Mycobacterium avium-intracellulare contamination of mammalian cell cultures.

Mycobacterium avium contamination has been described as a putative contaminant of nonphagocytic mammalian cells. Screening of numerous cultured nonphagocytic mammalian cell lines revealed the presence of intracellular bacteria that were identified as M. avium-intracellulare. An extensive and critical analysis of the origin of infection, of cure protocols, and of biological manifestations in M. avium-infected cells is presented. As no tremendous visible alteration of turbidity or pH of cell culture media, and no morphological change occurred in most M. avium-infected cell cultures, detection of an infection by these bacteria is rather difficult. Recommendations are given for treatment of irreplaceable cultures and prevention of mycobacterial contamination in a tissue culture facility.

Effects of culture conditions on taurine uptake by various variants of human endometrial carcinoma cells in culture.

The general properties of the taurine uptake in human endometrial tumoral Ishikawa cells were similar to those usually found in other tissues. Uptake was notably affected by the oxygen pressure, being higher at the physiological pO(2) of the endometrium (40 mm Hg, equivalent to 5% O(2)) compared to that used under standard experimental culture conditions (160 mm Hg or 20% O(2)). Uptake of taurine was also density-dependent in Ishikawa cells and was significantly decreased at confluence. Uptake regulation by PKC driven phosphorylation occurs only in growing cells and not in resting cells. The taurine uptake of three Ishikawa cell lines was very different. The taurine uptake of one of the cell lines was affected by estradiol, probably through a non-genomic pathway, whereas tamoxifen had no effect in all cell lines.

Establishment of human tumoral ependymal cell lines and coculture with tubular-like human endothelial cells.

Ependymomas, rare neoplasms of the central nervous system, occur predominantly in children. They are highly vascularized, and histological findings show many perivascular rosettes of tumoral cells radially organized around capillaries. Treatment of ependymomas relies on surgery combined with radio- or chemotherapy, but the efficiency of chemotherapy is limited, probably because of their multidrug resistance (MDR) phenotype. Progress in the therapy of these neoplasms is dramatically limited by the absence of cell line models. We established conditions for the long-term culture of human tumoral ependymocytes and their 3D coculture in Matrigel with endothelial cells. Histological, immunological, and ultrastructural studies showed that the morphological features (microvilli, cilia, and caveolae) of these cultured cells were similar to those of the tumor in vivo. The cells expressed potential oncological markers related to the immature state of tumoral cells (nestin and Notch-1), their tumorigenicity [caveolae and epidermal growth factor-receptor (EGF-R)], or the MDR phenotype [P-glycoprotein (P-gp)]. The expression of P-gp, EGF-R, and caveolin-1 by these tumoral ependymocytes could be useful in studies on new drugs. This coculture model might represent a new powerful tool to study new therapeutic delivery strategies in tumoral cells.

Sensitivity of taurine uptake to oxygen-derived reactive substances in MDR and non-MDR cells.

In human KB and LoVo cell lines, high affinity taurine uptake was strongly reduced in both a time and dose-dependent manner by cumene hydroperoxide (CH) and to a lesser extent by hydrogen peroxide (H2O2). Uptake-inhibition was greater in multidrug resistant (MDR) cells than in their non-MDR counterparts. Basal taurine efflux was unaffected by the oxidants. Lipid peroxidation levels closely correlated with the uptake inhibition levels, and were greater in MDR cells than in their non-MDR counterparts. The two oxidants reduced the Vmax and, to a lesser extent, the affinity of the transporter for taurine. They also reduced low affinity taurine uptake and, to a lesser extent, taurine diffusion. The composition of the medium used for cell treatment, especially its pyruvate content, greatly affected the H2O2 effect. H2O2- or CH-induced reduction of the high affinity taurine uptake was unaffected by protein kinase C (PKC) inhibitors and by the calmodulin antagonist W-13, ruling out the involvement of PKC and perhaps of calmodulin kinases in their effect.

Characterisation of taurine uptake in human KB MDR and non-MDR tumour cell lines in culture.

Studies on taurine transport in MDR (multidrug resistant) and non-MDR KB cancer cells show that both cell types contain Na(+)- and Cl(-)-dependent high-affinity and low-affinity transport systems selective for beta-amino acids and a taurine diffusion component. Good buffers, such as HEPES or MOPS, interfered with taurine uptake. The basal taurine Vmax measured in isoosmotic medium represented 1/5 of taurine captured by uptake in KB non-MDR, but was negligible in KB MDR cells. High-and low- affinity uptake systems were reduced by medium hyperosmolarity in both cell types. Although properties of low-and high-affinity transport systems were similar in both cell types, Vmax (but not Km) were reduced in MDR compared to non-MDR cells. Taurine uptake was unaffected by chemotherapeutic agents (doxorubicin, vinblastine) or MDR revertants (verapamil). Taurine did not affect cell proliferation of MDR or non-MDR cells nor did it alter the inhibitory effect of doxorubicin or vinblastine cell proliferation.

