Modelling calcium microdomains using homogenisation

Microdomains of calcium (i.e., areas on the nanometer scale that have qualitatively different calcium concentrations from that in the bulk cytosol) are known to be important in many situations. In cardiac cells, for instance, a calcium microdomain between the L-type channels and the ryanodine receptors, the so-called diadic cleft, is where the majority of the control of calcium release occurs. In other cell types that exhibit calcium oscillations and waves, the importance of microdomains in the vicinity of clusters of inositol trisphosphate receptors, or between the endoplasmic reticulum (ER) and other internal organelles or the plasma membrane, is clear. Given the limits of computational power, it is not currently realistic to model an entire cellular cytoplasm by incorporating detailed structural information about the ER throughout the entire cytoplasm. Hence, most models use a homogenised approach, assuming that both cytoplasm and ER coexist at each point of the domain. Conversely, microdomain models can be constructed, in which detailed structural information can be incorporated, but, until now, methods have not been developed for linking such a microdomain model to a model at the level of the entire cell. Using the homogenisation approach we developed in an earlier paper [Goel, P., Friedman, A., Sneyd, J., 2006. Homogenization of the cell cytoplasm: the calcium bidomain equations. SIAM J. Multiscale Modeling Simulation, in press] we show how a multiscale model of a calcium microdomain can be constructed. In this model a detailed model of the microdomain (in which the ER and the cytoplasm are separate compartments) is coupled to a homogenised model of the entire cell in a rigorous way. Our method is illustrated by a simple model of the diadic cleft of a cardiac half-sarcomere.     

Effects of artificial social stimuli on the reproductive schedule and hormone levels of yellow-eyed penguins (Megadyptes antipodes)

The effects of social stimuli on avian reproductive behaviors such as breeding schedules and courtship behaviors are well known due to numerous field studies. However, studies that have simultaneously examined the effects of social stimuli on reproductive behavior and the mediating endocrine mechanisms have been largely restricted to captive populations, which may not be representative of free-living populations. This study, conducted over two breeding seasons, aimed to simultaneously measure the effects of experimentally increasing auditory stimuli on the breeding schedule and endocrinology (levels of total androgen, estradiol, progesterone and prolactin) on free-living yellow-eyed penguins (Megadyptes antipodes). The yellow-eyed penguin is the least colonial of all penguins, nesting far apart from each other under dense vegetation, and, therefore, is presumed to experience much lower levels of social stimuli than other penguins. Egg laying was significantly more synchronous and tended to be earlier when birds were exposed to playbacks of the calls of conspecifics in 1 year of the study. We also found that levels of total androgen and estradiol of males in 1 year, and prolactin in another year, were proportionally higher among treated birds compared control birds that received no artificial auditory stimuli. These results show that even among supposedly solitary nesters, social stimuli could still play a role in influencing reproductive behavior and physiology. For the first time in free-living seabirds, we have demonstrated that behavioral responses to increased social stimuli are associated with hormonal changes.     

Induction of T cell responses and recruitment of an inflammatory dendritic cell subset following tumor immunotherapy with Mycobacterium smegmatis

Mycobacteria and their cell wall components have been used with varying degrees of success to treat tumors, and Mycobacterium bovis BCG remains in use as a standard treatment for superficial bladder cancer. Mycobacterial immunotherapy is very effective in eliciting local immune responses against solid tumors when administered topically; however, its effectiveness in eliciting adaptive immune responses has been variable. Using a subcutaneous mouse thymoma model, we investigated whether immunotherapy with Mycobacterium smegmatis, a fast-growing mycobacterium of low pathogenicity, induces a systemic adaptive immune response. We found that M. smegmatis delivered adjacent to the tumor site elicited a systemic anti-tumor immune response that was primarily mediated by CD8⁺ T cells. Of note, we identified a CD11c⁺CD40^intCD11b^hiGr-1⁺ inflammatory DC population in the tumor-draining lymph nodes that was found only in mice treated with M. smegmatis. Our data suggest that, rather than rescuing the function of the DC already present in the tumor and/or tumor-draining lymph node, M. smegmatis treatment may promote anti-tumor immune responses by inducing the involvement of a new population of inflammatory cells with intact function.     

