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1.  Platelet Specific Promoters Are Insufficient to Express Protease Activated Receptor 1 (PAR1) Transgene in Mouse Platelets 
PLoS ONE  2014;9(5):e97724.
The in vivo study of protease activated receptors (PARs) in platelets is complicated due to species specific expression profiles. Human platelets express PAR1 and PAR4 whereas mouse platelets express PAR3 and PAR4. Further, PAR subtypes interact with one another to influence activation and signaling. The goal of the current study was to generate mice expressing PAR1 on their platelets using transgenic approaches to mimic PAR expression found in human platelets. This system would allow us to examine specific signaling from PAR1 and the PAR1-PAR4 heterodimer in vivo. Our first approach used the mouse GPIbα promoter to drive expression of mouse PAR1 in platelets (GPIbα-Tg-mPAR1). We obtained the expected frequency of founders carrying the transgene and had the expected Mendelian distribution of the transgene in multiple founders. However, we did not observe expression or a functional response of PAR1. As a second approach, we targeted human PAR1 with the same promoter (GPIbα-Tg-hPAR1). Once again we observed the expected frequency and distributing of the transgene. Human PAR1 expression was detected in platelets from the GPIbα-Tg-hPAR1 mice by flow cytometry, however, at a lower level than for human platelets. Despite a low level of PAR1 expression, platelets from the GPIbα-Tg-hPAR1 mice did not respond to the PAR1 agonist peptide (SFLLRN). In addition, they did not respond to thrombin when crossed to the PAR4−/− mice. Finally, we used an alternative platelet specific promoter, human αIIb, to express human PAR1 (αIIb-Tg-hPAR1). Similar to our previous attempts, we obtained the expected number of founders but did not detect PAR1 expression or response in platelets from αIIb-Tg-hPAR1 mice. Although unsuccessful, the experiments described in this report provide a resource for future efforts in generating mice expressing PAR1 on their platelets. We provide an experimental framework and offer considerations that will save time and research funds.
doi:10.1371/journal.pone.0097724
PMCID: PMC4022678  PMID: 24830314
2.  Differential Effects of G- and F-Actin on the Plasma Membrane Calcium Pump Activity 
Cell biochemistry and biophysics  2013;66(1):187-198.
We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641–1644, 2007). Results show evidences for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca2+ extrusion through the membrane. Our results provide further evidence of the activation–inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts.
doi:10.1007/s12013-012-9467-6
PMCID: PMC3894748  PMID: 23152090
PMCA; Cytoskeleton; Actin; Regulation
3.  Correction: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets 
PLoS ONE  2013;8(12):10.1371/annotation/0956857d-ecf5-4fb7-a834-131915a38a6a.
doi:10.1371/annotation/0956857d-ecf5-4fb7-a834-131915a38a6a
PMCID: PMC3857928
4.  Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets 
PLoS ONE  2013;8(2):e55740.
Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30–100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3−/− mice had increased Gq signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca2+) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca2+ release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3+/−) had an intermediate increase in maximum Ca2+ mobilization. Treatment of PAR3−/− mice platelets with P2Y12 antagonist (2MeSAMP) did not affect Ca2+ mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G12/13 signaling in response to thrombin was not significantly different between wild type and PAR3−/− mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca2+ mobilization and PKC activation in mouse platelets by physical interaction.
doi:10.1371/journal.pone.0055740
PMCID: PMC3566007  PMID: 23405206
5.  Marker-based linkage map of Andean common bean (Phaseolus vulgaris L.) and mapping of QTLs underlying popping ability traits 
BMC Plant Biology  2012;12:136.
Background
Nuña bean is a type of ancient common bean (Phaseolus vulgaris L.) native to the Andean region of South America, whose seeds possess the unusual property of popping. The nutritional features of popped seeds make them a healthy low fat and high protein snack. However, flowering of nuña bean only takes place under short-day photoperiod conditions, which means a difficulty to extend production to areas where such conditions do not prevail. Therefore, breeding programs of adaptation traits will facilitate the diversification of the bean crops and the development of new varieties with enhanced healthy properties. Although the popping trait has been profusely studied in maize (popcorn), little is known about the biology and genetic basis of the popping ability in common bean. To obtain insights into the genetics of popping ability related traits of nuña bean, a comprehensive quantitative trait loci (QTL) analysis was performed to detect single-locus and epistatic QTLs responsible for the phenotypic variance observed in these traits.
