The Carboxyl Terminal Residues 220–283 Are Not Required for Voltage Gating of a Chimeric Connexin32 Hemichannel

Connexin hemichannels display two distinct forms of voltage-dependent gating, corresponding to the operation of V_j- or fast gates and loop- or slow gates. The carboxyl terminus (CT) of connexin 32 has been reported to be required for the operation of the V_j (fast) gates, but this conclusion was inferred from the loss of a fast kinetic component in macroscopic currents of CT-truncated intercellular channels elicited by transjunctional voltage. Such inferences are complicated by presence of both fast and slow gates in each hemichannel and the serial head-to-head arrangement of these gates in the intercellular channel. Examination of voltage gating in undocked hemichannels and V_j gate polarity reversal by a negative charge substitution (N2E) in the amino terminal domain allow unequivocal separation of the two gating processes in a Cx32 chimera (Cx32^∗43E1). This chimera expresses currents as an undocked hemichannel in Xenopus oocytes and provides a model system to study the molecular determinants and mechanisms of Cx32 voltage gating. Here, we demonstrate that both V_j- and loop gates are operational in a truncation mutation that removes all but the first four CT residues (ACAR²¹⁹) of the Cx32^∗43E1 hemichannel. We conclude that an operational Cx32 V_j (fast) gate does not require CT residues 220–283, as reported previously by others.     

The N Terminus of Connexin37 Contains an ��-Helix That Is Required for Channel Function

The cytoplasmic N-terminal domain of connexins has been implicated in multiple aspects of gap junction function, including connexin trafficking/assembly and channel gating. A synthetic peptide corresponding to the first 23 amino acids of human connexin37 was prepared, and circular dichroism and nuclear magnetic resonance studies showed that this N-terminal peptide was predominantly α-helical between glycine 5 and glutamate 16. The importance of this structure for localization of the protein at appositional membranes and channel function was tested by expression of site-directed mutants of connexin37 in which amino acids leucine 10 and glutamine 15 were replaced with prolines or alanines. Wild type connexin37 and both substitution mutants localized to appositional membranes between transfected HeLa cells. The proline mutant did not allow intercellular transfer of microinjected neurobiotin; the alanine mutant allowed transfer, but less extensively than wild type connexin37. When expressed alone in Xenopus oocytes, wild type connexin37 produced hemichannel currents, but neither of the double substitution mutants produced detectable currents. The proline mutant (but not the alanine mutant) inhibited co-expressed wild type connexin37. Taken together, our data suggest that the α-helical structure of the connexin37 N terminus may be dispensable for protein localization, but it is required for channel and hemichannel function.Gap junction channels allow intercellular passage of ions and small molecules up to 1000 Da. They are oligomeric assemblies of members of a family of related proteins called connexins (CX)² (reviewed in Ref. 1). Six connexin monomers assemble to form a hemichannel or connexon (Fig. 1, top panel), which, in turn, forms a complete gap junction channel by docking with a hemichannel from an adjacent cell. Based on sequence similarities, connexins have been separated into subfamilies designated by Greek characters (2, 3). The majority of connexins are members of the α- and β-subfamilies. Connexin polypeptides span the plasma membrane four times and have three cytoplasmic regions: the N terminus (NT), a cytoplasmic loop between the second and third transmembrane domains, and the C terminus (Fig. 1, middle panel). Structural studies of gap junctions have revealed that each hemichannel contains a ring of 24 transmembrane spanning helices (4, 5). Most topological models suggest that the NT of α-subfamily connexins contains 23 amino acids (illustrated for connexin37, CX37, in Fig. 1, bottom panel) and that of β-subfamily connexins contains 22 amino acids.Open in a separate windowFIGURE 1.Diagrams depicting the relationships between a gap junction hemichannel (top), the connexin polypeptide (middle), and the amino acid sequence of the CX37 N-terminal domain (bottom). Thick vertical lines represent the boundaries of the plasma membrane; the intracellular and extracellular spaces are indicated. The transmembrane (M1–M4), extracellular (E1 and E2), and cytoplasmic (NT, N terminus; CL, cytoplasmic loop; and CT, C terminus) domains within a connexin are indicated.The importance of the connexin NT has been emphasized by the identification of a number of connexin mutants that cause amino acid substitutions within this region and are linked to diseases including sensorineural deafness (CX26, CX30, and CX31), Charcot-Marie-Tooth disease (CX32), oculodentodigital dysplasia (CX43), and congenital cataracts (CX46 and CX50). Among the disease-linked mutants that have been studied, some show impaired protein trafficking to the cell surface, whereas others traffic properly, but show loss or alterations of channel function (6–16). Heterologous expression of site-directed mutants and chimeric connexins has demonstrated the influence of NT amino acids upon channel properties, including transjunctional voltage (V_j)-dependent gating, unitary conductance, permeability, and sensitivity to regulation by polyamines (17–22). Lagree et al. (23) have provided evidence that the NT influences the compatibility of connexin hetero-oligomerization.The structure of the NT domain of a β-group connexin, Cx26, has been investigated through circular dichroism (CD) and nuclear magnetic resonance (NMR) of a synthetic peptide corresponding to part of the predicted CX26NT (24, 25). Based on their data, Purnick et al. (24) proposed a model for the NT of CX26 with an α-helix extending from position 1 to 10 and a critical bend at positions 11 and 12 that was suggested to act as a “hinge” allowing the first 10 amino acids to swing into the pore and block the channel. Oshima et al. (5) have published structural studies of a “permeability” mutant (M34A) of CX26 (26) showing a density within the pore of the channel that they suggested might represent a bundle of N termini acting as a “plug” to close the channel.We have been studying CX37, an α-group connexin that is expressed in endothelial cells (27), which may be important for development of atherosclerotic disease (28) and that can form large conductance channels and hemichannels (27, 29). We have shown that as much as half the length of the CX37NT can be deleted without affecting formation of gap junction plaques, but a full-length N terminus is required for hemichannel gating and intercellular communication (30). These observations suggested that the CX37NT may have a structure that is required for function. Therefore, the present experiments were designed to determine the structure of the NT of CX37 and the importance of that structure for protein localization and formation of functional channels and hemichannels. Differences between our data and those previously reported in studies of CX26 suggest that the structure of the NT in α-group connexins may differ from that in β-group connexins.     

