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.     

Quantifying changes in gap junction structure as a function of lens fiber cell differentiation

The ocular lens is an ideal model system for studying gap junction structure-function relationships. Here we apply novel methods to quantitatively compare connexin expression over macroscopic distances while simultaneously resolving the intracellular distribution of gap junctions in sub-micron detail. Our approach has identified three distinct zones of connexin density and allowed changes in gap junction plaque size, number and dispersion to be quantified. Our analysis is the first to precisely correlate changes in gap junction plaque structure with the reported changes in gap junction function that occur as a consequence of fiber cell differentiation.     

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.     

Ryanodine block of calcium oscillations in heart muscle and the sodium-tension relationship

The control of tension is examined in cardiac Purkinje fibers. We show, in accordance with earlier results (14, 15), that tension produced by this preparation is a steep power function of intracellular sodium. With the aid of ryanodine, a pharmacological agent that blocks spontaneous and spatially asynchronous calcium release from the sarcoplasmic reticulum (SR), we investigate the influence that such calcium fluctuations have on tension. We find that even when we control for alterations of intracellular pH, the presence of such fluctuations reduces the dependence of tension on intracellular sodium. We present a simple model that can explain how the presence of oscillations of intracellular calcium leads to a reduction of the slope of the tension-calcium relationship. We show, furthermore, that when the oscillations are spatially asynchronous, this reduction of slope is even greater. The modeling takes account of the known relationships between tension and calcium and tension and sarcomere length. We conclude that the effect of ryanodine to steepen the tension-sodium relationship can be explained by ryanodine's blocking calcium release from the SR, thereby abolishing oscillations of intracellular calcium.     

The superficial buffer barrier in vascular smooth muscle.

Force development and fura-2 fluorescence were simultaneously measured in the rabbit inferior vena cava. Discharging SR Ca2+ with either caffeine or norepinephrine prior to stimulation of Ca2+ influx induced a delay of 30-70 s between the intracellular Ca2+ signal and development of force. This delay was abolished by the application of caffeine. These data support the superficial buffer barrier hypothesis, which holds that Ca2+ entry from the extracellular space proceeds via a restricted cytoplasmic region between the inner plasmalemmal surface and the peripheral sarcoplasmic reticulum (SR). Ca2+ accumulation by this SR fraction appears to be able to delay Ca2+ entry into the deeper myoplasm where it activates the myofilaments. Caffeine and thapsigargin elevated the steady-state [Ca2+]i, suggesting a contribution by the SR Ca2+ pump to Ca2+ extrusion from the cells. Norepinephrine enhanced myofilament Ca2+ sensitivity, while caffeine decreased it.     

Purification and characterization of pentagalloylglucose, and alpha-glucosidase inhibitor/antibiotic from the freshwater green alga Spirogyra varians.

An alpha-glucosidase inhibitor/antibiotic was purified from the freshwater green alga Spirogyra varians and was determined to be the pentagalloylglucose 3-O-digalloyl-1,2,6-trigalloylglucose.     

Microalgae and cyanobacteria as a source of glycosidase inhibitors

Culture filtrates and organic solvent extracts of over 500 freshwater and marine eukaryotic microalgae and cyanobacteria were screened for the presence of glycosidase inhibitors. Rapid colorimetric assays were used to detect inhibitors of alpha-glucosidase, alpha-amylase and beta-galactosidase. Inhibitors were found from 38 species. The results suggest that microalgae and cyanobacteria have potential as a source of glycosidase inhibitors which may have clinical applications.     

N-poor ecosystems may respond more to elevated [CO2] than N-rich ones in the long term. A model analysis of grassland.

The Hurley Pasture Model was used to examine the short and long-term responses of grazed grasslands in the British uplands to a step increase from 350 to 700 μmol mol^–1 CO₂ concentration ([CO₂]) with inputs of 5 or 100 kg N ha^–1 y^–1. In N-rich grassland, [CO₂] doubling quickly increased net primary productivity (NPP), total carbon (C_sys) and plant biomass by about 30%. By contrast, the N-poor grassland underwent a prolonged ‘transient’, when there was little response, but eventually NPP, C_sys and plant biomass more than doubled. The ‘transient’ was due to N immobilization and severe depletion of the soil mineral N pool. The large long-term response was due to slow N accumulation, as a result of decreased leaching, decreased gaseous N losses and increased N₂-fixation, which amplified the CO₂ response much more in the N-poor than in the N-rich grassland. It was concluded that (i) ecosystems use extra carbon fixed at high [CO₂] to acquire and retain nutrients, supporting the contention of Gifford et al. (1996 ), (ii) in the long term, and perhaps on the real timescale of increasing [CO₂], the response (in NPP, C_sys and plant biomass) of nutrient-poor ecosystems may be proportionately greater than that of nutrient-rich ones, (iii) short-term experiments on nutrient-poor ecosystems may observe only the transient responses, (iv) the speed of ecosystem responses may be limited by the rate of nutrient accumulation rather than by internal rate constants, and (v) ecosystem models must represent processes affecting nutrient acquisition and retention to be able to simulate likely real-world CO₂ responses.     

Climate as a driver of phenological change in southern seabirds

Seabirds are one of the most threatened groups of birds globally and, overall, their conservation status is deteriorating rapidly. Southern hemisphere countries are over-represented in the number of species of conservation concern yet long-term phenological data on seabirds in the southern hemisphere is limited. A better understanding of the implications of changes in the marine and terrestrial environments to seabird species is required in order to improve their management and conservation status. Here we conducted a meta-analysis of the phenological drivers and trends among southern hemisphere seabirds. Overall there was a general trend towards later phenological events over time (34 % of all data series, N?=?47; 67 % of all significant trends), though this varied by taxa and location. The strongest trends towards later events were for seabirds breeding in Australia, the Laridae (gulls, noddies, terns) and migratory southern polar seabirds. In contrast, earlier phenologies were more often observed for the Spheniscidae (penguins) and for other seabirds breeding in the Antarctic and subantarctic. Phenological changes were most often associated with changes in oceanographic conditions, with sea-ice playing an important role for more southerly species. For some species in some locations, such as the Little Penguin Eudyptula minor in south-eastern Australia, warmer oceans projected under various climate change scenarios are expected to correspond to increased seabird productivity, manifested through earlier breeding, heavier chicks, an increased chance of double brooding, at least in the short-term.