Replication of adenovirus type 7 in monkey cells: a new determinant and its transfer to adenovirus type 2.

A strain of human adenovirus type 7, adapted to replication in green-monkey kidney cells, requires the interaction of two particles to initiate plaque formation in the simian cells. One particle is a true adenovirion. The second, apparently defective, consists of a genome carrying amonkey-adapting component in an adenovirus capsid; this genome does not express known SV40 determinants. The addition of human adenovirus type 7 that is not adapted enhances the titer and changesconditions for plaque formation by the adapted virus to a one-particle requirement. Addition of nonadapted human adenovirus type 2 as helper virus results in the transfer of the monkey-adaptingcomponent from adenovirus type 7 to adenovirus type 2. The population containing the adenovirus 2 transcapsidant then has the ability to replicate in simian cells.

Limited brain diffusion of the glucocorticoid receptor agonist RU28362 following i.c.v. administration: implications for i.c.v. drug delivery and glucocorticoid negative feedback in the hypothalamic-pituitary-adrenal axis.

The experiments described herein present a method for tracking diffusion of the glucocorticoid receptor agonist RU28362 in brain following i.c.v. drug administration. A useful property of glucocorticoid receptor is that it is primarily cytoplasmic when unbound and rapidly translocates to the nucleus when bound by ligand. Thus, removal of endogenous glucocorticoids by adrenalectomy allows us to identify brain regions with activated glucocorticoid receptor after i.c.v. glucocorticoid receptor agonist treatment by examining the presence or absence of nuclear glucocorticoid receptor immunostaining. We have previously demonstrated that an i.p. injection of 150 microg/kg RU28362 1 h prior to restraint stress is sufficient to suppress stress-induced hypothalamic-pituitary-adrenal axis hormone secretion [Ginsberg AB, Campeau S, Day HE, Spencer RL (2003) Acute glucocorticoid pretreatment suppresses stress-induced hypothalamic-pituitary-adrenal axis hormone secretion and expression of corticotropin-releasing hormone hnRNA but does not affect c-fos mRNA or fos protein expression in the paraventricular nucleus of the hypothalamus. J Neuroendocrinol 15:1075-1083]. We report here, however, that in rats i.c.v. treatment with a high-dose of RU28362 (1 microg) 1 h prior to stressor onset does not suppress stress-induced hypothalamic-pituitary-adrenal axis activity. We then performed a series of experiments to examine the possible differences in glucocorticoid receptor activation patterns in brain and pituitary after i.c.v. or i.p. treatment with RU28362. In a dose-response study we found that 1 h after i.c.v. injection of RU28362 (0.001, 0.1 and 1.0 microg) glucocorticoid receptor nuclear immunoreactivity was only evident in brain tissue immediately adjacent to the lateral or third ventricle, including the medial but not more lateral portion of the medial parvocellular paraventricular nucleus of the hypothalamus. In contrast, i.p. injection of RU28362 produced a uniform predominantly nuclear glucocorticoid receptor immunostaining pattern throughout all brain tissue. I.c.v. injection of the endogenous glucocorticoid receptor agonist, corticosterone (1 microg) also had limited diffusion into brain tissue. Time-course studies indicated that there was not a greater extent of nuclear glucocorticoid receptor immunostaining present in brain after shorter (10 or 30 min) or longer (2 or 3 h) intervals of time after i.c.v. RU28362 injection. Importantly, time-course studies found that i.c.v. RU28362 produced significant increases in nuclear glucocorticoid receptor immunostaining in the anterior pituitary that were evident within 10 min after injection and maximal after 1 h. These studies support an extensive literature indicating that drugs have very limited ability to diffuse out of the ventricles into brain tissue after i.c.v. injection, while at the same time reaching peripheral tissue sites. In addition, these studies indicate that significant occupancy of some glucocorticoid receptor within the paraventricular nucleus of the hypothalamus and pituitary is not necessarily sufficient to suppress stress-induced hypothalamic-pituitary-adrenal axis activity.

Habituation to repeated restraint stress is associated with lack of stress-induced c-fos expression in primary sensory processing areas of the rat brain.

Rats repeatedly exposed to restraint show a reduced hypothalamic-pituitary-adrenal axis response upon restraint re-exposure. This hypothalamic-pituitary-adrenal axis response habituation to restraint does not generalize to other novel stressors and is associated with a decrease in stress-induced c-fos expression in a number of stress-reactive brain regions. We examined whether habituation to repeated restraint is also associated with adaptation of immediate early gene expression in brain regions that process and relay primary sensory information. These brain regions may not be expected to show gene expression adaptation to repeated restraint because of their necessary role in experience discrimination. Rats were divided into a repeated restraint group (five 1-hour daily restraint sessions) and an unstressed group (restraint naïve). On the sixth day rats from each group were either killed with no additional stress experience or at 15, 30 or 60 min during restraint. Immediate early gene expression (corticotrophin-releasing hormone heteronuclear RNA, c-fos mRNA, zif268 mRNA) was determined by in situ hybridization. A reduction in stress-induced hypothalamic-pituitary-adrenal axis hormone secretion (plasma corticosterone and adrenocorticotropic hormone) and immediate early gene expression levels in the paraventricular nucleus of the hypothalamus, the lateral septum and the orbital cortex was observed in repeated restraint as compared with restraint naïve animals. This reduction was already evident at 15 min of restraint. Unexpectedly, we also found in repeated restraint rats a reduction in restraint-induced c-fos expression in primary sensory-processing brain areas (primary somatosensory cortex, and ventroposteriomedial and dorsolateral geniculate nuclei of thalamus). The overall levels of hippocampal mineralocorticoid receptor heteronuclear RNA or glucocorticoid receptor mRNA were not decreased by repeated restraint, as may occur in response to severe chronic stress. We propose that repeated restraint leads to a systems-level adaptation whereby re-exposure to restraint elicits a rapid inhibitory modulation of primary sensory processing (i.e. sensory gating), thereby producing a widespread attenuation of the neural response to restraint.

