Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading. (C) 2003 Elsevier B.V. All rights reserved.

The use of simultaneous H-1 & P-31 magic angle spinning nuclear magnetic resonance measurements to characterize energy metabolism during the conversion of muscle to meat

In order to investigate the potential of magic angle spinning nuclear magnetic resonance (MAS NMR) in the elucidation of post-mortem metabolism in muscle biopsies, simultaneous H-1 and (31)p MAS NMR measurements were made continuously on postmortem (20 min to 24 h) muscle longissimus samples from rabbits. The animals had either been or not been given adrenaline (0.5 mg kg(-1) 4 h pre-slaughter) to deplete stores of muscle glycogen. The intracellular pH was calculated from H-1 spectra, and the post-mortem rate of formation of lactate was followed and quantified. Comparison of measurements made on muscle samples from rabbits treated with adrenaline with measurements made on muscle samples from untreated' rabbits revealed significant effects of adrenaline treatment on both pH (pH24 h = 6.42 vs. pH24 It = 5.60) and formation of lactate (16 mmol g(-1) vs. 65 mmol g(-1)). The P-31 NMR spectra were used to follow the rate of degradation of ATP and phosphocreatine. The present study clearly shows that MAS NMR has potential for the study of post-mortem energy metabolism.

Gene duplication and hierarchical modularity in intracellular interaction networks

Networks of interactions evolve in many different domains. They tend to have topological characteristics in common, possibly due to common factors in the way the networks grow and develop. It has been recently suggested that one such common characteristic is the presence of a hierarchically modular organization. In this paper, we describe a new algorithm for the detection and quantification of hierarchical modularity, and demonstrate that the yeast protein-protein interaction network does have a hierarchically modular organization. We further show that such organization is evident in artificial networks produced by computational evolution using a gene duplication operator, but not in those developing via preferential attachment of new nodes to highly connected existing nodes. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Intracellular compartmentation in planctomycetes

The phylum Planctomycetes of the domain Bacteria consists of budding, peptidoglycan-less organisms important for understanding the origins of complex cell organization. Their significance for cell biology lies in their possession of intracellular membrane compartmentation. All planctomycetes share a unique cell plan, in which the cell cytoplasm is divided into compartments by one or more membranes, including a major cell compartment containing the nucleoid. Of special significance is Gemmata obscuriglobus, in which the nucleoid is enveloped in two membranes to form a nuclear body that is analogous to the structure of a eukaryotic nucleus. Planctomycete compartmentation may have functional physiological roles, as in the case of anaerobic ammonium-oxidizing anammox planctomycetes, in which the anammoxosome harbors specialized enzymes and is wrapped in an envelope possessing unique ladderane lipids. Organisms in phyla other than the phylum Planctomycetes may possess compartmentation similar to that of some planctomycetes, as in the case of members of the phylum Poribacteria from marine sponges.

Specialization in pyramidal cell structure in the cingulate cortex of the Chacma baboon (Papio ursinus): An intracellular injection study of the posterior and anterior cingulate gyrus with comparative notes on the macaque and vervet monkeys

This study forms part of an ongoing investigation of pyramidal cell structure in the cingulate cortex of primates. Recently we have demonstrated that layer III pyramidal cells in the anterior cingulate gyrus are considerably larger, more branched and more spinous than those in the posterior cingulate gyrus (areas 24 and 23, respectively) in the macaque and vervet monkeys. Moreover, the extent of the interareal difference in specialization in pyramidal cell structure differed between the two species. These data suggest that pyramidal cell circuitry may have evolved differently in these closely related species. Presently there are too few data to speculate on what is selecting for this specialization in structure. Here we extend the basis for comparison by studying pyramidal cell structure in cingulate gyrus of the Chacma baboon (Papio ursinus). Methodology used here is the same as that for our previous studies: intracellular injection of Lucifer Yellow in flat-mounted cortical slices. We found that pyramidal cells in anterior cingulate gyrus (area 24) were more branched and more spinous than those in posterior cingulate gyrus (area 23). Moreover, the complexity in pyramidal cell structure in both the anterior and posterior cingulate gyrus of the baboon differed to that in the corresponding regions in either the macaque or vervet monkeys. (C) 2005 Elsevier Ireland Ltd. All rights reserved.

Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis

The aim was to investigate the roles of transmembrane domain 2 and the adjacent region of the first intracellular loop in determining human noradrenaline transporter (hNET) function by pharmacological and substituted-cysteine accessibility method (SCAM) analyses. It was first necessary to establish a suitable background NET for SCAM. Alanine mutants of endogenous hNET cysteines, hC86A, hC131A and hC339A, were examined and showed no marked effects on expression or function. hNET and the mutants were also resistant to methanethiosulfonate (MTS), ethylammonium (MTSEA) and MTStrimethylammonium (MTSET). Hence, wild-type hNET is an appropriate background for production of cysteine mutants for SCAM. Pharmacological investigation showed that all mutants except hT99C and hL109C showed reduced cell-surface expression, while all except hM107C showed a reduction in functional activity. The mutations did not markedly affect the apparent affinities of substrates, but apparent affinities of cocaine were decreased 7-fold for hP97C and 10-fold for hF101C and increased 12-fold for hY98C. [H-3]Nisoxetine binding affinities were decreased 13-fold for hP97C and 5-fold for hF101C. SCAM analysis revealed that only hL102C was sensitive to 1.25 mM MTSEA, and this sensitivity was protected by noradrenaline, nisoxetine and cocaine. The results suggest that this region of hNET is important for interactions with antidepressants and cocaine, but it is probably not involved in substrate translocation mechanisms.

Regional specialization in pyramidal cell structure in the visual cortex of the galago: An intracellular injection study of striate and extrastriate areas with comparative notes on New World and Old World monkeys

Recent studies have revealed marked differences in the basal dendritic structure of layer III pyramidal cells in the cerebral cortex of adult simian primates. In particular, there is a consistent trend for pyramidal cells of increasing complexity with anterior progression through occipitotemporal cortical visual areas. These differences in pyramidal cell structure, and their systematic nature, are believed to be important for specialized aspects of visual processing within, and between, cortical areas. However, it remains unknown whether this regional specialization in the pyramidal cell phenotype is unique to simians, is unique to primates in general or is widespread amongst mammalian species. In the present study we investigated pyramidal cell structure in the prosimian galago (Otolemur garnetti). We found, as in simians, that the basal dendritic arbors of pyramidal cells differed between cortical areas. More specifically, pyramidal cells became progressively more spinous through the primary (V1), second (V2), dorsolateral (DL) and inferotemporal ( IT) visual areas. Moreover, pyramidal neurons in V1 of the galago are remarkably similar to those in other primate species, in spite of large differences in the sizes of this area. In contrast, pyramidal cells in inferotemporal cortex are quite variable among primate species. These data suggest that regional specialization in pyramidal cell phenotype was a likely feature of cortex in a common ancestor of simian and prosimian primates, but the degree of specialization varies between species. Copyright (C) 2005 S. Karger AG, Basel.

Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons

1 The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca2+ concentration ([Ca2+](i)) and membrane currents were investigated in neonatal rat intracardiac neurons. 2 In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca2+](i), which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca2+](i) transients was 28 muM, close to the estimated clinical EC50 (clinically relevant (half-maximal) effective concentration) of thiopental. 3 In fura-2-loaded neurons, voltage clamped at -60mV to eliminate any contribution of voltage-gated Ca2+ channels, thiopental (25 muM) simultaneously inhibited nAChR-induced increases in [Ca2+](i) and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by - 40% at - 120, -80 and -40 mV holding potential, indicating that the inhibition is voltage independent. 4 The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC50 were also shown to inhibit nAChR-induced increases in [Ca2+](i) by similar to40%. 5 Thiopental (25 muM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca2+](i), indicating that inhibition of Ca2+ release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely. 6 Depolarization-activated Ca2+ channel currents were unaffected in the presence of thiopental (25 muM), pentobarbital (50 muM) and ketamine (10 muM). 7 In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca2+](i) transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions.

Regional specialization in pyramidal cell structure in the limbic cortex of the vervet monkey (Cercopithecus pygerythrus): an intracellular injection study of the anterior and posterior cingulate gyrus

The pyramidal cell phenotype varies quite dramatically in structure among different cortical areas in the primate brain. Comparative studies in visual cortex, in particular, but also in sensorimotor and prefrontal cortex, reveal systematic trends for pyramidal cell specialization in functionally related cortical areas. Moreover, there are systematic differences in the extent of these trends between different primate species. Recently we demonstrated differences in pyramidal cell structure in the cingulate cortex of the macaque monkey; however, in the absence of other comparative data it remains unknown as to whether the neuronal phenotype differs in cingulate cortex between species. Here we extend the basis for comparison by studying the structure of the basal dendritic trees of layer III pyramidal cells in the posterior and anterior cingulate gyrus of the vervet monkey (Brodmann's areas 23 and 24, respectively). Cells were injected with Lucifer Yellow in flat-mounted cortical slices, and processed for a light-stable DAB reaction product. Size, branching pattern, and spine density of basal dendritic arbors were determined, and somal areas measured. As in the macaque monkey, we found that pyramidal cells in anterior cingulate gyrus (area 24) were more branched and more spinous than those in posterior cingulate gyrus (area 23). In addition, the extent of the difference in pyramidal cell structure between these two cortical regions was less in the vervet monkey than in the macaque monkey.

