DMEM Cell Culture Medium Research Articles

Aminocarbyne-Alkyne Coupling in Diruthenium Complexes: Exploring the Anticancer Potential of the Resulting Vinyliminium Complexes and Comparison with Diiron Homologues.

New diruthenium complexes based on the scaffold Ru2Cp2(CO)2 (Cp = η5-C5H5) and containing a bridging vinyliminium ligand, [2a-d]CF3SO3, were synthesized through regioselective coupling of alkynes with an aminocarbyne precursor (85-90% yields). The reaction involving phenylacetylene proceeded with the formation of a diruthenacyclobutene byproduct, [4]CF3SO3 (10% yield). Complexes [2a-d]+ undergo partial alkyne extrusion in contact with alumina or CDCl3. All products were characterized by elemental analysis, infrared and multinuclear NMR spectroscopy, and single crystal X-ray diffraction in two cases. Complexes [2a-d]+ revealed an outstanding stability in DMEM cell culture medium at 37 °C (<1% degradation over 72 h). These complexes exhibited cytotoxicity in human colon colorectal adenocarcinoma HT-29 cells in the low micromolar range, with lower IC50 values than those obtained with the homologous diiron complexes previously reported. Evaluation of ROS (reactive oxygen species) production and O2 consumption rate (OCR) highlighted the higher potential of Ru2 complexes, compared to the Fe2 counterparts, to impact mitochondrial activity, with the heterometallic Ru2-ferrocenyl complex [2d]+ showing the best performance.

Bioactivity of cerium dioxide nanoparticles as a function of size and surface features.

Nano-dispersed cerium dioxide is promising for use in medicine due to its unique physicochemical properties, including low toxicity, the safety of in vivo usage, active participation in different redox processes occurring in living cells, and its regenerative potential, manifested in the ability of CeO2 to participate repeatedly in redox reactions. In this work, we examined the biological activity of cerium dioxide nanoparticles (CeO2 NPs) synthesized by precipitation in mixed water/alcohol solutions at a constant pH of 9. This synthesis method allowed controlling the size and Ce3+/Ce4+ proportion on the surface of NPs, changing the synthesis conditions and obtaining highly stable suspensions of "naked" CeO2 NPs. Changes in the surface properties upon contact of CeO2 NPs with protein-rich media, e.g., bovine serum albumin and DMEM cell culture medium supplemented with 10% fetal bovine serum, the characteristics of nanoparticle uptake by mouse aortic endothelial cells and the antioxidant activity of the nanoparticles of different sizes were investigated by various state-of-the-art analytical methods.

In vitro detection of nanoparticle cytotoxicity based on big data technology

Nanoparticles have been used in the cytotoxic test for many times because of their easy size control, good biocompatibility and other characteristics. Therefore, a method based on big data technology for the in vitro cytotoxic test of nanoparticles is proposed. The cells were evenly inoculated into 96 well plate, the prepared AuNPs was diluted with DMEM cell culture medium, and the cytotoxicity was divided into 5 levels by 5-point system. A database model of cytotoxic characteristics of gold nanoparticles (AuNPs) was built to mine the toxic characteristics of cultured cells. The results showed that when the diameter and size of AuNPs were 1.8 nm, the cytotoxicity rating was the highest, which was 4. When the diameters of AuNPs were 18 nm and 27 nm, the cytotoxicity rating was 0. When the high concentration DMEM solution of 90 μg/mL was used to treat AuNPs, the cytotoxicity reached the highest value, and the lowest activity was 12%. When the cells were treated with DMEM solution for 24 hours, the activity and toxicity of the cells were the least and the toxicity was the strongest, and the number of apoptosis in the cell community was significantly reduced under the action of AuNPs.

