ß1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory ß-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or ß1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) ß1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the ß1-subunit within the α/ß1-subunit complex.

Moving gating charges through the gating pore in a Kv channel voltage sensor.

Voltage sensor domains (VSDs) regulate ion channels and enzymes by transporting electrically charged residues across a hydrophobic VSD constriction called the gating pore or hydrophobic plug. How the gating pore controls the gating charge movement presently remains debated. Here, using saturation mutagenesis and detailed analysis of gating currents from gating pore mutations in the Shaker Kv channel, we identified statistically highly significant correlations between VSD function and physicochemical properties of gating pore residues. A necessary small residue at position S240 in S1 creates a "steric gap" that enables an intracellular access pathway for the transport of the S4 Arg residues. In addition, the stabilization of the depolarized VSD conformation, a hallmark for most Kv channels, requires large side chains at positions F290 in S2 and F244 in S1 acting as "molecular clamps," and a hydrophobic side chain at position I237 in S1 acting as a local intracellular hydrophobic barrier. Finally, both size and hydrophobicity of I287 are important to control the main VSD energy barrier underlying transitions between resting and active states. Taken together, our study emphasizes the contribution of several gating pore residues to catalyze the gating charge transfer. This work paves the way toward understanding physicochemical principles underlying conformational dynamics in voltage sensors.

Nano-positioning system for structural analysis of functional homomeric proteins in multiple conformations.

Proteins may undergo multiple conformational changes required for their function. One strategy used to estimate target-site positions in unknown structural conformations involves single-pair resonance energy transfer (RET) distance measurements. However, interpretation of inter-residue distances is difficult when applied to three-dimensional structural rearrangements, especially in homomeric systems. We developed a positioning method using inverse trilateration/triangulation to map target sites within a homomeric protein in all defined states, with simultaneous functional recordings. The procedure accounts for probe diffusion to accurately determine the three-dimensional position and confidence region of lanthanide LRET donors attached to a target site (one per subunit), relative to a single fluorescent acceptor placed in a static site. As first application, the method is used to determine the position of a functional voltage-gated potassium channel's voltage sensor. Our results verify the crystal structure relaxed conformation and report on the resting and active conformations for which crystal structures are not available.

Symmetry-constrained analysis of pulsed double electron-electron resonance (DEER) spectroscopy reveals the dynamic nature of the KcsA activation gate.

Distance determination from an echo intensity modulation obtained by pulsed double electron-electron resonance (DEER) experiment is a mathematically ill-posed problem. Tikhonov regularization yields distance distributions that can be difficult to interpret, especially in a system with multiple discrete distance distributions. Here, we show that by using geometric fit constraints in symmetric homo-oligomeric protein systems, we were able to increase the accuracy of a model-based fit solution based on a sum of Rice distributions. Our approach was validated on two different ion channels of known oligomeric states, KcsA (tetramer) and CorA (pentamer). Statistical analysis of the resulting fits was integrated within our method to help the experimenter evaluate the significance of a symmetry-constrained vs standard model distribution fit and to examine multidistance confidence regions. This approach was used to quantitatively evaluate the role of the C-terminal domain (CTD) on the flexibility and conformation of the activation gate of the K(+) channel KcsA. Our analysis reveals a significant increase in the dynamics of the inner bundle gate upon opening. Also, it explicitly demonstrates the degree to which the CTD restricts the motion of the lower gate at rest and during activation gating.

Fluorescence detection of the movement of single KcsA subunits reveals cooperativity.

The prokaryotic KcsA channel is gated at the helical bundle crossing by intracellular protons and inactivates at the extracellular selectivity filter. The C-terminal transmembrane helix has to undergo a conformational change for potassium ions to access the central cavity. Whereas a partial opening of the tetrameric channel is suggested to be responsible for subconductance levels of ion channels, including KcsA, a cooperative opening of the 4 subunits is postulated as the final opening step. In this study, we used single-channel fluorescence spectroscopy of KcsA to directly observe the movement of each subunit and the temporal correlation between subunits. Purified KcsA channels labeled at the C terminus near the bundle crossing have been inserted into supported lipid bilayer, and the fluorescence traces analyzed by means of a cooperative or independent Markov model. The analysis revealed that the 4 subunits do not move fully independently but instead showed a certain degree of cooperativity. However, the 4 subunits do not simply open in 1 concerted step.

CL22 - a novel cationic peptide for efficient transfection of mammalian cells.

Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.

Low incidence of acute graft-versus-host disease and recurrent leukaemia in patients undergoing allogeneic haemopoietic stem cell transplantation from sibling donors with methotrexate and dose-monitored cyclosporin A prophylaxis.

