Electrostatic fasteners hold the T cell receptor-CD3 complex together

Assembly of the T cell receptor includes the formation of trimers stabilized by electrostatic interactions inside the membrane (Call et al., 2003). Such interactions can strongly stabilize subunit associations while permitting conformation changes during signaling.     

Ischemic preconditioning attenuates apoptotic cell death associated with ischemia/reperfusion

Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60, 90 or 120 min of reperfusion. At the end of each experiment, the heart was processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG® in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 90 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were completely abolished by subjecting the myocardium to repeated short-term ischemia and reperfusion which also reduced the ischemic reperfusion injury as evidenced by better recovery of left ventricular performance in the preconditioned myocardium. The results of this study indicate that reperfusion of ischemic heart, but not ischemia, induces apoptotic cell death and DNA fragmentation which can be inhibited by myocardial adaptation to ischemia.     

An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences.

Nonoverlapping deletions that eliminated the 5' (HIV-1US/603del), middle (HIV-1U5/206del), and 3' (HIV-1U5/604del) thirds of the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) were studied for their effects on virus replication (transient transfection of HeLa cells) and infectivity (T-cell lines and peripheral blood mononuclear cells). All three mutants exhibited a wild-type phenotype in directing the production and release of virus particles from transfected HeLa cells. In infectivity assays, HIV-1U5/206del was usually indistinguishable from wild-type virus whereas HIV-1U%/603del was unable to infect human peripheral blood mononuclear cells or MT4 and CEM cells. Investigations of HIV-1U5/603del particles revealed a packaging defect resulting in a 10-fold reduction of encapsidated genomic RNA. The HIV-1U5/604del mutant either was noninfectious or exhibited delayed infection kinetics, depending on the cell type and multiplicity of infection. Quantitative competitive PCR indicated that HIV-1U5/604del synthesized normal amounts of viral DNA in newly infected cells. During the course of a long-term infectivity assay, a revertant of the HIV-1U5/604del mutant that displayed rapid infection kinetics emerged. Nucleotide sequence analysis indicated that the original 26-nucleotide deletion present in HIV-1U5/604del had been extended an additional 19 nucleotides in the revertant virus. Characterization of the HIV-1U5/604del mutant LTR in in vitro integration reactions revealed defective 3' processing and strand transfer activities that were partially restored when the revertant LTR substrate was used, suggesting that the reversion corrected a similar defect in the mutant virus.     

Improved postischemic ventricular functional recovery by amphetamine is linked with its ability to induce heat shock

Heat shock has been shown to increase the cellular tolerances to ischemic injury. In this study, we examined the effects of heat shock induced by amphetamine on postischemic myocardial functional recovery in a setting of coronary revascularization for acute myocardial infarction. Intramuscular injection of amphetamine (3 mg/kg, i.m.) to pigs increased the body temperature to 42.5°C within 1 h, and maintained this temperature for an additional 2 h. Fourty h after the amphetamine injection, the pigs were placed on by cardiopulmonary bypass and then isolated,in situ heart preparations were subjected to 1 h of global hypothermic cardioplegic arrest and 1 h of normothermic reperfusion. Postischemic myocardial performance was monitored by measuring left ventricular (LV) pressure, its dp/dt, myocardial segmental shortening (%SS), and coronary blood flow. Cellular injury was examined by measuring creatine kinase (CK) release. Biochemical measurements included quantification of plasma catecholamines and study of the induction of heat shock gene expression and antioxidative enzymes in the heart tissue. The results of this study indicated significantly greater recovery of LV contractile functions by amphetamine as demonstrated by improved recovery of LVDP (61% vs 52%), dp/dt_max (52% vs 44%), and segmental shortening (46.2% vs 10%). Myocardial CK release was significantly reduced in the amphetamine group. Furthermore, amphetamine pretreatment was associated with the induction of heat shock protein (HSP) 27 mRNA and stimulated Cu/Zn-superoxide dismutase and catalase levels, suggesting that amphetamine mediated improved postischemic ventricular recovery might be linked with its ability to induce heat shock and stimulate antioxidant enzymes.     

Reduced free radical generation during reperfusion of hypothermically arrested hearts

Several studies indicate the presence of hydroxyl radical (OH·) as well as its involvement in the myocardial reperfusion injury. A transition metal-like iron is necessary for the conversion of superoxide anion (O₂ ^–) to a highly reactive and cytotoxic hydroxyl radical (OH·). In the present study, we have examined the generation of OH· and free iron in reperfused hearts following either normothermic (37°C) or hypothermic ischemia (5°C). Employing the Langendorff technique, isolated rat hearts were subjected to global ischemia for 30 min at 37°C or 5°C and were then reperfused for 15 min at 37°C. The results of the study suggest that both the OH· generation in myocardium and free iron release into perfusate were significantly lower in hearts made ischemic at 5°C as compared to 37°C. Release of myoglobin and lactic acid dehydrogenase into perfusate also followed a similar pattern. Furthermore, in in vitro studies, chemically generated O₂ ^– at 5°C caused a significantly lower rate of oxidation of oxymyoglobin as well as generation of OH° and free iron as compared to 37°C. These results suggest that (1) reperfusion of hypothermic ischemic heart is associated with a reduction in the generation of OH· and cellular damage compared to that of normothermic ischemic heart, and (2) myoglobin, an intracellular protein, is a source of free iron and plays a role in the reperfusion injury mediated by free radicals.Abbreviations OH· hydroxyl radical - O₂ ^– superoxide anion - ODFR oxygen-derived free radicals - KHB Krebs-Henseleit buffer - LDH lactate hydrogenase - SOD superoxide dismutase     

Community control strategies for scabies: A cluster randomised noninferiority trial