Hypoxic preconditioning enhances the differentiation of bone marrow stromal cells into mature oligodendrocytes via the mTOR/HIF‐1α/VEGF pathway in traumatic brain injury

ObjectiveTraumatic brain injury (TBI) results not only in gray matter damage, but also in severe white matter injury (WMI). Previous findings support hypoxic preconditioning (HP) could augment the efficacy of bone marrow stromal cell (BMSC) transplantation in a TBI mouse model. However, whether HP‐treated BMSCs (H‐BMSCs) could overcome remyelination failure after WMI is unclear, and the molecular mechanisms remain to be explored. Here, we focused on the therapeutic benefits of H‐BMSC transplantation for treating WMI, as well as its underlying mechanisms.MethodsIn vitro, BMSCs were incubated at passage 4 in the hypoxic preconditioning (1.0% oxygen) for 8 hr. In vivo, a TBI mouse model was established, and DMEM cell culture medium (control), normal cultured BMSCs (N‐BMSCs), or H‐BMSCs were transplanted to mice 24 hr afterward. Neurobehavioral function, histopathological changes, and oligodendrogenesis were assessed for up to 35 days post‐TBI.ResultsCompared with the control group, improvement of cognitive functions and smaller lesion volumes was observed in the two BMSC‐transplanted groups, especially the H‐BMSC group. H‐BMSC transplantation resulted in a greater number of neural/glial antigen 2 (NG2)–positive and adenomatous polyposis coli (APC)–positive cells than N‐BMSC transplantation in both the corpus callosum and the striatum. In addition, we observed that the expression levels of hypoxia‐inducible factor‐1a (HIF‐1α), phosphorylated mechanistic target of rapamycin (p‐mTOR), and vascular endothelial growth factor (VEGF) were all increased in H‐BMSC–transplanted mice. Furthermore, the mTOR pathway inhibitor rapamycin attenuated the impact of HP both in vivo and in vitro.ConclusionThe results provided mechanistic evidences suggesting that HP‐treated BMSCs promoted remyelination partly by modulating the pro‐survival mTOR/HIF‐1α/VEGF signaling pathway.

Selection of extraction medium influences cytotoxicity of zinc and its alloys

Zinc (Zn) alloys have been considered as promising absorbable metals, mainly due to their moderate degradation rates ranging between magnesium alloys and iron alloys. The degradation behavior depends on the specific physiological environment. Released metallic ions and corrosion products directly influence biocompatibility. The initial contact of orthopedic implants or vascular stents after implantation will be with blood. In this study, fetal bovine serum (FBS) was used as a model system of blood components. We investigated the influence of FBS on in vitro degradation behavior and cytotoxicity of pure Zn, and Zn-4Ag and Zn-2Ag-1.8Au-0.2 V (wt%) alloys. The initial degradation rates in FBS were assessed and compared with the degradation and toxicity in four other common physiological model systems: DMEM cell culture medium ± FBS and McCoy's 5A medium ± FBS. Test samples in pure FBS showed the highest initial degradation rates, and accordingly, FBS supplemented media accelerated the degradation process as well. Moreover, an extract test according to ISO 10993-5 and -12 with L929 and Saos-2 cells was performed to investigate the role of FBS in the extraction medium. The cytotoxic effects observed in the tests were correlated with FBS-mediated Zn2+ release. These findings have significant implications regarding the selection of appropriate media for in vitro degradation and cytotoxicity evaluation of Zn and its alloys. Statement of SignificanceMetallic zinc and its alloys have been considered as promising biodegradable metals, mainly due to their moderate degradation rates. However, in vitro cytotoxicity tests according to the current ISO 10993 standard series are not suitable to predict biocompatibility of Zn alloys due to the inconsistent correlation between in vitro and in vitro biocompatibility. In this study, we show that the outcomes of standardized in vitro cytotoxicity tests of Zn and Zn alloys are influenced by fetal bovine serum in the extraction vehicle because FBS promotes Zn2+ release during the extraction process. The results of the study provide significant information for selection of appropriate model systems to evaluate in vitro degradation behavior and cytotoxicity.

P043 Secretome modulation of Caco-2 cell line induced by a multi-strain probiotic

