Theiler's virus-induced intrinsic apoptosis in M1-D macrophages is Bax mediated and restricts virus infectivity: a mechanism for persistence of a cytolytic virus

Theiler's murine encephalomyelitis virus (TMEV), a member of the Cardiovirus genus in the family Picornaviridae, is a highly cytolytic virus that produces necrotic death in rodent cells except for macrophages, which undergo apoptosis. In the present study we have analyzed the kinetics of BeAn virus infection in M1-D cells, in order to temporally relate virus replication to the apoptotic signaling events. Apoptosis was associated with early exponential virus growth from 1 to 12 h postinfection (p.i.); however, >/=80% of peak infectivity was lost by 16 to 24 h p.i. The pan-caspase inhibitor qVD-OPh led to significantly higher virus yields, while zVAD-fmk completely inhibited virus replication until 10 h p.i., precluding its assessment in apoptosis. In contrast, while zVAD-fmk significantly inhibited BeAn virus replication in BHK-21 cells at 12 and 16 h p.i., virus replication at these time points was not altered by qVD-OPh. Bax translocation into mitochondria, efflux of cytochrome c into the cytoplasm, and activation of caspases 9 and 3 between approximately 8 and 12 h p.i. (all hallmarks of the intrinsic apoptotic pathway) were transiently inhibited by expression of Bcl-2, which is not expressed in M1-D cells. Thus, BeAn virus infection in M1-D macrophages, which restricts virus replication, provides a potential mechanism for modulating TMEV neurovirulence during persistence in the mouse central nervous system.     

Enhanced release of sphingosine-1-phosphate from hypercholesterolemic platelets: role in development of hypercholesterolemic atherosclerosis

Although it is well known that sphingosine-1-phosphate (S1P), which induces many biological responses, is present in plasma and is mainly released from activated platelets, little is known whether the release of S1P is increased when platelets are activated in the hypercholesterolemic condition, and what are the roles of increased S1P generation in the development or progression of the atherosclerosis. Results show that 0.5% cholesterol diet for 16 weeks induces platelet hyperaggregability to low doses of agonists as well as development of hypercholesterolemic atherosclerosis in the rabbits. The generation and released level of S1P were significantly increased in the hypersensitized platelets and blood plasma in hypercholesterolemic rabbits. We also demonstrated that S1P increased VSMC proliferation via endothelial differentiation gene (EDG)-1 receptor dependent pathway. Our results indicate that release of S1P from activated platelets was increased by enhanced platelet sensitivity in hypercholesterolemia, which potentiated the ox-LDL-induced VSMC proliferation via EDG-1 receptor pathway.     

Tanshinone IIA from Salvia miltiorrhiza BUNGE inhibits human aortic smooth muscle cell migration and MMP-9 activity through AKT signaling pathway

Smooth muscle cell (SMC) migration plays an important role in normal angiogenesis and is relevant to disease-related vascular remodeling in conditions such as brain arteriovenous malformations, pulmonary hypertension, arteriosclerosis, and restenosis after angioplasty. In this present study, we showed that tanshinone IIA, the major lipid-soluble pharmacological constituent of Salvia miltiorrhiza BUNGE, inhibits human aortic smooth muscle cell (HASMC) migration and MMP-9 activity. Tanshinone IIA significantly inhibited IkappaBalpha phosphorylation and p65 nuclear translocation through inhibition of AKT phosphorylation. Tanshinone IIA inhibited TNF-alpha-induced ERK and c-jun phosphorylation, but not other MAPKs such as JNK and p38. Tanshinone IIA also inhibited NF-kappaB and AP-1 DNA-binding. Moreover, tanshinone IIA inhibited the migration of TNF-alpha-induced HASMCs. Our results provide evidence that tanshinone IIA has multiple effects in the inhibition of HASMC migration and may offer a therapeutic approach to block HASMC migration.     

Effects of beta-mercaptoethanol and hydrogen peroxide on enzymatic conversion of human proinsulin to insulin

Human insulin is a hormone well-known to regulate the blood glucose level. Recombinant preproinsulin, a precursor of authentic insulin, is typically produced in E. coli as an inactive inclusion body, the solubilization of which needs the addition of reducing agents such as beta-mercaptoethanol. To make authentic insulin, recombinant preproinsulin is modified enzymatically by trypsin and carboxypeptidase B. The effects of beta-mercaptoethanol on the formation of human insulin derivatives were investigated in the enzymatic modification by using commercially available human proinsulin as a substrate. Addition of 1 mM beta-mercaptoethanol induced the formation of various insulin derivatives. Among them, the second major one, impurity 3, was found to be identical to the insulin B chain fragment from Phe1 to Glu21. Minimization of the formation of insulin derivatives and concomitant improvement of the production yield of human insulin were achieved by the addition of hydrogen peroxide. Hydrogen peroxide bound with beta-mercaptoethanol and thereby reduced the negative effects of beta-mercaptoethanol considerably. Elimination of the impurity 3 and other derivatives by the addition of over 10 mM hydrogen peroxide in the presence of beta-mercaptoethanol led to a 1.3-fold increase in the recovery efficiency of insulin, compared with those for the case without hydrogen peroxide. The positive effects of hydrogen peroxide were also confirmed with recombinant human preproinsulin expressed in recombinant E. coli as an inclusion body.     

