Seeing is believing: Recent trends in the measurement of Ca2+ in subcellular domains and intracellular organelles

The role of Ca2+ in the regulation of the cell cycle has been investigated mostly in studies assessing global cytosolic free Ca2+. Recent studies, however, have used unique techniques to assess Ca2+ in subcellular organelles, such as mitochondria, and in discrete regions of the cytoplasm. These studies have used advanced fluorescence digital imaging techniques and Ca2+-sensitive fluorescence probes, and/or targeting of Ca2+-sensitive proteins to intracellular organelles. The present review describes the results of some of these studies and the techniques used. The novel techniques used to measure Ca2+ in microdomains and intracellular organelles are likely to be of great use in future investigations assessing Ca2+ homeostasis during the cell cycle.

Characterisation of Australian isolates of Fusarium oxysporum f. sp cubense by DNA fingerprinting analysis

Genetic variation among Australian isolates of the fungus Fusarium oxysporum f. sp. cubense (Foc), which causes Fusarium wilt in banana, was examined using DNA amplification fingerprinting (DAF). Ninety-four isolates which represented Races 1, 2, 3, and 4, and vegetative compatibility groups (VCGs) 0120, 0124, 0125, 0128, 0129, 01211, 01213/16, and 01220 were analysed. The genetic relatedness among isolates within each VCG, and between the 8 different VCGs of Foc present in Australia was determined. The DNA fingerprint patterns were VCG-specific, with each VCG representing a unique genotype. The genetic similarity among isolates within each VCG ranged from 97% to 100%. Among the different VCGs of Foc, 3 major clusters were distinguished which corresponded with race. All Race 1 and 2 isolates (VCGs 0124, 0125, 0128, and 01220) were closely related and clustered together, the Race 3 isolates from Heliconia clustered separately, and all Race 4 isolates (VCGs 0120, 0129, 01211, and 01213/16) clustered together. Fifteen isolates from Alstonville, NSW, were characterised because although they were classified as Race 2 based on their recovery from cooking banana cultivars, they belonged in VCG 0124, which had previously contained only Race 1 isolates. The occurrence of more than one race within a VCG means that vegetative compatibility grouping cannot be used to assign pathotype to pathogenic race as previously thought. It was possible to distinguish the Race 1 and Race 2 isolates within VCG 0124 using DNA fingerprinting, as each race produced a unique DNA fingerprint pattern. Among the Australian isolates, DNA fingerprinting analysis identified 9 different VCGs and genotypes of Foc.

Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains

We have previously demonstrated that or-smooth muscle (alpha -SM) actin is predominantly distributed in the central region and beta -non-muscle (beta -NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha -actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta -NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and ol-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha -SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha -actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sar-comere-like units with alpha -actinin-rich dense bodies analogous to Z-lines, are the contractile vimentin structures of cultured SMCs that link to the network of vimentin-containing intermediate alpha -actinin filaments through the dense bodies and dense plaques.

Truncation of the GABA(A)-receptor gamma 2 subunit in a family with generalized epilepsy with febrile seizures plus

Recent findings from studies of two families have shown that mutations in the GABA(A)-receptor gamma2 subunit are associated with generalized epilepsies and febrile seizures. Here we describe a family that has generalized epilepsy with febrile seizures plus (GEFS(+)), including an individual with severe myoclonic epilepsy of infancy, in whom a third GABA(A)-receptor gamma2-subunit mutation was found. This mutation lies in the intracellular loop between the third and fourth transmembrane domains of the GABA(A)-receptor gamma2 subunit and introduces a premature stop codon at Q351 in the mature protein. GABA sensitivity in Xenopus laevis oocytes expressing the mutant gamma2(Q351X) subunit is completely abolished, and fluorescent-microscopy studies have shown that receptors containing GFP-labeled gamma2(Q351X) protein are retained in the lumen of the endoplasmic reticulum. This finding reinforces the involvement of GABA(A) receptors in epilepsy.

