Cuidados intraoperatorios en cirugía palpebral / Intraoperative care during eyelid surgery

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Bebé colodión autorresolutivo: nueva mutación en el gen ALOX12B / Self-healing Collodion Baby: A New Mutation in the ALOX12BGene

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Entry of poliovirus into cells is blocked by valinomycin and concanamycin A.

Poliovirus contains a virus particle devoid of a lipid envelope that does not require an intact pH to enter into susceptible cells. Thus, the blockade of pH gradient generated in endosomes is not sufficient to impede the translocation of poliovirus particles to the cytoplasm, suggesting that translocation takes place at the plasma membrane. Measuring both viral protein synthesis and eIF4G-1 cleavage mediated by poliovirus protease 2A has been used to monitor productive entry of poliovirus into cells. Translation of the input poliovirus RNA produces enough 2A(pro) to cleave eIF4G-1, providing a sensitive assay to estimate poliovirus RNA delivery to the cytoplasm followed by its translation. Combination of concanamycin A, a vacuolar proton-ATPase inhibitor, and valinomycin, an ionophore that promotes K(+) efflux from cells, powerfully prevented poliovirus infection. Moreover, modifying the ionic conditions of the culture medium (increasing the concentration of K(+) and decreasing the concentration of Na(+)), together with concanamycin A, also significantly interfered with poliovirus entry. These findings suggest that poliovirus RNA requires an intact concentration of K(+) ions inside the cells to be uncoated and to gain access to the cytoplasm. In addition, the actual contribution of concanamycin A (as well as other inhibitors of endocytosis) to the total inhibition of productive poliovirus entry points to the idea that at least some percentage of polioviral subparticles translocates from the endosomes.

Antipoliovirus flavonoids from Psiadia dentata.

The search for antiviral agents against vesicular stomatitis virus, herpes simplex virus type 1 and poliovirus type 2 in plants extracts, led to the isolation of two antipoliovirus flavonoids from the medicinal plant Psiadia dentata (Cass.) DC, Asteraceae: 3-methylkaempferol and 3,4'-dimethylkaempferol. The antipoliovirus activity of both compounds was estimated by comparison with 3-methylquercetin, guanidine and Ro-090179. The most potent inhibitor of poliovirus replication was 3-methylkaempferol, and therefore we investigated its mechanism of action. We showed, using the inhibition of [3H]uridine incorporation in viral RNA and performing a dot-blot with one RNA probe specific for the poliovirus genomic strand RNA, that 3-methylkaempferol inhibits the genomic RNA synthesis of poliovirus.

Search for antiviral activity in higher plant extracts.

In the course of our search for plant natural products as antiviral agents, extracts of ten plants from the Iberian Peninsula were tested for antiviral activity against herpes simplex type I (HSV-1), vesicular stomatitis virus (VSV) and poliovirus type 1. Aqueous extracts of five of these medicinal plants, namely Nepeta nepetella (150-500 microg/mL), Nepeta coerulea (150-500 microg/mL), Nepeta tuberosa (150-500 microg/mL), Dittrichia viscosa (50-125 microg/mL) and Sanguisorba minor magnolii (50-125 microg/mL), showed a clear antiviral activity against two different DNA and RNA viruses, i.e. HSV-1 and VSV. Only the medicinal plant Dittrichia viscosa was active against an additional virus, poliovirus type 1.

Antiviral activity of Bolivian plant extracts.

Ethanolic and aqueous extracts of seven plant species used in the traditional medicine of Bolivia have been tested for their antiviral activity against herpes simplex type I (HSV-1), vesicular stomatitis virus (VSV), and poliovirus type 1. The aqueous extracts of most of the species investigated showed antiviral activity. Two of these plants-namely, Satureja boliviana and Baccharis genistelloides-were active against two different viruses-HSV-1 and VSV.

Poliovirus protein 2BC increases cytosolic free calcium concentrations.

Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis.