Detailed study of the different taurine uptake systems of colon LoVo MDR and non-MDR cell lines.

In human, physiological taurine requirement is partly dependent on nutrition. Study of the human carcinoma LoVo cells shows the presence of a high and a low affinity taurine uptake. Besides them, a diffusion system has been found. A detailed analysis of the properties of the three systems is presented. A comparison of LoVo chemosensitive cells, and LoVo chemoresistant (MDR) cells which overexpress the multidrug transporter P-glycoprotein, shows that the only difference between the two cell types belong to the kinetic properties of the high and low affinity taurine uptake systems.

pH drift of "physiological buffers" and culture media used for cell incubation during in vitro studies.

In pharmacological or toxicological studies performed at room atmosphere comparison of various media used for cell incubation revealed discrepancies among results due to pH instability when these media contain bicarbonate. With the classically used protocols, a relatively fast and notable rise of the pH of such media has been observed, and values higher than 8.5 could be reached after 1 h of incubation. A less important rise in pH was also observed for media containing low amounts of sodium bicarbonate, e.g., Hank's formula-derived media. Because Hepes-buffered media or media with abnormal osmolarity cannot always be used for such studies, our choice of media is limited.

In vitro taurine uptake into cell culture influenced by using media with or without CO2.

Buffers used to incubate cells for pharmacological or toxicological studies are usually of very simple composition, far from the composition of biological fluids or cell culture media. Comparative studies on taurine uptake levels by cultured cells show that a new CO2-Independent Medium (CIM) is suitable for incubating cells in place of the Krebs-Ringer buffer (KR) usually used. Basal uptake level of taurine was lower for cells incubated in CIM or in other culture media when compared to those incubated whether in KR or in other "physiological buffers." Isoproterenol depressed similarly the taurine uptake in cells incubated in CIM or KR. The same uptake modulation by beta-alanine, GES, GABA, or HEPES was observed for cells incubated in CIM or KR. C6 cells growth in CIM was dependent on the starting cell density when classically vented T-flasks were used, growth being notably reduced at low density. In tightly closed flasks cells grew in CIM similarly to control cultures maintained in M199 medium or DMEM.

Differential effect of ebselen on compound 48/80- and anti-IgE-induced histamine release from rat peritoneal mast cells.

2-Phenyl-1, 2-benzisoselenazol-3-(2H)one (ebselen), a nontoxic seleno-organic compound, exhibits anti-inflammatory activity through the inhibition of many enzymes involved in inflammation. In view of the role played by histamine in the pathophysiology of inflammation, we looked at the effect of ebselen on histamine secretion by rat peritoneal mast cells. It inhibited compound 48/80-induced histamine release in a concentration-dependent manner. Half-maximal and maximal (100%) inhibitory response occurred at 5.10(-7)M and 10(-5)M, respectively. In contrast, ebselen was without any effect on histamine release induced immunologically. Prevention of the inhibitory effect of ebselen by GSH suggests that it interacts with critical thiols involved in the compound 48/80 activation pathway.

Non-myotendinous force transmission in rat extensor digitorum longus muscle.

The extensor digitorum longus muscle (EDL) of the rat hindleg consists of four heads. The heads are named after their insertions on the digits of toes II, III, IV and V. The EDL heads share a proximal tendon and aponeurosis, but have separate distal aponeuroses and tendons. By cutting the distal tendons of selected heads, direct myotendinous force transmission within these heads is prevented. Therefore, force exerted by the muscle would be expected to decrease according to the physiological cross-sectional area disconnected if myotendinous force transmission were the only mechanism of force transmission. The results indicate that EDL force production remained at high levels after acute tenotomy: muscle length-force curves did not alter significantly following cutting of the tendons of heads II and III. Cutting the tendon of head IV as well leaves only head V in its original condition. After tenotomy of head IV, length-force characteristics were altered significantly, but optimum force was maintained at 84% of that of the intact muscle. After separation of head IV from head V intramuscularly for some distance along their interface, the force dropped to much lower levels, with optimum force approaching 50% of that of the intact muscle. The length of active proximal fibres (located within head II) did not remain constant but increased with increasing muscle lengths after tenotomy as well as after partial separation of heads IV and V. The amount of length change decreased after intramuscular separation of the heads, indicating declining reactive forces. It is concluded that force transmission occurred from tenotomized heads to their intact neighbours and vice versa. The magnitude of the force transmitted from head to head was dependent on the degree of integrity of the connective tissue at the interface between heads.