The N-Terminal Transmembrane Domain of λ S Is Required for Holin but Not Antiholin Function

The λ S gene encodes a holin, S105, and an antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology of S105, which is N-out, C-in, with three transmembrane domains (TMDs). Here we show that the N terminus of S105 retains its fMet residue but that the N terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into the membrane very rapidly but that in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107 has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized membrane. Moreover, an allele, S105_ΔTMD1, with TMD1 deleted, was found to be defective in lysis, insensitive to membrane depolarization, and dominant to the wild-type allele, indicating that the lysis-defective, antiholin character of S107 is due to the absence of TMD1 from the bilayer rather than to its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion protein was compromised by the substitution of early-lysis missense mutations in either the deletion protein or parental S105 but restored when both S105_ΔTMD1 and holin carried the substitution.In general, holins control the length of the infection cycle of double-stranded DNA phages (37). During late gene expression, the holin protein accumulates harmlessly in the bilayer until suddenly and spontaneously triggering the formation of holes in the membrane at an allele-specific time (13, 15). Holin genes are extremely diverse, but most can be grouped into two main classes based on the number of predicted transmembrane domains (TMDs): class I, with three TMDs and a predicted N-out, C-in topology, and class II, with two TMDs and a predicted N-in, C-in topology (38). Holin genes and function are subject to several levels of regulation, among which a particularly striking feature is the common occurrence of two potential translational starts, or dual-start motifs (5, 37), separated by only a few codons. Dual-start motifs are found in many holins of both of the two major classes; in nearly every case, the two starts are separated by at least one basic residue. The first dual-start motif to be characterized was that of λ S, the prototype class I holin gene (Fig. 1A and B). Translation initiation events occur at codons 1 and 3, giving rise to two products, S107 and S105, each named because of the length of its amino acid sequence; in the wild-type (wt) allele, two RNA structures define the ratio of initiations at the two start codons, resulting in an S105/S107 ratio of ∼2:1.Open in a separate windowFIG. 1.Gene, topology, and sequence of λ S. (A, top) The λ lysis cassette, including genes S, R, Rz, and Rz1, is shown, along with its promoter p_R′, and Q, encoding the late gene activator. The 5′ end of the class I holin gene S has two start codons, Met1, the start for S107, and Met3, the start for S105, and two RNA structures that regulate initiations at these codons. The S105 and S107 alleles have Leu (CUG) codons in place of the Met3 and Met1 codons, respectively. (B) Primary structure of S proteins. Missense changes relevant to the text are shown. Starts for S107 and S105 are indicated by asterisks. The three TMDs are boxed (13), and the extent of the ΔTMD1 deletion is indicated. (C) Model for the membrane topology of S105, S107, and S105_ΔTMD1. Topology and boundary residues for TMD1, -2, and -3 are based on Graschopf and Blasi (11) and Gründling et al. (13), respectively.Although they differ only by the Met-Lys N-terminal extension of S107, the two proteins have opposing functions; S105 is the holin and S107 the antiholin. The antiholin function is reflected by four principal features: first, when the Met3 start is inactivated, the mutant allele, designated S107 (Fig. ​(Fig.1A),1A), is lysis defective (26); second, the S107 protein binds and inhibits S105 specifically (3, 16); third, when S107 is produced in stoichiometric excess over S105, lysis is blocked for several times the length of the normal infection cycle (3, 4, 7, 16); and fourth, S107 antiholin function, i.e., inhibition of S105 hole formation, can be instantly subverted by collapsing the proton motive force, most easily done by addition of energy poisons to the medium (3). The predicted N-out, C-in topology and the requirement for the energized membrane led to a model in which S107 is initially inserted in the membrane with only two TMDs, with TMD1 being blocked from insertion by the presence of the positively charged residue, Lys2, whereas S105 has three TMDs (Fig. ​(Fig.1C)1C) (39). From this perspective, S105-S107 complexes, which are approximately twice as numerous as the S105 homodimers, are defective in triggering hole formation. An appealing feature of this model is that when an S105-mediated hole formation event does occur in a cell, the resultant collapse of the membrane potential allows insertion of TMD1 of S107 into the membrane, instantly tripling the amount of active holin by making the previously inactive pool of S105-S107 complexes functional (38).Some genetic and physiological evidence for the topology of the λ S proteins has been obtained using gene fusions. First, a fusion of the S gene at codon 105 with lacZ generates a functional, membrane-inserted β-galactosidase chimera, indicating, as expected, the cytoplasmic disposition of the highly charged C terminus of the S protein (40). Second, Graschopf and Bläsi (12) demonstrated that S-mediated hole formation could be obtained with constructs where a secretory signal sequence was fused to the N termini of both S105 and S107. Lysis required the cleavage of the signal sequence by leader peptidase, and export of the signal-S107 form was slower than for the signal-S105 form. However, evidence for the topology of native forms of S has not been available. Moreover, no basis for the inhibitory character of S107 has been established. In the simplest view, the antiholin function could be due to the absence of TMD1 from the bilayer or the ectopic localization of TMD1 in the cytoplasm, or both. Here, we report studies directed at dissecting the precise role of topology in S107 function and correlating antiholin activity with its ability to heterodimerize with S105. The results are discussed in terms of a general model for the formation of the holin lesion and the role of dynamic membrane topology in its temporal regulation.     

