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Regulation of calcium phosphate formation by amelogenins under physiological conditions.

ABSTRACT: Amelogenin is essential for proper enamel formation. The present in vitro study extends our previous work at low (10 mM) ionic strength (IS) by examining the effect of amelogenin on mineralization under higher (162 mM) IS conditions found in developing enamel. Full-length phosphorylated (P173) and non-phosphorylated (rP172) amelogenins were examined, along with P148 and rP147 that lack the hydrophilic C-terminus. Calcium phosphate formation was assessed by pH change, while the minerals formed were characterized using transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy. Amelogenin self-assembly was also studied using dynamic light scattering and TEM. The results indicate that IS does not influence the effects of rP147, rP172, and P173 on mineralization. However, in contrast to the findings for low IS, where both P173 and P148 stabilize initially formed amorphous calcium phosphate (ACP) nanoparticles for >1 d, elongated hydroxyapatite crystals were observed after 24 h using P148 at high IS, unlike that seen with P173. Differences in self-assembly help explain these findings, which suggest that P173 and P148 may play different roles in regulating enamel mineral formation. The present data support the notion that proteolytic processing of P173 is required in vivo to induce the transformation of initial ACP phases to apatitic enamel crystals.

SUBMITTER: Kwak SY

PROVIDER: S-EPMC3448280 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Regulation of calcium phosphate formation by amelogenins under physiological conditions.

Kwak Seo-Young SY Green Samantha S Wiedemann-Bidlack Felicitas B FB Beniash Elia E Yamakoshi Yasuo Y Simmer James P JP Margolis Henry C HC

European journal of oral sciences 20111201

Amelogenin is essential for proper enamel formation. The present in vitro study extends our previous work at low (10 mM) ionic strength (IS) by examining the effect of amelogenin on mineralization under higher (162 mM) IS conditions found in developing enamel. Full-length phosphorylated (P173) and non-phosphorylated (rP172) amelogenins were examined, along with P148 and rP147 that lack the hydrophilic C-terminus. Calcium phosphate formation was assessed by pH change, while the minerals formed we ...[more]

PMID: 22243235

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Project description:BackgroundCalcium phosphate mediated transfection has been used for delivering DNA into mammalian cells in excess of 30 years due to its most low cost for introducing recombinant DNA into culture cells. However, multiple factors affecting the transfect efficiency are commonly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as CHO and C2C12 limits its application on research.ResultsIn this paper, we systematically evaluated the possible factors affecting the transfection rate of this approach. Two categories, calcium phosphate-DNA co-precipitation and on-cell treatments were set for optimization of plasmid DNA transfection into CHO and C2C12 cell-lines. Throughout experimentation of these categories such as buffer system, transfection media and time, glycerol shocking and so on, we optimized the best procedure to obtain the highest efficiency ultimately. During calcium phosphate DNA-precipitation, the transfection buffer is critical condition optimized with HBS at pH 7.10 (P = 0.013 compared to HEPES in CHO). In the transfection step, FBS is a necessary component in transfection DMEM for high efficiency (P = 0.0005 compared to DMEM alone), and high concentration of co-precipitated particles applied to cultured cells in combination with intermittent vortexing is also crucial to preserve the efficiency. For 6-well culture plates, 800 µl of co-precipitated particles (11.25 µg/mL of cDNA) in 1 well is the optimal (P = 0.007 compared to 200 µl). For the highest transfection efficiency, the most important condition is glycerol in shock treatment (P = 0.002 compared to no shock treatment in CHO, and P = 0.008 compared to no shock treatment in C2C12) after a 6 h incubation (P = 0.004 compared to 16 h in CHO, and P = 0.039 compared to 16 h in C2C12) on cultured cells.ConclusionsCalcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.

Project description:Increased brain ammonium (NH4(+)/NH3) plays a central role in the manifestation of hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH4(+)/NH3 on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH4(+)/NH3-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH4(+)/NH3 for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼ 90%) experienced a persistent calcium increase by ∼ 50 nM. This persistent increase was already evoked at concentrations of 1-2 mM NH4(+)/NH3, developed within 10-20 minutes and was maintained as long as the NH4(+)/NH3 was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH4(+)/NH3, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of sodium/calcium exchange (NCX), AMPA- or mGluR5-receptors did not dampen NH4(+)/NH3-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH4(+)/NH3 causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE.

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Regulation of calcium phosphate formation by amelogenins under physiological conditions.

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Regulation of calcium phosphate formation by amelogenins under physiological conditions.

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