Major Entry Pathway Research Articles

Down-the-drain pathways for fipronil and imidacloprid applied as spot-on parasiticides to dogs: Estimating aquatic pollution

Fipronil and imidacloprid have been widely detected in UK surface waters in recent years, often at concentrations that ecotoxicological studies have shown can harm aquatic life. Down-the-drain (DTD) passage of pet flea and tick treatments are being implicated as an important source, with many of the UK's 22 million cats and dogs receiving routine, year-round preventative doses containing these parasiticides. The UK Water Industry's 3rd Chemical Investigation Programme (UKWIR CIP3) has confirmed wastewater as a major entry pathway for these chemicals into surface waters, but the routes by which they enter the wastewater system remain unclear. We addressed this knowledge gap by conducting the first quantification of DTD emissions from 98 dogs treated with spot-on ectoparasiticides containing fipronil or imidacloprid, through bathing, bed washing and washing of owners' hands. Both chemicals were detected in 100 % of washoff samples, with bathing accounting for the largest emissions per event (up to 16.8 % of applied imidacloprid and 24.5 % of applied fipronil). Modelled to account for the frequency of emitting activities, owner handwashing was identified as the largest source of DTD emissions from the population overall, with handwash emissions occurring for at least 28 days following product application and an estimated 4.9 % of imidacloprid and 3.1 % of fipronil applied in dog spot-ons passing down-the-drain via this route. The normalised daily per capita emissions for all routes combined were 8.7 μg/person/day for imidacloprid and 2.1 μg/person/day for fipronil, equivalent to 20–40 % of the daily per capita load in wastewater, as estimated from UKWIR CIP3 data. Within the current international regulatory framework adhered to by the UK, the environmental exposure of veterinary medicines intended for use in small companion animals is assumed to be low, and DTD pathways are not considered. We recommend a systematic review of regulations and practices to address this overlooked pollution pathway.

Pest categorisation of cowpea mosaic virus.

The EFSA Panel on Plant Health conducted a pest categorisation of cowpea mosaic virus (CPMV) for the EU territory. The identity of CPMV, a member of the genus Comovirus (family Secoviridae), is established and detection and identification methods are available. The pathogen is not included in the Commission Implementing Regulation (EU) 2019/2072. It has been reported from the Americas, and several countries in Africa and Asia and it is not known to be present in the EU in natural conditions. CPMV is considered a major pathogen of cowpea on which it causes symptoms ranging from mild to severe mosaic, chlorosis and necrosis. The virus has been reported sporadically on some other cultivated species of the family Fabaceae, including soybean and some common bean varieties. CPMV is transmitted by cowpea seeds, with uncertainty on the transmission rate. There is uncertainty on seed transmission by other Fabaceae host species due to lack of information. CPMV is also transmitted by several beetle species, one of which, Diabrotica virgifera virgifera, is present in the EU. Seeds for sowing of cowpea are identified as the major entry pathway. The cultivated area and production of cowpea in the EU territory are mainly limited to local varieties cultivated at a small scale in Mediterranean EU Member States. Should the pest establish in the EU, an impact is expected on cowpea crops at local scale. There is high uncertainty on the potential impact that CPMV would cause on other natural hosts cultivated in the EU due to the lack of information from the areas of CPMV's current distribution. Despite the uncertainty concerning the potential impact on bean and soybean crops in the EU, CPMV satisfies the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

Urban pathways of biocides towards surface waters during dry and wet weathers: Assessment at the Paris conurbation scale

Eighteen biocides used in building materials and domestic products were monitored in wastewater treatment plants (WWTPs) during dry weather and in combined sewer overflows (CSOs) during wet weather in the Paris conurbation. The aims of this study were to (i) acquire data on biocides in urban waters, which are very scarce up to now, (ii) identify their origins in CSOs with the perspective of reducing these contaminants at source, and (iii) compare and rank biocide pathways to the river (dry vs. wet weather) at the annual and conurbation scales. The results showed the ubiquity of the 18-targeted biocides in WWTP waters and CSOs. High concentrations of methylisothiazolinone, benzisothiazolinone (0.2−0.9 μg/L) and benzalkonium C12 (0.5−6 μg/L) were measured in wastewater. Poor WWTP removals (< 50 %) were observed for most of the biocides. Both wastewater (mainly domestic uses) and stormwater (leaching from building materials) contributed to the CSO contamination. However, benzisothiazolinone mainly came from wastewater whereas diuron, isoproturon, terbutryn, carbendazim, tebuconazole, and mecoprop mainly came from stormwater. Annual mass loads discharged by WWTPs and CSOs into the Seine River were estimated using a stochastic approach (Monte Carlo simulations) at the conurbation scale and showed that WWTP discharges are the major entry pathway.

