Into ion channel and transporter function. Caenorhabditis elegans ClC-type chloride channels: novel variants and functional expression

Six ClC-type chloride channel genes have been identified in Caenorhabditis elegans, termed clh-1 through clh-6. cDNA sequences from these genes suggest that clh-2, clh-3, and clh-4 may code for multiple channel variants, bringing the total to at least nine channel types in this nematode. Promoter-driven green fluorescent protein (GFP) expression in transgenic animals indicates that the protein CLH-5 is expressed ubiquitously, CLH-6 is expressed mainly in nonneuronal cells, and the remaining isoforms vary from those restricted to a single cell to those expressed in over a dozen cells of the nematode. In an Sf9 cell expression system, recombinant CLH-2b, CLH-4b, and CLH-5 did not form functional plasma membrane channels. In contrast, both CLH-1 and CLH-3b produced strong, inward-rectifying chloride currents similar to those arising from mammalian ClC2, but which operate over different voltage ranges. Our demonstration of multiple CLH protein variants and comparison of expression patterns among the clh gene family provides a framework, in combination with the electrical properties of the recombinant channels, to further examine the physiology and cell-specific role each isoform plays in this simple model system.     

Identification of further important residues within the Glut4 carboxy-terminal tail which regulate subcellular trafficking

The insulin-responsive glucose transporter, Glut4, exhibits a unique subcellular distribution such that in the absence of insulin >95% of the protein is stored within intracellular membranes. In response to insulin, Glut4 exhibits a large mobilisation to the plasma membrane. Studies of the amino acid motifs which regulate the unique trafficking of Glut4 have identified several key residues within the soluble cytoplasmic N- and C-terminal domains of Glut4. Of particular note is a Leu-498Leu-499 motif within the C-terminal domain that has been proposed to regulate both internalisation from the plasma membrane and sorting to an insulin-sensitive compartment. In this study, we have examined the role of the adjacent amino acids (Glu-491, Gln-492 and Glu-493) by their sequential replacement with Ala. Our results are consistent with the notion that Glu-491 and Glu-493 play an important role in the sub-endosomal trafficking of Glut4, as substitution of these residues with Ala results in increased levels of these proteins at the cell surface, reduced insulin-stimulated translocation and increased susceptibility to endosomal ablation. These residues, together with other identified sequences within the C-terminus of Glut4, are likely to be crucial targeting elements that regulate Glut4 subcellular distribution.     

Human CASK/LIN-2 Binds Syndecan-2 and Protein 4.1 and Localizes to the Basolateral Membrane of Epithelial Cells

In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction.     

Branch growth of riparian cottonwoods: a hydrologically sensitive dendrochronological tool

 The conservation of riparian (river valley flood plain) forests relies on the provision of instream flows that are sufficient to sustain tree growth. In the present study, annual branch growth increments were investigated as an indicator of environmental favorability for riparian cottonwoods. Trees of three species, Populus angustifolia, P. balsamifera, and P. deltoides, and their natural interspecific hybrids, were studied at five sites along the Oldman and South Saskatchewan rivers in Alberta, Canada. Annual branch growth increments for the interval from 1983 to 1992 were positively correlated with stream flows (r ² = 0.79 at Lethbridge) and slightly negatively correlated with weather variables that contribute to water demand: evaporation, temperature, wind, and/or sunshine. The combination of January to May stream flow (water supply) and June evaporation (water demand) almost entirely accounted for the branch growth variation across years (r ² = 0.91 at Lethbridge). Tree ring increments were also investigated but were less closely correlated than branch increments across trees or with stream flow. Branch growth increments thus provide an accurate but short duration (1 or 2 decades) record of environmental favorability for growth. The close correlation between branch growth and stream flow indicates that water is the principal limitation to growth of these riparian cottonwoods and that these trees obtained their water from a source linked to the stream, the riparian water table. Analyses of branch increments should provide a management tool for (i) determining instream flow needs for riparian cottonwoods and (ii) analyzing impacts of stream flow alterations due to river damming or water diversion. Received: 8 May 1997 / Accepted: 23 September 1997     

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon Q_B photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 Asn replacement on Q_B ^–/Q_B FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 Asp, Arg M233 Cys, or Arg H177 His suppressor mutation. The proton uptake by Glu L212 upon Q_B ^– formation, as reflected by the positive band at 1728 cm^–1, is increased in the Asn M44 Asp and Arg H177 His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 Asn mutant RCs. Only the Arg M233 Cys suppressor mutation affected the 1728 cm^–1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 Asn lesion.     