Protonation of Individual Histidine Residues Is Not Required for the pH-Dependent Entry of West Nile Virus: Evaluation of the “Histidine Switch” Hypothesis

Histidine residues have been hypothesized to function as sensors of environmental pH that can trigger the activity of viral fusion proteins. We investigated a requirement for histidine residues in the envelope (E) protein of West Nile virus during pH-dependent entry into cells. Each histidine was individually replaced with a nonionizable amino acid and tested functionally. In each instance, mutants capable of orchestrating pH-dependent infection were identified. These results do not support a requirement for any single histidine as a pH-sensing “switch,” and they suggest that additional features of the E protein are involved in triggering pH-dependent steps in the flavivirus life cycle.Flaviviruses are enveloped RNA viruses that cause a spectrum of illnesses in humans ranging from fever to encephalitis and hemorrhagic disease (20). These small (∼50 nM) spherical virions incorporate 180 envelope (E) proteins that orchestrate the process of virus entry (22). Flaviviruses bind cells via poorly characterized receptors, are internalized by clathrin-mediated endocytosis (10), and fuse with the membranes of endosomal compartments in a pH-dependent fashion (18, 33). Upon exposure to mildly acidic conditions (∼pH 6.5), E proteins undergo extensive changes in conformation and oligomeric state that serve to tether viral and cellular membranes and pull them into the close apposition required to promote lipid mixing (12, 29). While this transformation has been detailed using biochemical and structural approaches (14, 22, 29), how an acidic environment triggers these events is less clear. The “histidine switch” hypothesis identifies a critical role for histidine residues as sensors that trigger the activity of viral fusion proteins that direct pH-dependent entry into cells (16, 21). The rationale for this theory is that histidine is unique among amino acids in that it becomes protonated and charged upon exposure to acidic environments similar to those that support viral fusion (the pK_a of histidine in proteins is ∼6.4) (32). Evidence in support of a required role for histidine residues in the conformational changes of viral proteins that direct membrane fusion has been obtained using several groups of viruses that enter cells in a pH-dependent fashion (4, 5, 9, 15, 27, 31).West Nile virus (WNV) is a mosquito-borne encephalitic flavivirus that has emerged during the last decade as a threat to public health in the Western hemisphere (3). To investigate a requirement for the ionization of histidine residues during WNV entry, we constructed a panel of mutants with histidine substitutions in the E protein and evaluated their capacity to mediate infection. The E protein is composed of three distinct domains (DI to DIII) that are connected to the viral membrane by a helical region called the stem-anchor (17, 24). Each of these protein regions contains histidine residues that could be involved in triggering conformational changes that drive pH-dependent membrane fusion (Fig. ​(Fig.1A).1A). Five of the 13 histidine residues in the E protein are completely conserved among flaviviruses. Each individual histidine was mutated to two different amino acids using QuikChange mutagenesis (Stratagene, La Jolla, CA). Alanine and glutamine substitutions were selected because the side chains of these amino acids are nonionizable (Fig. 1B to D).Open in a separate windowFIG. 1.Impact of mutations at individual histidine residues in the WNV E protein on virus infectivity. (A) The WNV E protein is composed of three structurally distinct domains (DI-DIII). There are 13 histidine residues in the E protein; 3 of these are located in DI (red ribbons), 4 in DII (yellow ribbons), 4 in DIII (blue ribbons), and 2 in the stem-anchor region that anchors the E protein to the viral membrane (not pictured). Five of these histidine residues are conserved among flaviviruses (labeled in red). (B to D) RVPs composed of E proteins incorporating alanine (red) or glutamine (green) histidine substitutions were produced by genetic complementation. The infectious titer of each preparation was determined following infection of Raji-DC-SIGNR cells with serial twofold dilutions of RVPs. Infection was scored as a function of reporter gene expression 48 h postinfection and was measured using flow cytometry. (E) The infectious titer of each glutamine substitution mutant in Raji-DC-SIGNR cells was calculated for multiple independent RVP preparations using data from linear portions of the virus dose/infectivity curves. Hatched bars show results for mutants with substitutions at conserved histidine residues. The means of the results for four or five independent RVP preparations are shown; error bars identify the standard errors. (F) The titers of glutamine substitution mutants were determined following infection of Vero cells with serial twofold dilutions of RVPs. The infectious titer of each mutant was calculated as described above. Hatched bars show results for mutants with substitutions at conserved histidine residues. The means of titers from four independent experiments are shown; error bars represent the standard errors.WNV reporter virus particles (RVPs) that incorporate each E protein mutant were produced by complementation as described previously (25, 26). Briefly, BHK-21 cells that propagate a subgenomic WNV replicon encoding green fluorescent protein were transfected with plasmids encoding WNV capsid and precursor to membrane (prM)-E proteins, followed by incubation for 2 days at 37°C. The titers of RVP preparations in Raji cells expressing the attachment factor DC-SIGNR were determined following infection with six serial twofold dilutions of virions (7). Because RVPs are capable of only a single round of infection and do not encapsidate a genome that encodes the E protein, there is no opportunity for reversion to the wild-type (WT) sequence during