Biomedical subjects

R E Johnston

Publications and source records attributed to R E Johnston.

At least 19 recordsLinked to original sources

PubMed1992

Social influences on reproductive development and fertility in female Djungarian hamsters (Phodopus campbelli).

Social influences on the sexual maturation of female Djungarian hamsters were investigated in two experiments. In the first experiment females were housed from weaning with an adult male, by themselves, or with a weanling sister. Maturation was accelerated in females housed with males as indicated by younger age at first ovulation, increased rates of ovarian and uterine growth, and lower LH levels at some ages. Maturation was delayed in females housed with sisters compared to those housed alone as measured by time of first ovulation and by lower estradiol levels at some ages. The most marked differences between groups occurred 8 to 12 days after weaning, suggesting that events during this period are particularly important in the social mediation of sexual maturation. In the second experiment the effects of reproductive suppression (caused by living with a sister) on the subsequent fertility of females housed with males were examined. If male-female pairs were housed in clean cages, no effects were observed; however, pairs housed in cages previously soiled by the female and her sister had fewer young surviving until 1 week of age despite no differences in the age of pregnancy onset or in the initial litter size. Thus, even cues present in unrenewed soiled bedding may have subtle but long lasting effects on reproductive function.

Animals↗

PubMed1992

Socially stimulated androgen surges in male hamsters: the roles of vaginal secretions, behavioral interactions, and housing conditions.

The sources of cues necessary for elicitation of androgen surges and sexual behavior in male golden hamsters (Mesocricetus auratus) were investigated. Circulating androgen levels were measured in males after interactions with other males or several types of estrous females: intact females, vaginectomized females, or vaginectomized females scented with vaginal secretions. All groups of males that interacted with estrous females demonstrated significant elevations in androgens whereas those that interacted with other males did not. Thus, the presence of vaginal secretions is not necessary for the elicitation of androgen surges in sexually experienced male hamsters. Individual differences in sexual performance were not correlated with the degree of change in androgen levels, suggesting that such hormonal responses are not graded but are all-or-none. Housing males in isolation from females did not alter either baseline androgen levels or the magnitude of androgen responses caused by interactions with females.

Animals↗

PubMed1992

Vomeronasal and/or olfactory mediation of ultrasonic calling and scent marking by female golden hamsters.

The role of the olfactory and vomeronasal systems in mediating odor-stimulated ultrasonic calling, flank marking, and vaginal marking by female hamsters was investigated by selective lesions of either system. Removal of the vomeronasal organ resulted in reduced frequencies of ultrasonic calling by estrous and nonestrous females in response to conspecific odors but it had no influence on either scent marking behavior during the same tests. When tested immediately after separation from a male, ultrasonic calling was not reduced by vomeronasal removal, indicating that such surgery does not cause deficits in calling ability and that the vomeronasal organ specifically mediates odor-stimulated calling. Zinc sulfate treatment of the olfactory mucosa led to reduction in the frequency of ultrasonic calling, flank marking, and vaginal marking in response to conspecific odors. Females' ability to discover buried food was also impaired by this treatment. Thus, the stimulation of both scent marking behaviors due to the perception of conspecific odors appears to be mediated primarily by the olfactory system, whereas stimulation of ultrasonic calling is mediated by both olfactory and vomeronasal systems.

Animals↗

PubMed1992

Visual increment and decrement threshold curves as a function of luminance range and noise in simulated computed tomographic scans.

OBJECTIVES: The effect of luminance range compression on the visual threshold of a target in a computed tomographic (CT) scan was investigated in eight experiments. METHODS: Both visual increment thresholds of a hot target and visual decrement thresholds of a cold target were obtained. Realistic noisy CT images simulating a scan reconstruction and partial volume blurring were tested as representative of complex medical images. Negative versions, high luminance versions, and noise-free versions of these CT images also were tested. RESULTS AND CONCLUSIONS: In all the noisy images, as the luminance range of the image was compressed, proportionately smaller physical luminance differences between the target and its local background were needed to reach visual threshold. However, the thresholds were the same in terms of the difference in CT numbers between target and background. Noise in terms of CT numbers sets the threshold for a wide range of display conditions. In the noise-free CTs, as the luminance range was compressed, the luminance differences between the target and background needed to reach threshold also decreased, but only marginally. However, in terms of CT numbers, the thresholds were increased.

Analysis of Variance↗

PubMed1992

Conformational alteration of Sindbis virion glycoproteins induced by heat, reducing agents, or low pH.