Range-wide distribution of genetic diversity in the North American tree Juglans cinerea: a product of range shifts, not ecological marginality or recent population decline

The spatial distribution of genetic diversity is a product of recent and historical ecological processes, as well as anthropogenic activities. A current challenge in population and conservation genetics is to disentangle the relative effects of these processes, as a first step in predicting population response to future environmental change. In this investigation, we compare the influence of contemporary population decline, contemporary ecological marginality and postglacial range shifts. Using classical model comparison procedures and Bayesian methods, we have identified postglacial range shift as the clear determinant of genetic diversity, differentiation and bottlenecks in 29 populations of butternut, Juglans cinerea L., a North American outcrossing forest tree. Although butternut has experienced dramatic 20th century decline because of an introduced fungal pathogen, our analysis indicates that recent population decline has had less genetic impact than postglacial recolonization history. Location within the range edge vs. the range core also failed to account for the observed patterns of diversity and differentiation. Our results suggest that the genetic impact of large-scale recent population losses in forest trees should be considered in the light of Pleistocene-era large-scale range shifts that may have had long-term genetic consequences. The data also suggest that the population dynamics and life history of wind-pollinated forest trees may provide a buffer against steep population declines of short duration, a result having important implications for habitat management efforts, ex situ conservation sampling and population viability analysis.     

Role of Absolute Humidity in the Inactivation of Influenza Viruses on Stainless Steel Surfaces at Elevated Temperatures

Influenza virus has been found to persist in the environment for hours to days, allowing for secondary transmission of influenza via inanimate objects known as fomites. We evaluated the efficacy of heat and moisture for the decontamination of surfaces for the purpose of preventing of the spread of influenza. Aqueous suspensions of influenza A virus were deposited onto stainless steel coupons, allowed to dry under ambient conditions, and exposed to temperatures of 55°C, 60°C, or 65°C and relative humidity (RH) of 25%, 50%, or 75% for up to 1 h. Quantitative virus assays were performed on the solution used to wash the viruses from these coupons, and results were compared with the solution used to wash coupons treated similarly but left under ambient conditions. Inactivation of influenza virus on surfaces increased with increasing temperature, RH, and exposure time. Reductions of greater than 5 logs of influenza virus on surfaces were achieved at temperatures of 60 and 65°C, exposure times of 30 and 60 min, and RH of 50 and 75%. Our data also suggest that absolute humidity is a better predictor of surface inactivation than RH and allows the prediction of survival using two parameters rather than three. Modest amounts of heat and adequate moisture can provide effective disinfection of surfaces while not harming surfaces, electrical systems, or mechanical components, leaving no harmful residues behind after treatment and requiring a relatively short amount of time.In a recent publication, Shaman and Kohn concluded that absolute humidity, which can be calculated if temperature and relative humidity (RH) are known, is the controlling factor in both the inactivation of influenza virus and the transmission of influenza (27). To arrive at this conclusion, Shaman and Kohn reanalyzed experimental data collected by Lowen et al. and Harper (12, 17, 18), which covered a rather narrow range of temperatures typical of normal weather conditions, that is, in the range of 5 to 30°C. For this temperature range, the maximum absolute humidity that can occur is 24 g/m³. One question that comes to mind is whether this trend of decreasing influenza virus survival with increasing absolute humidity (AH) persists as temperature increases. If it does, then AH may also be the controlling factor when heat and moisture are used to decontaminate surfaces. In the present work, we tried to answer this question through a series of experiments in which absolute humidity was sufficiently high to result in effective surface decontamination.Effective and easily implemented public health interventions are needed to prevent the spread of infectious diseases such as influenza. Transmission of influenza virus, especially in the event of a pandemic with a highly virulent strain of influenza virus, such as avian influenza H5N1 virus or 2009 H1N1 influenza A virus, is of great concern due to widespread mortality and morbidity (7, 23). The significant morbidity and mortality associated with seasonal influenza should also not be discounted. There is compelling evidence for transmission of influenza viruses from infected individuals to uninfected individuals by direct contact, via fomites (inanimate objects capable of carrying infectious organisms), and through large droplets expelled during forceful exhalation, such as during coughing and sneezing (2-4, 19). Virtually any exposed surface can become contaminated with infectious viruses and can be a potential source of secondary virus transmission. The probability for transmission increases in situations where many people are in close proximity and in locations with highly transient populations, such as public transportation, air transportation, classrooms, theaters, and other public venues (16, 20, 22). Influenza virus has been found to persist in the environment for hours to days; it has been found on surfaces in day-care centers, hands, laboratory gowns, and in surface dust (3, 4, 9, 29). Efforts to prevent the spread of flu through contact transmission via fomites require methods of decontamination that are easy to use and will not disrupt critical services and operations.Laboratory research has shown that the moisture content of the air is an important factor for antimicrobial survival. Past research on influenza virus has focused on airborne viruses and generally suggests that survival and/or transmission is facilitated by low RH (10, 12, 15, 17, 26, 30). A limited number of surface inactivation studies have been performed at environmentally relevant temperatures, ranging from 7 to 32°C (30), and have shown modest reductions in numbers of influenza viruses (5, 21).We evaluated the efficacy of heat and moisture for decontaminating surfaces and controlling the spread of influenza virus infection. In this study, temperatures were maintained well above room temperature (55 to 65°C) but were still not expected to cause harm to most surfaces, mechanical components, or electrical systems. Furthermore, heat does not leave behind potentially harmful residues, as do most chemical decontaminants, and the use of heat requires a relatively brief period of time for surface treatment and allows resources to be quickly returned to service. To our knowledge no previous measurements of influenza virus inactivation rates on surfaces at these temperatures have been made.     