Abstract Background Probiotics are defined as live, non-pathogenic bacteria that confer health benefits beyond their nutritional value. Particularly VSL#3, a probiotic mix containing 4 strains of Lactobacilli (L. paracasei, L. plantarum, L. acidophilus and L. delbrueckii subsp. bulgaricus*), 3 strains of Bifidobacteria (B. longum**, B. infantis***, B. breve) and Streptococcus thermophilus, has demonstrated efficacy in the management of diseases characterised by increased intestinal permeability such as irritable bowel syndrome and ulcerative colitis. *Recently reclassified as L. helveticus. **Recently reclassified as B. longum subsp. lactis. ***Recently reclassified as B. infantis subsp. lactis. The aim of the present study was to study secreted bioactive factors to evaluate the mechanisms of action of VSL#3 to enhance intestinal epithelia. Methods Two different lots of VSL#3 ( Nutrilinea Srl, Gallarate (VA), Italy, lot #802097 and lot #802100) were used. Caco-2 cell line were treated with a conditioning media (CM) prepared using 1 g of probiotic formula grown in D-MEM cell culture medium (free of serum and antibiotics) at 37°C for 48 h without shaking and in anaerobic conditions. Caco-2 were treated with diluted CM at 1:10 and 1:25 for 24 and 48 h. Media culture for each conditions has been collected and analysed by a deeper proteomics approach. Differential protein expression in was evaluated by shotgun proteomics analysis based on nLC-HDMSE and carried out on Synapt G2-Si mass spectrometer. Protein identification and protein expression analysis were perfomed by Protein Linx Global Server (PLGS v. 3.0.3, Waters Corp). Results The analysis of supernatants from Caco-2, treated with CM, showed the presence of bacteria strain-specific proteins. Human proteins synthesised from CaCo-2 were also identified, such as caspase 1, IL8, HSP70, HSP70b, HSP90, HSP105. The production were time- and dose- dependent. In CM diluted 1:10, probiotic derived proteins have been shown to be more expressed at 24 h. Human caspase 1, IL8, HSP 70, HSP 70b, HSP 90, HSP 105 were also found up-regulated in CaCo-2 treated for 24 h with CM diluted 1:10. Conclusions This is the first time where a probiotic secretome was explored. The study on probiotic secretome is useful to understand if the probiotic was well reconstituted. Analysis of secretome from CaCo-2 treated with CM helped us to understand the mechanism by probiotics can enhance intestinal barrier: by strengthen the autophagy process, an arm of innate immunity, by overexpression of caspase 1, IL8 and HSP 70, and by HSPs dependent modulation of inflammation.

Spectral Characteristics of the DMEM Cell-Culture Medium

Spectral characteristics of luminescence of the DMEM cell-culture medium are studied. The luminescence spectrum of the medium is determined in the visible and near-IR spectral ranges at wavelengths of up to 1350 nm under excitation at 405 and 660 nm. The lifetimes of excited states of the medium are determined, and the absence of singlet oxygen phosphorescence at about 1270 nm is demonstrated.

P026 The impact of trophic effects of multi strain probiotic preparation on paracellular permeability

Abstract Background Probiotics are defined as live, non-pathogenic bacteria that confer health benefits beyond their nutritional value. Particularly VSL#3, a probiotic mix containing four strains of Lactobacilli (L. paracasei, L. plantarum, L. acidophilus, and L. delbrueckii subsp. bulgaricus*), three strains of Bifidobacteria (B. longum**, B. infantis**, B. breve) and Streptococcus thermophilus, has demonstrated efficacy in the management of diseases characterised by increased intestinal permeability such as irritable bowel syndrome and ulcerative colitis. The aim of the present study was to evaluate the mechanism of action of VSL#3 to reduce intestinal permeability, and its effect in modulating the activity on tight junctions, focusing on the effects of secreted bioactive factors. *Recently reclassified as L. helveticus. **Recently reclassified as B. animalis subsp. lactis. Methods Two different lots of VSL#3 (Manufacturer: Nutrilinea Srl, Gallarate (VA), Italy) were used. Caco2 and HT29 cell lines were treated with a conditioning media (CM) prepared using 1g of probiotic formula grown in D-MEM cell culture medium (free of serum and antibiotics) at 37°C for 48 h without shaking and in anaerobic conditions. The effects of probiotic on proliferations of cells were evaluated by cell growth curve and MTT assay. The effect on apoptosis will be analysed by cytometry using double staining with Annexin V-FITC and Propidium Iodide, as well as it is well established. Furthermore, the effect on expression of tight junctions, in particular claudin-2, occludin, and ZO-1, will be investigated by western blot analysis. Results The multistrain probiotic formula, in particular the more concentrate CM (1:10) increase Caco2 and HT29 cells proliferation and the percentage of metabolic cell activity (up 20%), together with a decrease of 90% of apoptotic cells in the presence of CM. These trophic effects may have an impact on paracellular permeability. The expression of tight junction proteins increase in 24 h for claudin and ZO-1, and after 48 h of treatment in case of occludin. Also the densitometry and the ratio between of relative density of protein and endogenous control (β-actin) confirms these data. The results are the same using two different lots. Conclusions These preliminary data in vitro could be able to explain how VSL#3 works at intestinal mucosa level, in particular by secretion of factors that enhances barrier integrity. The proliferative stimuli and the increase of expression of tight junction proteins are consistent with an effect on mucosa regeneration and re-epithelization. Other experiments, also on other intestinal cell line, are necessary to deeply understand the mechanism of action of this kind of probiotic formula.