Metabolism of prednisolone by the isolated perfused human placental lobule

Previous studies of the metabolism of 11 beta-hydroxy corticosteroids by placental tissue have indicated that the only product is the C11-oxidized metabolite. In the present study we have re-examined the metabolism of prednisolone in the isolated, perfused, dual recirculating human placental lobule, using a perfusate based on tissue culture medium 199. Four metabolites were identified in both the maternal and fetal compartments in 6 h perfusions by comparison of relative retention times measured by HPLC and capillary gas chromatography (GC) and of mass spectra recorded by capillary gas chromatography-mass spectrometry (GC-MS) with those of authentic reference standards. The steroids were derivatized as the MO-TMS ethers for mass spectral measurements. Analysis of samples from five perfusion experiments resulted in the following percentage conversions after 6 h perfusion (mean +/- SD, maternal and fetal perfusate, respectively): prednisone (49.1 +/- 7.8, 49.1 +/- 6.6), 20 alpha-dihydroprednisone (0.84 +/- 0.29, 0.81 +/- 0.35), 20 beta-dihydroprednisone (39.1 +/- 6.7, 39.2 +/- 5.9), 20 beta-dihydroprednisolone (6.8 +/- 2.7, 6.3 +/- 1.6) and unmetabolized prednisolone (4.1 +/- 1.8, 4.6 +/- 2.1). No evidence was found for metabolites formed by 6 beta-hydroxylation or cleavage of the C17-C20 bond.     

The influence of auxins on transformation of wheat and tritordeum and analysis of transgene integration patterns in transformants

 Although it is possible to transform wheat, broad application of the technology is limited because of the low overall efficiency and the lack of reliability of the technique. In addition there is little published data on transgene integration patterns and inheritance in wheat. We have generated a population of transgenic wheat and tritordeum lines under different auxin regimes and show that, under the conditions described, the presence of picloram results in higher transformation efficiencies than the presence of 2,4-D. Molecular analysis shows low-copy numbers and simple integration patterns to be prevalent in the transgenic lines. Mendelian inheritance of transgenes in T ₁ progeny was observed for the majority of lines. Received: 2 December 1997 / Accepted: 12 March 1998     

Phase Ia clinical evaluation of the safety and immunogenicity of the Plasmodium falciparum blood-stage antigen AMA1 in ChAd63 and MVA vaccine vectors

Background

Traditionally, vaccine development against the blood-stage of Plasmodium falciparum infection has focused on recombinant protein-adjuvant formulations in order to induce high-titer growth-inhibitory antibody responses. However, to date no such vaccine encoding a blood-stage antigen(s) alone has induced significant protective efficacy against erythrocytic-stage infection in a pre-specified primary endpoint of a Phase IIa/b clinical trial designed to assess vaccine efficacy. Cell-mediated responses, acting in conjunction with functional antibodies, may be necessary for immunity against blood-stage P. falciparum. The development of a vaccine that could induce both cell-mediated and humoral immune responses would enable important proof-of-concept efficacy studies to be undertaken to address this question.

Methodology

We conducted a Phase Ia, non-randomized clinical trial in 16 healthy, malaria-naïve adults of the chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient viral vectored vaccines encoding two alleles (3D7 and FVO) of the P. falciparum blood-stage malaria antigen; apical membrane antigen 1 (AMA1). ChAd63-MVA AMA1 administered in a heterologous prime-boost regime was shown to be safe and immunogenic, inducing high-level T cell responses to both alleles 3D7 (median 2036 SFU/million PBMC) and FVO (median 1539 SFU/million PBMC), with a mixed CD4⁺/CD8⁺ phenotype, as well as substantial AMA1-specific serum IgG responses (medians of 49 µg/mL and 41 µg/mL for 3D7 and FVO AMA1 respectively) that demonstrated growth inhibitory activity in vitro.

