak was enriched in adrenaline and phenylethanolamine-N-methyltransferase (PNMT), suggesting that over 90% of cells were adrenergic. The lighter peak was preferentially enriched in noradrenaline but not in PNMT. With this information, we could collect by gentle aspiration two main fraction layers of larger volumes; one at the bottom of the Percoll gradient, which contained essentially adrenaline-storing cells and the other at the top of the gradient, enriched in noradrenaline cells. Those cells could be maintained viable for at least 1 week in primary monolayer cultures, as shown by neutral red staining and trypan blue exclusion. This method will allow the identification of chemical components, receptors, or ionic channels present in one specific type of cell, to determine their relevance to the regulation of the differential secretion of specific materials present in one but not in the other cell type and to ascertain whether the released materials from one cell type affect the functions of the other.

Adrenal Medulla↗
PubMed1992

Application of the chemiluminescent assay to cytotoxicity test: detection of menadione-catalyzed H2O2 production by viable cells.

Menadione-catalyzed H2O2 production by viable cells is proportional to viable cell number. The correlations between the viable cell number and the concentration of H2O2 produced are determined with the rapid chemiluminescent assay (S. Yamashoji, T. Ikeda, and K. Yamashoji, 1989, Anal. Biochem. 181, 149-152). This chemiluminescent assay of viable cells requires only 10 min and is much faster than NR (neutral red) inclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays, which require 3-5 h. When viable cells are incubated with antitumor drugs, detergents, mycotoxins, and glycoalkaloids for 24-48 h, a decrease in menadione-catalyzed H2O2 production in a dose- or incubation time-dependent manner is observed. In general, the 50% inhibition concentration determined by the chemiluminescent assay is lower than that determined by NR inclusion and MTT reduction assays, and the order of relative cytotoxic effects of agents is the same among these assays. Furthermore, clear cytotoxic effects are observed by the chemiluminescent assay after 1 h exposure of trypsinized cells to toxic compounds. Therefore, the chemiluminescent assay is expected to be more useful for the rapid detection of cytotoxic compounds than NR inclusion and MTT reduction assays.

3T3 Cells↗
PubMed1984

Variable fluorescence of photosystem I particles and its application to the study of the structure and function of photosystem I.

Chlorophyll a fluorescence in Photosystem I (PSI) particles isolated according to the method of Bengis and Nelson [J. Biol. Chem. 252, 4564-4569 (1977)] was found to be dependent on the redox state of both P700 and X (an acceptor on the reducing side of PSI). Addition of dithionite plus neutral red to PSI caused an increase in fluorescence intensity and a shift of the main fluorescence peak from 689 to 674 nm. Addition of electron acceptors such as ferredoxin and methyl viologen decreased the fluorescence yield when added to PSI incubated under anaerobic conditions in the presence of excess dichlorophenol indophenol (DCIPH2). The Km for ferredoxin agreed with that determined from direct measurements of ferredoxin reduction, showing that X is a quencher of fluorescence. P700 was also found to be a quencher of fluorescence, since electron donors such as DCIPH2, TMPD, and plastocyanin decreased fluorescence with Km's nearly identical to those observed for P700+ reduction. Chemical modification of PSI (with ethylene diamine + a water-soluble carbodiimide) to make it positively charged increased the fluorescence yield and shifted the 689-nm peak to 674 nm. The Km's for DCIPH2 and ferredoxin were decreased. In contrast, modification of PSI with succinic anhydride, which increased the net negative charge, increased the Km for ferredoxin. Salts affected the interaction of methyl viologen with PSI. Both anion and cation selectivity were observed. Limited proteolysis increased the Km for both methyl viologen and ferredoxin, indicating that their binding site on PSI was altered. These results suggest that the binding site for ferredoxin is on either the 70- or the 20-kDa subunit of PSI.

Chlorophyll↗
PubMed1979

Absorption changes of P-700 reversible in milliseconds at low temperature in Triton-solubilized photosystem I particles.