Effects of Tacrolimus or Sirolimus on the adhesion of vascular wall cells: Controlled in-vitro comparison study.

In drug eluting stents the cytostatic drugs Sirolimus or Tacrolimus are used to inhibit blood vessel restenosis by limiting the proliferation of smooth muscle cells. However, the cytostatic activity of both drugs was shown to be not cell specific and could also affect the stent endothelialisation, respectively. Currently, only limited in vitro data are available about the impact of Sirolimus and Tacrolimus on endothelial cell proliferation over a broad concentration range. To answer this question the following study was performed.Commercially obtained HUVEC were expanded with DMEM cell culture medium (GIBCO, Germany) supplemented with 5 vol% fetal calf serum on non-coated regular polystyrene-based 24-multiwell plates. For drug testings 2×104 cells/cm2 were seeded and grown for 24 h until 30-40% of the multiwell surfaces were covered and then exposed to Sirolimus (1.0×10-11 - 1.0×10-5 mol/l) or Tacrolimus (2.0×10-8 - 6.2×10-5 mol/l), both dissolved in DMSO. 12, 24 and 48 h after adding the drugs cell numbers per area were quantified by counting the cells in six wells with four fields of view per well, representing 0.6 mm2, using a confocal laser microscope.After 48 h of cell growth in the drug-free cell culture medium, the HUVEC number increased from 2.0×104 to 3.55×104 cells/cm2 (mean cell doubling time: 53.6 h, n = 6). At lower concentrations (≤2.0×10-6 mol/l) Tacrolimus reduced the number of adherent HUVEC significantly less than Sirolimus (p < 0.05). However, at higher concentrations (≥2.07×10-5 mol/l) the effect of Tacrolimus on the number of adherent endothelial cells was significantly greater than that of Sirolimus (p < 0.05). At the highest concentration applied (6.22×10-5 mol/l), Tacrolimus induced detachment of all HUVECs within 12 h after drug application. The number of adherent HUVEC decreased only slightly (about 9%) after Sirolimus application at the highest concentration (1.09×10-5 mol/l).These data show that in a non-flow model the cytostatic drug Tacrolimus reduced the number of adherent endothelial cells less than Sirolimus, as long as the drug concentration did not surpass 10-6 mol/l. At the limits of solubility, Sirolimus (1×10-5 mol/l) reduced the number of adherent endothelial cells less than Tacrolimus (6×10-5 mol/l), which induced detachment of endothelial cells.

The photodynamic therapy activity of 3-(1-hydroxylethyl)-3-devinyl-131-(dicyanomethylene) pyropheophorbide-a methyl ester (HDCPPa) against HeLa cell in vitro

Photodynamic therapy (PDT) has been a potential therapeutic method for the treatment of various cancers, with photosensitizer being the key component in photodynamic therapy. In this paper, we prepared a photosensitizer 3-(1-hydroxylethyl)-3-devinyl-131-(dicyanomethylene) pyropheophorbide-a methyl ester (HDCPPa), based on chlorophyll pyropheophorbide-a according to the previous report, and systematically investigated the fluorescence emission spectrum and ultraviolet absorption spectrum HDCPPa has long absorption in the near-infrared spectral region (around 695 nm). The excitation wavelength and the emission wavelength were 415 nm and 699 nm respectively in dichloromethane, 1O2 quantum yield was 63.5%. HDCPPa also had high stability in PBS solution, DMEM cell culture medium and normal saline (NS) in vitro. After irradiation by the light of 675 nm (10 J.cm[Formula: see text]) for 70 min the degradation rate of HDCPPa was 12.5%, which indicated that the target compound showed high stability under light. The in vitrophotodynamic therapy activities against HeLa cells were also studied, which showed that HDCPPa had extremely low dark toxicity but great phototoxicity, and the cell viability is lower than 10% under the light irradiation of 675 nm (10 J.cm[Formula: see text]). Moreover, HDCPPa can quickly enter the cell after being incubated with HeLa cells in less than 30 min. We also evaluated the mechanism of the photochemical reaction, which had proved that Type II is primarily responsible for the cell death. Therefore HDCPPa could serve as a very promising photosensitizer for photodynamic therapy.