Colletotrichum gloeosporioides s.l. associated with Theobroma cacao and other plants in Panama: multilocus phylogenies distinguish host-associated pathogens from asymptomatic endophytes

Colletotrichum interacts with numerous plant species overtly as symptomatic pathogens and cryptically as asymptomatic endophytes. It is not known whether these contrasting ecological modes are optional strategies expressed by individual Colletotrichum species or whether a species' ecology is explicitly pathogenic or endophytic. We explored this question by inferring relationships among 77 C. gloeosporioides s.l. strains isolated from asymptomatic leaves and from anthracnose lesions on leaves and fruits of Theobroma cacao (cacao) and other plants from Panamá. ITS and 5'-tef1 were used to assess diversity and to delineate operational taxonomic units for multilocus phylogenetic analysis. The ITS and 5'-tef1 screens concordantly resolved four strongly supported lineages, clades A-D: Clade A includes the ex type of C. gloeosporioides, clade B includes the ex type ITS sequence of C. boninense, and clades C and D are unidentified. The ITS yielded limited resolution and support within all clades, in particular the C. gloeosporioides clade (A), the focal lineage dealt with in this study. In contrast the 5'-tef1 screen differentiated nine distinctive haplotype subgroups within the C. gloeosporioides clade that were concordant with phylogenetic terminals resolved in a five-locus nuclear phylogeny. Among these were two phylogenetic species associated with symptomatic infections specific to either cacao or mango and five phylogenetic species isolated principally as asymptomatic infections from cacao and other plant hosts. We formally describe two new species, C. tropicale and C. ignotum, that are frequent asymptomatic associates of cacao and other Neotropical plant species, and epitypify C. theobromicola, which is associated with foliar and fruit anthracnose lesions of cacao. Asymptomatic Colletotrichum strains isolated from cacao plants grown in China included six distinct C. gloeosporioides clade taxa, only one of which is known to occur in the Neotropics.     

Recovery of Populus tremuloides seedlings following severe drought causing total leaf mortality and extreme stem embolism

In contrast with other native Populus species in North America, Populus tremuloides (aspen) can successfully establish itself in drought‐prone areas, yet no comprehensive analysis has been performed on the ability of seedlings to withstand and recover from a severe drought resulting in complete leaf mortality. Here, we subjected 4‐month‐old aspen seedlings grown in two contrasting soil media to a progressive drought until total leaf mortality, followed by a rewatering cycle. Stomatal conductance (g_s), photosynthesis and transpiration followed a sigmoid decline with declining fraction of extractable soil water values. Cessation of leaf expansion occurred close to the end of the linear‐decrease phase, when g_s was reduced by 95%. Leaf mortality started after g_s reached the lowest values, which corresponded to a stem–xylem pressure potential (Ψ_xp) of ?2.0 MPa and a percent loss of stem hydraulic conductivity (PLC) of 50%. In plants with 50% leaf mortality, PLC values remained around 50%. Complete leaf mortality occurred at an average stem PLC of 90%, but all seedlings were able to resprout after 6–10 days of being rewatered. Plants decapitated at soil level before rewatering developed root suckers, while those left with a 4‐cm stump or with their stems intact resprouted exclusively from axillary buds. Resprouting was accompanied by recovery of stem hydraulic conductivity, with PLC values around 30%. The percentage of resprouted buds was negatively correlated with the stem %PLC. Thus, the recovery of stem hydraulic conductivity appears as an important factor in the resprouting capacity of aspen seedlings following a severe drought.