One of the major aims of allogeneic haemopoietic stem cell transplantation has been the effective suppression of graft-versus-host disease (GVHD) without loss of a graft-versus-leukaemia effect. For GVHD suppression, one of the most frequently used regimens has been the combination of cyclosporin (CsA) and a short course of methotrexate (MTX) although the optimal usage of these agents remains unclear. Here, we report the results of 55 patients with standard risk leukaemia who have undergone allogeneic transplantation using either bone marrow (n = 48) or G-CSF mobilised peripheral blood stem cells (n = 7) using CsA and MTX for GVHD prophylaxis where the dosage of CsA was regularly adjusted to maintain a trough whole blood level of 95-205 ng/ml for the first 50 days post-transplant. To achieve this level of CsA in the immediate post-transplant period, over 40% of patients required dose adjustments of CsA as a result of sub-therapeutic levels on day +1 post-transplant. The achievement of CsA levels within the therapeutic range was expedited following the introduction of a sliding scale for dose adjustment. With this regimen we have observed a low incidence of acute GVHD with only 11% of patients developing > or =grade II disease. With a median follow-up of 66 months (range 8-132) the probability of relapse is only 6.6%. The disease-free survival probability for all patients was 72% at 5 years. These results demonstrate that effective GVHD prevention with CsA and MTX can be achieved without a high risk of recurrent leukaemia provided that rapid attainment of therapeutic CsA levels is achieved and maintenance within a low therapeutic range may help to maximise this effect.

Gynaecology, forced sterilisation, and asylum in the USA.

PIP: On September 30, 1996, the US Congress enacted a law to grant asylum protection to victims of forced sterilization, forced abortion, and other forms of coerced population control and to opponents of these practices. Before this time, the US Board of Immigration Appeals judged forced sterilization insufficient grounds for political asylum unless it was performed for some discriminatory reason. The authors of this article have examined five women who claimed they were forcibly sterilized in China and were granted political asylum in the US as a result. The average age of these women was 32 years, while the average duration since forced sterilization was 8.6 years. Since asylum applications are not filed according to cause, there are no estimates of the number of women seeking asylum in the US as a result of forced sterilization. The Chinese government claims it does not authorize forced sterilization, but local officials acknowledge it sometimes occurs. In many cases, sterilization is not formally coerced but required as a condition of employment. Consistency between the woman's claim of forced sterilization and medical or psychological reports is essential to the success of asylum applications. US physicians should be prepared to play a documentary role in the evaluation of such requests.^ieng

Upregulation of intracellular glutathione by fibroblast-derived factor(s): enhanced survival of activated T cells in the presence of low Bcl-2.

Activated interleukin-2 (IL-2)-dependent T cells express high levels of Bcl-2 protein. On cytokine withdrawal, Bcl-2 expression decreases and the cells die rapidly by apoptosis. We have previously shown that the survival of IL-2-deprived T cells can be promoted by factor(s) secreted by fibroblasts. Here we report that reduced glutathione (GSH), but not its oxidized counterpart GSSG, also enhances the in vitro survival of these cells. Exogenous GSH mediates its effect intracellularly, as (1) endogenous glutathione concentrations are increased up to fivefold in the presence of GSH, and (2) acivicin, an inhibitor of transmembrane GSH transport, abrogates GSH-dependent survival. The GSH-rescued T cells do not proliferate and express only low levels of Bcl-2, resembling W138 fibroblast-rescued T cells. We, therefore, investigated a role for GSH in fibroblast-promoted T-cell survival. We show that W138-promoted survival results in elevated GSH levels in surviving T cells and is abrogated by buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. Furthermore, both W138-promoted T-cell survival and GSH upregulation are associated with large molecular weight molecules (>30 kD). Thus, the upregulation of GSH by W138 fibroblasts appears to be crucial in their ability to enhance the survival of cytokine-deprived activated T cells in vitro.

Inhibition of T cell apoptosis in the rheumatoid synovium.

Synovial T cells in rheumatoid arthritis are highly differentiated and express a phenotype suggesting susceptibility to apoptosis (CD45RB dull, CD45RO bright, Bcl-2 low, Bax high, Fas high). However, no evidence of T cell apoptosis was found in synovial fluid from any of 28 patients studied. In contrast, synovial fluid from 10 patients with crystal arthritis showed substantial levels of T cell apoptosis. The failre of apoptosis was not an intrinsic property of rheumatoid synovial T cells, as they showed rapid spontaneous apoptosis on removal from the joint. Synovial T cells from rheumatoid arthritis and gout patients could be rescued from spontaneous apoptosis in vitro either by IL-2R gamma chain signaling cytokines (which upregulate Bcl-2 and Bcl-XL) or by interaction with synovial fibroblasts (which upregulates Bcl-xL but not Bcl-2). The phenotype of rheumatoid synovial T cells ex vivo (Bcl-2 low, Bcl-xL high) suggested a fibroblast-mediated mechanism in vivo. This was confirmed by in vitro culture of synovial T cells with fibroblasts which maintained the Bcl-xL high Bcl-2 low phenotype. Synovial T cells from gout patients were Bcl-2 low Bcl-xL low and showed clear evidence of apoptosis in vivo. Inhibition experiments suggested that an integrin-ligand interaction incorporating the Arg-Gly-Asp motif is involved in fibroblast-mediated synovial T cell survival. We propose that environmental blockade of cell death resulting from interaction with stromal cells is a major factor in the persistent T cell infiltration of chronically inflamed rheumatoid synovium.