Triton-solubilized Photosystem I particles from spinach chloroplasts exhibit largely reversible P-700 absorption changes over the temperature range from 4.2 K to room temperature. For anaerobic samples treated with dithionite and neutral red at pH 10 and illuminated during cooling, a brief (1 microseconds) saturating flash produces absorption changes in the long wavelength region that decay in 0.95 +/- 0.2 ms from 4.2 to 50 K. Above 80 K a faster (100 +/- 30 microseconds) component dominates in the decay process, but this disappears again above about 180 K. The major decay at temperatures above 200 K occurs in about 1 ms. The difference spectrum of these absorption changes between 500 and 900 nm closely resembles that of P-700. Using ascorbate and 2.6-dichlorophenolindophenol as the reducing system with a sample of Photosystem I particles cooled in darkness to 4.2 K, a fully reversible signal is seen upon both the first and subsequent flashes. The decay time in this case is 0.9 +/- 0.3 ms.

Chlorophyll↗
PubMed1981

The thermodynamic properties of some commonly used oxidation-reduction mediators, inhibitors and dyes, as determined by polarography.

The oxidation-reduction midpoint potentials (Em) of the following compounds have been measured in the range of pH from 3 to 12 by polarography: methyl viologen; benzyl viologen; 2-hydroxy-1,4-naphthoquinone; 2-hydroxy-1,4-anthraquinone; N,N,N',N',-tetramethyl-p-phenylenediamine; 2,3,5,6-tetramethyl-p-phenylenediamine; phenazine; N-methylphenazonium methosulfate; N-methylphenazonium sulfonate methosulfate; N-ethylphenazonium ethosulfate; pyocyanine; neutral red; safranin; phenol red; chlorophenol red; cresol red; bromocresol purple; 2,5-dibromo-3-methyl-6-isopropylbenzoquinone and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. Many of these previously assumed to have a simple behavior in this range have proven to be rather more complicated, and several anomalous observations have been reconciled.

Dibromothymoquinone↗
PubMed1981

Fusion of Avena sativa mesophyll cell protoplasts by electrical breakdown.

Studies with the light microscope were carried out on mesophyll cell protoplasts of Avena sativa which had been made to undergo fusion by reversible electrical breakdown of the cell membrane. In order to establish close membrane contact between the cells, an important prerequisite for fusion, a method known as dielectrophoresis was used. In an inhomogeneous alternating electrical field the protoplasts adhere to the electrodes and to each other in the direction of the field lines. The cells which were thus brought into close contact with each other could be made to fuse by the application of a field pulse of high amplitude (about 750 V/cm) and short duration (20-50 mus). The field strength required for fusion exceeds the value necessary for the electrical breakdown of the cell membrane. Fusion took place within some minutes and led to a high yield of fused protoplasts. The fusion of cells being in the electric field occurred in a synchronous manner. In some of the fusion experiments part of the protoplasts of A. sativa were stained with neutral red. When these cells were fused with unstained protoplasts, the vacuoles from the different cells within the fused aggregate could be shown to remain separate for quite some time.

Cell Fusion↗
PubMed1989

Electric pulse induced membrane permeabilization. Spatial orientation and kinetics of solute efflux in freely suspended and dielectrophoretically aligned plant mesophyll protoplasts.

