GABAB receptors expressed in human aortic endothelial cells mediate intracellular calcium concentration regulation and endothelial nitric oxide synthase translocation

GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

Towards a quantitative theory of epidermal calcium profile formation in unwounded skin

We propose and mathematically examine a theory of calcium profile formation in unwounded mammalian epidermis based on: changes in keratinocyte proliferation, fluid and calcium exchange with the extracellular fluid during these cells' passage through the epidermal sublayers, and the barrier functions of both the stratum corneum and tight junctions localised in the stratum granulosum. Using this theory, we develop a mathematical model that predicts epidermal sublayer transit times, partitioning of the epidermal calcium gradient between intracellular and extracellular domains, and the permeability of the tight junction barrier to calcium ions. Comparison of our model's predictions of epidermal transit times with experimental data indicates that keratinocytes lose at least 87% of their volume during their disintegration to become corneocytes. Intracellular calcium is suggested as the main contributor to the epidermal calcium gradient, with its distribution actively regulated by a phenotypic switch in calcium exchange between keratinocytes and extracellular fluid present at the boundary between the stratum spinosum and the stratum granulosum. Formation of the extracellular calcium distribution, which rises in concentration through the stratum granulosum towards the skin surface, is attributed to a tight junction barrier in this sublayer possessing permeability to calcium ions that is less than 15 nm/s in human epidermis and less than 37 nm/s in murine epidermis. Future experimental work may refine the presented theory and reduce the mathematical uncertainty present in the model predictions.

Mathematical models of calcium and tight junctions in normal and reconstructed epidermis

This project investigated the calcium distributions of the skin, and the growth patterns of skin substitutes grown in the laboratory, using mathematical models. The research found that the calcium distribution in the upper layer of the skin is controlled by three different mechanisms, not one as previously thought. The research also suggests that tight junctions, which are adhesions between neighbouring skin cells, cannot be solely responsible for the differences in the growth patterns of skin substitutes and normal skin.

Exploration of the fundamental equilibrium behaviour of calcium exchange with weak acid cation resins

This study evaluated the complexity of calcium ion exchange with sodium exchanged weak acid cation resin (DOW MAC-3). Exchange equilibria recorded for a range of different solution normalities revealed profiles which were represented by conventional “L” or “H” type isotherms at low values of equilibrium concentration (Ce) of calcium ions, plus a superimposed region of increasing calcium uptake was observed at high Ce values. The loading of calcium ions was determined to be ca. 53.5 to 58.7 g/kg of resin when modelling only the sorption curve created at low Ce values,which exhibited a well-defined plateau. The calculated calcium ion loading capacity for DOWMAC-3 resin appeared to correlate with the manufacturer's recommendation. The phenomenon of super equivalent ion exchange (SEIX) was observed when the “driving force” for the exchange process was increased in excess of 2.25 mmol calcium ions per gram of resin in the starting solution. This latter event was explained in terms of displacement of sodium ions from sodium hydroxide solution which remained in the resin bead following the initial conversion of the as supplied “H+” exchanged resin sites to the “Na+” version required for softening studies. Evidence for hydrolysis of a small fraction of the sites on the sodium exchanged resin surface was noted. The importance of carefully choosing experimental parameters was discussed especially in relation to application of the Langmuir–Vageler expression. This latter model which compared the ratio of the initial calcium ion concentration in solution to resin mass, versus final equilibrium loading of the calcium ions on the resin; was discovered to be an excellent means of identifying the progress of the calcium–sodium ion exchange process. Moreover, the Langmuir–Vageler model facilitated standardization of various calcium–sodium ion exchange experiments which allowed systematic experimental design.

Characterization of the sulphate mineral coquimbite, a secondary iron sulphate from Javier Ortega mine, Lucanas Province, Peru – Using infrared, Raman spectroscopy and thermogravimetry

The mineral coquimbite has been analysed using a range of techniques including SEM with EDX, thermal analytical techniques and Raman and infrared spectroscopy. The mineral originated from the Javier Ortega mine, Lucanas Province, Peru. The chemical formula was determined as ðFe3þ 1:37; Al0:63ÞP2:00ðSO4Þ3 9H2O. Thermal analysis showed a total mass loss of 73.4% on heating to 1000 C. A mass loss of 30.43% at 641.4 C is attributed to the loss of SO3. Observed Raman and infrared bands were assigned to the stretching and bending vibrations of sulphate tetrahedra, aluminium oxide/hydroxide octahedra, water molecules and hydroxyl ions. The Raman spectrum shows well resolved bands at 2994, 3176, 3327, 3422 and 3580 cm 1 attributed to water stretching vibrations. Vibrational spectroscopy combined with thermal analysis provides insight into the structure of coquimbite.