Pest categorisation of the non-EU phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L.

Following a request from the European Commission, the EFSA Panel on Plant Health performed a pest categorisation of nine phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L. (hereafter “host plants”) known to occur only outside the EU or having a limited presence in the EU. This opinion covers the (i) reference strains of ‘Candidatus Phytoplasma australiense’, ‘Ca. P. fraxini’, ‘Ca. P. hispanicum’, ‘Ca. P. trifolii’, ‘Ca. P. ziziphi’, (ii) related strains infecting the host plants of ‘Ca. P. aurantifolia’, ‘Ca. P. pruni’, and ‘Ca. P. pyri’, and (iii) an unclassified phytoplasma causing Buckland valley grapevine yellows. Phytoplasmas can be detected by available methods and are efficiently transmitted by vegetative propagation, with plants for planting acting as a major entry pathway and a long‐distance spread mechanism. Phytoplasmas are also transmitted in a persistent and propagative manner by some insect families of the Fulgoromorpha, Cicadomorpha and Sternorrhyncha (order Hemiptera). No transovarial, pollen or seed transmission has been reported. The natural host range of the categorised phytoplasmas varies from one to more than 90 plant species, thus increasing the possible entry pathways. The host plants are widely cultivated in the EU. All the categorised phytoplasmas can enter and spread through the trade of host plants for planting, and by vectors. Establishment of these phytoplasmas is not expected to be limited by EU environmental conditions. The introduction of these phytoplasmas in the EU would have an economic impact. There are measures to reduce the risk of entry, establishment, spread and impact. Uncertainties result from limited information on distribution, biology and epidemiology. All the phytoplasmas categorised here meet the criteria evaluated by EFSA to qualify as potential Union quarantine pests, and they do not qualify as potential regulated non‐quarantine pests, because they are non‐EU phytoplasmas.

Pest categorisation of non-EU viruses of Rubus L.

The Panel on Plant Health of EFSA conducted a pest categorisation of 17 viruses of Rubus L. that were previously classified as either non‐EU or of undetermined standing in a previous opinion. These infectious agents belong to different genera and are heterogeneous in their biology. Blackberry virus X, blackberry virus Z and wineberry latent virus were not categorised because of lack of information while grapevine red blotch virus was excluded because it does not infect Rubus. All 17 viruses are efficiently transmitted by vegetative propagation, with plants for planting representing the major pathway for entry and spread. For some viruses, additional pathway(s) are Rubus seeds, pollen and/or vector(s). Most of the viruses categorised here infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Cherry rasp leaf virus, raspberry latent virus, raspberry leaf curl virus, strawberry necrotic shock virus, tobacco ringspot virus and tomato ringspot virus meet all the criteria to qualify as potential Union quarantine pests (QPs). With the exception of impact in the EU territory, on which the Panel was unable to conclude, blackberry chlorotic ringspot virus, blackberry leaf mottle‐associated virus, blackberry vein banding‐associated virus, blackberry virus E, blackberry virus F, blackberry virus S, blackberry virus Y and blackberry yellow vein‐associated virus satisfy all the other criteria to be considered as potential QPs. Black raspberry cryptic virus, blackberry calico virus and Rubus canadensis virus 1 do not meet the criterion of having a potential negative impact in the EU. For several viruses, the categorisation is associated with high uncertainties, mainly because of the absence of data on biology, distribution and impact. Since the opinion addresses non‐EU viruses, they do not meet the criteria to qualify as potential Union regulated non‐quarantine pests.