Asymmetric breakdown (occurring in only one hemisphere of the cell) was induced in freely suspended and dielectrophoretically aligned vacuole-containing or evacuolated plant protoplasts as well as in isolated vacuoles. In suspended cells breakdown was restricted to the hemisphere facing the anode and in isolated vacuoles to the opposite hemisphere. This difference in the orientation of the asymmetric breakdown can be explained by the opposite direction of the intrinsic membrane potentials of isolated vacuoles and of cells on which the generated potential difference is superimposed. The ensuing permeabilization of the membrane was microscopically monitored by dye uptake and by release of chloroplasts and of cytoplasmic and/or vacuolar solutes. The asymmetric release of intracellular substances (organic acids and/or amino acids) was detected by accumulation of chemotactic bacteria (Pseudomonas aeruginosa) close to the permeabilised membrane area of the cells or vacuoles. Maximum bacteria accumulation required about 5 min and subsequently disappeared after a further 20 min presumably because of the restoration of the original membrane impermeability. With vacuoles retention of the accumulated bacteria was shorter indicating that the resealing process of the tonoplast membrane was faster than that of the plasmalemma. From the kinetics of bacteria accumulation and retention it is therefore possible to deduce information about the life-span and the resealing properties of electropermeabilized membrane areas on the single-cell level. Symmetric breakdown in both hemispheres of the cells could be achieved by electric field-mediated cell rotation of about 180 degrees between two pulses of the same polarity or by application of two pulses of alternating polarity. In dielectrophoretically aligned protoplasts of comparable diameter, breakdown occurred in both hemispheres, even though the breakdown was still asymmetric. It could be demonstrated by the uptake of the vital dye neutral red that the size of the membrane area which was permeabilized was much larger in that hemisphere oriented to the anode than in the other one. The relevance of these observations for further improvement of electroinjection of macromolecules and of electrofusion is discussed. In particular, it is pointed out that positioning of differently sized cells in electric field-mediated hybridisation and the polarity of the breakdown pulse is of great importance with respect to hybrid yield.

Cell Membrane Permeability↗
PubMed1981

Enzymatic synthesis in biphasic aqueous-organic systems. II. Shift of ionic equilibria.

Ionic equilibria in "water-water-immiscible organic solvent" systems have been studied. It has been shown that in such systems shift of the apparent pK value of acids and bases takes place (compared to aqueous solutions), the value of the shift being rather high, up to 5 and more pH units (with 2,4-dinitrophenyltryptophan as an acid and neutral red as a base). The pK shift of ionogenic reagents observed in biphasic systems can be used in preparative organic synthesis for increasing the yield of end products in enzyme-catalyzed reactions. In connection with this, the physico-chemical reasons for the equilibrium shift in a chemical reaction that involves one or two ionogenic reagents are theoretically analyzed. The above approach has been tested with two alpha-chymotrypsin-catalyzed reactions, i.e., synthesis of N-benzoyl-L-phenylalanine ethyl ester (from NBz-LPhe-OH and ethanol) and synthesis of N-acetyl-L-tryptophanyl-L-leucine amide (from NAc-LTrp-OH and LLeu-NH2). In water the equilibria in these reactions are shifted almost entirely towards the starting reagents with the yield of end product being negligibly low. In biphasic systems consisting of chloroform +5% (v/v) water or ethyl acetate +2% (v/v) water, the yield of both the ester and the dipeptide reaches 100%.

Animals↗
PubMed1986

Leydig cells: octopaminergic neurons in the leech.

Leydig cells are electrically-coupled neurons in the segmental ganglia of the leech. This study reports that they stain specifically with high concentrations (0.05 mg/ml) of the dye neutral red, and that they contain octopamine, as demonstrated by radioenzymatic assay. Individual cell bodies were pooled and found to contain 0.37 pmol octopamine/cell body, giving an approximate intracellular concentration of 7.75 mM. Leydig cell bodies contain approximately 75% of the octopamine content of a segmental ganglia. Intracellular injection of large amounts of Lucifer yellow, coupled with long diffusion times, revealed a previously-undescribed Leydig cell process. The probability that Leydig cells are octopaminergic neurosecretory cells is discussed.

Action Potentials↗
PubMed1993

Photochemically induced focal cerebral ischemia in rat: time dependent and global increase in expression of basic fibroblast growth factor mRNA.