Autonomic control of the respiratory-hemodynamic system in 8-to 11-year-old children with different baroreflex sensitivities

The efficiency of baroreflex control depends on the baroreflex sensitivity (BRS), which is defined as the ratio of the change in the heart rate (HR) to the change in the blood pressure (BP). The BRS value may be used for assessing the autonomic control of the cardiovascular system and the degree of autonomic dysfunction. Until recently, the baroreflex had not been assessed in a large population of healthy subjects. In this study, the BRS was estimated by the ratio of the low-frequency component of the HR spectrum and the low-frequency component of the rhythm of the systolic BP. For assessing the arterial baroreflex in children, the BRSs for spontaneous and induced baroreflexes were compared. Sex-and age-related differences in BRS were found in 8-to-11-year-old children, and correlations between BRS and some spectral components of HR variability (HRV) and BP rhythm variability were determined. Cluster analysis of the BRS calculated for the spontaneous baroreflex at rest was used to distinguish three clusters of subjects (with high, medium, and low BRSs). These clusters differed in the variability of the basic parameter and size and showed sex-related differences.     

Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes

This study examines whether fluid pressure (FP) modulates the L-type Ca²⁺ channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (16 dyn/cm²) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca²⁺ current (I_Ca) and cytosolic Ca²⁺ signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed I_Ca (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of I_Ca. The level of I_Ca suppression by FP depended on the level and duration of pressure. The Ba²⁺ current through the Ca²⁺ channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca²⁺ channel during FP stimulation. The cytosolic Ca²⁺ transients and the basal Ca²⁺ in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the I_Ca and on the Ca²⁺ transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca²⁺ buffer, eliminated the FP-induced acceleration of I_Ca inactivation and reduced the inhibitory effect of the FP on I_Ca by 80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca²⁺ release, eliminated the accelerating effect of FP on the I_Ca inactivation, and they reduced the inhibitory effect of FP on the I_Ca. These results suggest that the fluid pressure indirectly suppresses the Ca²⁺ channel by enhancing the Ca²⁺-induced intracellular Ca²⁺ release in rat ventricular myocytes. L-type Ca²⁺ current; fluid pressure; ventricular myocytes; cytosolic Ca²⁺ transient     

Seedling Growth and Heavy Metal Accumulation of Candidate Woody Species for Revegetating Korean Mine Spoils

Selecting plant species that can overcome harsh soil and microclimatic conditions and speed the recovery of degraded minelands remains a worldwide restoration challenge. This study evaluated the potential of three woody species and various organic and inorganic fertilization treatments for revegetating abandoned metalliferous mines in Korea. We compared survival, growth, and heavy metal uptake of species common to Korean minelands in two spoil types and a reference forest soil. Substrate type and fertilization both influenced seedling growth and metal concentrations substantially, but they had little effect on seedling survival. Fertilization increased the growth of all three species when grown in mine spoils but influenced the growth of seedlings grown in forest soil only marginally. Initial seedling survival and growth indicate that the study species can tolerate the heavy metal concentrations and other soil constraints of metalliferous spoil types. We estimate that plants can stabilize 2–22% of various heavy metals contained in spoil materials into plant biomass during 20 years of plantation growth. Combined with the erosion control and site amelioration benefits of mineland reforestation, stabilization of heavy metals in forest biomass justifies this treatment on abandoned Korean metalliferous mines.     

The SARS-CoV-2 RNA interactome

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Characterization of an oviposition stimulant from the surface of sweet potato Ipomoea batatas storage roots for the sweet potato weevil, Cylas formicarius elegantulus

An improved laboratory bioassay was used to characterize an oviposition stimulant from the surface of sweet potato Ipomoea batatas (L.) Lam. storage roots for the sweetpotato weevil, Cylas formicarius elegantulus (Summers). Filter paper discs impregnated with a methylene chloride surface extract of sweet potato storage roots induced significantly (p<0.05) higher oviposition on root cores than those treated with solvent only. Significantly higher oviposition was also observed in the nonpolar fractions, especially one that contains a tentatively identified triterpenoid present in susceptible cultivars.

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Caractérisation d'une substance extraite de la surface des racines tubéreuses d'Ipomoea batatas induisant la ponte de Cylas formicarius elegantulus

Résumé La caractérisation des substances extraites de la surface des racines tubéreuses d'I. batatas Lam et stimulant la ponte de C. formicarious elegantulus Summers, a été effectuée à partir d'une technique améliorée. Différents substrats ont été essayés: extraits au chlorure de méthylène ou péridermes intacts de cultivars résistants ou sensibles, racines tubéreuses après élimination du périderme, morceaux de Solanum tuberosum ou de papier filtre présentés au milieu de plaques à 24 plots pour culture de tissus. Le meilleur substrat s'est révélé être de petits disques de papier filtre (diamètre 0,4 cm), fixés au milieu d'un morceau de racine avec encore un peu de périderme sur lequel le coléoptère pouvait pondre. Cette méthode a été utilisée dans les tests ultérieurs pour caractériser la nature du stimulant.Des disques de papier filtre inhibés d'extraits au chlorure de méthylène du contenu des structures superficielles de racines tubéreuses de cultivars sensibles induisent une ponte significativement plus importante (p<0,05) sur les morceaux de racines par comparaison avec ceux traités au chlorure de méthylène pur. Une ponte significativement plus importante a aussi été obtenue avec des fractions non-polaires, particulièrement celles qui contiennent un triterpènoïde de cultivars dont l'identification a été tentée.