Pest categorisation of non-EU viruses of Ribes L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses of Ribes L. determined as being either non‐EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. Alaska vitivirus 1 and Ribes virus F were excluded from categorisation because these are very poorly characterised viruses. The pest categorisation was completed for seven viruses with clear identity and for which detection methods are available. All these viruses are efficiently transmitted by vegetative propagation techniques, with plants for planting representing the major pathway for long‐distance dispersal and thus considered as the major pathway for entry. Depending on the virus, additional pathway(s) can also be Ribes seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but tomato ringspot virus (ToRSV) has a wide host range, thus extending the possible entry pathways. ToRSV meets all the criteria evaluated by EFSA to qualify as potential Union quarantine pest (QP). With the exception of impact in the EU territory, on which the Panel was unable to conclude, Actinidia virus X, blackcurrant leaf chlorosis‐associated virus, blackcurrant leafroll‐associated virus, black currant‐associated rhabdovirus, blackcurrant waikavirus A and Ribes americanum virus A satisfy all the other criteria to be considered as potential Union QPs. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically the non‐EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non‐quarantine pests.

Pest categorisation of non-EU viruses and viroids of Prunus L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Prunus L. determined as being either non‐EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of Ilarvirus S1 and Ilarvirus S2, for which very limited information exists, the pest categorisation was completed for 26 viruses and 1 viroid having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative plant propagation techniques, with plants for planting representing the major pathway for long‐distance dispersal and thus considered as the major pathway for entry. Depending on the virus, additional pathway(s) can also be Prunus seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Apple scar skin viroid, American plum line pattern virus, cherry mottle leaf virus, cherry rasp leaf virus, cherry rosette virus, cherry rusty mottle‐associated virus, cherry twisted leaf‐associated virus, peach enation virus, peach mosaic virus, peach rosette mosaic virus, tobacco ringspot virus and tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). With the exception of impact in the EU territory, on which the Panel was unable to conclude, apricot vein clearing virus, Asian prunus virus 1, Asian prunus virus 2, Asian prunus virus 3, Caucasus prunus virus, cherry virus B, Mume virus A, nectarine stem pitting‐associated virus, nectarine virus M, peach chlorotic mottle virus, peach leaf pitting‐associated virus, peach virus D, prunus virus F and prunus virus T satisfy all the other criteria to be considered as potential Union QPs. Prunus geminivirus A does not meet the criterion of having negative impact in the EU. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically the non‐EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non‐quarantine pests.

Pest categorisation of non-EU viruses of Fragaria L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Fragaria L. determined as being either non‐EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of strawberry latent virus and strawberry latent C virus for which very limited information exists, the pest categorisation was completed for 12 viruses having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative propagation techniques, with plants for planting representing the major pathway for long‐distance dispersal and thus considered as the major pathway for entry. Depending on the virus, additional pathway(s) can also be Fragaria seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Strawberry chlorotic fleck‐associated virus, strawberry leaf curl virus, strawberry necrotic shock virus, strawberry pallidosis‐associated virus, strawberry vein banding virus (SVBV) and tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). For SVBV, the Panel considered that following its entry and establishment into the EU territory, an impact of uncertain magnitude is expected mainly because a synergistic effect may occur in strawberry in case of mixed infections with viruses already present in the EU . Strawberry crinivirus 3, strawberry crinivirus 4 and strawberry polerovirus 1 meet all criteria for being considered as potential Union QPs, except for the impact in the EU territory, on which the Panel was unable to conclude. Fragaria chiloensis cryptic virus, Fragaria chiloensis latent virus and strawberry pseudo mild yellow edge virus do not meet the criterion of having potential negative impact in the EU. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically the non‐EU viruses, in general, these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non‐quarantine pests.

Pest categorisation of non-EU viruses and viroids of Cydonia Mill., Malus Mill. and Pyrus L.