Properties of the vertebrate skeletal muscle tubular system as a sealed compartment

Confocal imaging of impermeant fluorescent dyes trapped in the tubular (t-) system of skeletal muscle fibres of rat and cane toad was used to examine changes in the morphology of the t-system upon mechanical skinning, the time course of dye loss from the sealed t-systern in mechanically skinned fibres and the influence of rapid application and removal of glycerol on the morphology of the sealed t-system. In contrast to intact fibres, which have a t-systern open to the outside, the sealed t-systern of toad mechanically skinned fibres consistently displayed local swellings (vesicles). The occurrence of vesicles in the sealed t-system of rat-skinned fibres was infrequent. Application and removal of 200-400 mM glycerol to the sealed t-system did not produce any obvious changes in its morphology. The dyes fluo-3, fura-2 and Oregon green 488 were lost from the sealed t-system of toad fibres at different rates suggesting that the mechanism of organic anion transport across the tubular wall was not by indiscriminate bulk transport. The rate of fluo-3 and fura-2 loss from the sealed t-system of rat fibres was greater in rat than in toad fibres and could be explained by differences in surface area: volume ratio of the t-system in the two fibre types. Based on the results presented here and on other results from this laboratory, an explanation is given for the formation of numerous vesicles in toad-skinned fibres and lack of vesicle formation in rat-skinned fibres. This explanation can also help with better understanding the mechanism responsible for vacuole formation in intact fibres. (C) 2002 Elsevier Science Ltd. All rights reserved.

Tomosyn interacts with the t-SNAREs Syntaxin4 and SNAP23 and plays a role in insulin-stimulated GLUT4 translocation

The Sec1p-like/Munc18 (SM) protein Munc18a binds to the neuronal t-SNARE Syntaxin1A and inhibits SNARE complex assembly. Tomosyn, a cytosolic Syntaxin1A-binding protein, is thought to regulate the interaction between Syntaxin1A and Munc18a, thus acting as a positive regulator of SNARE assembly. In the present study we have investigated the interaction between b-Tomosyn and the adipocyte SNARE complex involving Syntaxin4/SNAP23/VAMP-2 and the SM protein Munc18c, in vitro, and the potential involvement of Tomosyn in regulating the translocation of GLUT4 containing vesicles, in vivo. Tomosyn formed a high affinity ternary complex with Syntaxin4 and SNAP23 that was competitively inhibited by VAMP-2. Using a yeast two-hybrid assay we demonstrate that the VAMP-2-like domain in Tomosyn facilitates the interaction with Syntaxin4. Overexpression of Tomosyn in 3T3-L1 adipocytes inhibited the translocation of green fluorescent protein-GLUT4 to the plasma membrane. The SM protein Munc18c was shown to interact with the Syntaxin4 monomer, Syntaxin4 containing SNARE complexes, and the Syntaxin4/Tomosyn complex. These data suggest that Tomosyn and Munc18c operate at a similar stage of the Syntaxin4 SNARE assembly cycle, which likely primes Syntaxin4 for entry into the ternary SNARE complex.

Response selection deficits in melancholic but not nonmelancholic unipolar major depression

One consistent functional imaging finding from patients with major depression has been abnormality of the anterior cingulate cortex (ACC). Hypoperfusion has been most commonly reported, but some studies suggest relative hyperperfusion is associated with response to somatic treatments. Despite these indications of the possible importance of the ACC in depression there have been relatively few cognitive studies ACC function in patients with major depression. The present study employed a series of reaction time (RT) tasks involving selection with melancholic and nonmelancholic depressed patients, as well as age-matched controls. Fifteen patients with unipolar major depression (7 melancholic, 8 nonmelancholic) and 8 healthy age-matched controls performed a series of response selection tasks (choice RT, spatial Stroop, spatial stimulus-response compatibility (SRC), and a combined Stroop + SRC condition). Reaction time and error data were collected. Melancholic patients were significantly slower than controls on all tasks but were slower than nonmelancholic patients only on the Stroop and Stroop + SRC conditions. Nonmelancholic patients did not differ from the control group on any task. The Stroop task seems crucial in differentiating the two depressive groups, they did not differ on the choice RT or SRC tasks. This may reflect differential task demands, the SRC involved symbolic manipulation that might engage the dorsal ACC and dorsolateral prefrontal cortex (DLPFC) to a greater extent than the, primarily inhibitory, Stroop task which may engage the ventral ACC and orbitofrontal cortex (OFC). This might suggest the melancholic group showed a greater ventral ACC-OFC deficit than the nonmelancholic group, while both groups showed similar dorsal ACC-DLPFC deficit.