Vibrational spectroscopic characterization of the sulphate-carbonate mineral burkeite: Implications for evaporites

Burkeite formation is important in saline evaporites and in pipe scales. Burkeite is an anhydrous sulphate-carbonate with an apparent variable anion ratio. Such a formula with two oxyanions lends itself to vibrational spectroscopy. Two symmetric sulphate stretching modes are observed, indicating at least at the molecular level the nonequivalence of the sulphate ions in the burkeite structure. The strong Raman band at 1065 cm−1 is assigned to the carbonate symmetric stretching vibration. The series of Raman bands at 622, 635, 645, and 704 cm−1 are assigned to the ν4 sulphate bending modes. The observation of multiple bands supports the concept of a reduction in symmetry of the sulphate anion from T d to C 3v or even C 2v.

Cortisol and dehydroepiandrosterone-sulphate levels correlate with symptom severity in first-episode psychosis

Dehydroepiandrosterone (DHEA) and its sulphate form (DHEA) are neuroactive steroids with antiglucocorticoid properties. An imbalance in the ratio of cortisol to DHEA(S) has been implicated in the pathophysiology of stress-related psychiatric disorders. This study prospectively investigated circulating cortisol, DHEAS and their ratio in first-episode psychosis (FEP) patients compared to healthy controls, and their relationship to perceived stress, psychotic, negative and mood symptoms. METHODS: Blood cortisol and DHEAS levels were obtained in 39 neuroleptic-naïve or minimally-treated FEP patients and 25 controls. Twenty-three patients and 15 controls received repeat assessments after 12 weeks. Perceived stress was assessed using the Perceived Stress Scale and symptoms were assessed in patients using standard rating scales. RESULTS: At baseline, no differences were observed in cortisol, DHEAS or the cortisol/DHEAS ratio between patients and controls. There were also no group differences in the change in these biological variables during the study period. Within FEP patients, decreases in cortisol and the cortisol/DHEAS ratio over time were directly related to the improvement in depression (r = 0.45; p = 0.031, r = 0.52; p = 0.01), negative (r = 0.51; p = 0.006, r = 0.55; p = 0.008) and psychotic symptoms (cortisol only, r = 0.53; p = 0.01). Perceived stress significantly correlated with DHEAS (r = 0.51; p = 0.019) and the cortisol/DHEAS ratio (r = -0.49; p = 0.024) in controls, but not patients, possibly reflecting an impaired hormonal response to stress in FEP patients. CONCLUSIONS: These findings further support the involvement of the stress system in the pathophysiology of psychotic disorders, with implications for treatment strategies that modulate these neurosteroids.

A Raman and infrared spectroscopic study of the sulphate mineral aluminite Al2(SO4)(OH)4·7H2O

The mineral aluminite has been studied using a number of techniques, including scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDX) and Raman and infrared spectroscopy. Raman spectroscopy identifies multiple sulphate symmetric stretching modes in line with the three sulphate crystallographically different sites. Raman spectroscopy also identifies a low intensity band at 1069 cm−1 which may be attributed to a carbonate symmetric stretching mode, indicating the presence of thaumasite. The observation of multiple bands in this ν4 spectral region offers evidence for the reduction in symmetry of the sulphate anion from Td to C2v or even lower symmetry. The Raman band at 3588 cm−1 is assigned to the OH unit stretching vibration and the broad feature at around 3439 cm−1 to water stretching bands. Water stretching vibrations are observed at 3157, 3294, 3378 and 3439 cm−1. Vibrational spectroscopy enables an assessment of the molecular structure of aluminite to be made.

Comparison of silicate-substituted calcium phosphate (Actifuse®) with rhBMP-2 (Infuse®) in posterolateral instrumented lumbar fusion

Study Design This was a randomised controlled trial in patients with degenerative disc disease (DDD) who underwent instrumented posterolateral lumbar fusion (PLF) surgery. Objective The aim of this study was to assess the efficacy of the bone grafting substitute, silicate-substituted calcium phosphate (SiCaP) compared with bone morphogenetic protein (rhBMP-2) and to evaluate clinical outcomes over a period of two years. Methods Patients undergoing PLF surgery for DDD at a single centre were recruited and randomised to one of two groups; SiCaP (n=9) or rhBMP-2 (n=10). One patient withdrew prior to randomisation and another from the rhBMP-2 group after randomisation. The radiological and clinical outcomes were examined and compared. Fusion was assessed at 12 months with computed tomography (CT) and plain radiographs. Clinical outcomes were evaluated by recording measures of pain, quality of life, disability and neurological status from six weeks to two years postoperatively. Results In the SiCaP and rhBMP-2 groups, fusion was observed in 9/9 and 8/9 patients respectively. Pain and disability scores were reduced and quality of life increased in both groups. Leg pain, disability and satisfaction scores were similar between the groups at each postoperative time point, however, back pain was less at six weeks and quality of life was higher at six months in the SiCaP group than the rhBMP-2 group. Conclusions SiCaP and rhBMP-2 were comparable in terms of achieving successful bone growth and fusion. Both groups similarly alleviated pain and improved quality of life, neurological, satisfaction and return to work outcomes following PLF surgery.