Optimal Paths and Costs of Adjustment in Dynamic DEA Models: With Application to Chilean Department Stores

In this paper we propose a range of dynamic data envelopment analysis (DEA) models which allow information on costs of adjustment to be incorporated into the DEA framework. We first specify a basic dynamic DEA model predicated on a number or simplifying assumptions. We then outline a number of extensions to this model to accommodate asymmetric adjustment costs, non-static output quantities, non-static input prices, and non-static costs of adjustment, technological change, quasi-fixed inputs and investment budget constraints. The new dynamic DEA models provide valuable extra information relative to the standard static DEA models-they identify an optimal path of adjustment for the input quantities, and provide a measure of the potential cost savings that result from recognising the costs of adjusting input quantities towards the optimal point. The new models are illustrated using data relating to a chain of 35 retail department stores in Chile. The empirical results illustrate the wealth of information that can be derived from these models, and clearly show that static models overstate potential cost savings when adjustment costs are non-zero.

Type III secretion contact-dependent model for the intracellular development of Chlamydia

The medically significant genus Chlamydia is a class of obligate intracellular bacterial pathogens that replicate within vacuoles in host eukaryotic cells termed inclusions. Chlamydia's developmental cycle involves two forms; an infectious extracellular form, known as an elementary body (EB), and a non-infectious form, known as the reticulate body (RB), that replicates inside the vacuoles of the host cells. The RB surface is covered in projections that are in intimate contact with the inclusion membrane. Late in the developmental cycle, these reticulate bodies differentiate into the elementary body form. In this paper, we present a hypothesis for the modulation of these developmental events involving the contact-dependent type III secretion (TTS) system. TTS surface projections mediate intimate contact between the RB and the inclusion membrane. Below a certain number of projections, detachment of the RB provides a signal for late differentiation of RB into EB. We use data and develop a mathematical model investigating this hypothesis. If the hypothesis proves to be accurate, then we have shown that increasing the number of inclusions per host cell will increase the number of infectious progeny EB until some optimal number of inclusions. For more inclusions than this optimum, the infectious yield is reduced because of spatial restrictions. We also predict that a reduction in the number of projections on the surface of the RB (and as early as possible during development) will significantly reduce the burst size of infectious EB particles. Many of the results predicted by the model can be tested experimentally and may lead to the identification of potential targets for drug design. © Society for Mathematical Biology 2006.

Coordinate activation of intracellular signaling pathways by insulin-like growth factor-1 and platelet-derived growth factor in rat hepatic stellate cells

Proliferation of activated hepatic stellate cells (HSC) is an important event in the development of hepatic fibrosis. Insulin-like growth factor-1 (IGF-1) has been shown to be mitogenic for HSC, but the intracellular signaling pathways involved have not been fully characterized. Thus, the aims of the current study were to examine the roles of the extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (P13-K) and p70-S6 kinase (p70-S6-K) signaling pathways in IGF-1- and platelet-derived growth factor (PDGF)-induced mitogenic signaling of HSC and to examine the potential crosstalk between these pathways. Both IGF-1 and PDGF increased ERK, P13-K and p70-S6-K activity. When evaluating potential crosstalk between these signaling pathways, we observed that P13-K is required for p70-S6-K activation by IGF-1 and PDGF, and is partially responsible for PDGF-induced ERK activation. PDGF and IGF-1 also increased the levels of cyclin D1 and phospho-glycogen synthase kinase-30. Coordinate activation of ERK, P13-K and p70-S6-K is important for perpetuating the activated state of HSC during fibrogenesis.

Energy and calcium stores are preserved in girls and young women with CF, independent of disease severity: An explanation for the 'gender gap'?

Bioenergetics differ between males and females of many species. Human females apportion a substantial proportion of energy resources towards gynoid fat storage, to support the energetic burden of reproduction. Similarly, axial calcium accrual is favoured in females compared with males. Nutritional status is a prognostic indicator in cystic fibrosis (CF), but girls and young women are at greater risk of death despite equivalent nutritional status to males. The aim of this study was to compare fat (energy) and calcium stores (bone density) in males and females with CF over a spectrum of disease severity. Methods: Fat as % body weight (fat%) and lumbar spine (LS) and total body (TB) bone mineral density (BMD) were measured using dual absorption X-ray photometry in 127(59M) control and 101(54M) CF subjects, aged 9–25 years. An equation for predicted age at death had been determined using survival data and history of pulmonary function for the whole clinic, based on a trivariate normal model using maximum likelihood methods (1). For the CF group, a disease severity index (predicted age at death) was calculated from the derived equations according to each subjects history of pulmonary function, current age, and gender. Disease severity was classified according to percentile of predicted age at death (‘mild’ ≥75th, ‘moderate’ 25th–75th, ‘severe’ ≤25th percentile). Wt for age z-score was calculated. Serum testosterone and oestrogen were measured in males and females respectively. Fat% and LSBMD were compared between the groups using ANOVA. Results: There was an interaction between disease severity and gender: increasing disease severity was associated with greater deficits in TB (p=0.01), LSBMD (p