Inflation targeting and macroeconomic stability in a Post Keynesian economy

This paper examines the compatibility of inflation targeting with an economy that is Post Keynesian in character. We show that in a Post Keynesian environment, policymakers can both set and achieve an inflation target without adverse consequences for the real economy, as long as an appropriate policy mix is chosen. The latitude that policymakers have in making this choice is investigated. One of our key results is that orthodox policy regimes do not provide appropriate policy mixes. Indeed, the more orthodox the policy regime becomes, the less viable is inflation targeting in a Post Keynesian economy.

Reproductive biology of Echinodorus longipetalus (Alismataceae): Sexual morphs, breeding system and pollinators

The floral biology, pollinators and breeding system of Echinodorus longipetalus Micheli were studied in a marshy area of the district of Taquaritinga (State of Sao Paulo), southeastern Brazil. E. longipetalus is gynodioecious and as far as is known, this is the first record of unisexual flowers, besides perfect flowers, in Echinodorus. Proportion of female individuals in the studied population is 50% and produces 31% more flowers than hermaphrodites. Perfect and pistillate flowers of E. longipetalus are similar in appearance and are pollinated by several species of Hymenoptera (mainly by Xylocopa (Neoxylocopa) suspecta Moure & Camargo). Perfect flowers offer pollen as a reward. Pistillate flowers attract floral visitors by deceit with their staminodes that resemble the stamens of the perfect flowers. Visits to pistillate flowers are quick (1-2 s), while visits to perfect flowers last up to 120 s. The perfect flowers are self-compatible and produce fruits through spontaneous self-pollination (control flowers), whereas the pistillate ones only set fruits through cross-pollinations. Perfect and pistillate flowers set more fruits under natural conditions than in manual treatments, respectively. Although the pistillate and perfect flowers bear a strong similarity, the selective pollinator behavior seems to be responsible for the increase of fruit set in perfect flowers. (C) 2008 Elsevier B.V. All rights reserved.

Male-by-female interactions influence fertilization success and mediate the benefits of polyandry in the sea urchin Heliocidaris erythrogramma

Numerous studies have reported that females benefit from mating with multiple males (polyandry) by minimizing the probability of fertilization by genetically incompatible sperm. Few, however, have directly attributed variation in female reproductive success to the fertilizing capacity of sperm. In this study we report on two experiments that investigated the benefits of polyandry and the interacting effects of males and females at fertilization in the free-spawning Australian sea urchin Heliocidaris erythrogramma. In the first experiment we used a paired (split clutch) experimental design and compared fertilization rates within female egg clutches under polyandry (eggs exposed to the sperm from two males simultaneously) and monandry (eggs from the same female exposed to sperm from each of the same two males separately). Our analysis revealed a significant fertilization benefit of polyandry and strong interacting effects of males and females at fertilization. Further analysis of these data strongly suggested that the higher rates of fertilization in the polyandry treatment were due to an overrepresentation of fertilizations due to the most compatible male. To further explore the interacting effects of males and females at fertilization we performed a second factorial experiment in which four mates were crossed with two females (in all eight combinations). In addition to confirming that fertilization success is influenced by male X female interactions, this latter experiment revealed that both sexes contributed significant variance to the observed patterns of fertilization. Taken together, these findings highlight the importance of male X female interactions at fertilization and suggest that polyandry will enable females to reduce the cost of fertilization by incompatible gametes.

