Calcium influx inhibits MT1-MMP processing and blocks MMP-2 activation

We have previously reported that concanavalin A (ConA)-induced MMP-2 activation involves both transcriptional and non-transcriptional mechanisms. Here we examined the effects of calcium influx on MT1-MMP expression and MMP-2 activation in MDA-MB-231 cells. The calcium ionophore ionomycin caused a dose-dependent inhibition of ConA-induced MMP-2 activation, but had no effect on MT1-MMP mRNA levels. However, Western analysis revealed an accumulation of pro-MT1-MMP (63 kDa), indicating that ionomycin blocked the conversion of pro-MT1-MMP protein to the active 60 kDa form. This suggests that increased calcium levels inhibit the processing of MT1-MMP. This finding may help to elucidate the mechanism(s) which regulates MT1-MMP activation.

Gonadotropin-induced ovarian cancer cell migration and proliferation require extracellular signal-regulated kinase 1/2 activation regulated by calcium and protein kinase Cδ

The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCδ inhibitor rottlerin, and downregulation of PKCδ was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCδ in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCδ siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCδ-dependent manner.

Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis.

Active regulation of the epidermal calcium profile

A distinct calcium profile is strongly implicated in regulating the multi-layered structure of the epidermis. However, the mechanisms that govern the regulation of this calcium profile are currently unclear. It clearly depends on the relatively impermeable barrier of the stratum corneum (passive regulation) but may also depend on calcium exchanges between keratinocytes and extracellular fluid (active regulation). Using a mathematical model that treats the viable sublayers of unwounded human and murine epidermis as porous media and assumes that their calcium profiles are passively regulated, we demonstrate that these profiles are also actively regulated. To obtain this result, we found that diffusion governs extracellular calcium motion in the viable epidermis and hence intracellular calcium is the main source of the epidermal calcium profile. Then, by comparison with experimental calcium profiles and combination with a hypothesised cell velocity distribution in the viable epidermis, we found that the net influx of calcium ions into keratinocytes from extracellular fluid may be constant and positive throughout the stratum basale and stratum spinosum, and that there is a net outflux of these ions in the stratum granulosum. Hence the calcium exchange between keratinocytes and extracellular fluid differs distinctly between the stratum granulosum and the underlying sublayers, and these differences actively regulate the epidermal calcium profile. Our results also indicate that plasma membrane dysfunction may be an early event during keratinocyte disintegration in the stratum granulosum.

Synthesis of 4-arm star poly(L-Lactide) oligomers using an in situ-generated calcium-based initiator

Using an in situ-generated calcium-based initiating species derived from pentaerythritol, the bulk synthesis of well-defined 4-arm star poly(L-lactide) oligomers has been studied in detail. The substitution of the traditional initiator, stannous octoate with calcium hydride allowed the synthesis of oligomers that had both low PDIs and a comparable number of polymeric arms (3.7 – 3.9) to oligomers of similar molecular weight. Investigations into the degree of control observed during the course of the polymerization found that the insolubility of pentaerythritol in molten L-lactide resulted in an uncontrolled polymerization only when the feed mole ratio of L-lactide to pentaerythritol was 13. At feed ratios of 40 and greater, a pseudo-living polymerization was observed. As part of this study, in situ FT-Raman spectroscopy was demonstrated to be a suitable method to monitor the kinetics of the ring-opening polymerization (ROP) of lactide. The advantages of using this technique rather than FT-IR-ATR and 1H NMR for monitoring L-lactide consumption during polymerization are discussed.

Deposition of hydroxyapatite and calcium oxalate dihydrate on a heat exchanger tube

Most studies on the characterisation of deposits on heat exchangers have been based on bulk analysis, neglecting the fine structural features and the compositional profiles of layered deposits. Attempts have been made to fully characterise a fouled stainless steel tube obtained from a quintuple Roberts evaporator of a sugar factory using X-ray diffraction and scanning electron microscopy techniques. The deposit contains three layers at the bottom of the tube and two layers on the other sections and is composed of hydroxyapatite, calcium oxalate dihydrate and an amorphous material. The proportions of these phases varied along the tube height. Energy-dispersive spectroscopy and XRD analysis on the surfaces of the outermost and innermost layers showed that hydroxyapatite was the major phase attached to the tube wall, while calcium oxalate dihydrate (with pits and voids) was the major phase on the juice side. Elemental mapping of the cross-sections of the deposit revealed the presence of a mineral, Si-Mg-Al-Fe-O, which is probably a silicate mineral. Reasons for the defects in the oxalate crystal surfaces, the differences in the crystal size distribution from bottom to the top of the tube and the composite fouling process have been postulated.

