cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin

The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.     

Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells

Bone marrow stromal cells (MSCs) differentiation and proliferation are controlled by numerous growth factors and hormones. Continuous parathyroid hormone (PTH) treatment has been shown to decrease osteoblast differentiation, whereas pulsatile PTH increases osteoblast differentiation. However, the effects of PTH treatments on MSCs have not been investigated. This study showed continuous PTH treatment in the presence of dexamethasone (DEX) promoted osteogenic differentiation of rat MSCs in vitro, as demonstrated by increased alkaline phosphatase (ALP) activity, number of ALP expressing cells, and up-regulation of PTH receptor-1, ALP, and osteocalcin mRNA expressions. In contrast, pulsatile PTH treatment was found to suppress osteogenesis of rat MSCs, possibly by promoting the maintenance of undifferentiated cells. Additionally, the observed effects of PTH were strongly dependent on the presence of DEX. MSC proliferation however was not influenced by PTH independent of treatment regimen and presence or absence of DEX. Furthermore, our work raised the possibility that PTH treatment may modulate stem/progenitor cell activity within MSC cultures.     

Characterization of the N-terminal structure of pertussis toxin subunit S1 and hybridization of oligodeoxyribonucleotide probes with a Bordetella pertussis DNA fragment

Abstract N-terminal amino acid sequence analysis of the enzymatic subunit S1 of pertussis toxin from Bordetella pertussis showed the structure of the first 25 residues. 2 different oligodeoxyribonucleotide probes were synthesized as mixed 32-mers corresponding to the DNA sequence deduced. Southern blot analysis of pertussis DNA digested with restriction endonuclease Cla I showed that both mixed probes hybridized with a DNA fragment of about 10 kb, thus identifying a B. pertussis gene corresponding to the protein structure determined.     

Specificity and detection limit of a dermal temperature histamine sensitization test for absence of residual pertussis toxin in vaccines

Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.     

Transport of protein toxins into cells: pathways used by ricin,cholera toxin and Shiga toxin

Ricin, cholera, and Shiga toxin belong to a family of protein toxins that enter the cytosol to exert their action. Since all three toxins are routed from the cell surface through the Golgi apparatus and to the endoplasmic reticulum (ER) before translocation to the cytosol, the toxins are used to study different endocytic pathways as well as the retrograde transport to the Golgi and the ER. The toxins can also be used as vectors to carry other proteins into the cells. Studies with protein toxins reveal that there are more pathways along the plasma membrane to ER route than originally believed.     

Susceptibility of primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin in two- and three-dimensional culture conditions

The human pathogen Bordetella pertussis targets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC^– with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC^–. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin–cell interaction using primary hAEC.     

Four distinct chondrocyte populations in the fetal bovine growth plate: highest expression levels of PTH/PTHrP receptor,Indian hedgehog,and MMP-13 in hypertrophic chondrocytes and their suppression by PTH (1-34) and PTHrP (1-40)

Differentiation and growth of chondrocytes in fetal growth plates of vertebrate long bones and ribs appear to occur in a gradual, continuous manner between the resting zone through the proliferation zone, maturation zone, and upper and lower hypertrophic zones, with a continuous increase in cell size up to 10-fold of the volume of a resting chondrocyte. Here we provide evidence, however, that after centrifugation through a continuous Percoll gradient growth plate chondrocytes separate into four distinct cell populations (B1 to B4) which differ markedly in density, size, and gene expression. These populations collect in the absence of any phase borders in the gradient which might serve as concentration barriers. Fractions B1 and B2 contained the largest cells with the lowest buoyant density and showed the highest expression levels for type X collagen (Col X), but only the B1 population expressed high levels of matrix metalloproteinase-13 (collagenase 3). Cells in fraction B3 were significantly smaller and expressed little Col X, while cells in fraction B4 were of similar size to cells in the resting zone without significant Col X expression. The highest levels of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR-1), and Indian hedgehog (Ihh) expression were also found in the hypertrophic fractions B1 and B2 and not in the prehypertrophic fraction B3, as expected from in situ hybridization data on PTHR-1 expression in fetal rodent or chicken growth plates. Incubation of fractions B1 to B3 with the amino-terminal fragments PTH (1-34) or PTHrP (1-40) suppressed the expression of Col X and PTHR-1 by more than 50% and the expression of Ihh nearly completely. In contrast, the mid-regional PTH fragment PTH (28-48) and PTH (52-84) consistently stimulated the expression of PTHR-1 by 10-20% in fractions B1 to B3. These findings confirm the existence of distinct differentiation stages within chondrocytes of the growth plate and support the hypothesis proposed by Vortkamp et al. (Science 273(1996)613) of a regulatory feedback loop of Ihh and PTH/PTHrP fragments controlling the differentiation of proliferating to prehypertrophic chondrocytes, but extend the ability to respond to PTH/PTHrP hypertrophic chondrocytes.     