Calcium phosphate flocs and the clarification of sugar cane juice from whole of crop harvesting

Sugar cane biomass is one of the most viable feedstocks for the production of renewable fuels and chemicals. Therefore, processing the whole of crop (WC) (i.e., stalk and trash, instead of stalk only) will increase the amount of available biomass for this purpose. However, effective clarification of juice expressed from WC for raw sugar manufacture is a major challenge because of the amounts and types of non-sucrose impurities (e.g., polysaccharides, inorganics, proteins, etc.) present. Calcium phosphate flocs are important during sugar cane juice clarification because they are responsible for the removal of impurities. Therefore, to gain a better understanding of the role of calcium phosphate flocs during the juice clarification process,the effects of impurities on the physicochemical properties of calcium phosphate flocs were examined using small-angle laser light scattering technique, attenuated total reflectance Fourier transformed infrared spectroscopy, and X-ray powder diffraction. Results on synthetic sugar juice solutions showed that the presence of SiO2 and Na+ ions affected floc size and floc structure. Starch and phosphate ions did not affect the floc structure; however, the former reduced the floc size, whereas the latter increased the floc size. The study revealed that high levels of Na+ ions would negatively affect the clarification process the most, as they would reduce the amount of suspended particles trapped by the flocs. A complementary study on prepared WC juice using cold and cold/intermediate liming techniques was conducted. The study demonstrated that, in comparison to the one-stage (i.e., conventional) clarification process, a two-stage clarification process using cold liming removed more polysaccharides (≤19%),proteins (≤82%), phosphorus (≤53%), and SiO2 (≤23%) in WC juice but increased Ca2+ (≤136%) and sulfur (≤200%)

Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5pl5. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

Inhibition by poly(acrylic acid) and morphological changes in calcium carbonate and calcium carbonate/calcium sulfate crystallization on silica fibers

An intrinsic exposed core optical fiber sensor (IECOFS) made from fused silica was used to monitor the crystallization of calcium carbonate (CaCO3) and CaCO3/calcium sulfate (CaSO4) composite at 100 and 120 °C in the absence and presence of low-molar-mass (Mn ≤ 2000) poly(acrylic acid) (PAA) with different end groups. The IECOFS responded only to deposition and growth processes on the fiber surface rather than changes occurring in the bulk of the solution. Hexyl isobutyrate-terminated PAA (Mn = 1400) and hexadecyl isobutyrate-terminated PAA (Mn = 1700) were the most effective species in preventing CaCO3 deposition. Phase transformation from vaterite to aragonite/calcite decreased with increasing hydrophobicity of the PAA end group. Low-molar-mass PAA at 10 ppm showed very significant inhibition of CaCO3/CaSO4 composite formation for all end groups investigated.

Novel ANKH amino terminus mutation (Pro5Ser) associated with early-onset calcium pyrophosphate disease with associated phosphaturia

This report describes a 32-year-old woman presenting since childhood with progressive calcium pyrophosphate disease (CPPD), characterized by severe arthropathy and chondrocalcinosis involving multiple peripheral joints and intervertebral disks. Because ANKH mutations have been previously described in familial CPPD, the proband's DNA was assessed at this locus by direct sequencing of promoter and coding regions and revealed 3 sequence variants in ANKH. Sequences of exon 1 revealed a novel isolated nonsynonymous mutation (c.13 C>T), altering amino acid in codon 5 from proline to serine (CCG>TCG). Sequencing of parental DNA revealed an identical mutation in the proband's father but not the mother. Subsequent clinical evaluation demonstrated extensive chondrocalcinosis and degenerative arthropathy in the proband's father. In summary, we report a novel mutation, not previously described, in ANKH exon 1, wherein serine replaces proline, in a case of early-onset severe CPPD associated with metabolic abnormalities, with similar findings in the proband's father.

Genetic studies of disorders of calcium crystal deposition

In summary, although many factors are likely to be involved in regulating calcification and ossification processes, studies of the causation of articular chondrocalcinosis and disorders of spinal ossification, such as DISH and OPLL, implicate control over inorganic pyrophosphate levels as being one of the most important factors in their aetiopathogenesis. The findings of these studies may prove relevant to other rheumatic diseases in which ectopic ossification occurs, such as AS.

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [P-32] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [P-32] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [P-32] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.