Studies on the in vitro uncoating of poliovirus. II. Characteristics of the membrane-modified particle.

Interaction of type I poliovirus with an extract of isolated HeLa cell membranes resulted in a modification of the viral capsid. As a consequence of the modification, the capsid became sensitive to proteases and detergents, and the sedimentation value of the virion was slightly diminished. Treatment of the modified particle with chymotrypsin reduced its sedimentation value and, although the RNA genome was still encapsidated, it was degradable by RNase. Treatment of the membrane-modified particle with detergent (sodium dodecyl sulfate, SDS) disengaged the viral RNA as an intact (i.e., 35 S) molecule. Each of the three serotypes of poliovirus was modified when treated with membrane extract. They differed, however, in the effect of secondary treatment with chymotrypsin, viz., type I was further modified, but types II and III underwent degradation. When secondary treatment was with SDS, all three released 35 S RNA. The above in vitro reactions of the membrane-modified particle are discussed as possible counterparts of the in vivo uncoating phenomenon.     

Inhibition of uncoating of poliovirus by arildone, a new antiviral drug.

The antiviral effects of a new drug, arildone (4-[6-(2-chloro-4-methoxyphenoxy)hexyl]-3,5-heptanedione, on poliovirus type 2 replication and host cell functions are described. Arildone inhibits poliovirus replication at a minimal inhibitory concentration (MIC) of 0.2 μM, while transport of radioactively labeled precursors and synthesis of DNA, RNA, and protein in uninfected HeLa cells are not inhibited. This drug is not virucidal and does not interfere with adsorption or penetration. Arildone inhibits uncoating of poliovirus and thereby prevents virus-induced shutoff of host cell protein synthesis. The possible mechanisms by which arildone interacts with the poliovirus icosahedral capsid to prevent uncoating are discussed.     

Studies on the in vitro uncoating of poliovirus : I. Characterization of the modifying factor and the modifying reaction

A method is described for obtaining a membrane-rich fraction from HeLa cells that can induce a modification in poliovirus characterized by (a) the loss of infectivity and (b) the development of sensitivity of the viral RNA to ribonuclease after treatment with chymotrypsin.Physico-chemical characterization of the modifying factor indicates that it is, or is bound to, a high molecular weight component of the plasma membrane. Modifying activity is lost after exposure to ether, deoxycholate, low pH, trypsin, or brief sonication. Treatment of intact cells with trypsin prior to fractionation, yields a final product devoid of activity. Activity is less readily lost after treatment with alkali. About 50% of the activity is lost after several cycles of freeze-thaw, without further reduction on continued treatment. At 5°, activity gradually diminishes to an undetectable level in about 1 week. At higher temperatures, e.g., 25–45°, activity is lost more rapidly. Neither DNAse nor RNAse has any effect on activity.The modifying reaction proceeds optimally at pH 8. The reaction is readily inhibited by nonionic and ionic compounds. Sucrose and glycerol at a concentration of 5% inhibit almost completely. Ionic compounds at a molarity of 0.05 are strongly inhibitory. On a molar basis, divalent cations are 10-fold more inhibitory than monovalent cations. Kinetic studies indicate that at 25° there is no measurable activity, but between 30 and 37° the reaction has an activation energy of 60 kcal/mole.The modifying factor is active on a wild-type and an attenuated type I poliovirus, but inactive on types II and III viruses.Evidence has been obtained that the membrane extract contains two factors, one with modifying activity, and another that can competitively inhibit the modification reaction.     