BackgroundScabies is a neglected tropical disease hyperendemic to many low- and middle-income countries. Scabies can be successfully controlled using mass drug administration (MDA) using 2 doses of ivermectin-based treatment. If effective, a strategy of 1-dose ivermectin-based MDA would have substantial advantages for implementing MDA for scabies at large scale.Methods and findingsWe did a cluster randomised, noninferiority, open-label, 3-group unblinded study comparing the effectiveness of control strategies on community prevalence of scabies at 12 months. All residents from 35 villages on 2 Fijian islands were eligible to participate. Villages were randomised 1:1:1 to 2-dose ivermectin-based MDA (IVM-2), 1-dose ivermectin-based MDA (IVM-1), or screen and treat with topical permethrin 5% for individuals with scabies and their household contacts (SAT). All groups also received diethylcarbamazine and albendazole for lymphatic filariasis control. For IVM-2 and IVM-1, oral ivermectin was dosed at 200 μg/kg and when contraindicated substituted with permethrin. We designated a noninferiority margin of 5%.We enrolled 3,812 participants at baseline (July to November 2017) from the 35 villages with median village size of 108 (range 18 to 298). Age and sex of participants were representative of the population with 51.6% male and median age of 25 years (interquartile range 10 to 47). We enrolled 3,898 at 12 months (July to November 2018). At baseline, scabies prevalence was similar in all groups: IVM-2: 11.7% (95% confidence interval (CI) 8.5 to 16.0); IVM-1: 15.2% (95% CI 9.4 to 23.8); SAT: 13.6% (95% CI 7.9 to 22.4). At 12 months, scabies decreased substantially in all groups: IVM-2: 1.3% (95% CI 0.6 to 2.5); IVM-1: 2.7% (95% CI 1.1 to 6.5); SAT: 1.1% (95% CI 0.6 to 2.0). The risk difference in scabies prevalence at 12 months between the IVM-1 and IVM-2 groups was 1.2% (95% CI −0.2 to 2.7, p = 0.10). Limitations of the study included the method of scabies diagnosis by nonexperts, a lower baseline prevalence than anticipated, and the addition of diethylcarbamazine and albendazole to scabies treatment.ConclusionsAll 3 strategies substantially reduced prevalence. One-dose was noninferior to 2-dose ivermectin-based MDA, as was a screen and treat approach, for community control of scabies. Further trials comparing these approaches in varied settings are warranted to inform global scabies control strategies.Trial registrationClinitrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03177993","term_id":"NCT03177993"}}NCT03177993 and ANZCTR N12617000738325.

In a cluster randomized trial, Myra Hardy and colleagues, compare mass drug administration of one-dose and two-dose ivermectin-based treatment for community control of scabies.     

Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor

Growth factor receptors are typically activated by the binding of soluble ligands to the extracellular domain of the receptor, but certain viral transmembrane proteins can induce growth factor receptor activation by binding to the receptor transmembrane domain. For example, homodimers of the transmembrane 44-amino acid bovine papillomavirus E5 protein bind the transmembrane region of the PDGF beta receptor tyrosine kinase, causing receptor dimerization, phosphorylation, and cell transformation. To determine whether it is possible to select novel biologically active transmembrane proteins that can activate growth factor receptors, we constructed and identified small proteins with random hydrophobic transmembrane domains that can bind and activate the PDGF beta receptor. Remarkably, cell transformation was induced by approximately 10% of the clones in a library in which 15 transmembrane amino acid residues of the E5 protein were replaced with random hydrophobic sequences. The transformation-competent transmembrane proteins formed dimers and stably bound and activated the PDGF beta receptor. Genetic studies demonstrated that the biological activity of the transformation-competent proteins depended on specific interactions with the transmembrane domain of the PDGF beta receptor. A consensus sequence distinct from the wild-type E5 sequence was identified that restored transforming activity to a non-transforming poly-leucine transmembrane sequence, indicating that divergent transmembrane sequence motifs can activate the PDGF beta receptor. Molecular modeling suggested that diverse transforming sequences shared similar protein structure, including the same homodimer interface as the wild-type E5 protein. These experiments have identified novel proteins with transmembrane sequences distinct from the E5 protein that can activate the PDGF beta receptor and transform cells. More generally, this approach may allow the creation and identification of small proteins that modulate the activity of a variety of cellular transmembrane proteins.     

A view of dynamics changes in the molten globule-native folding step by quasielastic neutron scattering

In order to understand the changes in protein dynamics that occur in the final stages of protein folding, we have used neutron scattering to probe the differences between a protein in its folded state and the molten globule states. The internal dynamics of bovine alpha-lactalbumin (BLA) and its molten globules (MBLA) have been examined using incoherent, quasielastic neutron scattering (IQNS). The IQNS results show length scale dependent, pico-second dynamics changes on length scales from 3.3 to 60 A studied. On shorter-length scales, the non-exchangeable protons undergo jump motions over potential barriers, as those involved in side-chain rotamer changes. The mean potential barrier to local jump motions is higher in BLA than in MBLA, as might be expected. On longer length scales, the protons undergo spatially restricted diffusive motions with the diffusive motions being more restricted in BLA than in MBLA. Both BLA and MBLA have similar mean square amplitudes of high frequency motions comparable to the chemical bond vibrational motions. Bond vibrational motions thus do not change significantly upon folding. Interestingly, the quasielastic scattering intensities show pronounced maxima for both BLA and MBLA, suggesting that "clusters" of atoms are moving collectively within the proteins on picosecond time scales. The correlation length, or "the cluster size", of such atom clusters moving collectively is dramatically reduced in the molten globules with the correlation length being 6.9 A in MBLA shorter than that of 18 A in BLA. Such collective motions may be important for the stability of the folded state, and may influence the protein folding pathways from the molten globules.