NAD+-induced inhibition of phosphate transport in canine renal brush-border membranes Mediation through a process other than or in addition to NAD+ hydrolysis

We previously demonstrated inhibition of Na⁺-dependent ³²P_i transport in canine renal brush-border membranes in association with NAD⁺-induced ADP ribosylation of membrane protein(s) and postulated that NAD⁺ inhibits P_i transport across the brush-border membrane via ADP ribosylation. Recently it was shown that incubation of rat brush-border membrane with NAD⁺ resulted in release of P_i which was prevented by EDTA. It was proposed that NAD⁺-mediated inhibition of ³²P_i transport might occur through this mechanism. To determine whether NAD⁺ inhibited ³²P_i transport by a mechanism other than or in addition to release of P_i, we compared Na⁺-dependent ³²P_i counterflow in brush-border membrane equilibrated with P_i or with P_i generated from NAD⁺. Release of P_i from NAD⁺ incubated with brush-border membrane was confirmed. The increased uptake of ³²P_i which was demonstrated in brush-border membrane equilibrated with P_i was not measured when intravesicular P_i was generated from a concentration of NAD⁺ which effected ADP-ribosylation of brush border membranes (100 μM NAD⁺). In contrast, increased uptake of ³²P_i was demonstrated when intravesicular P_i was generated from 1 μM NAD⁺ which did not effect ADP ribosylation. Mg²⁺-dependent ADP ribosylation of brush-border membrane incubated with NAD⁺ was demonstrated which persisted during the time interval of ³²P_i uptake measurements. Our findings are compatible with the hypothesis that NAD⁺-induced ADP ribosylation of brush-border membrane protein(s) results in inhibition of P_i transport across the membrane in vivo. EDTA may act to prevent this inhibition in brush-border membrane by chelation of Mg²⁺ and decreased ADP ribosylation.     

Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome

Quantitative confocal microscopic analyses of living, polarized MDCK cells demonstrate different pH profiles for apical and basolateral endocytic pathways, despite a rapid and extensive intersection between the two. Three-dimensional characterizations of ligand trafficking demonstrate that the apical and basolateral endocytic pathways share early, acidic compartments distributed throughout the medial regions of the cell. Polar sorting for both pathways occurs in these common endosomes as IgA is sorted from transferrin to alkaline transcytotic vesicles. While transferrin is directly recycled from the common endosomes, IgA is transported to a downstream apical compartment that is nearly neutral in pH. By several criteria this compartment appears to be equivalent to the previously described apical recycling endosome. The functional significance of the abrupt increase in lumenal pH that accompanies IgA sorting is not clear, as disrupting endosome acidification has no effect on polar sorting. These studies provide the first detailed characterizations of endosome acidification in intact polarized cells and clarify the relationship between the apical and basolateral endocytic itineraries of polarized MDCK cells. The extensive mixing of apical and basolateral pathways underscores the importance of endocytic sorting in maintaining the polarity of the plasma membrane of MDCK cells.     