Effect of poly(acrylic acid) end-group functionality on inhibition of calcium oxalate crystal growth

A number of series of poly(acrylic acids) (PAA) of differing end-groups and molecular weights prepared using atom transfer radical polymerization were used as inhibitors for the crystallization of calcium oxalate at 23 and 80°C. As measured by turbidimetry and conductivity and as expected from previous reports, all PAA series were most effective for inhibition of crystallization at molecular weights of 1500–4000. However, the extent of inhibition was in general strongly dependent on the hydrophobicity and molecular weight of the end-group. These results may be explicable in terms of adsorption/desorption of PAA to growth sites on crystallites. The overall effectiveness of the series didn't follow a simple trend with end-group hydrophobicity, suggesting self-assembly behavior or a balance between adsorption and desorption rates to crystallite surfaces may be critical in the mechanism of inhibition of calcium oxalate crystallization.

Effect of poly(acrylic acid) molecular mass and end-group functionality on calcium oxalate crystal morphology and growth

A number of series of poly(acrylic acids) (PAA) of differing end-groups and molecular mass were used to study the inhibition of calcium oxalate crystallization. The effects of the end-group on crystal speciation and morphology were significant and dramatic, with hexyl-isobutyrate end groups giving preferential formation of calcium oxalate dihydrate (COD) rather than the more stable calcium oxalate monohydrate (COM), while both more hydrophobic end-groups and less-hydrophobic end groups led predominantly to formation of the least thermodynamically stable form of calcium oxalate, calcium oxalate trihydrate. Conversely, molecular mass had little impact on calcium oxalate speciation or crystal morphology. It is probable that the observed effects are related to the rate of desorption of the PAA moiety from the crystal (lite) surfaces and that the results point to a major role for end-group as well as molecular mass in controlling desorption rate.

Formation of thermodynamically unstable calcium oxalate dihydrate in sugar mill evaporators

Calcium oxalate (CaOX) is the most intractable scale component to remove in sugar mill evaporators by either mechanical or chemical means. The operating conditions of sugar mill evaporators should preferentially favour the formation of the thermodynamically stable calcium oxalate monohydrate (COM), yet analysis of scale deposit from different sugar factories have shown that calcium oxalate dihydrate (COD) is usually the predominant phase, and in some cases is the only hydrate formed. The effects of trans-aconitic, succinic and acetic acids, all of which are present in sugarcane juice, and ethylenediamine tetraacetic acid disodium salt (EDTA) on the growth of CaOX crystals have been examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray powder diffraction (XRD) and thermogravimetric analysis (TGA). trans-Aconitic acid, which constitutes two-thirds of the organic acid component in sugarcane juice, in the presence of sugar resulted in the formation of COD and COM in a 3:1 ratio. EDTA was the most effective acid to promote the formation of COD followed by trans-aconitic acid, then acetic acid and lastly succinic acid.

Some aspects of calcium phosphate chemistry in sugarcane clarification

This paper reviews some aspects of calcium phosphate chemistry since phosphate in juice is an important parameter in all sugar juice clarification systems. It uses basic concepts to try and explain the observed differences in clarification performance obtained with various liming techniques. The paper also examines the current colorimetric method used for the determination of phosphate in sugar juice. In this method, a phosphomolybdate blue complex formed due to the addition of a dye is measured at 660 nm. Unfortunately, at this wavelength there is interference of the colour arising from within the juice and results in the underestimation of the amount of soluble inorganic phosphate content of juice. It is suggested that phosphate analysis be conducted at the higher wavelength of 875 nm where the interference of the juice colour is minimised.