Induction of basic fibroblast growth factor (bFGF) mRNA expression was studied in a Rose bengal induced focal cerebral ischemia during a time course of 2, 4, 24, 72 h and 7 days. Focal cerebral ischemia induced by Rose bengal resulted in a global upregulation in bFGF gene expression at the 24 h time-interval. This upregulation in bFGF gene expression was due to an upregulation in glial bFGF expression in most of the areas studied as seen by means of non-radioactive in situ hybridization in combination with immunocytochemistry for glial fibrillary acidic protein. However, in the piriform cortex a putative neuronal upregulation of bFGF could be detected by combination of non-radioactive in situ hybridization, immunohistochemistry for glial fibrillary acidic protein and nuclear staining with Neutral red. Semiquantitative data concerning bFGF mRNA expression were obtained by use of computer-assisted microdensitometry and revealed substantial increases in bFGF mRNA expression in the cingulate cortex, the neostriatum, a 1 mm marginal zone close to the external capsule and the olfactory tubercle at bregma levels 1 to 2 mm rostral to the lesion. No changes in bFGF gene expression were seen in field CA1 of Ammon's horn on the lesioned side and in dentate gyrus at bregma levels between -2.12 to -3.30 mm. We observed significant changes in bFGF upregulation in the caudate putamen, the piriform cortex and the amygdaloid region and the frontoparietal cortex at bregma levels -2.12 to -3.30 mm. These data indicate that photochemically induced focal cerebral ischemia leads to an early and global response in bFGF gene expression, which is due to an upregulation mainly in astrocytes. The observed widespread upregulation of the bFGF gene transcription rostral and caudal to the lesion is suggested to be due in part to neuronal glutaminergic connections between the areas investigated and in part due to increases in extracellular fluid signals (volume transmission).

Animals↗
PubMed1988

Studies on the relationship of human natural killer and lymphokine-activated killer cells with lysosomal staining and analysis of surface marker phenotypes.

We have previously demonstrated that natural killer (NK) cells are lysosome-rich and stain more intensely with lysosomotropic agents such as neutral red and quinacrine (Qu) than do non-NK cells. In this study we combined the quantitation of Qu staining with surface marker staining to define subpopulations of NK cells. While all NK activity was contained within the Qu+ population, most but not all NK cells expressed the surface marker CD16. A subpopulation of NK cells was found to be Qu+CD16- composed of medium- to large-sized cells with a granular appearance on Giemsa staining. Culture with interleukin-2 (IL-2) induced enhanced cytotoxicity in peripheral blood lymphocytes (PBL) against NK-sensitive and NK-resistant tumor cells. Like NK cells, these lymphokine-activated killer (LAK) cells were predominantly Qu+CD16+. However, some LAK cells were Qu+CD16-. The Qu+CD16+ cells were typical large granular lymphocytes (LGL). The Qu+CD16- cells were also large lymphocytes, more than 50% of which were proliferating. However, the granulation in some Qu+CD16- cells, as detected by Giemsa staining, was more prominent and numerous than others in the same population. No LAK activity was ever detected in Qu- cells, which were uniformly small lymphocytes. Quantitation of Qu staining in effector cells was therefore demonstrated to have a good correlation with NK and LAK functions, and with surface markers can help to characterize both types of cells. Moreover, these results indicate that both NK and LAK populations include a small subset of CD16- cells in each.

Antigens, Surface↗
PubMed1992

Thawed human hepatocytes in primary culture.

In drug metabolism studies, isolated and cultured human hepatocytes provide a useful model for overcoming the difficulty of extrapolating from animal data. In vitro studies with human hepatocytes are scarce because of the lack of livers and suitable methods of storage. After developing a new method for cryopreservation of human hepatocytes, we evaluated the effects of deep freezing storage on their viability, morphology, and functional and toxicological capabilities in classical culture conditions. Freshly isolated human hepatocytes were cryopreserved in medium containing 10% Me2SO and 20% fetal calf serum, using a Nicool ST20 programmable freezer (-1.9 degrees C/min for 18 min and -30 degrees C/min for 4 min). Cells were stored in liquid nitrogen. Viability of thawed human hepatocytes was 50-65% as assessed by erythrosin exclusion test prior to purification on a Percoll density gradient. Morphological criteria showed that thawed human hepatocytes require an adaptation period to the medium after seeding. Functional assessments showed that human hepatocytes which survive freezing and thawing preserve their protein synthesis capabilities and are able to secrete a specific protein, anionic peptidic fraction, which is involved in the hepatic uptake of bile-destined cholesterol. We then studied Midazolam biotransformation to test metabolic functions, and erythromycin toxicity by Neutral Red test (cell viability) and 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide test (cell metabolism). All of these experiments indicated that thawed human hepatocytes should be used 38 h after seeding for optimum recovery of their functions: membrane integrity, protein synthesis, and stabilization of drug metabolism enzymes.