Following a request from the EU Commission, the Panel on Plant Health performed a pest categorisation of 17 viruses and viroids, herein called viruses, of Cydonia Mill., Malus Mill. and Pyrus L. determined as being either non‐EU or of undetermined standing in a previous EFSA opinion. These viruses belong to different genera and are heterogeneous in their biology. They can be detected by available methods and are efficiently transmitted by vegetative propagation techniques, with plants for planting representing a major long‐distance spread mechanism and, potentially, a major entry pathway. Depending on the viruses, additional pathway(s) can also be seed, pollen and/or vector transmission. Most of the viruses categorised here are known to infect only one of few related plant genera, but some of them have a wider host range, thus extending the possible entry pathways. Three viruses (apple necrotic mosaic virus, cherry rasp leaf virus, temperate fruit decay‐associated virus) and one viroid (apple fruit crinkle viroid) satisfy all the criteria to be considered as Union quarantine pests. Five viruses (apple green crinkle‐associated virus, blackberry chlorotic ringspot virus, eggplant mottled crinkle virus, tobacco ringspot virus and tomato ringspot virus) and one viroid (apple scar skin viroid), satisfy the criteria to be considered as Union quarantine pests with the possible exception of being absent from the EU territory or having a restricted presence and being under official control. The remaining six viruses (apple geminivirus, apple latent spherical virus, apple‐associated luteovirus, Pyrus pyrifolia cryptic virus, Pyrus pyrifolia partitivirus 2 and Tulare apple mosaic virus) and one viroid (apple hammerhead viroid) were not found to satisfy one or more of these criteria. The Panel highlights that for several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly linked to the absence of data on biology and distribution. Since this opinion addresses specifically the non‐EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as a potential Union regulated non‐quarantine pests.

ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its naturalin vivotarget cells, by lipid raft-dependent macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane, and released capsid is transported to the nuclear vicinity, resulting in the nuclear entry of viral DNA. The endosomal sorting complexes required for transport (ESCRT) proteins, which include ESCRT-0, -I, -II, and -III, play a central role in endosomal trafficking and sorting of internalized and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d by macropinocytosis. Knockdown of Hrs by short hairpin RNA (shRNA) transduction resulted in significant decreases in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and plasma membrane isolation and proximity ligation assay (PLA) demonstrated the translocation of Hrs from the cytosol to the plasma membrane of infected cells and association with α-actinin-4. In addition, infection induced the plasma membrane translocation and activation of the serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown reduced these associations, suggesting that the recruitment of ROCK1 is an Hrs-mediated event. Interaction between Hrs and ROCK1 is essential for the ROCK1-induced phosphorylation of NHE1 (Na(+)/H(+)exchanger 1), which is involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs- and ROCK1-dependent mechanisms and that ROCK1-mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV. Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has been extensively studied with respect to endosomal movement and sorting of ubiquitinated proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we demonstrate for the first time that upon KSHV infection, the endogenous Hrs localizes to the plasma membrane and the membrane-associated Hrs facilitates assembly of signaling molecules, macropinocytosis, and virus entry. Hrs recruits ROCK1 to the membrane, which is required for the activation of NHE1 and an increase in submembranous intracellular pH occurring during macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the plasma membrane is important for coupling membrane dynamics to the cytosolic signaling events during macropinocytosis of KSHV.

Heparan sulfate proteoglycan as a cell-surface endocytosis receptor

How various macromolecules are exchanged between cells and how they gain entry into recipient cells are fundamental questions in cell biology with important implications e.g. non-viral drug delivery, infectious disease, metabolic disorders, and cancer. The role of heparan sulfate proteoglycan (HSPG) as a cell-surface receptor of diverse macromolecular cargo has recently been manifested. Exosomes, cell penetrating peptides, polycation–nucleic acid complexes, viruses, lipoproteins, growth factors and morphogens among other ligands enter cells through HSPG-mediated endocytosis. Key questions that partially have been unraveled over recent years include the respective roles of HSPG core protein and HS chain structure specificity for macromolecular cargo endocytosis, the down-stream intracellular signaling events involved in HSPG-dependent membrane invagination and vesicle formation, and the biological significance of the HSPG transport pathway. Here, we discuss the intriguing role of HSPGs as a major entry pathway of macromolecules in mammalian cells with emphasis on recent in vitro and in vivo data that provide compelling evidence of HSPG as an autonomous endocytosis receptor.