production. Analysis of the panel of mutants revealed three patterns (Fig. 1B to D and data not shown). Near-WT levels of infectivity were observed for mutants with both substitutions at four different histidine residues (H81, H320, H395, and H398) (Fig. ​(Fig.1B).1B). Analysis of multiple independent RVP preparations with glutamine substitutions indicated that, on average, the titer of RVPs incorporating these mutants was reduced by only ∼25% (n = 4 or 5 preparations) in comparison to the titer in preparations with WT E protein (Fig. ​(Fig.1E).1E). In contrast, neither substitution at position H144 or H246 yielded infectious virus particles (Fig. 1D and E). A third group of mutants displayed an intermediate phenotype with the degree of attenuation dependent upon the amino acid substitution (Fig. 1C and E). A similar pattern was observed when Vero cells were used to determine RVP titers (Fig. ​(Fig.1F).1F). While in several instances (H144 and H246) a glutamine substitution resulted in a significant reduction in the number of virus particles released from transfected cells, quantitative analysis of the E protein content of RVP preparations indicated that a simple failure to release virus particles is not sufficient to explain the reduction in infectious titer observed with each mutant (data not shown). A limitation of any mutagenesis approach is that negative results must be interpreted with caution. While the identification of functional substitutions that encode nonionizable amino acids rules out a requirement for histidine protonation at a given position during virus entry, mutations may also subtly affect E protein structure or functions not relating to sensing changes in endosomal pH.We next expanded our efforts to identify functional substitutions at positions H144 and H246. Additional mutants were constructed by site-directed mutagenesis employing primers encoding a random sequence (NNN) at the codons of H144 or H246. Because mutations may affect virus infectivity by modulating the efficiency of virion maturation, RVPs were produced in cells cotransfected with a plasmid expressing human furin, a condition shown previously to dramatically enhance the efficiency of prM cleavage (7, 23). Augmented furin expression in RVP-producing cells resulted in complete maturation of each mutant tested (data not shown), suggesting that the low-pH-mediated conformational changes that regulate exposure of the furin cleavage site are not dependent upon a single histidine residue. RVPs were also produced at both 37°C and 28°C; experiments at the latter temperature were performed to minimize the potential impact of mutations on E protein folding and stability. For substitutions at most histidine residues, overexpression of furin in producer cells had only a modest effect on the release of infectious RVPs relative to the level in control experiments with WT E proteins (data not shown; n = 3 or 4 preparations). Notably, conditions that promoted more efficient maturation resulted in a significant increase in the production of infectious RVPs incorporating the H144Q substitution (25-fold, P < 0.05), albeit at very low titers (Fig. ​(Fig.2A2A and data not shown; ∼1.4 × 10³ infectious units/ml at 37°C, n = 9). Analysis of seven additional H144 mutants identified asparagine (Fig. 2A and B) and methionine (data not shown) substitutions that could be incorporated into infectious virions. RVPs composed of the H144N mutant were produced at ∼1.4% and 31% (37°C and 28°C, respectively; n = 4) of the level observed in paired experiments with WT RVPs, and the levels were not significantly different in the presence or absence of furin overexpression despite relatively inefficient cleavage of prM of H144N in the absence of furin expression (data not shown). All 19 amino acid substitutions at H246 were analyzed, and substitutions with aromatic side chains (tryptophan, phenylalanine, and tyrosine) were identified as capable of orchestrating virus entry (Fig. 2C and D and data not shown). When produced at 28°C, the titer of H246F RVPs was reduced only ∼twofold relative to the titers in studies with WT RVPs. In agreement with these findings, a neutralization escape mutant of WNV encoding an H246Y mutation has recently been described (35). Altogether, these studies identified individual substitutions with nonionizable side chains at each of the 13 E protein histidine residues that can be incorporated into RVPs with significant infectious titers.Open in a separate windowFIG. 2.Identification of functional substitutions for H144 and H246. WNV RVPs incorporating E protein histidine substitution mutants were produced by complementation in the presence or absence of a plasmid expressing human furin, followed by incubation at 37°C (A and C) or 28°C (B and D) (1, 7, 23). The infectious titers of RVP populations were determined as described in the Fig. ​Fig.11 legend. All titer experiments were performed at 37°C, even when lower temperatures were used to produce the RVPs. The titers of RVPs produced in the presence of exogenous furin expression are shown by hatched bars. The means of the results of three or four independent experiments are displayed; error bars identify the standard errors. (E and F) Entry of WNV RVPs incorporating histidine mutants is pH dependent. Raji-DC-SIGNR cells were treated with serial dilutions of the weak base NH₄Cl (from 0.2 to 50 mM) for 5 to 10 min at room temperature prior to infection with RVPs. Cells were washed at 12 h postinfection and cultured for an additional 36 h in fresh medium. Infectivity was measured by flow cytometry at 48 h postinfection. The concentration of NH₄Cl required to inhibit infection by 50% (IC₅₀) was calculated by nonlinear regression. (F) The means of two or three independent measurements are shown; error bars represent the standard errors. Hatched bars show results for mutants with substitutions at conserved histidine residues.To confirm that virions composed of each functional mutant entered cells in a pH-dependent fashion, we next