In vitro ion adsorption and cytocompatibility of dicalcium phosphate ceramics

BackgroundIn vitro cell testing of degradable bioceramics such as brushite or monetite is often challenging due to the ion release into or adsorption from the culture medium. These ionic changes are then mostly responsible for cell proliferation and activity, which prohibits the investigation of effects originating from surface topography or further material modifications.MethodsHere, we aimed to solve this problem by developing a pre-conditioning regime following the repeated immersion of brushite and monetite samples in various Ca2+, Mg2+ and PO43− containing electrolytes, followed by studying ion adsorption / release as well as changes in phase composition and in vitro cytocompatibility with MG63 cells.ResultsThe results demonstrated that by using DMEM cell culture medium in a ratio of 10 ml/sample was sufficient to minimize changes of ionic composition after 7 d with a daily change of the medium. This leads to changes of the surface composition with dissolution of the brushite phase. In turn, this also positively influences the in vitro cytocompatibility with a 2–3 fold higher cell number and cell activity on the DMEM pretreated surfaces.ConclusionsControlled sample washing prior to cell testing using DMEM medium seems to be a valuable procedure not only to stabilize the pH during cell culture but also to maintain ion concentrations within a cell friendly range.

Hypoxic Preconditioning Augments the Therapeutic Efficacy of Bone Marrow Stromal Cells in a Rat Ischemic Stroke Model.

Transplantation of bone marrow stromal cells (BMSCs) is a promising therapy for ischemic stroke, but the poor oxygen environment in brain lesions limits the efficacy of cell-based therapies. Here, we tested whether hypoxic preconditioning (HP) could augment the efficacy of BMSC transplantation in a rat ischemic stroke model and investigated the underlying mechanism of the effect of HP. In vitro, BMSCs were divided into five passage (P0, P1, P2, P3, and P4) groups, and HP was applied to the groups by incubating the cells with 1% oxygen for 0, 4, 8, 12, and 24h, respectively. We demonstrated that the expression of hypoxia-inducible factor-1α (HIF-1α) was increased in the HP-treated BMSCs, while their viability was unchanged. We also found that HP decreased the apoptosis of BMSCs during subsequent simulated ischemia-reperfusion (I/R) injury, especially in the 8-h HP group. In vivo, a rat transient focal cerebral ischemia model was established. These rats were administered normal cultured BMSCs (N-BMSCs), HP-treated BMSCs (H-BMSCs), or DMEM cell culture medium (control) at 24h after the ischemic insult. Compared with the DMEM control group, the two BMSC-transplanted groups exhibited significantly improved functional recovery and reduced infarct volume, especially the H-BMSC group. Moreover, HP decreased neuronal apoptosis and enhanced the expression of BDNF and VEGF in the ischemic brain. Survival and differentiation of transplanted BMSCs were also increased by HP, and the quantity of engrafted BMSCs was significantly correlated with neurological function improvement. These results suggest that HP may enhance the therapeutic efficacy of BMSCs in an ischemic stroke model. The underlying mechanism likely involves the inhibition of caspase-3 activation and an increasing expression of HIF-1α, which promotes angiogenesis and neurogenesis and thereby reduces neuronal death and improves neurological function.

In vitro selection and characterization of deoxyribonucleic acid aptamers against connective tissue growth factor

Connective tissue growth factor (CTGF) is a secreted matricellular protein possessing complex biological functions. CTGF modulates a number of signaling pathways that are involved in cell adhesion, migration, angiogenesis, myofibroblast activation, extracellular matrix deposition and tissue remodeling. Aptamers are oligonucleic acid chains or polypeptides that bind with specific target molecules hence have the potential to be used in the detection and blockade of the targets. In this study, we selected CTGF-targeting DNA aptamers by using systematic evolution of ligands by exponential enrichment (SELEX). After 8 iterative rounds of selection, cloning, DNA sequencing and affinity determination, six aptamers with high affinities to CTGF were obtained. Among them, one (C-ap17P) binds with the N-terminal region (aa 1-190) and the other five (C-ap11, 12, 14, 15 and 18) bind with the C-terminal region (aa 191-350) of hCTGF specifically. The biological stability assay indicated that a representative aptamer, C-ap17P, could keep its integrity at a rather high level for at least 24 h in complete DMEM cell culture medium. These CTGF aptamers might be used as a easy and fast detection tool for CTGF and be developed as CTGF-specific inhibitors for both research works and clinical applications.