Photoperiod-temperature and neuroblast proliferation-migration in the adult lizard cortex.

The lizard medial cortex (a zone homologous to the mammalian fascia dentata) shows delayed postnatal neurogenesis throughout the lifetime of these animals. Experimental lesioning of this area is followed by neuronal regeneration, a unique phenomenon in the adult amniote telencephalon. The differential effects of temperature and photoperiod on postnatal neurogenetic activity were studied using tritiated thymidine pulses and posterior autoradiography as well as proliferating cell nuclear antigen (PCNA) immunostaining. Long (summer) photoperiods increased the number of proliferating neuroblasts in the ependymal neuroepithelium. Cold (winter) temperature prevented migration of the newly generated immature neurones.

Entry of Semliki forest virus into cells: effects of concanamycin A and nigericin on viral membrane fusion and infection.

Semliki forest virus (SFV) was biosynthetically labeled with pyrene phospholipids and used to investigate two alternative routes of entry of SFV into BHK-21 cells: (1) receptor-mediated endocytosis followed by fusion of the viral envelope with the endosomal membrane and (2) direct fusion of SFV with the plasma membrane induced by low pH treatment. The selective inhibitor of the vacuolar proton-ATPase, concanamycin A, abolished fusion and subsequent infection only when the virus utilized the endocytic route to enter cells. The inhibitory effect of this macrolide antibiotic was bypassed by low pH treatment of cells. However, the ionophore nigericin was inhibitory irrespective of the route used by the virus to infect cells, suggesting the necessity of a transmembrane pH gradient for the entry process. According to our results, concanamycin A emerges as a suitable tool for selectively investigating the involvement of endosomal function in animal virus entry.

Monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage.

Addition of monensin or nigericin after poliovirus entry into HeLa cells prevents the inhibition of host protein synthesis by poliovirus. The infected cells continue to synthesize cellular proteins at control levels for at least 8 h after infection in the presence of the ionophore. Cleavage of p220 (gamma subunit of eukaryotic initiation factor 4 [eIF-4 gamma]), a component of the translation initiation factor eIF-4F, occurs to the same extent in poliovirus-infected cells whether or not they are treated with monensin. Two hours after infection there is no detectable intact p220, but the cells continue to translate cellular mRNAs for several hours at levels similar to those in uninfected cells. Nigericin or monensin prevented the arrest of host translation at all the multiplicities of poliovirus infection tested. At high multiplicities of infection, an unprecedented situation was found: cells synthesized poliovirus and cellular proteins simultaneously. Superinfection of vesicular stomatitis virus-infected HeLa cells with poliovirus led to a profound inhibition of vesicular stomatitis virus protein synthesis, while nigericin partially prevented this blockade. Drastic inhibition of translation also took place in influenza virus-infected Vero cells treated with nigericin and infected with poliovirus. These findings suggest that the translation of newly synthesized mRNAs is dependent on the integrity of p220, while ongoing cellular protein synthesis does not require an intact p220. The target of ionophore action during the poliovirus life cycle was also investigated. Addition of nigericin at any time postinfection profoundly blocked the synthesis of virus RNA, whereas viral protein synthesis was not affected if nigericin was added at 4 h postinfection. These results agree well with previous findings indicating that inhibitors of phospholipid synthesis or vesicular traffic interfere with poliovirus genome replication. Therefore, the action of nigericin on the vesicular system may affect poliovirus RNA synthesis. In conclusion, monensin and nigericin are potent inhibitors of poliovirus genome replication that prevent the shutoff of host translation by poliovirus while still permitting cleavage of p220.

Enhanced intracellular calcium concentration during poliovirus infection.