established that infectious entry could be inhibited by neutralization of endosomal compartments. Ammonium chloride is a lysosomotropic agent shown to block membrane fusion by pH-dependent viruses, including flaviviruses (10, 11, 13, 28, 34). Infection by each mutant was completely inhibited by NH₄Cl; the concentrations required to inhibit 50% of infection were calculated by nonlinear regression and found to be similar for WT WNV and histidine mutants (Fig. 2E and F).While our data suggest that no single histidine residue is required for E protein-mediated entry of WNV, it is possible that several act in concert. For example, ionization of multiple histidine residues is thought to trigger pH-dependent conformational changes in the fusion proteins of vesicular stomatitis virus and baculovirus (4, 15). The antiparallel arrangement of E proteins on the virion clusters H144, H152, and H320 at the hinge between DI and DII along the dimer interface of the E protein. Previous structural studies speculated that ionization of these three residues may trigger dimer disassociation following exposure to an acidic environment (2, 9, 24). Therefore, we constructed double and triple mutants containing substitutions in histidine residues at the DI-DII hinge and were able to identify combinations of mutations that allow for the production of infectious RVPs with significant titers (Fig. ​(Fig.3).3). While these data indicate that this cluster of histidine residues hypothesized previously to function as a pH sensor are not required for infection, it is certainly possible that other combinations of histidine (and nonhistidine) residues may coordinate to function in this regard.Open in a separate windowFIG. 3.Production of infectious RVPs incorporating multiple histidine substitutions at the DI-DII hinge. RVPs incorporating substitutions at one or more histidine positions clustered around the DI-DII hinge were produced by genetic complementation. The infectious titers of multiple independent preparations of RVPs were calculated as described in the Fig. ​Fig.11 legend. The means of two or three independent measurements are shown; error bars represent the standard errors.Recent studies of tick-borne encephalitis virus (TBEV) identified the conserved histidine at position 323 (corresponding to H320 of WNV) as the critical pH sensor required for the initiation of viral membrane fusion (9). Using a recombinant subviral particle (SVP) system (6, 8, 30), Fritz and colleagues found that TBEV SVPs incorporating a H323A mutation form unstable E protein trimers and are unable to fuse with synthetic liposomes when exposed to low pH (9). SVPs are small (∼30 nM), noninfectious virus-like particles composed of 60 E proteins that have been used extensively as a model of flavivirus fusion (29). Because our studies with WNV RVPs and a replication-competent infectious clone (data not shown) indicate that this residue is dispensable for pH-dependent infection, we explored this discrepancy by introducing mutations into the E protein of Langat virus (LGTV), a flavivirus that shares roughly 80% amino acid identity with TBEV. A panel of H146 and H323 mutants were produced using degenerate primers as described above and incorporated into RVPs composed of the replicon RNA and capsid protein of WNV. LGTV RVPs composed of H323L and H146R mutants were produced with titers similar to or greater than the titer of those incorporating WT LGTV E proteins (1.1 and 4.6 times the WT titer, respectively) (Fig. ​(Fig.4A).4A). In each case, RVP infection remained pH dependent (Fig. ​(Fig.4B).4B). How to reconcile the differences between our findings with WNV and LGTV and those reported for TBEV is unclear. One possibility is that the sensor of acidic pH differs between SVPs and infectious virions, perhaps due to differences in the geometry of these two classes of virus particles (T = 1 and pseudo-T = 3, respectively) (8, 19). Alternatively, the lipid-mixing assay used in vitro may be differentially sensitive to factors that affect the requirements for and efficiency of viral membrane fusion.Open in a separate windowFIG. 4.Mutation of conserved histidine residues in the tick-borne flavivirus LGTV. (A) A panel of mutants at positions H146 and H323 in LGTV was produced by QuikChange mutagenesis employing redundant primers. Mutants were assayed for their capacity to direct virus entry of RVPs produced by complementation. Multiple substitutions for positions H146 and H323 were capable of mediating entry of RVPs (data not shown). The titers of RVPs composed of H146R and H323L were calculated as described in the Fig. ​Fig.11 legend. The mean titers from three independent RVP preparations are shown; error bars represent the standard errors. (B) The pH dependence of LGTV RVP entry was confirmed as described in the Fig. ​Fig.22 legend. The means of two or three independent measurements are shown; error bars represent the standard errors.Histidine residues acquire a positive charge when exposed to the mildly acidic conditions known to trigger the fusion machinery of flaviviruses. In principle, these residues are uniquely suited to serve as sensors of environmental pH. However, our results indicate that there is no requirement for protonation of a particular “histidine switch” during the infectious entry of WNV, as mutagenesis studies identified viable substitutions with nonionizable side chains at all 13 histidine residues within the E protein. While not all the mutants identified were as infectious as WT RVPs, this is not particularly surprising, as any change away from the naturally selected WT sequence has the potential to affect protein structure or function. How the E protein senses a low-pH environment remains unclear and appears to require the contribution of additional amino acids. Because amino acid side-chain pK_a is influenced locally by protein structure, it is possible that nonhistidine residues are coordinated such that they are ionized at ∼pH 6.5. Resolving this complexity awaits further study.     