Inhibition of poliovirus uncoating by disoxaril (WIN 51711)

Disoxaril [WIN 51711, 5-[7-[4(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3- methylisoxazole] inhibits the replication of polioviruses types 1 and 2 in HeLa cells by stabilizing the virus capsid, which results in the inhibition of the pH-dependent viral uncoating in endosomes and/or lysosomes. As shown by electron microscopy the virus entered into the cell by receptor-mediated endocytosis via coated pits and coated vesicles into endosomes irrespective of the presence or absence of the compound. Measurements of viral RNA synthesis showed that disoxaril completely inhibited the arrival of viral RNA in the cytoplasm for new RNA synthesis only when the inocula were preincubated with disoxaril for 15 min at 37 degrees at 0.3 microgram disoxaril/ml for poliovirus type 1 and 0.03 microgram disoxaril/ml for poliovirus type 2. Simultaneous addition of the compound and virus resulted in reduced inhibition of viral RNA synthesis. The inhibitory effect of the compound could be partially reversed up to 25 min p.i. if the compound was eluted from the cells.     

Burkitt's lymphoma: new insights into molecular pathogenesis

The World Health Organisation classification reports three subcategories of Burkitt's lymphoma (BL)--endemic, non-endemic, and immunodeficiency associated--proposed to reflect the major clinical and genetic subtypes of this disease. These different types of BL have been reviewed and studied by immunohistochemistry and molecular methods. The results point out the heterogeneity of BL and suggest that AIDS related BL may have a different pathogenesis from that of classic BL.     

Childhood asthma: new insights into management.

Recently, a concerted effort has been made to reverse the trend of increasing asthma mortality and morbidity. One additional strategy might be to recognize patients at risk for persistent asthma and to intervene early. This review summarizes new information on asthma pathogenesis that has helped shape a new direction in managing childhood asthma. At the core is the recognition that asthma is a chronic inflammatory disease. Subsequently, inhaled steroids, the most potent anti-inflammatory asthma medications, have emerged as the cornerstone of the management of persistent asthma. The recent report of the National Heart, Lung, and Blood Institute's Childhood Asthma Management Program provides a comprehensive "profile of performance" for 3 treatment choices for the management of persistent asthma. This study answers questions regarding the benefits and shortcomings of the medications evaluated and prompts a closer evaluation of the long-term effects of other treatment strategies, including medications currently being developed. Although intervention with inhaled steroids offers new opportunities to control the development of asthma, one must be cognizant of potential risks in early and long-term therapeutic intervention. This review provides a perspective on our present knowledge, the rationale for early intervention, and opportunities for more aggressive therapy, as well as speculation on how ongoing clinical research will continue to play a role in advancing asthma care and moving toward a "cure" for this life-threatening disease.     

Infections and asthma: new insights into old ideas

Cite this as: C. M. Sevin and R. S. Peebles Jr, Clinical & Experimental Allergy, 2010 (40) 1142–1154. A relationship between infections and allergic airway disease has long been recognized, and many reviews have been written on this topic. However, both clinical and basic science studies published in the last 3 years provide new insights into the relationship between infection and allergic conditions. In this review, we focus on these very recent studies, which address the role of infection in the development, maintenance, and exacerbation of asthma. Bacterial, viral, fungal, and parasitic infections have each been examined and provide a framework for these novel concepts.     

Characterization of intermediates in the uncoating of vaccinia virus DNA

The fate of parental vaccinia virions in mouse L fibroblasts was studied. Four particulate subviral intermediates of uncoating were detected and were partially characterized. SVC (subviral component) 1 had the “mulberry” appearance of virions and possessed the same polypeptide complement, but sedimented slightly slower. SVC2 resembled, but was not identical with, cores prepared in vitro. It possessed 18 of the 30 virion polypeptides, which differed from those present in cores principally by including both glycopolypeptides. Like cores, it exhibited DNA-dependent RNA polymerase activity. Morphologically, it resembled cores surrounded by a layer of surface spikes 100 Å long and 50 Å in diameter, which probably consisted largely of the glycopolypeptides. SVC3 contained very little if any DNA and contained most but not all of the polypeptides also present in SVC2; it probably corresponded to the empty “shell” of SVC2 which remained after DNA had been liberated. SVC4 contained few if any vaccinia polypeptides; its DNA was still partially resistant to deoxyribonuclease. Eventually all vaccinia DNA was converted to a form which was completely digestible by this enzyme.     