一例母 JUE TI 及其亲子DNA指纹与血清蛋白、酯酶遗传的研究

采用DNA指纹分析和聚丙烯酰胺凝胶电泳法, 对一例与公驴交配生育了后代的母|JUE||TI|后代进行了亲缘鉴定和血清蛋白、酯酶遗传的分析。结果可以认定其亲子关系并证实母骡生育的事实。虽然本例母|JUE|| TI|的后代在Pr、Al、Pa和Hb、Es等基因座位上的基因表达倾向于驴,但其外貌仍明显地带有种间杂种的特征。因此,尚不能简单地认为其已“回归”为纯种的驴。 Abstract:By adopting DNA fingerprints analysis and polyacrylamide gel electrophoresis simultaneously,the author made an genetical study on analytical evaluation of kindred relationship and contents of serum protein and esterase of a female hinny and her offspring bred after her mating with a masculine ass.As a result,the parental relationship together with the child-bearing fact of the hinny is hereby clearly verified.The appearance of the offsping,however,still showed some prominent characters of interspecific hybrid,though in the gene demonstration the offsping was proved inclining to the ass especially on those gene loci as Pr,AL,Pa,Hb and Es.Therefore,in the author′s opinion,the offsping could not be simply regarded as a “regressed” ass pure-line.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 (PTH_1–34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [³H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylae activity appeared to follow Michaelis-Menten kintics, and 1,25-dihydroxyvitamin D-3 treatment increased the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 24-hydroxylase from 33 to 95 pmol/h per 10⁶ cells without affecting the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 · 10^?10 M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 μM cycloheximide. Treatment of the cells with PTH_1–34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 · 10^?9 M PTH_1–34. When 2.4 · 10^?9 M PTH_1–34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to $\text{70.7 ± 2.9\%}$ of control. Higher concentrations of PTH_1–34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH_1–34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH_1–34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

Parathyroid hormone (PTH) and hematopoiesis: new support for some old observations

Forty-seven years ago, the parathyroid hormone (PTH) in one injection of Lilly's old bovine parathyroid extract, PTE, was found to greatly increase the 30-day survival of heavily X-irradiated rats when given from 18 h before to as long as 3 h after irradiation but no later. This was the first indication that PTH might stimulate hematopoiesis. Recent studies have confirmed the relation between PTH and hematopoiesis by showing that hPTH-(1-34)OH increases the size of the hematopoietic stem cell pool in mice. The peptide operates through a cyclic AMP-mediated burst of Jagged 1 production in osteoblastic cells lining the stem cells' niches on trabecular bone surfaces. The osteoblastic cells' Jagged 1 increases the hematopoietic stem cell pool by activating Notch receptors on attached stem cells. PTH-triggered cyclic AMP signals also directly stimulate the proliferation of the hematopoietic stem cells. However, the single PTH injection in the early experiments using PTE probably increased the survival of irradiated rats mainly by preventing the damaged hematopoietic progenitors from irreversibly initiating self-destructive apoptogenesis during the first 5 h after irradiation. It has also been shown that several daily injections of hPTH-(1-34)OH enable lethally irradiated mice to survive by stimulating the growth of transplanted normal bone marrow cells. If the osteogenic PTHs currently entering or on the verge of entering the market for treating osteoporosis can also drive hematopoiesis in humans as well as rodents, they could be potent tools for reducing the damage inflicted on bone marrow by cytotoxic cancer chemotherapeutic drugs and ionizing radiation.     

Apical and basolateral Na/H exchange in cultured murine proximal tubule cells (MCT): Effect of parathyroid hormone (PTH)

Summary Kidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10^–8 m; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10^–8 m); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10^–8 m) resulted in a rapid and transient increase in [Ca²⁺] _i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca²⁺ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/P _i cotransport.This work was supported by the Swiss National Science Foundation (Grant No. 32-30785.91), the Stiftung für wissenschaftliche Forschung an der Universität Zürich, the Hartmann-Müller Stiftung, the Sandoz-Stiftung, the Roche Research Foundation and the Geigy-Jubiläumsstiftung. We are grateful to Denise Rossi and Christa Knellwolf for their excellent secretarial assistance.     

Transport of methotrexate in L1210 cells: Mechanism for inhibition by p-chloromercuriphenylsulfonate and N-ethylmaleimide

Methotrexate transport in L1210 cells is highly sensitive to inhibition by p-chloromercuriphenylsulfonate (CMPS) and, to a lesser extent, by N-ethylmaleimide. A 50% reduction in the methotrexate influx rate occurred upon exposure of cells to 3 μM CMPS or 175 μM N-ethylmaleimide, while complete inhibition was achieved at higher levels of these agents. Dithiothreitol reversed the inhibition by CMPS, suggesting that a sulfhydryl residue is involved. This residue is apparently not located at the substrate binding site of the transport protein, since methotrexate failed to protect the system from inactivation by either CMPS or N-ethylmaleimide, and the transport protein retained the ability to bind substrate (at 4°C) after exposure to these inhibitors (at 37°C). Methotrexate efflux was also inhibited by CMPS (50% at 4 μM), indicating that both the uptake and efflux of methotrexate in L1210 cells occur via the same transport system. High concentrations of CMPS (greater than 20 μM) increased the efflux rate, apparently by damaging the cell membrane and allowing the passive diffusion of methotrexate out of the cell.     

