Actin assembly by lithium ions.

The ability of Li+ to promote the assembly of actin has been compared with the more common cations used in actin assembly assays, K+, Mg2+, and Ca2+. The principal assay of actin assembly utilized was fluorescence photobleaching recovery (FPR), from which it is possible to determine the fraction of actin protomers incorporated into filaments and the average diffusion coefficients of the filaments. In addition, critical concentrations of actin over a range of concentrations of all of these cations have been determined using an assay that involves sonication and dilution of assembled actin filaments containing trace amounts of pyrene-labeled actin. The results demonstrate that Li+ is a more potent promoter of actin assembly than is K+. The more rapid assembly of actin in the presence of Li+ is attributable to an increased rate of filament elongation. Filaments assembled in equivalent concentrations of Li+ or K+ have the same diffusion coefficients, and thus presumably the same average lengths. The critical concentration of actin is about three times less in the presence of Li+ than in the presence of an equal concentration of K+. Cytochalasin D accelerates the rate of Li+-promoted actin assembly and reduces slightly the total fraction of actin assembly. However, cytochalasin D causes less shortening of filaments in the presence of Li+ than in the presence of K+ or Mg2+. By the criteria of assembly kinetics and critical concentration, Li+ is much less potent as a promoter of actin assembly than either Mg2+ or Ca2+. These results are discussed in terms of the role of electrostatic forces in the actin assembly mechanism and in terms of possible relationships to therapeutic and toxicity mechanisms for Li+.     

Discrete signaling regions in the lymphotoxin-beta receptor for tumor necrosis factor receptor-associated factor binding, subcellular localization, and activation of cell death and NF-kappaB pathways

Lymphotoxin-beta receptor (LTbetaR), a member of the tumor necrosis factor receptor superfamily, is essential for the development and organization of secondary lymphoid tissue. Wild type and mutant LTbetaR containing successive truncations of the cytoplasmic domain were investigated by retrovirus-mediated gene transfer into HT29.14s and in 293T cells by transfection. Wild type receptors accumulated in perinuclear compartments and enhanced responsiveness to ligand-induced cell death and ligand-independent activation of NFkappaB p50 dimers. Coimmunoprecipitation and confocal microscopy mapped the TRAF3 binding site to amino acids PEEGDPG at position 389. However, LTbetaR truncated at position Pro(379) acted as a dominant positive mutant that down-modulated surface expression and recruited TRAF3 to endogenous LTbetaR. This mutant exhibited ligand-independent cell death and activated NF-kappaB p50 dimers. By contrast, truncation at Gly(359) created a dominant-negative mutant that inhibited ligand-induced cell death and activation of NF-kappaB p50/p65 heterodimers. This mutant also blocked accumulation of wild type receptor into perinuclear compartments, suggesting subcellular localization may be crucial for signal transduction. A cryptic TRAF-independent NF-kappaB activating region was identified. These mutants define discrete subregions of a novel proline-rich domain that is required for subcellular localization and signal transduction by the LTbetaR.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

Integrated Delivery of Antiretroviral Treatment and Pre-exposure Prophylaxis to HIV-1–Serodiscordant Couples: A Prospective Implementation Study in Kenya and Uganda

BackgroundAntiretroviral-based interventions for HIV-1 prevention, including antiretroviral therapy (ART) to reduce the infectiousness of HIV-1 infected persons and pre-exposure prophylaxis (PrEP) to reduce the susceptibility of HIV-1 uninfected persons, showed high efficacy for HIV-1 protection in randomized clinical trials. We conducted a prospective implementation study to understand the feasibility and effectiveness of these interventions in delivery settings.ConclusionsIntegrated delivery of time-limited PrEP until sustained ART use in African HIV-1-serodiscordant couples was feasible, demonstrated high uptake and adherence, and resulted in near elimination of HIV-1 transmission, with an observed HIV incidence of <0.5% per year compared to an expected incidence of >5% per year.     

Physical heterogeneity of mouse thymus lymphocytes.

Mouse thymocytes have been separated by velocity sedimentation in a density gradient. The resulting fractions have been analyzed using electrophoretic light scattering. The electrophoretic distributions of the individual sedimentation fractions reveal the presence of physically distinct subpopulations. Comparison of the mean mobilities of each fraction indicates that the faster-sedimenting cells tend to have a higher electrophoretic mobility.     

Inhibition of cytotoxic T lymphocyte and natural killer cell-mediated lysis by O,S,S,-trimethyl phosphorodithioate is at an early postrecognition step

O,S,S,-Trimethyl phosphorodithioate (OSS-TMP), an organophosphate esterase inhibitor, has been shown to block the effector phase of the cytolytic reaction mediated by murine and human cytotoxic T lymphocytes (CTL) and human natural killer cells. The murine interleukin 2-dependent CTLL-1 (anti-Iad) clone was used to determine the phase of the cytolytic pathway inhibited by OSS-TMP. Pretreatment of the CTL or target cell with OSS-TMP was not effective at blocking lysis; however, inhibition of lysis was achieved if the reaction was carried out in the continuous presence of OSS-TMP (IC50 = 55 microM) or when CTL-target conjugates were performed and incubated with OSS-TMP (IC50 = 640 microM). Two structural analogues of OSS-TMP were unable to inhibit CTL-mediated lysis. In contrast to OSS-TMP, N-alpha-p-tosyl-L-lysine chloromethylketone required only a 5-min preincubation with the CTL to inhibit lysis. OSS-TMP did not block recognition-adhesion step(s) of the reaction since the ability to form conjugates was not impaired; however, the lytic efficiency of individual CTL-target pairs were blocked. OSS-TMP did not appear to be an inhibitor of the major granule-associated protease that cleaves the substrate, N-alpha-benzyloxycarbonyl-L-lysine thiobenzylester. Ca2+ pulse and kinetic experiments indicated that the OSS-TMP-sensitive site was at a pre-Ca2+-dependent phase but after recognition-adhesion. Human CTL and natural killer cell activity was also inhibited by OSS-TMP, suggesting the presence of a common site of action among these cytolytic systems. The results indicate that OSS-TMP may be a useful reagent in characterizing the early post-recognition events in the cytolytic pathway of CTL and natural killer effector cells.     

Lymphotoxin is expressed as a heteromeric complex with a distinct 33-kDa glycoprotein on the surface of an activated human T cell hybridoma.

We characterized the membrane-associated form of lymphotoxin (surface LT) on the activated II-23.D7 T cell hybridoma. Antibodies to rLT precipitated both surface LT and a distinct 33-kDa glycoprotein (p33). Because p33 and surface LT were antigenically unrelated, their coprecipitation suggested a physical association of p33 and surface LT on the membrane. Pulse-chase analysis indicated that LT and p33 associate with each other early in the LT biosynthetic pathway, precluding the possibility that LT is secreted and bound to p33 or a surface receptor. Furthermore, no p33 was associated with the secreted form of LT. Isoelectric focusing of surface LT and p33 under nondenaturing and denaturing conditions confirmed that surface LT and p33 existed as a complex. Treatment of cells with a high concentration of salt or with acid indicated that surface LT is a peripheral membrane protein. Although secreted LT is a homologous trimer, protein cross-linking studies revealed that surface LT existed as a monomer associated with a dimer of p33. Together the results demonstrate a novel mechanism for stable membrane expression of LT by activated T cells.