How Many Nucleotides Are Required to Resolve a Phylogenetic Problem? The Use of a New Statistical Method Applicable to Available Sequences

The evolution of bootstrap proportions (BP) with sequence length was studied using a 28S ribosomal RNA data set. For different sequence lengths, informative sites were jackknifed several times. Bootstrapping was subsequently performed on each of these subsamples. For each node, BPs so obtained were plotted against sequence length, showing the evolution of the robustness with increasing number of informative sites. For robust nodes (BP of 100%), the pattern of BPs is unvarying and is described by a simple function BP = 100(1− e^{−b(x − x′)}), where x is the number of informative sites and b and x′ are two parameters estimated using a nonlinear regression procedure. When a node has a BP <100% and the pattern of BPs fits this function, it is possible to estimate the number of informative sites required to obtain a given average BP. The method also identifies nonrobust nodes (nonascending clusters of BP dots), for which it seems to be more cost effective and fruitful to turn to other species and/or genes rather than to continue sequencing longer gene lengths from the same species to reach a BP of 95%.     

The quest for a mechanistic understanding of biodiversity–ecosystem services relationships

Ecosystem services (ES) approaches to biodiversity conservation are currently high on the ecological research and policy agendas. However, despite a wealth of studies into biodiversity''s role in maintaining ES (B–ES relationships) across landscapes, we still lack generalities in the nature and strengths of these linkages. Reasons for this are manifold, but can largely be attributed to (i) a lack of adherence to definitions and thus a confusion between final ES and the ecosystem functions (EFs) underpinning them, (ii) a focus on uninformative biodiversity indices and singular hypotheses and (iii) top-down analyses across large spatial scales and overlooking of context-dependency. The biodiversity–ecosystem functioning (B–EF) field provides an alternate context for examining biodiversity''s mechanistic role in shaping ES, focusing on species'' characteristics that may drive EFs via multiple mechanisms across contexts. Despite acknowledgements of a need for B–ES research to look towards underlying B–EF linkages, the connections between these areas of research remains weak. With this review, we pull together recent B–EF findings to identify key areas for future developments in B–ES research. We highlight a means by which B–ES research may begin to identify how and when multiple underlying B–EF relationships may scale to final ES delivery and trade-offs.     

The development of a radioimmunoassay for Tamm–Horsfall glycoprotein in serum

The Tamm–Horsfall glycoprotein prepared by salt precipitation from urine was found to comprise a heterogeneous collection of aggregates. These could be disaggregated with 8m-urea, following which chromatography on a column of Bio-Gel A.15m yielded a homogeneous glycoprotein of mol.wt. 73000 together with several unidentified impurities. Gel filtration of normal plasma showed the glycoprotein to exist predominantly in a form that is eluted identically with the purified preparation. In one case, material of higher molecular weight was also detected. The purified glycoprotein was used to develop a rapid specific radioimmunoassay for its measurement in human serum or plasma by the use of the Tamm–Horsfall glycoprotein, labelled with ¹²⁵I by the chloramine-t method as the tracer, an antiserum raised in rabbits, and separation of the bound and free fractions by a second antibody covalently linked to magnetizable particles. Parallelism was demonstrated between the standard preparation and samples. Recovery of added standard to serum varied between 99 and 109%. Total assay time was less than 4h with an intra-assay and inter-assay coefficient of variation of less than 10%. There were no significant differences in the ranges covered with regard to either age or sex, and no circadian rhythm was observed in normal subjects. A physiological range of 70–540ng/ml was established based on serum samples from 95 subjects with normal renal function, as defined by their serum creatinine and urea concentrations. No Tamm–Horsfall glycoprotein was detected in the serum of six anephric patients.     

The cichlid–Cichlidogyrus network: a blueprint for a model system of parasite evolution

Hydrobiologia - Species interactions are a key aspect of evolutionary biology. Parasites, specifically, are drivers of the evolution of species communities and impact biosecurity and public health....     