Biotransformation↗
PubMed1995

Flupirtine protects neurons against excitotoxic or ischemic damage and inhibits the increase in cytosolic Ca2+ concentration.

We tested the effect of flupirtine against ischemic and excitotoxic neuronal damage as well as on the glutamate-induced rise in cytosolic calcium ion concentration (= [Ca2+]i). For in vivo experiments we used a model of focal cerebral ischemia in mice. The middle cerebral artery was permanently occluded and 48 h afterwards brain tissue was stained with neutral red, perfusion-fixed and the infarct surface was determined planimetrically. Pretreatment with flupirtine significantly reduced the infarct area (controls: 24.3 +/- 4.8 mm2, 1 mg/kg flupirtine: 20.1 +/- 3.6 mm2 and 10 mg/kg flupirtine: 19.5 +/- 3.9 mm2; P < 0.05), whereas postischemic application of flupirtine failed to reduce the infarct area. For in vitro studies, primary neuronal cultures were prepared from the hippocampi of newborn rats and excitotoxic damage was induced by exposing the cells to 500 mu M L-glutamate for 30 min. We could demonstrate that flupirtine (1-10 microM) was capable of protecting neurons against glutamate-induced cytotoxicity. In order to elucidate the underlying mechanism of action, we tested the effect of flupirtine on the glutamate-induced rise in [Ca2+]i using the Ca2+-indicator fura-2. L-Glutamate added in a final concentration of 100 microM to the cultured cells for 16 s caused a rise in [Ca2+]i from about 100 nM to 900 nM. Flupirtine (0.1-10 microM) reduced the glutamate-induced rise in [Ca2+]i concentration dependently.

Aminopyridines↗
PubMed1995

Evidence for a cGMP gated cation channel in photoreceptor cell membranes of Sepia officinalis.

It is assumed that cyclic nucleotides are involved in signal transduction of invertebrate photoreceptors. In this study, membranes of photoreceptor cells from freshly caught cuttlefish were isolated, and the membrane proteins were reconstituted into proteoliposomes. With the dye Neutral red it was possible to measure cyclic nucleotide induced Na+ fluxes into the liposomes. cGMP and cAMP concentrations for half maximal activation of Na+ fluxes are 77 microM and 224 microM, respectively, with Hill coefficients of 2.0 for cGMP and 2.4 for cAMP. These fluxes may demonstrate the presence of at least one cyclic nucleotide gated cation channel in the membranes of the photoreceptor cells of the invertebrate Sepia officinalis.

Animals↗
PubMed1981

A modified 'low pH' lignocaine method to isolate human monocytes: a comparison with other separation procedures.

A modified 'low pH' lignocaine method for the recovery of human monocytes from untreated plastics is described and compared with other adherence separation procedures, viz. mechanical scraping by a rubber policeman, microexudate-coated plastic method and pretreatment of plastics by the fetal calf serum (FCS). Monocytes separated by each of the above-mentioned techniques carried out in parallel were characterised by morphological and functional criteria: non-specific esterase staining, contamination by T- and B-lymphocytes, viability, adherence, neutral red uptake, phagocytosis of yeast particles, random mobility, chemotaxis, and tumoricidal activity both in the absence and in the presence of interferon and lymphokines. The 'low pH' lignocaine method appears to be a simple and reproducible technique for the isolation of human monocytes. It seem preferable to other adherence procedures inasmuch as it yields viable, pure and functionally intact monocytes without requiring preconditioned plastics.

Blood↗