Specific and time-dependent effects of glucocorticoid receptor agonist RU28362 on stress-induced pro-opiomelanocortin hnRNA, c-fos mRNA and zif268 mRNA in the pituitary.

This study examined the effects of the glucocorticoid receptor (GR) agonist RU28362 on stress-induced gene expression in the pituitary of rats to investigate mechanisms of glucocorticoid negative feedback in vivo. In an initial experiment, acute restraint stress produced rapid (within 15 min) induction of c-fos mRNA, zif268 mRNA and pro-opiomelanocortin (POMC) hnRNA within the anterior and intermediate/posterior pituitary as determined by quantitative real-time polymerase chain reaction. Treatment with RU28362 (150 microg/kg, i.p.) 60 min before restraint inhibited adrenocorticotrophic hormone (ACTH) and corticosterone secretion and selectively suppressed the stress-induced increase in POMC hnRNA in the anterior pituitary gland. The failure of RU28362 to surpress the stress-induced rise in c-fos and expression of zif268 mRNA suggests that the central release of ACTH secretagogues was not affected at this time point by treatment with the GR agonist. Rather, the inhibition of ACTH release appeared to be due to a direct effect of RU28362 within the pituitary. A follow-up time-course study varied the interval (10, 60 or 180 min) between RU28362 pretreatment and the onset of restraint. The stress-induced increase in POMC hnRNA was completely blunted by RU28362 treatment within 10 min of treatment, although the stress induced hormone secretion, c-fos mRNA and zif268 mRNA were unaffected. The rapid inhibition of the stress-induced rise in POMC hnRNA in the anterior pituitary appears to reflect direct, GR-mediated suppression of POMC gene expression. RU28362 pretreatment 180 min before restraint onset was sufficient to suppress the stress-induced expression in the anterior pituitary gland of all three genes examined. Thus, the delayed negative feedback effects on hypothalamic-pituitary-adrenal axis activity that emerged after 180 min after glucocorticoid treatment were not evident at 60 min. Taken together, the data suggest that the inhibition of the stress-induced release of ACTH apparent within the first hour of glucocorticoid exposure is effected at the level of the pituitary gland. The delayed glucocorticoid effects evident 180 min after RU28362 treatment may include glucocorticoid actions in the brain and additional actions within the pituitary.

Experimental neuritis induced by a mixture of neural antigens and influenza vaccines. A possible model for Guillain-Barré syndrome.

We describe the development in rats of a possible model for Guillain-Barré syndrome (GBS): experimental neuritis (EN). The clinical symptoms, histopathology and the presence of antibody to nervous tissue are features that EN has in common with both GBS and experimental allergic neuritis (EAN), another GBS model. However, EN may be a more appropriate model than EAN for studying the role of autoimmune reactions in diseases such as GBS, which are triggered by various viruses or antigens, since EN depends on such agents being administered concomitantly with the syngeneic tissue.

[Comparative study of the antiviral properties of histones of animal and plant origin]. / Sravnitel'noe izuchenie antivirusnykh svoistv gistonov zhivotnogo i rastitel'nogo proiskhozhdeniia.

The antiviral properties of histones of animal (thymus) and plant (French beans) origin were studied in plants and with a plant virus, tobacco mosaic virus (TMV). Histones of the thymus and French beans were shown to be able to inhibit TMV reproduction. The antiviral properties of histones were found to depend on their concentration, pH, and to be determined by the modes of their introduction into leaves. The manifestation of the antiviral properties of histones seems to require not only their direct contact with virus but also a certain exposure on the leaf. The similarities of antiviral protective mechanisms of plants and animals determined by substances of the protein nature are discussed.

Clinical picture and epidemiology of adenovirus infections (a review).

Adenoviruses produce a variety of serious diseases in people of all ages. The mode of transmission of adenovirus infections includes respiratory, fomite, droplet, venereal, and faecal-oral routes. They have been shown to spread with ease in AIDS patients, in young children and in hospitalized patients. A constant rate of about 8% of world-wide reported virus infections were observed to be due to adenovirus infections. In the military, it can cause serious respiratory disease (ARD) of epidemic proportion in new recruits. The present commercial vaccine is prepared in unique triple-layered tablets containing live lyophilized virus. This vaccine has been taken by more than ten million subjects during the past 25 years with no adverse reaction and with near total eradication of ARD epidemics among new recruits at training centers. As the epidemiology of adenovirus diseases becomes more clearly defined, the need for and the possible role of potential vaccines, is becoming more evident.