Sindbis virions undergo a conformational rearrangement after attachment to cells but prior to entry, as detected by exposure of epitopes on virus-cell complexes which are not accessible to their cognate monoclonal antibodies on native virions (D. C. Flynn, W. J. Meyer, and R. E. Johnston, J. Virol. 64:3643-3653, 1990). The rearrangement did not appear to require transit of virions through a low-pH environment, and the altered virions participated in a productive infection. This naturally occurring structural alteration could be mimicked, although not precisely duplicated, by any of the three artificial treatments of purified virions in vitro: brief incubation at 51 degrees C, treatment with 1 to 5 mM dithiothreitol, or incubation of pH 5.8 to 6.0. Infectivity was maintained after all three treatments, suggesting that Sindbis virions are metastable and can exist in at least two infectious conformations. The integrity of external, neutralizing epitopes was maintained on cell-associated virions and in the altered conformations induced by heat and dithiothreitol, whereas these epitopes were unreactive under low-pH conditions that induced an analogous exposure of previously inaccessible epitopes. The pH at which the conformational change was induced and the pH at which virions could mediate cell-cell fusion from without were coordinately shifted when these two parameters were determined for another strain of Sindbis virus. This coordinate shift in pH optima suggests that the conformational change in virion structure observed at the cell surface may be causally related to fusion.

Antibodies, Monoclonal↗

PubMed1991

Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: construction of single and multiple mutants in a full-length cDNA clone.

Attenuated mutants of Venezuelan equine encephalitis virus (VEE) were isolated by selection for rapid penetration of cultured cells (R. E. Johnston and J. F. Smith, 1988, Virology 162, 437-443). Sequence analysis of these mutants identified candidate attenuating mutations at four loci in the VEE E2 glycoprotein gene: a double mutation at E2 codons 3 and 4, and single substitutions at E2 76, 120, and 209. Each candidate mutation was reproduced in an isogenic recombinant VEE strain using site-directed mutagenesis of a full-length cDNA clone of VEE. Characterization of these molecularly cloned mutant viruses showed that mutation at each of the four loci in the E2 gene was sufficient to confer both the accelerated penetration and attenuation phenotypes. Inoculation of the molecularly cloned viruses into rodent models that differ in their response to VEE suggested that individual mutations affected different aspects of VEE pathogenesis. Full-length clones containing multiple mutations were produced by combining independently attenuating mutations. Molecularly cloned viruses carrying two or three mutations were more attenuated in sensitive animal models than viruses which contained any single mutation alone. However, these highly attenuated strains still retained the ability to induce an immune response sufficient to protect against a high dose challenge with virulent VEE. These results indicate that production of a molecularly cloned live virus vaccine for VEE is feasible.

Animals↗

PubMed1991

Mutational analysis of a virulence locus in the E2 glycoprotein gene of Sindbis virus.

The substitution of arginine for serine at position 114 of glycoprotein E2 in several biological and recombinant Sindbis virus mutants was shown previously to attenuate the virus for neonatal mice and also to accelerate virus penetration into BHK cells. To further examine the genetically linked effects on both virus penetration into cultured cells and pathogenesis in vivo, mutants containing each of 16 different amino acid coding changes at this position were generated by site-directed mutagenesis of a full-length cDNA clone of the Sindbis virus genome. Viable virus was recovered following transfection of RNA transcripts from 14 of the clones. Phenotypic analysis of these virus mutants revealed that specific amino acid residues affected either the pathogenesis or penetration phenotype independently or both phenotypes simultaneously. Thus, both the position of a mutation within the E2 sequence and the particular amino acid encoded at that position are important determinants of the mutant phenotypes.

Amino Acid Sequence↗

PubMed1991

Proteolytic processing of the Sindbis virus membrane protein precursor PE2 is nonessential for growth in vertebrate cells but is required for efficient growth in invertebrate cells.

We have shown previously that processing of the Sindbis virus envelope protein precursor PE2 to envelope protein E2 is not required for virus maturation in cultured vertebrate fibroblast cells and that unprocessed PE2 can be incorporated into infectious virus in place of E2 (J. F. Presley and D. T. Brown, J. Virol. 63:1975-1980, 1989; D. L. Russell, J. M. Dalrymple, and R. E. Johnston, J. Virol. 63:1619-1629, 1989). To better understand the role of this processing event in the invertebrate vector portion of the alphavirus life cycle, we have examined the maturation of Sindbis virus mutants defective in PE2 processing in cultured mosquito cells. We found that although substantial amounts of structural proteins PE2, E1, and C were produced in infected mosquito (aedine) cell lines, very little infectious virus was released. When the period of infection was extended, plaque size variants appeared, some of which exhibited a restored ability to grow in mosquito cells. The nucleotide sequences of two such variants were determined. These variants contained point mutations that restored PE2 cleavage, indicating a genetic linkage between failure to cleave PE2 and failure to grow in mosquito cells.

Aedes↗

PubMed1990

A study of radiologists viewing multiple computed tomography examinations using an eyetracking device.

Understanding the scan patterns radiologists use to view medical images is critical to the design of image viewing devices. In this study, an eyetracker, a device for recording eye and head movement, was used to determine the scan patterns during the interpretation of single and multiple computed tomographic (CT) examinations presented on a four-over-four viewbox. CT examinations were used because they represent complex viewing situations. In two separate studies, radiologists viewed patient folders containing single or multiple CT chest examinations and dictated a report. Eye movement was recorded with an eyetracker and video camera. After mounting the films in order, radiologists generally started with a sequential scan through the entire examination, followed by careful viewing of two to four clusters of three to six images, followed by dictation. These results indicate that a well designed radiology workstation should provide an image index, sufficient display area to simultaneously view 10 or more images, random and sequential movement through the examination, image comparison, and image marking.

