Further Evidence for Multiple Forms of an N-Methyl-d-Aspartate Recognition Domain in Rat Brain Using Membrane Binding Techniques

Abstract— Pretreatment with sulfhydryl-reactive agents, such as N-ethylmaleimide and p-chloromercuriphenylsul-fonic acid, invariably resulted in marked inhibition of the binding of dl -(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acid ([³H]CGP 39653), a competitive antagonist at an N-methyl-d -aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and treated with Triton X-100, but did not significantly affect the binding of L-[³H]-glutamic acid ([³H]Glu), an endogenous agonist. The pre-treatment was effective in reducing the binding of [³H]-CGP 39653 at equilibrium, without altering the initial association rate, and decreased the affinity for the ligand. Pretreatment with sulfhydryl-reactive agents also enhanced the potencies of NMDA agonists to displace [³H]-CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [³H]Glu binding. The binding of both [³H]CGP 39653 and [³H]Glu was similarly sensitive to pretreatment with four different proteases in Tritontreated membranes, whereas pretreatment with phospho-lipase A₂ or C markedly inhibited [³H]CGP 39653 binding without altering [³H]Glu binding. Moreover, both phospho-lipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [³H]CGP 39653 and [³H]Glu, but also caused diminution of those of NMDA antagonists. These results suggest that both sulfhydryl-reactive agents and phospholipases may predominantly interfere with radiolabeling of the NMDA recognition domain in a state favorable to an antagonist by [³H]CGP 39653, with concomitant facilitation of that in an agonist-preferring form by [³H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.     

Incorporation of the transmembrane hydrophobic domain of glycophorin into small unilamellar phospholipid vesicles Ion Flux studies

Human erythrocyte glycophorin is one of the best characterized integral membrane proteins. Reconstitution of the membrane-spanning hydrophobic segment of glycophorin (the tryptic insoluble peptide released when glycophorin is treated with trypsin) with liposomes results in the production of freeze-fracture intrabilayer particles of 80 Å diameter (Segrest, J.P., Gulik-Krzywicki, T. and Sardet, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3294–3298), with particles appearing at or above a tryptic insoluble peptide concentration of 4 mmol per mol phosphatidylcholine. In the present study, increasing concentrations of tryptic insoluble peptide were added to sonicated small unilamellar egg phosphatidylcholine vesicles and the rate of efflux of ²²Na⁺ was examined by rapid (30 s) gel filtration on Sephadex G-50. Below a concentation of 3–5 mmol tryptic insoluble peptide/mol phosphatidylcholine, ²²Na⁺ efflux occurs at a constant slow rate at given tryptic insoluble peptide concentrations. Above a concentration of 3–5 mM, the rate of efflux is biphasic at given tryptic insoluble peptide concentrations, exhibiting both an initial fast and a subsequent slow component. On the basis of graphic and computer curve-fitting analysis, with increasing tryptic insoluble peptide concentration, the rate of the slow component reaches a plateau at a tryptic insoluble peptide concentration of 3–5 mM and remains essentially constant until much higher concentrations are reached; the fast component increases linearly with increasing tryptic insoluble peptide concentration well beyond 5 mM. The most consistent interpretation of this data is as follows. The slow ²²Na⁺ efflux component is due to perturbations of small unilamellar vesicle integrity by tryptic insoluble peptide monomers. At a tryptic insoluble peptide concentration of 3–5 mmol/mol, a critical concentration is reached following which there is intrabilayer tryptic insoluble peptide self-association. The fast ²²Na⁺ efflux component is due to the increasing presence of tryptic insoluble peptide self-associated multimers the 80-Å particles seen by freeze-fracture electron microscopy) which results in a significantly larger bilayer defect than do tryptic insoluble peptide monomers. The failure of complete saturation of efflux by the fast component is ascribed to the presence of two populations of small unilamellar vesicles, some of which contain tryptic insoluble peptide multimers and some of which do not.Addition of cholesterol to the tryptic insoluble peptide/phosphatidylcholine vesicles decreases the rate of ²²Na⁺ efflux by inhibiting primarily the fast component. Freeze-fracture electron microscopy indicates that the presence of cholesterol has no effect on the size, number or distribution of 80-Å intra-bilayer particles in the tryptic insoluble peptide/phosphatidylcholine vesicles. These results are consistent with a mechanism to explain the fast Na⁺ efflux component involving protein-lipid boundary perturbations.Efflux of ⁴⁵Ca²⁺ from phosphatidylcholine vesicles is also enhanced by incorporation of tryptic insoluble peptide, but only if divalent cations (Ca²⁺ or Mg²⁺) are present in the external bathing media as well as inside the sonicated vesicles. If monovalent Na⁺ only is present in the bathing media no ⁴⁵Ca²⁺ efflux is seen. Under conditions where ⁴⁵Ca²⁺ efflux is seen, both a fast and a slow component are present, although both appear lower than corresponding rate constants for ²²Na⁺ efflux. These results suggest a coordinated mechanism for ion efflux induced by tryptic insoluble peptide and, together with the ²²Na⁺ efflux studies, may have mechanistic implications for the transbilayer phospholipid exchange (flip-flop) suggesed to be induced at glycophorin/phospholipid interfaces (de Kruiff, B., van Zoelen, E.J.J. and van Deenen, L.L.M. (1978) Biochim. Biophys. Acta 509, 537–542).     