HPLC-FLD methods to quantify chloroaluminum phthalocyanine in nanoparticles, plasma and tissue: application in pharmacokinetic and biodistribution studies

Analytical and bioanalytical methods of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) were developed and validated for the determination of chloroaluminum phthalocyanine in different formulations of polymeric nanocapsules, plasma and livers of mice. Plasma and homogenized liver samples were extracted with ethyl acetate, and zinc phthalocyanine was used as internal standard. The results indicated that the methods were linear and selective for all matrices studied. Analysis of accuracy and precision showed adequate values, with variations lower than 10% in biological samples and lower than 2% in analytical samples. The recoveries were as high as 96% and 99% in the plasma and livers, respectively. The quantification limit of the analytical method was 1.12 ng/ml, and the limits of quantification of the bioanalytical method were 15 ng/ml and 75 ng/g for plasma and liver samples, respectively. The bioanalytical method developed was sensitive in the ranges of 15-100 ng/ml in plasma and 75-500 ng/g in liver samples and was applied to studies of biodistribution and pharmacokinetics of AlClPc. (C) 2011 Elsevier B.V. All rights reserved.

Fluorescence spectroscopy of small peptides interacting with microheterogeneous micelles

Many peptides containing tryptophan have therapeutic uses and can be studied by their fluorescent properties. The biological activity of these peptides involves interactions with many cellular components and micelles can function as carriers inside organisms. We report results from the interaction of small peptides containing tryptophan with several microheterogeneous systems: sodium dodecyl sulphate (SDS) micelles; sodium dodecyl sulphate-poly(ethylene oxide) (SDS-PEO) aggregates; and neutral polymeric micelles. We observed that specific parameters, such as wavelength of maximum emission and fluorescence anisotropy, could be used to ascertain the occurrence of interactions. Affinity constants were determined from changes in the intensity of emission while structural modifications in rotameric conformations were verified from time-resolved measurements. Information about the location and diffusion of peptides in the microheterogeneous systems were obtained from tryptophan emission quenching experiments using N-alkylpyridinium ions. The results show the importance of electrostatic and hydrophobic effects, and of the ionization state of charged residues, in the presence of anionic and amphiphilic SDS in the microheterogeneous systems. Conformational stability of peptides is best preserved in the interaction with the neutral polymeric micelles. (C) 2009 Elsevier B.V. All rights reserved.

Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation Of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean N-15-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.

Targeting Antigen-loaded Liposomes to Myeloid Antigen Presenting Cells

Objective: To target antigen-loaded liposomes to myeloid APC in vivo for immunotherapy and to manipulate immune function through liposome composition. Method: Liposomes were loaded with ovalbumin, the lipophilic red fluorescent marker, DiI, with or without QuilA adjuvant then injected either i.v. or s.c. to naı¨ ve C57Bl/6 mice. Spleen, liver and draining LN were stained with MHC class II and various myeloid markers to determine the uptake of liposomes. Frozen sections of spleen and draining LN were stained with FITC-labeled mAb to determine which cells take up the liposomes. To determine the effect on OVA-specific T cell responses, liposomes were administered to Balb/c mice which received DO11.10 OVAspecific TCR transgenic T cells labelled with CFSE. Results: The DiI fluorescence was visualized in MHC class II+ macrophages and DC in draining lymph nodes after s.c. injection and in spleen and liver after i.v injection. Immunofluorescence microscopy shows liposome uptake in marginal zone macrophages and some DC in the T cell areas of the spleen after i.v. injection. Administration of ova-liposomes with or without QuilA stimulated a specific T cell response as measured by CFSE dilution. Conclusion: APC of liver, spleen and LN, and subsequent antigen presentation to T cells can be targeted for immunotherapy by the administration of liposomes encapsulating antigen and adjuvant. Varying the composition and routes of liposome administration is expected to alter the function of the targeted APC and the T cell response.

Inhibition of NF kappa B leads to an intracellular reducing environment in human monocyte-derived immature dendritic cells

Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.