Conclusions

ChAd63-MVA is a safe and highly immunogenic delivery platform for both alleles of the AMA1 antigen in humans which warrants further efficacy testing. ChAd63-MVA is a promising heterologous prime-boost vaccine strategy that could be applied to numerous other diseases where strong cellular and humoral immune responses are required for protection.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01095055","term_id":"NCT01095055"}}NCT01095055     

Numerical simulation of local calcium movements during L-type calcium channel gating in the cardiac diad.

Computer simulation was used to investigate the calcium levels after sarcolemmal calcium influx through L-type calcium channels (DHPRs) into the narrow diadic space of cardiac muscle. The effect of various cytosolic and membranebound buffers, diad geometry, DHPR properties (open time and current), and surface charge were examined. The simulations showed that phospholipid binding sites on the sarcolemmal membrane are the major buffer affecting free calcium ([Ca2+]) levels in the diad. The inclusion of surface charge effects calculated from Gouy-Chapman theory resulted in a marked decrease in [Ca2+] levels at all times and a faster decay of [Ca2+] after termination of DHPR influx. For a DHPR current of 200 fA, [Ca2+] at the center of the diad reached peak levels of approximately 73 microM. In larger diads (> or = 400 nm diameter), [Ca2+] decayed more slowly than in smaller diads (100-200 nm diameter), although peak [Ca2+] levels reached during typical DHPR open times were similar. For a wide range of DHPR single-channel current magnitudes (Ica = 25-200 fA), [Ca2+] levels in the diad were approximately proportional to ICa. The decrease in calculated [Ca2+] levels due to the effects of surface charge can be interpreted as resulting from an effective "volume expansion" of the diad space. Furthermore, the layer of increased [Ca2+] close to the sarcolemmal membrane can act as a fast buffer.     

Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro

Acyl glucuronides are reactive metabolites of carboxylate drugs, able to undergo a number of reactions in vitro and in vivo, including isomerization via intramolecular rearrangement and covalent adduct formation with proteins. The intrinsic reactivity of a particular acyl glucuronide depends upon the chemical makeup of the drug moiety. The least reactive acyl glucuronide yet reported is valproic acid acyl glucuronide (VPA-G), which is the major metabolite of the antiepileptic agent valproic acid (VPA). In this study, we showed that both VPA-G and its rearrangement isomers (iso-VPA-G) interacted with bovine brain microtubular protein (MTP, comprised of 85% tubulin and 15% microtubule associated proteins {MAPs}). MTP was incubated with VPA, VPA-G and iso-VPA-G for 2 h at room temperature and pH 7.5 at various concentrations up to 4 mM. VPA-G and iso-VPA-G caused dose-dependent inhibition of assembly of MTP into microtubules, with 50% inhibition (IC₅₀) values of 1.0 and 0.2 mM respectively, suggesting that iso-VPA-G has five times more inhibitory potential than VPA-G. VPA itself did not inhibit microtubule formation except at very high concentrations (≥2 mM). Dialysis to remove unbound VPA-G and iso-VPA-G (prior to the assembly assay) diminished inhibition while not removing it. Comparison of covalent binding of VPA-G and iso-VPA-G (using [¹⁴C]-labelled species) showed that adduct formation was much greater for iso-VPA-G. When [¹⁴C]-iso-VPA-G was reacted with MTP in the presence of sodium cyanide (to stabilize glycation adducts), subsequent separation into tubulin and MAPs fractions by ion exchange chromatography revealed that 78 and 22% of the covalent binding occurred with the MAPs and tubulin fractions respectively. These experiments support the notion of both covalent and reversible binding playing parts in the inhibition of microtubule formation from MTP (though the acyl glucuronide of VPA is less important than its rearrangement isomers in this regard), and that both tubulin and (perhaps more importantly) MAPs form adducts with acyl glucuronides.