The Zinc Transporter Slc30a8/ZnT8 Is Required in a Subpopulation of Pancreatic α-Cells for Hypoglycemia-induced Glucagon Secretion

SLC30A8 encodes a zinc transporter ZnT8 largely restricted to pancreatic islet β- and α-cells, and responsible for zinc accumulation into secretory granules. Although common SLC30A8 variants, believed to reduce ZnT8 activity, increase type 2 diabetes risk in humans, rare inactivating mutations are protective. To investigate the role of Slc30a8 in the control of glucagon secretion, Slc30a8 was inactivated selectively in α-cells by crossing mice with alleles floxed at exon 1 to animals expressing Cre recombinase under the pre-proglucagon promoter. Further crossing to Rosa26:tdRFP mice, and sorting of RFP⁺: glucagon⁺ cells from KO mice, revealed recombination in ∼30% of α-cells, of which ∼50% were ZnT8-negative (14 ± 1.8% of all α-cells). Although glucose and insulin tolerance were normal, female αZnT8KO mice required lower glucose infusion rates during hypoglycemic clamps and displayed enhanced glucagon release (p < 0.001) versus WT mice. Correspondingly, islets isolated from αZnT8KO mice secreted more glucagon at 1 mm glucose, but not 17 mm glucose, than WT controls (n = 5; p = 0.008). Although the expression of other ZnT family members was unchanged, cytoplasmic (n = 4 mice per genotype; p < 0.0001) and granular (n = 3, p < 0.01) free Zn²⁺ levels were significantly lower in KO α-cells versus control cells. In response to low glucose, the amplitude and frequency of intracellular Ca²⁺ increases were unchanged in α-cells of αZnT8KO KO mice. ZnT8 is thus important in a subset of α-cells for normal responses to hypoglycemia and acts via Ca²⁺-independent mechanisms.     

ERRγ Is Not Required for Skeletal Development but Is a RUNX2-Dependent Negative Regulator of Postnatal Bone Formation in Male Mice

To assess the effects of the orphan nuclear Estrogen receptor-related receptor gamma (ERRγ) deficiency on skeletal development and bone turnover, we utilized an ERRγ global knockout mouse line. While we observed no gross morphological anomalies or difference in skeletal length in newborn mice, by 8 weeks of age ERRγ +/− males but not females exhibited increased trabecular bone, which was further increased by 14 weeks. The increase in trabecular bone was due to an increase in active osteoblasts on the bone surface, without detectable alterations in osteoclast number or activity. Consistent with the histomorphometric results, we observed an increase in gene expression of the bone formation markers alkaline phosphatase (Alp) and bone sialoprotein (Bsp) in bone and increase in serum ALP, but no change in the osteoclast regulators receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) or the resorption marker carboxy-terminal collagen crosslinks (CTX). More colony forming units-alkaline phosphatase and -osteoblast (CFU-ALP, CFU-O respectively) but not CFU-fibroblast (CFU-F) formed in ERRγ +/− versus ERRγ +/+ stromal cell cultures, suggesting that ERRγ negatively regulates osteoblast differentiation and matrix mineralization but not mesenchymal precursor number. By co-immunoprecipitation experiments, we found that ERRγ and RUNX2 interact in an ERRγ DNA binding domain (DBD)-dependent manner. Treatment of post-confluent differentiating bone marrow stromal cell cultures with Runx2 antisense oligonucleotides resulted in a reduction of CFU-ALP/CFU-O in ERRγ +/− but not ERRγ +/+ mice compared to their corresponding sense controls. Our data indicate that ERRγ is not required for skeletal development but is a sex-dependent negative regulator of postnatal bone formation, acting in a RUNX2- and apparently differentiation stage-dependent manner.     

Structure of the Arabidopsis thaliana DCL4 DUF283 domain reveals a noncanonical double-stranded RNA-binding fold for protein–protein interaction

Dicer or Dicer-like (DCL) protein is a catalytic component involved in microRNA (miRNA) or small interference RNA (siRNA) processing pathway, whose fragment structures have been partially solved. However, the structure and function of the unique DUF283 domain within dicer is largely unknown. Here we report the first structure of the DUF283 domain from the Arabidopsis thaliana DCL4. The DUF283 domain adopts an α-β-β-β-α topology and resembles the structural similarity to the double-stranded RNA-binding domain. Notably, the N-terminal α helix of DUF283 runs cross over the C-terminal α helix orthogonally, therefore, N- and C-termini of DUF283 are in close proximity. Biochemical analysis shows that the DUF283 domain of DCL4 displays weak dsRNA binding affinity and specifically binds to double-stranded RNA-binding domain 1 (dsRBD1) of Arabidopsis DRB4, whereas the DUF283 domain of DCL1 specifically binds to dsRBD2 of Arabidopsis HYL1. These data suggest a potential functional role of the Arabidopsis DUF283 domain in target selection in small RNA processing.     