Regulation of taurine transport in rat astrocytes by protein kinase C: role of calcium and calmodulin.

Phorbol 12-myristate 13-acetate, a potential stimulator of protein kinase C (PKC), inhibited taurine uptake in rat astrocytes. This effect was mimicked by 1-oleoyl-2-acetyl-sn-glycerol, an endogenous stimulator of PKC, and by r-59949, an inhibitor of diacylglycerol kinase. Maximal inhibition was obtained at microM phorbol 12-myristate 13-acetate (PMA) after 1 h of treatment. This effect was prevented by pretreatment of the cells with chelerythrine, a potent and selective inhibitor of PKC. The transport of beta-alanine, an amino acid that shares the same transporter as taurine, was inhibited to a comparable extent. The effect of PMA was potentiated by cotreatment of the cells with thapsigargin or the Ca2+ ionophore A-23187. However, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N1,N1-tetraacetic acid and verapamil did not prevent the PMA effect. Pretreatment of the cells with calmodulin antagonists W-13 or calmidazolium, prevented the PMA-induced inhibition of taurine uptake. This inhibition was not affected by cycloheximide, actinomycin D, colchicine, or cytochalasin D. The Na(+)-to-Cl(-)-to-taurine coupling ratio was unaffected. Dimethyl amiloride, a selective inhibitor of Na+/H+ antiport, was unable to prevent the effects of PMA. These effects were associated with a decrease in the maximal velocity and an increase in the Michaelis-Menten constant.

Activation of protein kinase C down-regulates glial but not neuronal taurine uptake.

Phorbol myristate acetate (PMA), a protein kinase C (PKC) activator significantly decreased in a time- and dose-dependent manner taurine uptake by rat astroglial but not neuronal cells. The PMA-induced inhibition of taurine uptake by rat astrocytes was prevented by chelerythrine, a potent and selective inhibitor of PKC. The differential effect of PMA on rat neuronal and astroglial taurine transport was also obtained with the protein phosphatase inhibitor okadaic acid. This was not only the feature of rat cells since the same differential effects were obtained with human glioma GL15 and human neuroblastoma IMR32 cell lines. The results suggest that the neuronal and astroglial taurine transporter may be structurally different.

Characterization of taurine transport in human glioma GL15 cell line: regulation by protein kinase C.

Data describing characteristics of taurine transport system in human brain cells are not currently available. We have used GL15 cells, a cell line of human brain origin that keeps some properties of normal glial cells, to investigate these characteristics. The human glioma cell line GL15 was found to take up taurine. The uptake was strictly sodium-dependent. Replacement of NaCl with choline chloride almost totally abolished the uptake. There was also an anion requirement for the uptake system, and Cl- was the most potent among several monovalent anions tested. The uptake process was specific for beta-amino acids such as taurine, hypotaurine and beta-alanine. The kinetics of uptake were studied. Apparently, a single transport system with a K(m) of 8.95 +/- 0.26 microM was responsible for the uptake. A maximal velocity of 1.32 +/- 0.03 nmol/mg of protein/10 min was found. Stoichiometric analysis revealed that two Na+ and one Cl- ions were involved in the translocation of one taurine molecule. Phorbol 12-myristate 13-acetate (PMA), a potent stimulator of protein kinase C (PKC), inhibited taurine uptake. Maximal inhibition was obtained at 50 nM after 1 hr of treatment. This effect was prevented by pretreatment of the cells with chelerythrine, a potent and selective inhibitor of PKC. The transport of beta-alanine was inhibited to a comparative extent. The mechanism of this inhibition was not investigated, but it was found that this inhibitory effect was not prevented by cycloheximide, actinomycin D, colchicine or cytochalasin D, indicating that neither protein synthesis, nor microfilament function were involved. The effect of PMA was associated with an impairment of kinetic constants. It is concluded that human GL15 cells have a taurine transporter similar to that expressed in rodent glial cells, and that the activation of PKC can modulate the activity of this transporter.

Effect of ammonia on endocytosis and cytokine production by immortalized human microglia and astroglia cells.