Apical to basolateral transcytosis and apical recycling of immunoglobulin G in trophoblast-derived BeWo cells: effects of low temperature, nocodazole, and cytochalasin D

The murine neonatal Fc receptor, FcRn, carries out two functions: materno-fetal IgG delivery and maintenance of serum IgG homeostasis. During human pregnancy maternal IgG is transferred across placental syncytiotrophoblasts presumably by the human homolog of FcRn, hFcRn. Trophoblast-derived BeWo cells express hFcRn endogenously and can be considered as a model system to investigate IgG transport in syncytiotrophoblasts. Using a pulse-chase protocol, we here demonstrate that polarized BeWo cells exhibit not only apical to basolateral transcytosis but also apical IgG recycling. Thus, for the first time we demonstrate that epithelial cells can be involved in both materno-fetal IgG transmission and regulation of serum IgG levels. Lowering the temperature from 37 to 16 degrees C reduced, but did not block, IgG recycling and transcytosis. Microtubule-disruption by nocodazole did not influence transcytosis or apical recycling. Disassembly of filamentous actin by cytochalasin D stimulated apical endocytosis and recycling, while transcytosis remained unaffected. In summary, in BeWo cells apically internalized IgG enters both a transcytotic and recycling pathway. While the transcytotic route is temperature-sensitive but independent from microtubules and actin filaments, the apical recycling pathway is temperature-influenced and stimulated by actin disassembly, suggestive for the involvement of distinct endosome subcompartments in transcytosis and recycling.     

Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

Effects of the calcium-sensing receptor A986S polymorphism on serum calcium and parathyroid hormone levels in healthy individuals: a meta-analysis

The calcium-sensing receptor (CaSR) is involved in maintaining calcium homeostasis via the regulation of parathyroid hormone (PTH) secretion. The associations between serum calcium concentrations, parathyroid hormone (PTH) level and CaSR polymorphism A986S have been studied, but results are inconsistent. Therefore, we performed a meta-analysis to clarify the role of this polymorphism on this topic. Weighted mean difference (WMD) and 95% confidence interval (CI) were calculated with random-effects model or fixed-effects model based on the heterogeneity analysis. Overall 2820, 1135 and 3149 healthy individuals were included for total calcium concentration, ionized calcium concentration and PTH level meta-analyses, respectively. Most of the individuals in this meta-analysis were healthy women. Healthy individuals with the AS + SS genotype had significantly higher total and ionized calcium concentrations than those with the AA genotype. However, there was no statistical significance concerning serum PTH level. The pooled WMD for total calcium concentration was 0.028 (95% CI: 0.012-0.045, P = 0.001); for ionized calcium concentration was 0.016 (95% CI: 0.013-0.020, P < 0.0001); and for PTH level was − 0.027 (95% CI: −0.360-0.306, P = 0.874). In conclusion, our meta-analysis indicates that the CaSR A986S polymorphism might be associated with total and ionized calcium concentrations in healthy individuals.     

Growth characteristics and toxin production in batch cultures of Anabaena flos-aquae: effects of culture media and duration

The effects of media and culture duration on growth, macromolecular composition and toxicity of an anatoxin- a-producing freshwater cyanobacterium Anabaena flos-aquae (UTEX 2383) were evaluated. The four media A₃M₇, CB, MA and B-12 influenced growth in terms of cell number, chlorophyll-a content and specific growth rate. A₃M₇ medium supported the best growth. The macromolecular composition of cultured cells, viz. total carbohydrate, protein and lipid content varied with media and culture duration reaching maximum concentration at various growth periods. The differences were significant due to interaction of the culture medium and duration. Toxicity of cells grown in different media was compared by Artemia salina bioassay and mouse units. The cells grown in A₃M₇ medium showed highest toxicity and the optimum culture duration was 5 weeks. In terms of both growth characteristics and toxicity the media can be ranked as A₃M₇, MA, CB and B-12 in decreasing order.