Endogenous and exogenous Ca2+ buffers differentially modulate Ca2+-dependent inactivation of Ca(v)2.1 Ca2+ channels

Voltage-gated Ca2+ channels undergo a negative feedback regulation by Ca2+ ions, Ca2+-dependent inactivation, which is important for restricting Ca2+ signals in nerve and muscle. Although the molecular details underlying Ca2+-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca2+-dependent inactivation of Ca(v)2.1 (P/Q-type) Ca2+ channels is suppressed by strong cytoplasmic Ca2+ buffering, we investigated how factors that regulate cellular Ca2+ levels affect inactivation of Ca(v)2.1 Ca2+ currents in transfected 293T cells. We found that inactivation of Ca(v)2.1 Ca2+ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mm), but not with high concentrations of the fast Ca2+ buffer BAPTA (10 mm). However, when the concentration of BAPTA was reduced to 0.5 mm, inactivation of Ca2+ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca2+-dependent inactivation of Ca(v)2.1. Cotransfection of Ca(v)2.1 with the EF-hand Ca2+-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca2+ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca2+ buffers. We conclude that Ca2+-dependent inactivation of Ca(v)2.1 depends on a subplasmalemmal Ca2+ microdomain that is affected by the amplitude of the Ca2+ current and differentially modulated by distinct Ca2+ buffers.     

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.     

Sex-specific effects of <Emphasis Type="Italic">ACE</Emphasis> I/D and <Emphasis Type="Italic">AGT</Emphasis>-M235T on pulse pressure: the HyperGEN Study

Evidence shows that an elevated pulse pressure (PP) may lead to an increased risk of cardiovascular morbidity and mortality. There is also evidence that PP is a sexually dimorphic trait, and that genetic factors influence inter-individual variation in PP. The aim of this project was to assess the genotype-by-sex interaction on PP in a sample of mostly hypertensive African American and White participants using candidate genes involved in the renin–angiotensin–aldosterone system. Subjects were participants in the HyperGEN Study, including men (43%) and women (57%) over the age of 55 years (mean age = 65). Candidate gene polymorphisms used were ACE insertion/deletion (1,789 subjects genotyped) and AGT-M235T (1,800 subjects genotyped). We employed linear regression methods to assess the genotype-by-sex interaction. For ACE, genotype-by-sex interaction on PP was detected (P = 0.04): the “D/D” genotype predicted a 2.2 mmHg higher pulse pressure among women, but a 1.2 mmHg lower PP among men, compared to those with an “I” allele, after adjusting for age, weight, height, ethnicity, and antihypertension medication use. A similar interaction was found for systolic blood pressure. The genotype-by-sex interaction was consistent across ethnicity. The interaction was evident among those on antihypertensive medications (P = 0.05), but not among those not taking such medications (P = 0.55). In our analysis of AGT, no evidence of a genotype-by-sex interaction affecting PP, SBP, or DBP was detected. This evidence for a genotype-by-sex interaction helps our understanding of the complex genetic underpinnings of blood pressure phenotypes.     

Targeting CreERT2 expression to keratin 8-expressing murine simple epithelia using bacterial artificial chromosome transgenesis

Keratin 8 (K8) is a type II keratin that is associated with the type I keratins K18 or K19 in single layered epithelia. We generated a bacterial artificial chromosome (BAC) transgenic mouse line that expresses the tamoxifen inducible CreER(T2) inserted into the endogenous murine K8 gene. The transgenic mouse line contains two copies of the BAC transgene. To determine the expression specificity and inducibility of CreER(T2), the K8-CreER(T2) mice were bred with a Gt(ROSA 26)( ACTB-tdTomato-EGFP ) fluorescent protein-based reporter transgenic mouse line. We demonstrated that CreER(T2) and the endogenous K8 gene share the same patterns of expression and that the enzymatic activity of CreER(T2) can be efficiently induced by tamoxifen in all K8-expressing tissues. This mouse line will be useful for studying gene function in development and homeostasis of simple epithelia, and investigating both tissue lineage hierarchy and the identity of the cells of origin for epithelial cancers.     

Acute inducible ablation of GRP78 reveals its role in hematopoietic stem cell survival, lymphogenesis and regulation of stress signaling

GRP78, a master regulator of the unfolded protein response (UPR) and cell signaling, is required for inner cell mass survival during early embryonic development. However, little is known about its role in adult hematopoietic stem cells (HSCs) and hematopoiesis. Here we generated a conditional knockout mouse model that acutely deletes Grp78 in the adult hematopoietic system. Acute GRP78 ablation resulted in a significant reduction of HSCs, common lymphoid and myeloid progenitors, and lymphoid cell populations in the mutant mice. The GRP78-null induced reduction of the HSC pool could be attributed to increased apoptosis. Chimeric mice with Grp78 deletion only in the hematopoietic cells also showed a loss of HSCs and lymphopenia, suggesting a cell intrinsic effect. Analysis of GRP78 deficient bone marrow (BM) cells showed constitutive activation of all the major UPR signaling pathways, including activation of eIF2α, ATF6, xbp-1 splicing, as well as caspase activation. A multiplex cytokine assay further revealed alteration in select cytokine and chemokine serum levels in the mutant mice. Collectively, these studies demonstrate that GRP78 plays a pleiotropic role in BM cells and contributes to HSC survival and the maintenance of the lymphoid lineage.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of unsymmetrical 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives

Substituted 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives were investigated as inhibitors of genotype 1 HCV polymerase. Structure-activity relationship patterns for this class of compounds are discussed. It was found that the saturated alkane dialkyl units provided the most active analogs.     

Four putative entomopathogenic fungi of armoured scale insects on <Emphasis Type="Italic">Citrus</Emphasis> in Australia

This study led to the discovery of four putative entomopathogenic fungi of armoured scale insects on citrus trees in coastal New South Wales. Two of these species belong in Podonectria as P. coccicola (Ellis & Everh.) Petch (syn. Tetracrium coccicola (Höhn.) Ellis & Everh.) and P. novae-zelandiae Dingley. Members of this genus are grown in culture for the first time. Formerly placed in the Pleosporales, Tubeufiaceae, or more recently in the Tubeufiales, these species are herein placed in the new family Podonectriaceae fam. nov., Pleosporales. Another species is placed in the Hypocreales, Bionectriaceae as Clonostachys coccicola (J.A. Stev.) H.T. Dao comb. nov. (basionym Tubercularia coccicola J.A. Stev., syn. Nectria tuberculariae Petch). The fourth species is Myriangium citri Henn. (Myriangiales, Myriangiaceae). Each fungal species is characterized and the phylogenetic placement confirmed by molecular analyses of the ITS and 28 s rDNA regions. In addition, their biology is noted, including location of the fungi within tree canopies.     

Gene copy number effects in the mer operon of plasmid NR1.

The level of resistance to Hg2+ determined by the inducible mer operon of plasmid NR1 was essentially the same for three gene copy number variants in Escherichia coli, less in Proteus mirabilis, and intermediate in P. mirabilis "transitioned" to a high r-determinant gene copy number. Cell-free volatilization rates of radioactive mercury indicated increasing levels of intracellular mercuric reductase enzyme from low- to high-gene copy number forms in P. mirabilis and from low- to high-copy number forms in E. coli, but the additional enzyme in E. coli was effectively cryptic.