Release of the inhibition of messenger RNA translation in extracts of interferon-treated Ehrlich ascites tumor cells by added transfer RNA

In an extract of Ehrlich ascites tumor (EAT) cells which had been “preincubated” for 45 min to lower endogenous protein synthesis (S30_C) the translation of exogenous encephalomyocarditis (EMC) viral mRNA proceeds at a constant rate for over 90 min. In a similarly treated extract of interferon-treated EAT cells (S30_INT) the translation proceeds at a lower rate than in the S30_C for about 30 min and then stops. The impairment of the translation in the S30_INT is mediated by one or more inhibitors. After the cessation of translation the viral mRNA in the S30_INT is in large polysomes. The size of these changes little (if any) during a further 15 min incubation. The addition of mouse tRNA (but not ribosomal RNA or $\text{E. coli}$ tRNA) to the S30_INT after the cessation of viral mRNA translation results in the restart of translation at a rate close to that in the S30_C. This effect of tRNA is diminished by pactamycin, which inhibits peptide chain initiation but not elongation. These results indicate that addition of tRNA allows the elongation of incomplete peptide chains and the initiation of new chains. The need for added tRNA may be due to the fact that in S30_INT the amino acid acceptance of some of the endogenous tRNA species (but not of added tRNAs) is impaired. This impairment is pronounced for leucine and very slight, if any, for five other amino acids tested (i.e. isoleucine, methionine, phenylalanine, threonine, and valine).     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b₅ at the N-terminal of interferon (b₅-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b₅ at the C-terminal of interferon (Met-IFN-b₅-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b₅-chimera), resolved this problem and gave enhanced biological activity.     

Association of a cellular 21-kDa transmembrane protein (VAP21) with enveloped viruses.

We reported previously that the rabies virions contained a 21-kDa cellular transmembrane protein (referred to as VAP21) as a minor component (Sagara, J. et al, Microbiol. Immunol. 41(12): 947-955, 1997). In this study, we further examined the possible interactions of VAP21 with other enveloped viruses, including the vesicular stomatitis virus (VSV; negative-stranded RNA virus), Sindbis virus (positive-stranded RNA virus) and herpes simplex virus type 1 (HSV-1; double-stranded DNA virus). An immunoblot analysis demonstrated that all of these enveloped viruses contained VAP21 in the virion as a minor component. Immunoprecipitation studies suggested that VAP21 was associated with certain viral proteins in the cell, such as the matrix (M) protein of VSV, a capsid protein of Sindbis virus, and at least a capsid protein (VP5) of HSV-1. The association was disrupted by treatment with 0.5% sodium dodecyl sulfate, but resistant to the treatment with 1% NP-40 plus 1% deoxycholate. These results suggest that: 1) VAP21 is not primarily associated with the viral transmembrane glycoprotein but rather with the internal viral protein, and, 2) this association would cause the efficient incorporation of VAP21 into the virion.     

The OSBP-related proteins: a novel protein family involved in vesicle transport,cellular lipid metabolism,and cell signalling

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.     

Denture-related stomatitis managed with tissue conditioner and hard autopolymerising reline material

doi: 10.1111/j.1741–2358.2010.00398.x
Denture related stomatitis managed with tissue conditioner and hard autopolymerising reline material. Objective: To compare the response of denture‐related stomatitis (DS) under management with a tissue conditioner (TC) and autopolymerising hard reline material (AHRM). Background data: Denture‐related stomatitis affects up to 75% of denture wearers; not wearing the denture at night, using TC or prescribing topical or systemic antifungal agents could reduce its incidence. Materials and methods: This was a double‐blind study consisting of 44 participants with DS who wear denture; they were randomly divided into two unmatched groups according to the material used for the management of DS. The TC was replaced weekly, and the AHRM was placed at the beginning of the study and was not changed for 4 weeks. A dentist performed an initial and a weekly clinical diagnosis for DS; the clinical situation was recorded by means of photographs for each week. Results: Both TC and AHRM were effective in the management of DS. Significant differences were found in the DS resolution time (p < 0.001), taking longer for the TC. Conclusions: Both the tissue conditioner and AHRM are effective for the management of DS, but AHRM requires less time for recovery and as a result fewer appointments are required for the patient.     