The infection of human fibroblasts by poliovirus leads to a notable increase in the intracellular calcium concentration, [Ca2+]i, measured by microfluorimetry or by flow cytometry. [Ca2+]i increases from 2 to 3 h postinfection, and by the fifth hour there is a 5- to 10-fold increase in [Ca2+]i. At this time postinfection there is active viral protein synthesis. The modifications in [Ca2+]i are not observed in the presence of cycloheximide, guanidine, or Ro 09-0179, indicating that virus gene expression is required for the increase in [Ca2+]i. Attempts to identify the source of the intracellular Ca2+ by using different inhibitors of calcium fluxes suggest that calcium enters from the culture medium through voltage-sensitive calcium channels.

Action of brefeldin A on translation in Semliki Forest virus-infected HeLa cells and cells doubly infected with poliovirus.

Brefeldin A (BFA) is a macrolide antibiotic that blocks membrane traffic through the vesicular system and affects the glycosylation of viral glycoproteins. Treatment of HeLa cells infected with Semliki Forest virus (SFV) with BFA enhances the synthesis of late viral proteins. Proteolytic cleavage of p107 is partially blocked and viral glycoproteins accumulate in BFA-treated cells. This enhanced synthesis of late SFV proteins is due, at least in part, to an increase in the formation of the subgenomic SFV 26S mRNA. Since BFA blocks the replication of poliovirus genomes without affecting the cleavage of the translation initiation factor p220, protein synthesis was analysed in doubly infected cells. HeLa cells infected with SFV and poliovirus at the same multiplicity predominantly synthesize poliovirus proteins. But if these cells are treated with BFA they synthesize significant amounts of SFV capsid protein C for several hours, despite the fact that p220 has been degraded.

Brefeldin A blocks protein glycosylation and RNA replication of vesicular stomatitis virus.

Brefeldin A is a macrolide antibiotic that interferes with membrane traffic and blocks the growth of several animal viruses including vesicular stomatitis virus (VSV). The inhibition of VSV by brefeldin A takes place at least at two different steps during the growth cycle: the glycosylation of VSV G protein and the replication of viral genomes. Our results indicate that interference with membrane traffic leads not only to inhibition of viral protein glycosylation, but also to the blockade of virus genome replication in several cytoplasmic RNA-containing viruses.

Enhancement of phospholipase activity during poliovirus infection.

Infection of human cells with poliovirus leads to modification of phospholipase activity. Phospholipase C, which generates inositol triphosphate, is stimulated, whereas the activation of phospholipase A2 by the calcium ionophore A23187 is inhibited. Analysis of phospholipid moieties in media of HeLa cells infected with poliovirus indicates that the release of fatty acids is not enhanced during infection, suggesting that phospholipase A1 and A2 activities are not stimulated. The release of choline into the medium is significantly higher 3 h after infection, indicating that a phospholipase that has phosphatidylcholine as its substrate becomes activated. This activation requires viral gene expression because inhibitors of poliovirus gene expression added at the beginning of infection block choline release, but continuous viral protein synthesis is not required. Choline and phosphorylcholine are released into the medium, but the pools of both are gradually depleted in poliovirus-infected cells, perhaps as a consequence of their release into the medium and the increased synthesis of phospholipids that takes place in poliovirus-infected cells. Inhibitors of phospholipase activity such as mepacrine, zinc or cadmium ions significantly reduce this increased release of choline from poliovirus-infected cells. Labelling of cells with [3H]phosphatidylcholine suggests that the choline released from infected cells comes, at least in part, from the hydrolysis of this compound. These results indicate that, in addition to the activation of the phospholipase C which hydrolyses phosphatidylinositol in poliovirus-infected cells, a phospholipase C that acts on a phosphatidylcholine is also activated.

Activation of phospholipase activity during Semliki Forest virus infection.