Two-dimensional immuno-osmophoresis of adenovirus type 7 antigens.

A test is described in which adenovirus type 7 antigens are separated by two successive perpendicular applications of electrophoresis through agar, causing precipitin formation with cationic immune globulins. Three bands produced by adenovirus cultures distinguish among hexon, fiber, and penton antigens.

[Localization of the Meler's reaction with ethanol catalase trap in the chain of photosynthetic electron transport]. / O lokalizatsii reaktsii Melera s etanolkatalaznoi lovushkoi v tsepi fotosinteticheskogo transporta elektronov

The common view of photosystem I as the action site of catalase and ethanol at oxygen uptake in chloroplasts are based on indirect data on this reaction. That is why the question on Mehler reaction localization in electron transport chain with ethanolcatalase trap has been investigated anew. It has been demonstrated that oxygen uptake with catalase and ethanol does not decrease in presence of dibromothymoquinone (2,5-dibromo-3-methyl-6 isopropyl-p-benzoquinone--DBTQ) which blocks electron transfer to photosystem I at plastoquinones level. The summation of oxygen uptake activities is observed on the combined action of catalase and ethanol with any of the Mehler reagents functioning in photosystem I (methylviologen,FMN, epinephrine, ferredoxin). Catalase and ethanol in contrast to methylviologen have no effect on photooxidation rate of reduced dichlorphenolindophenol in photosystem I. The quatum yield of oxygen uptake with catalase and ethanol versus wave length of actinic light shows a distinct maximum in the photosystem II absorption area and a "red drop" in the longware area. The obtained data show that the Mehler reaction with catalase and ethanol takes place in photosystem II only.

Polymyxin B release of unnicked cholera toxin subunit A.

Polymyxin B treatment of Vibrio cholerae 569B grown with or without lincomycin released an extracytoplasmic pool of free unnicked cholera toxin subunit A.

Development of a purified cholera toxoid. I. Purification of toxin.

The enterotoxin from Vibrio cholerae is selectively concentrated from cell-free culture supernatant by co-precipitation with hexametaphosphate and is further purified by adsorption on aluminum hydroxide powder. The bulk of residual somatic antigen becomes insoluble upon lyophilization of the toxin preparation and is removed by centrifugation of the rehydrated material. Other contaminants are eliminated by treatment with activated carbon. Preparations of toxin, purified by this method, have been characterized by: (i) a single immunoprecipitin line against polyvalent antisera; (ii) homogeneity on acrylamide gels; (iii) specific activities on the order of 22 limit-of-bluing doses/mug; (iv) ultraviolet spectra characteristic of pure protein; and (v) overall yields on the order of 50%, irrespective of purification scale. Such preparations, however, have been shown to contain trace amounts of somatic antigen when they are intensively tested either for their ability to elevate serum vibriocidal antibody titers in immunized rabbits or for their ability to increase resistance of immunized mice to live vibrio challenge. In the latter test system, the level of residual somatic antigen per 50 mug of toxin (toxoid) antigen generally did not exceed 0.025% of the Division of Biological Standards reference vaccine, V. cholerae Inaba IN-12. Methods for elimination of this small amount of somatic antigen have been investigated and are discussed. The particular combination of purification steps which are presently described have been easily and reproducibly applied on a production scale to prepare gram amounts of toxin with a high degree of purity, even under a variety of initial conditions.

Effect of formamide on the capsid morphology of adenovirus types 4 and 7.

Treatment of adenovirus types 4 and 7 with formamide disrupted the virions, degrading the capsids into predominantly single capsomers. As shown by electron microscopic observation, disruption proceeded in the following sequence: (i) reduction of the electron density of the virions, suggesting release of an internal component; (ii) progressive cleavage of the capsid into two or more segments and the formation (type 7 only) of capsomer "sheets"; (iii) final cleavage of the capsid into single or groups of a few capsomers. The sequence appeared similar for both adenoviruses; for both types, the rate and extent of disruption were dependent on the formamide concentration, but type 7 was more easily disrupted than type 4 by short-term (5 to 10 sec) treatment at the low (10%) concentration. At 30% formamide, the intercapsomer bonds of either type were fully cleaved, and the capsids were completely degraded into predominantly single capsomers. Pretreatment with formaldehyde did not prevent this degradation. Under suitable conditions, virus-derived remnants can be observed among the breakdown products. These remnants have been shorn of capsomers and presumably represent intact internal nucleoprotein.

Disruption of adenovirus type 7 by lithium iodide resulting in the release of viral deoxyribonucleic acid.

Adenovirus type 7 exposed to solutions of LiI was progressively converted into slower sedimenting deoxyribonucleic acid (DNA)-containing particles, and, ultimately, under proper conditions, DNA free or almost free from protein was released from the virus. The degree of viral degradation was dependent on the time of treatment, on the temperature, and on the concentration of the reagent.