A Re-Evaluation of the Role of hCTR1, the Human High-Affinity Copper Transporter, in Platinum-Drug Entry into Human Cells

Cisplatin (cDDP) is an anticancer drug used in a number of malignancies, including testicular, ovarian, cervical, bladder, lung, head, and neck cancers. Its use is limited by the development of resistance, often rationalized via effects on cellular uptake. It has been claimed that human copper transporter 1 (hCTR1), the human high-affinity copper transporter, is the major entry pathway for cDDP and related drugs via a mechanism that mimics copper. This is an unexpected property of hCTR1, a highly selective copper (I) transporter. We compared the uptake rates of copper with cDDP (and several analogs) into human embryonic kidney 293 cells overexpressing wild-type or mutant hCTR1, mouse embryonic fibroblasts that do or do not express CTR1, and human ovarian tumor cells that are sensitive or resistant to cDDP. We have also compared the effects of extracellular copper, which causes regulatory endocytosis of hCTR1, to those of cDDP. We confirm the correlation between higher hCTR1 levels and higher platinum drug uptake in tumor cells sensitive to the drug. However, we show that hCTR1 is not the major entry route of platinum drugs, and that the copper transporter is not internalized in response to extracellular drug. Our data suggest the major entry pathway for platinum drugs is not saturable at relevant concentrations and not protein-mediated. Clinical trials have been initiated that depend upon regulating membrane levels of hCTR1. If reduced drug uptake is a major factor in resistance, hCTR1 is unlikely to be a productive target in attempts to enhance efficacy, although the proteins involved in copper homeostasis may play a role.

Abstract 813: Mechanism of cellular uptake of Pt drugs: The role of hCTR1

Abstract Cisplatin is an anti-cancer drug used to treat various human malignancies including breast, testicular, ovarian, prostrate, lung, head, neck, and colon cancers. While this drug has been shown to successfully treat tumors, its use is limited by the rapid development of resistance. This drug has been used since the 70's to treat different forms of cancer and recent studies have attempted to understand the mechanism of resistance by examining how this anti-cancer drug is taken into the cell, distributed within the cell, and exported. It has been claimed that hCTR1, the human high affinity Cu transporter, is the major entry pathway for cis-Pt and related anti-cancer drugs in human cells. Furthermore, that the entry mechanism via hCTR1 mimics that of Cu. Since hCTR1 is a very selective transporter for Cu(I), its function as a cisplatin entry pathway is unexpected. We will describe the results of our recent studies on HEK293 cells that over-express wild-type hCTR1 and a number of mutant Cu transporters, ovarian tumor cells that are sensitive or resistant to cis-Pt, and a number of human breast cell tumor lines. We have compared the relative entry rates of Cu and Pt in these cells and, via cell surface biotinylation and imaging, the effects of extracellular Cu, which causes regulatory endocytosis of the transporter, to that of the cis-Pt drugs. Our results suggest that hCTR1 is not the major entry route of Pt drugs into human cells and that the response of the transporter, in a number of these cells, to the presence of extracellular drug is different from its response to Cu. Supported by NIH Grant P01 GM 067166. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 813. doi:1538-7445.AM2012-813

Flux through hepatic pyruvate carboxylase and phosphoenolpyruvate carboxykinase detected by hyperpolarized 13 C magnetic resonance