Ammonium acetate decreased in a concentration-dependent manner the phagocytic uptake of mannosylated latex microspheres and of yeast by immortalized human microglia (CHME-5) and astroglioma (GL-15) cells. In both cell lines ammonium acetate affected also the secretion of certain cytokines. The most conspicuous effects were the following: in both cell lines ammonium acetate enhanced greatly the secretion of tumor necrosis factor-alpha in the absence of any other stimulus. in the human microglia cells ammonia decreased the constitutive secretion of interleukin-6, but it enhanced the stimulated (interleukin-1 alpha, tumor necrosis factor-alpha, gamma-interferon and gamma-interferon + tumor necrosis factor-alpha) secretion of interleukin-8. In the astroglioma cell line, the stimulated release of tumor necrosis factor-alpha, interleukin-6 and interleukin-8 was diminished by ammonium acetate. The magnitude of the ammonia-effect depended on the stimulating agent (lipopolysaccharide, interleukin-1 alpha, tumor necrosis factor-alpha, gamma-interferon). The results are discussed with regard to their potential importance in the pathogenesis of human diseases with elevated blood and brain ammonia concentrations.

Membrane properties of cultured glial cells obtained mechanically or by enzymatic dissociation: differential alterations by gangliosides.

Rat astroglial cells were obtained either mechanically or by enzymatic dissociation and some properties of the primary and secondary cultures were studied. Differences in the ganglioside amount, taurine uptake, membrane fluorescence anisotropy, or their respective modulation by total brain gangliosides confirmed that although the two types of cultures are morphologically similar, they are biochemically distinct.

Taurine analog modulation of taurine uptake by two different mechanisms in cultured glial cells.

Previous data have shown that HEPES, a taurine structural analog, inhibits the uptake of taurine by cultured cells differently, depending on its addition either to the culture medium or to the Krebs-Ringer buffer used for cell incubation during taurine uptake measurements (Lleu and Rebel, J Neurosci Res 23: 78-86, 1989). An extensive study of the effect of numerous other taurine structural analogs on taurine uptake by cultured glial cells was carried out. Our results show that taurine uptake modulation by structural analogs follows two different mechanisms. For the first mechanism, observable after the simultaneous presence of taurine and of its analog during the incubation time of the uptake experiment (10 min), the amine function on the molecule is essential. The sulfonate group could be replaced either by a sulfinic group or by a carboxylic group. beta-Alanine, hypotaurine, acetyltaurine, guanidinoethanesulfonate and guanidinopropionate are the most potent inhibitors in this first mechanism. For the second mechanism, which requires the presence of the analog in the culture medium during the 48 hr preceding the taurine uptake measurement, the simultaneous presence of an amine and of a sulfonate group or of an amine and a sulfinate group is required. Carboxylates are ineffective in modulating taurine uptake in this mechanism. The sulfonate buffers synthesized by Good et al. (Biochemistry 5: 467-477, 1966) also affect taurine uptake in both mechanisms.

Changes in S-type lectin localization in neuroblastoma cells (N1E115) upon differentiation.

The distribution of a 14.4 kDa S-type lectin was examined in murine neuroblastoma cells, either undifferentiated or after differentiation induced by dibutyryl-cyclic adenosine monophosphate. In undifferentiated cells the immunoreactivity was detected extracellularly, associated with the plasma membrane and in bulges released into the extracellular milieu. Important modifications of the lectin localization were associated with the differentiation process that induced an increased cytosolic expression and a decreased externalization. Possible functions for the lectin expressed intracellularly in the differentiated cells are also considered.

Effect of ammonia on endocytosis, cytokine production and lysosomal enzyme activity of a microglial cell line.

Ammonia is a natural lysosomotropic compound. Concentrations of ammonium acetate > 2 mM impaired the phagocytic activity of BV-2 cells, an immortalized microglial cell line, as was determined by the uptake of fluorescent latex microspheres of different sizes. In contrast, an increase in the uptake of fluorescent dextran was observed with the elevation in ammonium acetate concentrations. This indicates that ammonia affects phagocytotic and pinocytotic activities of BV-2 cells differently. Interferon-gamma- and polyinosinic-polycytidylic acid-stimulated secretion of IL1 alpha as well as LPS-stimulated secretion of IL6 decreased with an elevation in ammonium acetate concentrations. The constitutive secretion of IL1 alpha was not significantly affected by ammonium acetate. However, an increase in LPS-stimulated IL1 alpha secretion was observed at 10 mM and 20 mM ammonium acetate. High concentrations of ammonia affected the activity of lysosomal enzymes of the BV-2 cells. Acid phosphatase and alpha-glucosidase activities increased with the increase in ammonium acetate up to 20 mM. The activity of cathepsin D was increased at 5 mM, but decreased at higher ammonia concentrations. The effects of ammonia on microglial functions are discussed with respect to pathogenetic mechanisms of dementia of the Alzheimer type.