Infection of animal cells by a number of cytolytic viruses leads to increased membrane permeability. Thus, Semliki Forest virus (SFV) infection of susceptible cells modifies the permeability of the membrane for a number of cations and metabolites (Muñoz et al. (1985), Virology 146, 203-212). The molecular basis of this modification of the cell membrane has not been investigated in detail. We report that during the infection of HeLa cells with SFV, or BHK cells with vesicular stomatitis virus, there is a significant increase in the release of choline and arachidonic acid into the culture medium, suggesting that both phospholipases (PLases) C and A2 become activated during infection. Both choline and phosphorylcholine are released into the medium as expected when PLase C is activated. Cells prelabeled with arachidonic acid release a significant amount of radioactivity from the third hour postinfection. Most of this radioactivity is present in the medium of SFV-infected cells in the form of free fatty acid, suggesting that phospholipid hydrolysis has occurred; no intact phospholipids are detected in the culture medium. Finally, the action of several inhibitors of PLases, such as zinc and cadmium ions, chloroquine, chlorpromazine, amantadine, and dansylcadaverine were assayed. Our findings indicate that the release of choline or arachidonic acid is potently blocked by some of these lipase inhibitors. Following infection by SFV HeLa cells become susceptible to the inhibition of protein synthesis by hygromycin B due to increased uptake of this antibiotic. Entry of hygromycin B was prevented by zinc ions or chloroquine, suggesting that the increase in membrane permeability in SFV-infected cells may be mediated in part by lipase activation.

Involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin A.

Brefeldin A (BFA) is a macrolide antibiotic that has multiple targets in vesicular transport and blocks membrane traffic between the cis- and trans-Golgi compartments, leading to the disruption of the trans-Golgi apparatus (for a review see Pelham, 1991, Cell 67, 449-451). Consequently, BFA interferes with the maturation of viral glycoproteins and suppresses the formation of infectious viruses that contain a lipid envelope. We report that this antibiotic strongly inhibits poliovirus replication even though this virus lacks a lipid envelope and does not encode any glycoproteins. Addition of BFA from the beginning of poliovirus infection blocks the synthesis of late proteins but has no effect on p220 cleavage, indicating that the input viral RNA is translated to produce active 2Apro. The presence of BFA at later times has no effect on poliovirus protein synthesis, indicating that this step is not a direct target for the antibiotic. Indeed, the target of BFA is viral RNA synthesis, because addition of the antibiotic at any time after poliovirus infection drastically reduces the incorporation of labeled uridine into poliovirus RNA. Both plus- and minus-stranded RNA syntheses are diminished when BFA is present from the beginning of infection, but plus-stranded RNA synthesis is more affected when the inhibitor is added at later times. The replication of poliovirus RNA takes place in close association with membrane vesicles that fill the cytoplasm of the infected cells. Little is known about the origin and function of these vesicles that form part of the viral replication complexes. Our findings suggest that the replication of poliovirus genomes may require the maturation of membranous vesicles from a vesicular compartment that is affected by BFA. The effects of BFA on late protein synthesis by other animal viruses varies according to the virus species examined. Among picornaviruses, rhinoviruses are sensitive to the antibiotic, whereas encephalomyocarditis virus is resistant. A negative-stranded RNA virus such as vesicular stomatitis is blocked by BFA, whereas vaccinia virus, a cytoplasmic DNA virus, is resistant.

Contractile response of human omental arteries to endothelin.

The effects of endothelin have been studied in isolated arterial segments (0.8-1 mm in external diam.) of human omental arteries obtained during the course of abdominal operations (15 patients, 7 men and 8 women). Paired segments, one normal and the other de-endothelized, were mounted for isometric recording of tension in organ baths. Endothelin produced concentration-dependent contractions with an EC50 value of 5.4 x 10(-9) M. Removal of endothelium did not affect significantly endothelin-induced contractions (EC50, 6.7 x 10(-9) M). Removal of extracellular calcium or addition of the calcium channel blocker nicardipine (10(-6) M) diminished but did not abolish responses to endothelin. These results indicate that endothelin exerts powerful contractile effects on human isolated omental arteries which are independent of the presence of an intact endothelial cell layer; this contraction cannot be explained solely by voltage-dependent calcium channels.