Results
A mapping population of 185 recombinant inbred lines (RILs) derived from a cross between two Andean common bean genotypes was evaluated for three popping related traits, popping dimension index (PDI), expansion coefficient (EC), and percentage of unpopped seeds (PUS), in five different environmental conditions. The genetic map constructed included 193 loci across 12 linkage groups (LGs), covering a genetic distance of 822.1 cM, with an average of 4.3 cM per marker. Individual and multi-environment QTL analyses detected a total of nineteen single-locus QTLs, highlighting among them the co-localized QTLs for the three popping ability traits placed on LGs 3, 5, 6, and 7, which together explained 24.9, 14.5, and 25.3% of the phenotypic variance for PDI, EC, and PUS, respectively. Interestingly, epistatic interactions among QTLs have been detected, which could have a key role in the genetic control of popping.
Conclusions
The QTLs here reported constitute useful tools for marker assisted selection breeding programs aimed at improving nuña bean cultivars, as well as for extending our knowledge of the genetic determinants and genotype x environment interaction involved in the popping ability traits of this bean crop.
doi:10.1186/1471-2229-12-136
PMCID: PMC3490973  PMID: 22873566
6.  X-ray Crystallography and Isothermal Titration Calorimetry Studies of the Salmonella Zinc Transporter ZntB 
SUMMARY
The ZntB Zn2+ efflux system is important for maintenance of Zn2+ homeostasis in Enterobacteria. We report crystal structures of ZntB cytoplasmic domains from Salmonella enterica serovar Typhimurium (StZntB) in dimeric and physiologically relevant homopentameric forms at 2.3 Å and 3.1 Å resolutions, respectively. The funnel-like structure is similar to that of the homologous Thermotoga maritima CorA Mg2+ channel and a Vibrio parahaemolyticus ZntB (VpZntB) soluble domain structure. However, the central α7 helix forming the inner wall of the StZntB funnel is oriented perpendicular to the membrane instead of the marked angle seen in CorA or VpZntB. Consequently, the StZntB funnel pore is cylindrical, not tapered, which may represent an “open” form of the ZntB soluble domain. Our crystal structures and isothermal titration calorimetry data indicate that there are three Zn2+ binding sites in the full-length ZntB, two of which could be involved in Zn2+ transport.
doi:10.1016/j.str.2011.02.011
PMCID: PMC3094545  PMID: 21565704
7.  Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells 
Molecular Vision  2011;17:279-290.
Purpose
Nanoparticles are a promising alternative for ocular drug delivery, and our group has proposed that they are especially suited for ocular mucosal disorders. The goal of the present study was to determine which internalization pathway is used by cornea-derived and conjunctiva-derived cell lines to take up hyaluronic acid (HA)-chitosan oligomer (CSO)-based nanoparticles (HA-CSO NPs). We also determined if plasmids loaded onto the NPs reached the cell nucleus.
Methods
HA-CSO NPs were made of fluoresceinamine labeled HA and CSO by ionotropic gelation and were conjugated with a model plasmid DNA for secreted alkaline phosphatase. Human epithelial cell lines derived from the conjunctiva and the cornea were exposed to HA-CSO NPs for 1 h and the uptake was investigated in living cells by fluorescence microscopy. The influence of temperature and metabolic inhibition, the effect of blocking hyaluronan receptors, and the inhibition of main endocytic pathways were studied by fluorometry. Additionally, the metabolic pathways implicated in the degradation of HA-CSO NPs were evaluated by lysosome identification.
Results
There was intracellular localization of plasmid-loaded HACSO NPs in both corneal and conjunctival cells. The intracellular presence of NPs diminished with time. HA-CSO NP uptake was significantly reduced by inhibition of active transport at 4 °C and by sodium azide. Uptake was also inhibited by blocking hyaluronan receptors with anti-CD44 Hermes-1 antibody, by excess HA, and by filipin, an inhibitor of caveolin-dependent endocytosis. HA-CSO NPs had no effect on cell viability. The transfection efficiency of the model plasmid was significantly higher in NP treated cells than in controls.
Conclusions
HA-CSO NPs were internalized by two different ocular surface cell lines by an active transport mechanism. The uptake was mediated by hyaluronan receptors through a caveolin-dependent endocytic pathway, yielding remarkable transfection efficiency. Most of HA-CSO NPs were metabolized within 48 h. This uptake did not compromise cell viability. These findings further support the potential use of HA-CSO NPs to deliver genetic material to the ocular surface.
PMCID: PMC3030601  PMID: 21283563

Results 1-7 (7)