Fibroblasts prevent apoptosis of IL-2-deprived T cells without inducing proliferation: a selective effect on Bcl-XL expression.

The apoptosis of human cytokine-deprived activated T cells can be prevented by a soluble mediator secreted by fibroblasts, epithelial and endothelial cells, and this rescue occurs with fibroblasts from different species. Fractionation of W138 fibroblast-conditioned medium indicated that the survival-promoting agent(s) were > 30,000 MW. The continuous presence of the survival factor was required for prevention of apoptosis, which did not involve the induction of proliferation. Nevertheless, the co-cultured T cells remained in a primed state. The expression of the apoptosis-inducing proteins Bax and CD95 (Fas/Apo-1) was either unchanged or slightly increased in fibroblast-rescued T cells, suggesting that constraints on survival still existed after co-culture. A fundamental observation in the present study was that although Bcl-2 was reduced, the levels of Bcl-XL was maintained in cytokine-deprived T cells by fibroblast co-culture. This suggests that fibroblasts and/or other stromal cells may promote activated T-cell survival by a selective effect on Bcl-XL expression, which is consistent with histological examination of activated T cells within lymphoid tissue in vivo. The rescued T cell could be re-activated by CD3 antibody, but only in the presence of CD28 co-stimulation, which induced both Bcl-2 and Bcl-XL expression and also proliferation. Thus, survival signals from stromal cells in tissue microenvironments may enable activated T-cell persistence in a primed but quiescent state, and our data suggest that the regulation of Bcl-XL expression may be central in this process. The further characterization of this process is essential to clarify how signals from stromal cells can influence the resolution and/or chronicity of immune responses in different tissues in vivo.

Resolution of recombination intermediates by a mammalian activity functionally analogous to Escherichia coli RuvC resolvase.

A mammalian endonuclease that resolves Holliday junctions has been partially purified from extracts of calf thymus and Chinese hamster ovary cells. The activity acts upon (i) synthetic Holliday junctions and (ii) recombination intermediates made by the Escherichia coli RecA protein and appears to be functionally analogous to the E. coli RuvC protein. Cleavage occurs by the introduction of symmetrically related nicks in strands of like polarity to produce nicked duplex DNA products. The nicks can be repaired by DNA ligase. The resolvase is specific for Holliday junctions and does not act upon Y junctions, G/A mismatches, or heterologous loops. The substrate specificity is therefore similar to that of E. coli RuvC protein and contrasts with the broad range specificity of other junction resolvases such as T4 endonuclease VII. The mammalian resolvase activity has been observed at normal levels in extracts prepared from a series of DNA repair-defective cells. These include the x-ray or UV-sensitive hamster lines xrs-5, xrs-6, and Chinese hamster ovary 43-3B (defective in ERCC-1), and murine cells that are severely immunodeficient and defective in both V(D)J rejoining and DNA repair.

Conjugal mobility of the multicopy plasmids NTP1 and NTP16.

Mobilization of the multicopy plasmid NTP16, like that of ColE1, is promoted by a range of conjugal plasmids. However, the mechanisms employed for NTP16 mobilization differ between groups. Mobilization by the IncI1 plasmid R64 requires trans-acting products from NTP16 plus a cis-acting region of the small plasmid. In contrast, this system is used inefficiently by the F plasmid and instead, a high-frequency conduction process occurs. Analysis of exconjugant cells reveals that F-mediated mobilization of NTP16 frequently involves rearrangements of NTP16 DNA, promoted by the Tn1000 transposon of F and/or by the kanamycin resistance transposon (Tn4352) of NTP16. Possible mechanisms for the high-frequency F-mediated mobilization of NTP16 are discussed. The plasmid NTP1, which is closely related to NTP16, is also mobilized efficiently by R64. It is not however efficiently mobilized by F, demonstrating the requirement for the Tn4352 element, which is not present in this plasmid, for effective F-mediated transfer.

Acute reversible renal failure secondary to renal candidiasis.

A diabetic patient presenting with bilateral enlarged kidneys and acute renal failure was found to have renal parenchymal candidiasis without obstruction. Treatment with amphotericin B resulted in return of renal function and decrease in renal size.