RNA silencing in viral infections: insights from poliovirus

RNA interference or RNA silencing is a dsRNA guided mechanism that mediates sequence specific degradation of RNA. The recent demonstration that RNA interference can be used to inhibit virus replication has initiated an exciting field of research: first, as a potential novel antiviral therapeutic approach and, second, as a tool for dissecting virus-host interactions. Here we review and discuss the current data and perspectives on the use of RNA interference in the study of poliovirus as a model for positive strand RNA viruses.     

Growing insights into the safety of bacteriocins: the case of enterocin S37

Very few studies have been reported on the cytotoxicity and impact of bacteriocins, and especially enterocins, upon eukaryotic cells. In order to gain more information on the safety of bacteriocins, we focused this study on enterocin S37, a bacteriocin produced by Enterococcus faecalis S37. We observed dose-dependent cytotoxicity toward undifferentiated Caco-2/TC7 cells. Moreover, no significant effect on differentiated monolayer Caco-2/TC7 and no apoptotic features were observed when cells were treated with 10 μg/ml of enterocin S37. The results obtained indicate possible safe use of enterocin S37 in the gastrointestinal tract of animals to prevent pathogen invasion and/or infection.     

Garlic and aging: new insights into an old remedy

There has been an impressive gain in individual life expectancy with parallel increases in age-related chronic diseases of the cardiovascular, brain and immune systems. These can cause loss of autonomy, dependence and high social costs for individuals and society. It is now accepted that aging and age-related diseases are in part caused by free radical reactions. The arrest of aging and stimulation of rejuvenation of the human body is also being sought. Over the last 20 years the use of herbs and natural products has gained popularity and these are being consumed backed by epidemiological evidence. One such herb is garlic, which has been used throughout the history of civilization for treating a wide variety of ailments associated with aging. The role of garlic in preventing age-related diseases has been investigated extensively over the last 10-15 years. Garlic has strong antioxidant properties and it has been suggested that garlic can prevent cardiovascular disease, inhibit platelet aggregation, thrombus formation, prevent cancer, diseases associated with cerebral aging, arthritis, cataract formation, and rejuvenate skin, improve blood circulation and energy levels. This review provides an insight in to garlic's antioxidant properties and presents evidence that it may either prevent or delay chronic diseases associated with aging.     

Transcranial magnetic stimulation: new insights into representational cortical plasticity

In the last decade, transcranial magnetic stimulation (TMS) has been used increasingly as a tool to explore the mechanisms and consequences of cortical plasticity in the intact human cortex. Because the spatial accuracy of the technique is limited, we refer to this as plasticity at a regional level. Currently, TMS is used to explore regional reorganization in three different ways. First, it can map changes in the pattern of connectivity within and between different cortical areas or their spinal projections. Important examples of this approach can be found in the work on motor cortex representations following a variety of interventions such as immobilization, skill acquisition, or stroke. Second, TMS can be used to investigate the behavioural relevance of these changes. By applying TMS in its "virtual lesion" mode, it is possible to interfere with cortical function and ask whether plastic reorganization within a distinct cortical area improves function. Third, TMS can be used to promote changes in cortical function. This is achieved by using repetitive TMS (rTMS) to induce short-term functional reorganization in the human cortex. The magnitude and the direction of rTMS-induced plasticity depend on extrinsic factors (i.e. the variables of stimulation such as intensity, frequency, and total number of stimuli) and intrinsic factors (i.e. the functional state of the cortex targeted by rTMS). Since conditioning effects of rTMS are not limited to the stimulated cortex but give rise to functional changes in interconnected cortical areas, rTMS is a suitable tool to investigate plasticity within a distributed functional network. Indeed, the lasting effects of rTMS offer new possibilities to study dynamic aspects of the pathophysiology of a variety of diseases and may have therapeutic potential in some neuropsychiatric disorders. Electronic Publication