Abstract 413: Genome-wide microarray expression and array-CGH analysis in neuroblastoma stem-like cells by use of the neurosphere-formation assay

Abstract In this work, we aimed at analyzing genome-wide gene expression and DNA gains and losses in neuroblastoma cell lines and in neurospheres containing stem-like cells. SKNDZ and SIMA neuroblastoma cell lines were grown in DMEM cell culture medium, and in DMEM-F12 selective serum free medium (with EGF, bFGF and B27; to induce the neurosphere-forming phenotype). After RNA and genomic DNA extractions from the 4 cell lines (2 standard, 2 neurosphere-forming cell lines), expression microarrays and array-CGH analyses were performed (Aligent Technologies). Array-CGH did not show any significant differences between standard and neurosphere-forming cell lines, both in SKNDZ and in SIMA. Microarray expression analysis demonstrated a total of 425 upregulated and 170 downregulated genes in neurosphere-forming cell lines. The differentially expressed genes were classified using the PANTHER classification system (www.pantherdb.org). As a result, apoptosis, cell adhesion, cell communication, cell cycle, and immune system processes appeared upregulated and downregulated in neurospheres. Some of those genes participate in pathways related with cancer (Table 1). In conclusion, the stem-like phenotype does not seem to be linked to anatomic changes at the level of deletions/gains of DNA, but to changes in expression of genes, like those of the TGF-beta, Notch and Sonic Hedgehog pathways. Those pathways, specially TGF-beta, widely described as an important therapeutic target against cancer, should be further studied to determine their real implication in the neurosphere-formation process in neuroblastoma, and therefore, as candidate targets for the treatment of neuroblastoma stem-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 413. doi:1538-7445.AM2012-413

Delta-Opioid Receptor Activation Promotes Mesenchymal Stem Cell Survival via PKC/STAT3 Signaling Pathway

The survival of stem cells upon transplantation into ischemic myocardium is a major concern in cell-based therapy. In this study, we tested the hypothesis that activation of opioid receptors would enhance the survival of mesenchymal stem cells (MSCs) upon exposure to an injury stimulus. MSCs were obtained from rat bone marrow and cultured in basal DMEM cell culture medium. Delta-opioid receptor (DOR) was present in MSCs as examined by reverse transcription-polymerase chain reaction and immunochemistry. Activation of DOR with 5µmol/L SNC80 (DOR agonist) for 24h significantly enhanced MSC viability upon exposure to 5µg/ml actinomycin D as determined by TUNEL and MTT assays. The cytoprotection was abolished with 20µmol/L naltrindole hydrochloride (a DOR antagonist). Treatment of the cells with 1.5µmol/L chelerythrine (protein kinase C inhibitor) and 1.25µmol/L WP1066 (signal transducer and activator of transcription 3 (STAT3) inhibitor) blocked SNC80-induced cytoprotection. Furthermore, treatment of the cells with chelerythrine also blocked STAT3-phosphorylation and Mcl-1 gene expression. Taken together, the results indicate that DOR plays a critical role in MSC survival upon exposure to actinomycin D through activation of protein kinase C and its downstream signaling molecules STAT3 and Mcl-1. DOR may be a novel therapeutic target for stem cell survival during cell-based therapy.