The use of electromyography for the assessment of sense of muscular effort: a test–retest reliability study

This study sought to examine the test–retest reliability to measure sense of muscular effort with electromyography (EMG). The EMG activity of the tibialis anterior muscle from 23 participants was recorded. Targets of EMG amplitudes produced at 10 and 20% of the maximum voluntary contraction (MVC) were calculated. Participants matched the target EMG level with and without visual feedback (FB). With NFB, the reliability was good to excellent when errors were represented as the average standard deviation (SD) of the error from the target (ICC_1,2 = 0.75 and 0.69 for 10 and 20% targets, respectively). Also, reliability was good when errors were presented as the average SD as a percentage of the MVC EMG (intraclass correlation coefficient (ICC_1,2) = 0.67 and 0.66, respectively, for 10 and 20% targets). Standard deviation around the target was the most reliable method to represent the error. This approach could be used as a simple cost-effective method to assess the sense of muscular effort.     

The β1-Subunit of Nav1.5 Cardiac Sodium Channel Is Required for a Dominant Negative Effect through α-α Interaction

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Several mutations on the cardiac sodium channel Na_v1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane. In order to mimic patient heterozygosity, a trafficking defective mutant, R1432G was co-expressed with Wild Type (WT) Na_v1.5 channels in HEK293T cells. This mutant significantly decreased the membrane Na current density when it was co-transfected with the WT channel. This dominant negative effect did not result in altered biophysical properties of Na_v1.5 channels. Luminometric experiments revealed that the expression of mutant proteins induced a significant reduction in membrane expression of WT channels. Interestingly, we have found that the auxiliary Na channel β₁-subunit was essential for this dominant negative effect. Indeed, the absence of the β₁-subunit prevented the decrease in WT sodium current density and surface proteins associated with the dominant negative effect. Co-immunoprecipitation experiments demonstrated a physical interaction between Na channel α-subunits. This interaction occurred only when the β₁-subunit was present. Our findings reveal a new role for β₁-subunits in cardiac voltage-gated sodium channels by promoting α-α subunit interaction which can lead to a dominant negative effect when one of the α-subunits shows a trafficking defective mutation.     

The Use of a Coaxial Electrodynamic System for Amplification of Microwave Range Waves During the Development of Beam–Plasma Instability

Plasma Physics Reports - A coaxial electrodynamic system for the amplification of microwaves with plasma filling, through which a relativistic electron beam moves, is studied theoretically. The...     

The development of a material model and wheel–tissue interaction for simulating wheeled surgical robot mobility

In vivo surgical robot wheel and tissue interaction was studied using a nonlinear finite element model. A liver material model, derived from laboratory experiments, was implemented as a viscoelastic material. A finite element simulation of this laboratory test confirmed the accuracy of the liver material model. This material model was then used as the tissue model to study wheel performance. A helical wheel moving on the liver model was used to replicate laboratory experiments that included several different slip ratios and applied loads. The drawbar force produced in this model showed good agreement with the physical tests. These results have provided the baseline for studying how changes in wheel geometry, such as tread height, tread spacing and wheel diameter, affect drawbar force and ultimately wheel performance. These results will be used in future surgical robot wheel designs.     

The efficacy of cognitive–behavioral therapy for older adults with ADHD: a randomized controlled trial

Older adults with ADHD exhibit significant functional impairment, yet there is little research to guide clinicians in evidence-based care of these adults. This study examined response to treatment in older adults who participated in a previous study of the efficacy of cognitive–behavioral treatment (CBT) in adult ADHD. It was hypothesized that older adults would respond less well to CBT than younger adults, given the cognitive demands of the treatment. As described in the original publication, 88 adults who met DSM-IV criteria for ADHD were randomized to receive either a manualized 12-week CBT group intervention targeting executive dysfunction or a parallel Support group. In the current study, outcomes for 26 adults, aged 50 or older, were compared with those of 55 younger adults with respect to inattentive symptoms assessed on a structured interview by a blind clinician, as well as on ratings by self and/or collateral on measures of attention, executive dysfunction, and comorbidity. Contrary to the hypothesis, older and younger adults were equally responsive to CBT on measures of attention. The older adults also responded as well to Support as to CBT on several outcome measures. The results provide preliminary evidence that CBT is an effective intervention for older adults with ADHD. The unexpected response to support highlights a possible age-specificity of effective therapeutic intervention that requires further investigation.     

The ecological role of moss in a polar desert: implications for aboveground–belowground and terrestrial–aquatic linkages

The McMurdo Dry Valleys of Antarctica is one of the coldest and driest habitats on the planet. As vascular plants are absent in this region, moss is the main form of aboveground primary production with a potentially important contribution to biogeochemical cycling, yet little is known about their ecological role. To determine the relationship between moss and soil properties relevant to biogeochemistry, we sampled both from a variety of locations in the Dry Valleys. Moss presence was compared to soil properties, and we measured the plasticity of moss stoichiometry (C:N:P) across gradients in nutrient availability. Results demonstrate that many soil properties significantly differed with moss presence, particularly conductivity and pH, but there is no strong evidence that this is caused by the moss presence and not the conditions inherent to the microsites where moss was found. There is great variability in moss stoichiometry, with some significant differences between sites, but generally variability within sites is larger than variation among sites. Results suggest that the main source of moss nutrients is from the soil, rather than water, but correlations with any one nutrient source are weak, suggesting a great deal of plasticity in moss stoichiometry and nutrient uptake.