Interactions of Folch-Lees proteolipid apoprotein with planar lipid bilayers

Summary Water-soluble Folch-Lees proteolipid apoprotein from bovine CNS white matter induces a voltage-dependent conductance in black lipid membranes. Na⁺ is required for the induced conductance change but the established conductance has very low ionic selectivity. The induced conductance fluctuates with a minimum amplitude of 10^–11–10^–10 mho. The magnitude of the conductivity change is dependent on protein concentration and on the composition of lipid bilayers. At a fixed voltage the induced conductance of a phosphatidylcholine-cholesterol membrane is proportional to the sixth power of the protein concentration and the first power of Na⁺ concentration. The interactions between the apoprotein and the lipids are both electrostatic and hydrophobic, but the interaction leading to the conductance increase appears to be mainly hydrophobic. Both the increase in conductance and the current fluctuations remain after extensive washing of the chambers to remove the protein. Furthermore, pronase or glutaraldehyde added to either the cis or trans side of the membrane does not affect the apoprotein-established conductance. However, if the bilayer is formed in the presence of both the apoprotein and pronase or if the apoprotein is treated with pronase prior to its addition to the chamber, no conductance change is observed. The association of the apoprotein with the membrane thus appears to render the protein inaccessible to proteolytic digestion, suggesting that the apoprotein is at least partially imbedded in the membrane interior.     

Analyzing the role of the putative inositol 1,3,4,5-tetrakisphosphate receptor GAP1IP4BP in intracellular Ca2+ homeostasis

Inositol 1,3,4,5-tetrakisphosphate (IP(4)) has been linked to a potential role in the regulation of intracellular free Ca(2+) concentration ([Ca(2+)](i)) following cellular stimulation with agonists that activate phosphoinositide-specific phospholipase C. However, despite many studies, the function of IP(4) remains unclear and indeed there is still some debate over whether it has a function at all. Here we have used various molecular approaches to address whether manipulation of the potential IP(4) receptor, GAP1(IP4BP), affects [Ca(2+)](i) following cellular stimulation. Using single cell imaging, we show that the overexpression of a constitutively active and a potential dominant negative form of GAP1(IP4BP) appear to have no effect on Ca(2+) mobilization or Ca(2+) entry following stimulation of HeLa cells with histamine. In addition, through the use of small interfering RNA duplexes, we have examined the effect of suppressing endogenous GAP1(IP4BP) production on [Ca(2+)](i). In HeLa cells in which the endogenous level of GAP1(IP4BP) has been suppressed by approximately 95%, we failed to observe any effect on Ca(2+) mobilization or Ca(2+) entry following histamine stimulation. Thus, using various approaches to manipulate the function of endogenous GAP1(IP4BP) in intact HeLa cells, we have been unable to observe any detectable effect of GAP1(IP4BP) on [Ca(2+)](i).     

Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference

Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.     

Lymphocytes protect against and are not required for reovirus-induced myocarditis.

Many studies suggest that host lymphocytes are damaging, rather than protective, in virally induced myocarditis. We have investigated the role of lymphocyte-based immunity in murine myocarditis by using a myocarditic reovirus (reovirus serotype 3 8B), nonmyocarditic reoviruses, adoptive transfer experiments, and mice with severe combined immunodeficiency (SCID mice). Prior to infection, passive transfer of monoclonal antibodies specific for 8B capsid proteins protected neonatal mice against 8B-induced myocarditis, indicating that humoral immunity can protect against myocarditis. Some monoclonal antibodies acted by blocking viral spread to and/or replication in the heart. Passive transfer of reovirus-immune, but not naive, spleen cells prior to infection protected neonatal mice from 8B-induced myocarditis. Depletion of either CD4 or CD8 T cells resulted in increased viral titer in the heart but did not abrogate immune cell-mediated protection against myocardial injury. This shows that both CD4 and CD8 T cells can act independently to protect myocardial tissue from reovirus infection. In addition, reovirus 8B caused extensive myocarditis in SCID mice. This confirms a prior report (B. Sherry, F. J. Schoen, E. Wenske, and B. N. Fields, J. Virol. 63:4840-4849, 1989) that T cells are not required for reovirus-induced myocarditis and demonstrates for the first time that B cells are not required for reovirus-induced myocarditis. We used SCID mice and a panel of reoviruses to assess (i) the relationship between growth in the heart and myocardial damage and (ii) the possibility that nonmyocarditic reoviruses exhibit a myocarditic phenotype in the absence of functional lymphocytes. Growth in the heart was not the sole determinant of myocarditic potential in SCID mice. Although 8B induced myocarditis in SCID mice, no or minimal myocarditis was found in SCID mice infected with four reovirus strains previously shown (B. Sherry and B. N. Fields, J. Virol. 63:4850-4856, 1989) to be nonmyocarditic or poorly myocarditic in normal neonatal mice. We conclude that (i) humoral immunity and cellular immunity are protective against, and not required for, reovirus-induced myocarditis and (ii) the potential to induce cardiac damage is a property of the virus independent of lymphocyte-based immunity.     

