Role of procoagulant lipids in human prothrombin activation. 2. Soluble phosphatidylserine upregulates and directs factor X(a) to appropriate peptide bonds in prothrombin.

Activation of human prothrombin to thrombin (II(a)) by factor X(a) during blood coagulation requires proteolysis of two bonds and thus involves two possible activation pathways (parallel-sequential activation model). Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). A soluble lipid, dicaproylphosphatidylserine (C6PS), enhances activation by 60-fold [Koppaka et al. (1996) Biochemistry 35, 7482]. We report here that C6PS binding to factor X(a) not only enhances the rate of activation but also alters the pathway. Activation was monitored using a chromogenic substrate (S-2238) to detect both II(a) and MzII(a) active site formation and SDS-PAGE to detect Pre2-F1.2 as well as II(a) and MzII(a). Of the four kinetic constants needed to describe activation, two (MzII(a) and Pre2-F1.2 consumption) were measured directly, and two (MzII(a) and Pre2-F1.2 formation) were obtained by fitting the three time courses simultaneously to the parallel-sequential reaction model. The time courses of II(a), MzII(a), and Pre2-F1.2 formations were all well described below the C6PS critical micelle concentration (CMC) by this activation model. The rate of Arg(322)-Ile cleavage leading to MzII(a) formation increased by 150-fold, while the rate of Arg(273)-Thr cleavage leading to Pre2-F1.2 formation was inhibited slightly. At concentrations of water-soluble C6PS above its CMC, all four proteolytic reactions increased in rate by 2-5-fold at the C6PS CMC. We conclude that soluble C6PS differentially affects the rate of individual bond cleavages during prothrombin activation in solution such that activation occurs almost exclusively via MzII(a) formation. Finally, C6PS enhanced the rates of all proteolytic reactions to within a factor of 3 of the enhancement seen with PS-containing membranes. We conclude that PS-containing membranes regulate prothrombin activation by factor X(a) mainly via interaction of individual PS molecules with factor X(a).     

Mitochondrial quality control by the ubiquitin-proteasome system

Mitochondria perform multiple functions critical to the maintenance of cellular homoeostasis and their dysfunction leads to disease. Several lines of evidence suggest the presence of a MAD (mitochondria-associated degradation) pathway that regulates mitochondrial protein quality control. Internal mitochondrial proteins may be retrotranslocated to the OMM (outer mitochondrial membrane), multiple E3 ubiquitin ligases reside at the OMM and inhibition of the proteasome causes accumulation of ubiquitinated proteins at the OMM. Reminiscent of ERAD [ER (endoplasmic reticulum)-associated degradation], Cdc48 (cell division cycle 42)/p97 is recruited to stressed mitochondria, extracts ubiquitinated proteins from the OMM and presents ubiquitinated proteins to the proteasome for degradation. Recent research has provided mechanistic insights into the interaction of the UPS (ubiquitin-proteasome system) with the OMM. In yeast, Vms1 [VCP (valosin-containing protein) (p97)/Cdc48-associated mitochondrial-stress-responsive 1] protein recruits Cdc48/p97 to the OMM. In mammalian systems, the E3 ubiquitin ligase parkin regulates the recruitment of Cdc48/p97 to mitochondria, subsequent mitochondrial protein degradation and mitochondrial autophagy. Disruption of the Vms1 or parkin systems results in the hyper-accumulation of ubiquitinated proteins at mitochondria and subsequent mitochondrial dysfunction. The emerging MAD pathway is important for the maintenance of cellular and therefore organismal viability.     

Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes.     

Leishmania: role of P glycoprotein in drug resistance and reversion strategies

Protozoan parasites are important causative agents of morbidity and mortality throughout the world--a problem further complicated by the emergence of drug resistance in these parasites. Mechanisms of drug resistance include the following: decreased uptake of the drug into the cell, loss of drug activation, alterations in the drug target, and over-expression of a well-known multiple drug transporter proteins. In this review, two critical components of resistance are stressed: (1) the role of ATP binding cassette proteins, such as P-glycoproteins, in mediating drug resistance in Leishmania and other protozoans, followed by development of cross-resistance to many structurally and functionally unrelated drugs, and (2) some concepts concerning the reversal mechanism of multidrug resistance by drugs and natural products. Several modulators or chemosensitizers alter the capacity of P-glycoproteins to maintain subtoxic intracellular drug concentrations. Calcium channel blockers such as verapamil act in this mode; however, high concentrations are required for an efficient and effective inhibition and, in addition, produce undesirable side effects. The discovery of new, natural product modulators of P-glycoproteins is stressed. This category of modulators offer potentially improved efficacy and lowered toxicity for the mammalian host.     

Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state

Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.     

Local polarity and hydrogen bonding inside the Sec14p phospholipid-binding cavity: high-field multi-frequency electron paramagnetic resonance studies

Sec14p promotes the energy-independent transfer of either phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) between lipid bilayers in vitro and represents the major PtdIns/PtdCho transfer protein in the budding yeast Saccharomyces cerevisiae. Herein, we employ multi-frequency high-field electron paramagnetic resonance (EPR) to analyze the electrostatic and hydrogen-bonding microenvironments for series of doxyl-labeled PtdCho molecules bound by Sec14p in a soluble protein-PtdCho complex. A structurally similar compound, 5-doxyl stearic acid dissolved in a series of solvents, was used for experimental calibration. The experiments yielded two-component rigid limit 130- and 220-GHz EPR spectra with excellent resolution in the gx region. Those components were assigned to hydrogen-bonded and nonhydrogen-bonded nitroxide species. Partially resolved 130-GHz EPR spectra from n-doxyl-PtdCho bound to Sec14p were analyzed using this two-component model and allowed quantification of two parameters. First, the fraction of hydrogen-bonded nitroxide species for each n-doxyl-PtdCho was calculated. Second, the proticity profile along the phospholipid-binding cavity of Sec14p was characterized. The data suggest the polarity gradient inside the Sec14p cavity is a significant contributor to the driving molecular forces for extracting a phospholipid from the bilayer. Finally, the enhanced g-factor resolution of EPR at 130 and 220 GHz provides researchers with a spectroscopic tool to deconvolute two major contributions to the x-component of the nitroxide g-matrix: hydrogen-bond formation and local electrostatic effects.     

Abscisic acid and CO2 signalling via calcium sensitivity priming in guard cells, new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses

Background

Stomatal guard cells are the regulators of gas exchange between plants and the atmosphere. Ca²⁺-dependent and Ca²⁺-independent mechanisms function in these responses. Key stomatal regulation mechanisms, including plasma membrane and vacuolar ion channels have been identified and are regulated by the free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt).

Scope

Here we show that CO₂-induced stomatal closing is strongly impaired under conditions that prevent intracellular Ca²⁺ elevations. Moreover, Ca²⁺ oscillation-induced stomatal closing is partially impaired in knock-out mutations in several guard cell-expressed Ca²⁺-dependent protein kinases (CDPKs) here, including the cpk4cpk11 double and cpk10 mutants; however, abscisic acid-regulated stomatal movements remain relatively intact in the cpk4cpk11 and cpk10 mutants. We further discuss diverse studies of Ca²⁺ signalling in guard cells, discuss apparent peculiarities, and pose novel open questions. The recently proposed Ca²⁺ sensitivity priming model could account for many of the findings in the field. Recent research shows that the stomatal closing stimuli abscisic acid and CO₂ enhance the sensitivity of stomatal closing mechanisms to intracellular Ca²⁺, which has been termed ‘calcium sensitivity priming’. The genome of the reference plant Arabidopsis thaliana encodes for over 250 Ca²⁺-sensing proteins, giving rise to the question, how can specificity in Ca²⁺ responses be achieved? Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca²⁺ signal transduction, a topic of central interest in cell signalling research. In this article we further propose an individual stomatal tracking method for improved analyses of stimulus-regulated stomatal movements in Arabidopsis guard cells that reduces noise and increases fidelity in stimulus-regulated stomatal aperture responses ( Box 1). This method is recommended for stomatal response research, in parallel to previously adopted blind analyses, due to the relatively small and diverse sizes of stomatal apertures in the reference plant Arabidopsis thaliana.