Autophagy during Early Virus–Host Cell Interactions

Autophagy refers to the conserved, multi-step mechanism that delivers cytosolic cargoes to vesicles of the endo-lysosomal system for degradation. It maintains cellular homeostasis by ensuring the continuous degradation of misformed/senescent intracellular components and the associated recycling of nutrients. Autophagy also represents an important cell-intrinsic defense mechanism against invasion by intracellular pathogens, including viruses. Autophagy might oppose viral invasion by targeting viral particles or viral components for degradation. It can also promote the interaction of viral constituents with receptors specialized in the activation of innate immunity pathways or facilitate the activation of anti-viral adaptive immunity. In response to such pressures, viruses have evolved various sophisticated strategies to avoid anti-viral autophagic responses or to manipulate the autophagic machinery to promote their own replication. This review focuses on our current knowledge of autophagy-related events that take place at early stages during interaction of viruses with host cells as well as on their associated consequences in terms of virus replication and cell fate.     

Molecular Basis for Immunity Protein Recognition of a Type VII Secretion System Exported Antibacterial Toxin

Gram-positive bacteria deploy the type VII secretion system (T7SS) to facilitate interactions between eukaryotic and prokaryotic cells. In recent work, we identified the TelC protein from Streptococcus intermedius as a T7SS-exported lipid II phosphatase that mediates interbacterial competition. TelC exerts toxicity in the inner wall zone of Gram-positive bacteria; however, intercellular intoxication of sister cells does not occur because they express the TipC immunity protein. In the present study, we sought to characterize the molecular basis of self-protection by TipC. Using sub-cellular localization and protease protection assays, we show that TipC is a membrane protein with an N-terminal transmembrane segment and a C-terminal TelC-inhibitory domain that protrudes into the inner wall zone. The 1.9-Å X-ray crystal structure of a non-protective TipC paralogue reveals that the soluble domain of TipC proteins adopts a crescent-shaped fold that is composed of three α-helices and a seven-stranded β-sheet. Subsequent homology-guided mutagenesis demonstrates that a concave surface formed by the predicted β-sheet of TipC is required for both its interaction with TelC and its TelC-inhibitory activity. S. intermedius cells lacking the tipC gene are susceptible to growth inhibition by TelC delivered between cells; however, we find that the growth of this strain is unaffected by endogenous or overexpressed TelC, although the toxin accumulates in culture supernatants. Together, these data indicate that the TelC-inhibitory activity of TipC is only required for intercellularly transferred TelC and that the T7SS apparatus transports TelC across the cell envelope in a single step, bypassing the cellular compartment in which it exerts toxicity en route.     

Cytological changes in oral mucosa in denture stomatitis

The cytological patterns associated with the atrophic and hyperplastic forms of denture stomatitis (DS) were studied in 94 patients with DS and 33 controls. Forty percent of patients with DS and 30% of patients in the control group had a positive culture for Candida. When compared to the smears from the control group patients, the smears from patients with DS presented a higher amount of: i) cytological cellular material; ii) fungal cells; iii) cells of the intermediate and parabasal types; iv) cells of the intermediate type with a positive culture for Candida; and v) polymorphonuclear leukocytes, preferentially in association with a positive culture for Candida. Conversely, smears from the control group showed a higher percentage of cells of the superficial type than those of the patients with DS. Although no specific changes in the DS-affected mucosa have been observed by cytology, we consider that this is a useful, easy and inexpensive technique that gives important information about the inflamed mucosa it can be used in the treatment and control of these patients.     

The molecular weight determination of proteins and glycoproteins of RNA enveloped viruses by polyacrylamide gel electrophoresis in SDS

The apparent molecular weights for glycoproteins of four RNA enveloped viruses — influenza virus, NDV, VSV and AMV, calculated relative to protein standards depend upon the percent of acrylamide used. Such anomaly is not observed for other proteins of these viruses. The irregular behaviour of glycoproteins resulted from their lesser capacity to bind SDS.