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.Subject terms: Cell death, Molecular biology     

Depression compromises antiviral innate immunity via the AVP-AHI1-Tyk2 axis

Depression is a serious public-health issue. Recent reports have suggested higher susceptibility to viral infections in depressive patients. However, how depression affects antiviral innate immune signaling remains unknown. Here, we revealed a reduction in expression of Abelson helper integration site 1 (AHI1) in the peripheral blood mononuclear cells (PBMCs) and macrophages from the patients with major depressive disorder (MDD), which leads to attenuated antiviral immune response. We found that depression-related arginine vasopressin (AVP) induces reduction of AHI1 in macrophages. Further studies demonstrated that AHI1 is a critical stabilizer of basal type-I-interferon (IFN-I) signaling. Mechanistically, AHI1 recruits OTUD1 to deubiquitinate and stabilize Tyk2, while AHI1 reduction downregulates Tyk2 and IFN-I signaling activity in macrophages from both MDD patients and depression model mice. Interestingly, we identified a clinical analgesic meptazinol that effectively stimulates AHI1 expression, thus enhancing IFN-I antiviral defense in depression model mice. Our study promotes the understanding of the signaling mechanisms of depression-mediated antiviral immune dysfunction, and reveals meptazinol as an enhancer of antiviral innate immunity in depressive patients.Subject terms: Innate immunity, Ubiquitylation, Cell signalling     

Rhabdomyolysis happened after the start of dabigatran etexilate treatment: A case report

There are few reports of rhabdomyolysis caused by anticoagulants, and it is extremely rare for it to be caused by dabigatran etexilate. An 86-year-old female experienced sudden muscle weakness and pain, a significant increase in Creatine kinase, and renal impairment after oral administration of dabigatran etexilate for 3 weeks. The enhanced thigh MRI showed abnormal signal in multiple thigh muscle groups, indicating that the lesions should be considered inflammatory diseases. In conclusion, the possibility of rhabdomyolysis should be ruled out when muscle weakness and myalgia occur at the beginning of dabigatran etexilate treatment.     

青藏公路沿线白昼交通运输等人类活动对藏羚羊迁徙的影响

To study effects of traffic during daytime and other human activities, we conducted surveys on the migration of Tibetan antelope Pantholops hodgsoni along the Qinghai-Tibet highway between Kunlun Mountain Pass to Wudaoliang,using line transects (LT) from June to September in 2001 and 2002, Data were collected for 64 days with 17 days in 2001, and 47days in 2002. The Stationary Observation Method (SOM) was also adopted for the investigation of status of the disturbance from traffic when antelopes passing the highway and the traffic situation from August 6 to 28 (6 : 30-19: 00), 4 to 20 (6: 00- 18: 00) and from December 21 to 29 (9：00- 19: 00) in 2002. The results showed that,the period of migration in 2002 was more prolonged than in 2001, and it took at least 45 min for each group to cross the highway, with success ratio of only 30.2 %, and the traffic flow of summer daytime was 76/h, while the winter‘s traffic flow was 29/h. Highway and rail traffic should be managed during daylight hours from June to August, to allow for the migration of the antelopes. Also conservation education and a further survey should be emphasized for the protection on the species [Acta Zoologica Sinica 50 (4) : 669-674, 2004].