Box 1. Improved resolution of stimulus-induced stomatal movements in guard cells by tracking of individual stomatal apertures

Arabidopsis guard cells have become a prime model system for analysing signal transduction, since early research combining genetic and ion channel analyses in this system (Ichida et al., 1997; Pei et al., 1997, 1998; Roelfsema and Prins, 1997). Arabidopsis stomata are small relative to other stomatal model systems and stomatal apertures of various plant types including Arabidopsis are known to show variability in the size of individual stomatal complexes and also variability in the opening apertures of stomata of similar size in a given leaf (Gorton et al., 1988; Mott and Buckley, 2000; Mott and Peak, 2007). Thus stomatal aperture measurements are expected to show a clear degree of statistical variation. Use of blind experiments, in which the genotype and, when possible, the stimulus being applied to guard cells is unknown to the experimenter (Murata et al., 2001) has been employed by several laboratories, has become a standard in the field and has aided in addressing the above limitations of the range of stomatal aperture sizes found under any given condition.Research in our laboratory has shown that a major additional improvement in experiments can be made, by adding imaging of the same individual stomatal apertures over time (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Siegel et al., 2009), while performing blind experiments. In such ‘stomatal tracking’ experiments the lower side of a leaf is attached to a glass coverslip in an extracellular incubation medium (Webb et al., 2001; Young et al., 2006). The mesophyll and upper leaf epidermis are removed surgically for better optical resolution of stomatal apertures in the intact lower leaf epidermis (Young et al., 2006). For stimulus-induced stomatal closing analyses, a field of well-opened stomata is located and images are captured (e.g. using Scion Image software) for later analyses and data storage. The bottom (dry side) of coverslips can be marked with colour marker pens to label grids in the regions where apertures where imaged, for finding these same stomata subsequently if needed. Images of the same stomatal apertures are taken over time and can be stored for later analyses of individual stomatal apertures and for deposition of image files. While this approach has been used as a standard for imposed Ca²⁺ oscillation studies (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Fig. 4), we have found that this method also substantially improves stomatal movement response analyses to any given stimulus (Siegel et al., 2009; see Figs 1 and 4 and, Box Fig. 1). For example, while individual stomata are known to have diverse apertures (e.g. Box Fig. 1C), the relative responses of wide open stomata and smaller stomatal apertures to ABA or to CO₂ were comparable (Fig. 1 and Box Fig. 1; Siegel et al., 2009). Note that this method has previously been proposed and used in Vicia faba (Gorton et al., 1988), for which stomata exhibit relatively weak ABA and CO₂ responses, compared with, for example, Arabidopsis. We propose that this simple image-capturing approach, together with blind analyses, be used as a standard for stomatal response research in arabidopsis. Our research experience with this method shows that this approach will aid in greatly improving resolution and robustness and in defining the functions of individual Ca²⁺-independent and Ca²⁺-dependent components and mechanisms in stomatal response analyses. Open in a separate windowBox Fig. 1.ABA-induced stomatal closing of individually tracked stomatal apertures. (A) Average individually tracked stomatal apertures in the presence of 50 µm Ca²⁺ (open triangles) and in the presence of 200 nm free Ca²⁺ (open squares) in the bath solution from three experiments are shown and were normalized to the stomatal apertures at time = 0. (B, C) ABA-induced stomatal closing in the presence of 50 µm Ca²⁺ in five individually tracked stomatal apertures. In (A; open triangles) normalized stomatal apertures of the same stomata depicted in (B) and (C) are shown. Methods used in these experiments tracking individual stomatal apertures are described in Siegel et al. (2009). ABA-induced stomatal closing experiments are reproduced from Siegel et al. (2009) with permission of the publisher.     

Do ectotherms partition thermal resources? We still do not know

Partitioning of the niche space is a mechanism used to explain the coexistence of similar species. Ectotherms have variable body temperatures and their body temperatures influence performance and, ultimately, fitness. Therefore, many ectotherms use behavioral thermoregulation to avoid reduced capacities associated with body temperatures far from the optimal temperature for performance. Several authors have proposed that thermal niche partitioning in response to interspecific competition is a mechanism that allows the coexistence of similar species of ectotherms. We reviewed studies on thermal resource partitioning to evaluate the evidence for this hypothesis. In almost all studies, there was insufficient evidence to conclude unequivocally that thermal resource partitioning allowed species coexistence. Future studies should include sites where species are sympatric and sites where they are allopatric to rule out alternative mechanisms that cause differences in thermal traits between coexisting species. There is evidence of conservatism in the evolution of most thermal traits across a wide range of taxa, but thermal performance curves and preferred temperatures do respond to strong selection under laboratory conditions. Thus, there is potential for selection to act on thermal traits in response to interspecific competition. Nevertheless, more stringent tests of the thermal resource partitioning hypothesis are required before we can assess whether it is widespread in communities of ectotherms in nature.