In the heart, detection of hyperpolarized [(13)C]bicarbonate and (13)CO(2) by magnetic resonance (MR) after administration of hyperpolarized [1-(13)C]pyruvate is caused exclusively by oxidative decarboxylation of pyruvate via the pyruvate dehydrogenase complex (PDH). However, liver mitochondria possess alternative anabolic pathways accessible by [1-(13)C]pyruvate, which may allow a wider diagnostic range for hyperpolarized MR compared with other tissue. Metabolism of hyperpolarized [1-(13)C]pyruvate in the tricarboxylic acid (TCA) cycle was monitored in the isolated perfused liver from fed and fasted mice. Hyperpolarized [1-(13)C]pyruvate was rapidly converted to [1-(13)C]lactate, [1-(13)C]alanine, [1-(13)C]malate, [4-(13)C]malate, [1-(13)C]aspartate, [4-(13)C]aspartate, and [(13)C]bicarbonate. Livers from fasted animals had increased lactate:alanine, consistent with elevated NADH:NAD(+). The appearance of asymmetrically enriched malate and aspartate indicated high rates of anaplerotic pyruvate carboxylase activity and incomplete equilibration with fumarate. Hyperpolarized [(13)C]bicarbonate was also detected, consistent with multiple mechanisms, including cataplerotic decarboxylation of [4-(13)C]oxaloacetate via phosphoenolpyruvate carboxykinase (PEPCK), forward TCA cycle flux of [4-(13)C]oxaloacetate to generate (13)CO(2) at isocitrate dehydrogenase, or decarboxylation of [1-(13)C]pyruvate by PDH. Isotopomer analysis of liver glutamate confirmed that anaplerosis was sevenfold greater than flux through PDH. In addition, signal from [4-(13)C]malate and [4-(13)C]aspartate was markedly blunted and signal from [(13)C]bicarbonate was completely abolished in livers from PEPCK KO mice, indicating that the major pathway for entry of hyperpolarized [1-(13)C]pyruvate into the hepatic TCA cycle is via pyruvate carboxylase, and that cataplerotic flux through PEPCK is the primary source of [(13)C]bicarbonate. We conclude that MR detection of hyperpolarized TCA intermediates and bicarbonate is diagnostic of pyruvate carboxylase and PEPCK flux in the liver.

In vitro characterization of aldosterone and cAMP effects in mouse distal convoluted tubule cells.

The distal nephron plays a capital role in the fine regulation of sodium reabsorption. Compared with the cortical collecting duct, much less information is available on the hormonal regulation of sodium transporter genes in the distal convoluted tubule (DCT), where the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is the major entry pathway for Na(+). The purpose of this study was to characterize the in vitro effects of aldosterone (Aldo; 1 microM) and cAMP (8-BrcAMP; 0.5 mM) on mouse DCT (mDCT) by using an immortalized mDCT cell line. Western blot analysis and semiquantitative RT-PCR were performed to analyze the expression of genes involved in sodium transport. The mDCTcell line expressed the 11 beta-hydroxysteroid dehydrogenase type 2 gene and both the mineralocorticoid and glucocorticoid receptor genes, suggesting Aldo responsiveness. In this sense, we found that mDCT cells expressed the amiloride-sensitive Na(+) channel (ENaC) and responded to Aldo by upregulating the alpha-subunit protein. Similarly, alpha(1) Na(+)-K(+)-ATPase protein was upregulated by Aldo and 8-BrcAMP. In addition, the Aldo intermediate gene sgk1 mRNA was increased in response to both Aldo and 8-BrcAMP, and the transcription factor HNF-3 alpha mRNA was induced by 8-BrcAMP. With respect to NCC regulation, although Aldo induced NCC protein levels in mice in vivo, neither Aldo nor 8-BrcAMP significantly induced the NCC mRNA or protein levels in mDCT cells. These results suggest that in mDCT, Aldo and cAMP modulate some downstream mediators and effectors in vitro but do not influence the expression of NCC in this cell model.

The Cationic Amphipathic α-Helix of HIV-1 Viral Protein R (Vpr) Binds to Nucleic Acids, Permeabilizes Membranes, and Efficiently Transfects Cells

Viral protein R (Vpr) is a small protein of 96 amino acids that is conserved among the lentiviruses human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. We recently sought to determine whether the karyophilic properties of Vpr, as well as its ability to bind nucleic acids, could be used to deliver DNA into cells. We have found that the C-terminal domain of Vpr-(52-96) is able to efficiently transfect various cell lines. Here, we show that the shortest active sequence for gene transfer corresponds to the domain that adopts a alpha-helix conformation. DNA binding studies and permeabilization assays performed on cells demonstrated that the peptides that are efficient in transfection condense plasmid DNA and are membranolytic. Electron microscopy studies and transfection experiments performed in the presence of inhibitors of the endocytic processes indicated that the major entry pathway of Vpr-DNA complexes is through endocytosis. Taken together, the results show that the cationic C-terminal alpha-helix of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection.