Best environmental predictors of breeding phenology differ with elevation in a common woodland bird species

Temperatures in mountain areas are increasing at a higher rate than the Northern Hemisphere land average, but how fauna may respond, in particular in terms of phenology, remains poorly understood. The aim of this study was to assess how elevation could modify the relationships between climate variability (air temperature and snow melt‐out date), the timing of plant phenology and egg‐laying date of the coal tit (Periparus ater). We collected 9 years (2011–2019) of data on egg‐laying date, spring air temperature, snow melt‐out date, and larch budburst date at two elevations (~1,300 m and ~1,900 m asl) on a slope located in the Mont‐Blanc Massif in the French Alps. We found that at low elevation, larch budburst date had a direct influence on egg‐laying date, while at high‐altitude snow melt‐out date was the limiting factor. At both elevations, air temperature had a similar effect on egg‐laying date, but was a poorer predictor than larch budburst or snowmelt date. Our results shed light on proximate drivers of breeding phenology responses to interannual climate variability in mountain areas and suggest that factors directly influencing species phenology vary at different elevations. Predicting the future responses of species in a climate change context will require testing the transferability of models and accounting for nonstationary relationships between environmental predictors and the timing of phenological events.     

Exploration of Ellsworth Subglacial Lake: a concept paper on the development, organisation and execution of an experiment to explore, measure and sample the environment of a West Antarctic subglacial lake

Antarctic subglacial lakes have, over the past few years, been hypothesised to house unique forms of life and hold detailed sedimentary records of past climate change. Testing this hypothesis requires in situ examinations. The direct measurement of subglacial lakes has been considered ever since the largest and best-known lake, named Lake Vostok, was identified as having a deep water-column. The Subglacial Antarctic Lake Environments (SALE) programme, set up by the Scientific Committee on Antarctic Research (SCAR) to oversee subglacial lakes research, state that prior exploration of smaller lakes would be a “prudent way forward”. Over 145 subglacial lakes are known to exist in Antarctica, but one lake in West Antarctica, officially named Ellsworth Subglacial Lake (referred to hereafter as Lake Ellsworth), stands out as a candidate for early exploration. A consortium of over 20 scientists from seven countries and 14 institutions has been assembled to plan the exploration of Lake Ellsworth. An eight-year programme is envisaged: 3 years for a geophysical survey, 2 years for equipment development and testing, 1 year for field planning and operation, and 2 years for sample analysis and data interpretation. The science experiment is simple in concept but complex in execution. Lake Ellsworth will be accessed using hot water drilling. Once lake access is achieved, a probe will be lowered down the borehole and into the lake. The probe will contain a series of instruments to measure biological, chemical and physical characteristics of the lake water and sediments, and will utilise a tether to the ice surface through which power, communication and data will be transmitted. The probe will pass through the water column to the lake floor. The probe will then be pulled up and out of the lake, measuring its environment continually as this is done. Once at the ice surface, any water samples collected will be taken from the probe for laboratory analysis (to take place over subsequent years). The duration of the science mission, from deployment of the probe to its retrieval, is likely to take between 24 and 36 h. Measurements to be taken by the probe will provide data about the following: depth, pressure, conductivity and temperature; pH levels; biomolecules (using life marker chips); anions (using a chemical analyzer); visualisation of the environment (using cameras and light sources); dissolved gases (using chromatography); and morphology of the lake floor and sediment structures (using sonar). After the probe has been retrieved, a sediment corer may be dropped into the lake to recover material from the lake floor. Finally, if time permits, a thermistor string may be left in the lake water to take time-dependent measurements of the lake’s water column over subsequent years. Given that the comprehensive geophysical survey of the lake will take place in two seasons during 2007–2009, a two-year instrument and logistic development phase from 2008 (after the lake’s bathymetry has been assessed) makes it possible that the exploration of Lake Ellsworth could take place at the beginning of the next decade.     