Impact of the Streptococcus pyogenes Mga regulator on human matrix protein binding and interaction with eukaryotic cells

The Streptococcus pyogenes (group A streptococci, GAS) stand-alone Mga regulator has been shown to positively control surface-expressed virulence factors like the antiphagocytic M protein during exponential growth phase and thus, was implicated to contribute to the acute infection process. In the present study, we generated mga mutants as well as mga promoter – luciferase reporter fusions in weakly and strongly encapsulated serotype M2 and M49 GAS strains. Employing the luc reporter fusions, we showed that the complex growth medium THY-broth decreased mga expression and identified albumin as one component responsible for this effect. Fibrinogen and c U50980omponents of the complex DMEM cell culture medium induced the mga transcription rate. The attachment of mga mutants to immobilized human matrix proteins (collagen type I, fibronectin, keratin, laminin) and serum proteins (albumin, fibrinogen) was consistently reduced. Changing the Mn 2+ or Ca 2+ growth medium concentrations did not affect the fibronectin/collagen binding of M49 GAS wild-type and mga mutant strains. Medium supplementation with the oxidative stressor paraquat or anaerobic growth on THY-agar led to a relatively increased human matrix protein binding of the mga mutant. Opposite to their matrix protein-binding behaviour, the M2 and M49 mga mutants displayed an increased attachment and internalization rate for eukaryotic cells. The host cell viability was considerably reduced after prolonged exposure to mga mutants. By generating and testing corresponding M protein gene ( emm) mutants, features of the eukaryotic cell interaction could not be associated to the Mga – M protein regulatory axis. In conclusion, the present results support the postulated central role of Mga regulation for GAS host colonization and acute infection stages.

Evidence for a Serum Factor That Initiates the Re-calcification of Demineralized Bone

The present studies show for the first time that demineralized bone re-calcifies rapidly when incubated at 37 degrees C in rat serum: re-calcification can be demonstrated by Alizarin Red and von Kossa stains, by depletion of serum calcium, and by uptake of calcium and phosphate by bone matrix. Re-calcification is specific for the type I collagen matrix structures that were calcified in the original bone, with no evidence for calcification in periosteum or cartilage. Re-calcification ceases when the amount of calcium and phosphate introduced into the matrix is comparable to that present in the original bone prior to demineralization, and the re-calcified bone is palpably hard. Re-calcified bone mineral is comparable to the original bone mineral in calcium to phosphate ratio and in Fourier transform infrared and x-ray diffraction spectra. The serum activity responsible for re-calcification is sufficiently potent that the addition of only 1.5% serum to Dulbecco's modified Eagle's medium causes bone re-calcification. This putative serum calcification factor has an apparent molecular mass of 55-150 kDa and is inactivated by trypsin or chymotrypsin. The serum calcification factor must act on bone for 12 h before re-calcification can be detected by Alizarin Red or von Kossa staining and before the subsequent growth of calcification will occur in the absence of serum. The speed, matrix-type specificity, and extent of the serum-induced re-calcification of demineralized bone suggest that the serum calcification factor identified in these studies may participate in the normal calcification of bone.

Silver Electrode as a Sensor for Determination of Zinc in Cell Cultivation Medium

Use of the silver electrode as a sensor for the monitoring of zinc in cell growth medium is described. Zinc at silver electrodes provides specific voltammetric signal, which is affected by solution components. Signals of zinc ions in phosphate buffer solutions with and without cell growth medium were compared. Common DMEM cell culture medium was used for the cultivation of a cell line of v-myb-transformed chicken monoblasts and its variants expressing v-jun and c-jun in a zinc-dependent manner. Electrochemical results showed zinc concentrations in the medium coincide very well with the jun expression. With respect to the low toxicity of silver for eukaryotic cells, silver electrodes represent promising tools for the determination of zinc concentrations in vivo without the potential risk of a cell culture damage.

Swimbladder gas gland cells of the European eel cultured in a superfusion system.

Swimbladder gas gland cells are polar epithelial cells which release acidic metabolites through the membranes of an extensive basolateral labyrinth, and secret surfactant via exocytosis at apical membranes. We have developed a method to establish primary cell cultures of gas gland cells in order to establish a model system for physiological analysis of gas gland cell function in vitro. Isolated gas gland cells attach to collagen S coated surfaces. Cells cultured in collagen S coated petri dishes were flat and showed no histological polarity. Cells cultured on Anodisc membranes in a superfusion system, in which the apical and basal side of the cells was supplied with a saline solution and with glucose containing DMEM cell culture medium, respectively, showed a clear polarity similar to the in vivo situation. Measurement of lactate release at the apical side and at the basal side revealed that these cells were functionally polar and secreted at least 70% of the lactate at their basal membranes. Gas gland cells could also be cultured in an air/liquid system, in which the apical membrane was exposed to humidified air. Cells cultured under these conditions released lactate only on the basal side and histologically were similar to cells cultured in the superfusion system.