L-type Ca 2+ channels of the embryonic mouse heart

In the heart, where Ca 2+ influx across the sarcolemma is essential for contraction, L-type Ca 2+ channels represent the major entry pathway of Ca 2+. Mice with a homozygous deletion of the L-type Ca v1.2 Ca 2+ channel gene die before day 14.5 p.c. Electrophysiological and pharmacological investigations on Ca v1.2−/− cardiomyocytes demonstrated that contractions depended on the influx of Ca 2+ through an L-type-like Ca 2+ channel. We analyzed now the expression pattern of various L-type Ca 2+ channels. Amplification of the alternative exons 1a and 1b revealed that embryonic cardiac cells express both Ca v1.2a and Ca v1.2b subunits. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplifications indicated the expression of Ca v1.1 and Ca v1.3 in about a 1:10 ratio in Ca v1.2−/− embryos. Two different amino termini of the Ca v1.3 cDNA were found in the embryonic heart, which both gave rise to functional channels. Ca v1.3(1a) and Ca v1.3(1b) channels have similar current kinetics and voltage-dependencies as described for Ca v1.3 8A channels [J. Biol. Chem. 276 (2001) 22100], but the properties of Ca v1.3(1a) or Ca v1.3(1b) channels are different from that of the L-type-like current in Ca v1.2−/− cardiomyocytes. The I Ba of Ca v1.3(1a) was blocked by the dihydropyridine nisoldipine with an IC 50 value of 0.13 μM at a holding potential of −80 mV. In embryonic Ca v1.2+/+ cardiomyocytes, I Ba was blocked by nisoldipine with an IC 50 value of 0.1 μM. Although the expressed Ca v1.3 channel has a similar affinity for nisoldipine as Ca v1.2+/+ cardiomyocytes, the L-type-like Ca 2+ channel found in Ca v1.2+/+ and −/− cardiomyocytes is not identical with the new Ca v1.3 splice variants.

Nonindigenous species introductions: a threat to Canada's forests and forest economy1

When organisms are moved from their natural range to new ecosystems, they are considered nonindigenous, invasive, or exotic species. Movement of exotic or native species may be international or from areas within Canada. Historically, Canada's forests have felt the effects of nonindigenous species introductions, as for example, Dutch elm disease, white pine blister rust, gypsy moth, and pine shoot beetle. With changes in global trade patterns, novel introductions will continue to occur. Although most of the research and regulatory efforts to control the movement of nonindigenous species has focused on insects, projects are underway to study fungal organisms and their association with insect vectors. International quarantine standards are being developed to minimize the risk associated with solid wood packing materials, a major entry pathway for nonindigenous organisms. Research needs include the development of enhanced detection capabilities, improved diagnostic tools, effective mitigation measures, as well as socio-economic impact assessments and basic biological information about nonindigenous species and their interactions with hosts.

Expression, regulation and function of carrier proteins for cationic amino acids.

Different carrier proteins exhibiting distinct transport properties participate in cationic amino acid transport. There are sodium-independent systems, such as b+, y+, y+L and b0,+, and a sodium-dependent system B0,+, most of which have now been identified at the molecular level. In most non-epithelial cells, members of the cationic amino acid transporter (CAT) family mediating system y+ activity seem to be the major entry pathway for cationic amino acids. CAT proteins underlie complex regulation at the transcriptional, post-transcriptional and activity levels. Recent evidence indicates that individual CAT isoforms are necessary for providing the substrate for nitric oxide synthesis, for example CAT-1 for Ca2+-independent nitric oxide production in endothelial cells and CAT-2B for sustained nitric oxide production in macrophages.

Genetics and pathology of voltage-gated Ca2+ channels.

Neurotransmitter release, neuronal excitation, and a whole variety of other neuronal functions are controlled by the intra/extra cellular Ca2+ gradient. The major pathway for entry of Ca2+ into the excitable cells is mediated by voltage-gated Ca2+ channels. Several functional subclasses of voltage-dependent Ca2+ channels have been identified, based on their pharmacological, biophysical properties, and molecular cloning. Recently, three human diseases (familial hemiplegic migraine, episodic ataxia type 2, and spinocerebellar ataxia 6) were added to the growing list of ion-channel disorders, all caused by different mutations in the P/Q-type Ca2+ channel alpha 1 subunit. Molecular analysis of the Ca2+ channelopathies will provide new insights into the role, function and pathology of these voltage-gated Ca2+ channels.