A divalent major histocompatibility complex/IgG1 fusion protein induces antigen-specific T cell activation in vitro and in vivo

Activation of antigen-specific T cell clones in vivo might be possible by generating soluble MHC molecules; however, such molecules do not induce effective T cell responses unless cross-linked. As a first step in generating a soluble MHC molecule that could function as an antigen-specific immunostimulant, the extracellular domains of the murine H-2Kb MHC class I molecule were fused to the constant domains of a murine IgG1 heavy chain, resulting in a divalent molecule with both a TCR-reactive and an Fc receptor (FcR)-reactive moiety. The fusion protein can be loaded with peptide and can induce T cell activation in a peptide-specific, MHC-restricted manner following immobilization on plastic wells or following cross-linking by FcR+ spleen cells. The fusion protein induces partial T cell activation in vivo in a mouse transgenic for a TCR restricted to H-2Kb. This fusion protein molecule may be useful to study peptide-MHC interactions and may provide a strategy for boosting in vivo antigen-specific T cell responses, such as to viral or tumor antigens.     

A single-chain class II MHC-IgG3 fusion protein inhibits autoimmune arthritis by induction of antigen-specific hyporesponsiveness

T cells play a central role in many autoimmune diseases. A method to specifically target the function of autoreactive T cell clones would avoid the global immunosuppression associated with current therapies. To develop a molecule capable of inhibiting autoreactive T cell responses in vivo, single-chain peptide-I-A-IgG3 fusion proteins were constructed and expressed in both mammalian and insect cells. The fusion proteins were designed with an IgG3 Fc moiety to make them divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The fusion proteins stimulated T cell hybridomas in vitro in a peptide-specific, MHC-restricted manner but failed to do so in soluble form. In vivo administration of an I-A(q) fusion protein, containing an immunodominant collagen II peptide, significantly delayed the onset and reduced the severity of collagen-induced arthritis in DBA/1 mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins may be useful to study novel therapeutic approaches for T cell-mediated autoimmune diseases.     

Identification and Characterization of γ-Glutamylamine Cyclotransferase,an Enzyme Responsible for γ-Glutamyl-?-lysine Catabolism

γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-ϵ-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ-glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-α-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031–22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu⁸²). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.     

Evolution of dispersal in density regulated populations: A haploid model

The evolution of dispersal is explored in a density-dependent framework. Attention is restricted to haploid populations in which the genotypic fitnesses at a single diallelic locus are decreasing functions of the changing number of individuals in the population. It is shown that migration between two populations in which the genotypic response to density is reversed can maintain both alleles when the intermigration rates are constant or nondecreasing functions of the population densities. There is always a unique symmetric interior equilibrium with equal numbers but opposite gene frequencies in the two populations, provided the system is not degenerate. Numerical examples with exponential and hyperbolic fitnesses suggest that this is the only stable equilibrium state under constant positive migration rates (m) less than . Practically speaking, however, there is only convergence after a reasonable number of generations for relatively small migration rates ( ). A migration-modifying mutant at a second, neutral locus, can successfully enter two populations at a stable migration-selection balance if and only if it reduces the intermigration rates of its carriers at the original equilibrium population size. Moreover, migration modification will always result in a higher equilibrium population size, provided the system approaches another symmetric interior equilibrium. The new equilibrium migration rate will be lower than that at the original equilibrium, even when the modified migration rate is a nondecreasing function of the population sizes. Therefore, as in constant viability models, evolution will lead to reduced dispersal.