Ergonomics↗

PubMed1990

Olfactory but not vomeronasal mediation of scent marking by male golden hamsters.

Although olfactory and vomeronasal projections to the olfactory bulb and to the rest of the brain are quite distinct, it is not clear how the functions of these two chemosensory systems differ. In these experiments we attempted to determine the roles of the main olfactory and vomeronasal sensory systems in mediating scent marking by male golden hamsters, a behavior known to be stimulated by odors of other hamsters. Lesions of the main olfactory mucosa by irrigation of the nasal cavity with a solution of zinc sulfate dramatically reduced the frequency of marking. The degree of reduction in marking was correlated with an independent test of ability to detect volatile odorants. In contrast, surgical removal of the vomeronasal organ had no effect on marking frequency. Thus scent marking by male hamsters is primarily mediated by the main olfactory system and does not require the presence of the vomeronasal system. These results are discussed in the context of a theory that stresses the importance of the main olfactory system in pattern recognition and, in particular, in many types of social recognition.

Animals↗

PubMed1990

Antigenic and genetic characterization of Sindbis virus monoclonal antibody escape mutants which define a pathogenesis domain on glycoprotein E2.

The Sindbis virus mutant SB-RL, in contrast to its parent, Sindbis strain AR339 (SB), is attenuated in neonatal mice, has an increased rate of penetration in tissue culture cells, and is more sensitive to neutralization by E2-specific monoclonal antibodies (MCAbs) R6 and R13. These phenotypic differences are controlled by substitution of an arginine for serine at amino acid 114 of the E2 glycoprotein. To explore these relationships further, MCAb R6 and R13 neutralization escape mutants of both SB and SB-RL were isolated and characterized. All mutants bound both MCAb R6 and R13 significantly less effectively in ELISA, and were more resistant to complement-mediated neutralization than their respective parental strains. Single coding changes in the E2 glycoprotein gene of each 11 mutants were identified. SB/R6, SB/R13, and SB-RL/R13 mutants contained a mutation at either E2 codon 96 or 159. SB-RL/R6 mutants contained changes at E2 codon 62, 96, or 159. These coding changes included two intragenic suppressor mutations. Mutation of E2 codon 159 from lysine to glutamate or codon 62 from asparagine to aspartate suppressed the attenuated phenotype conferred by E2 arginine 114 in SB-RL. However, only the change at E2 codon 62 significantly suppressed the rapid penetration phenotype of SB-RL. Mutation in E2 codon 96 of SB, replacing tyrosine with histidine, reduced the virulence of SB for neonatal mice but had no effect on penetration of cultured cells. Therefore, mutation in E2 codons 62, 96, 114, or 159 affected both virulence in animals and the binding or biological activity of these E2c-specific MCAbs. These results suggest that an E2 antigenic site (E2c), defined by MCAbs R6 and R13, is conformational in nature and may constitute a surface domain on Sindbis virions important for virulence in neonatal mice.

Animals↗

PubMed1990

A conformational change in Sindbis virus glycoproteins E1 and E2 is detected at the plasma membrane as a consequence of early virus-cell interaction.

A conformational change in the structure of Sindbis (SB) virus was detected after virion attachment to baby hamster kidney cells but before internalization. The alteration was manifested as increased virion binding of certain glycoprotein E1 and E2 monoclonal antibodies (MAbs) that recognized transitional epitopes. These epitopes were inaccessible to MAb on native virions but became accessible to their cognate MAbs in the early stages of infection. Transit of virions through a low-pH compartment apparently was not required for the conformational change. Exposure of transitional epitopes was unaffected by treatment of BHK cells with NH4Cl and occurred normally in Chinese hamster ovary cells temperature sensitive for endosomal acidification. However, the rearrangement was correlated with both the time course and temperature dependence of SB virus penetration, and the rearrangement occurred earlier with an SB virus mutant having an accelerated penetration phenotype. In addition, MAb to a transitional epitope, a probe specific for rearranged particles, retarded penetration of infectious virions. These results suggested that the SB virus E1/E2 glycoprotein spike undergoes a structural rearrangement as a consequence of virion interaction with the cell surface and that this altered virion form may be an important early intermediate in an entry pathway leading to productive infection.

Ammonium Chloride↗

PubMed1990

Attenuating mutations in glycoproteins E1 and E2 of Sindbis virus produce a highly attenuated strain when combined in vitro.

Alterations in either the E1 or the E2 glycoprotein of Sindbis virus can affect pathogenesis in animals. Previously, we identified two distinct E1 glycoprotein gene sequences which differed in their effect on pathogenesis. One had an attenuation phenotype following subcutaneous inoculation of neonatal mice (E1 Ala-72, Gly-75, and Ser-237), while the other was virulent (E1 Val-72, Asp-75, and Ala-237). In this study, we examined the basis for this difference in pathogenesis by using a full-length cDNA clone of Sindbis virus from which infectious RNA could be transcribed in vitro. The relative contribution of each E1 residue to the pathogenesis phenotype was determined by using site-directed mutagenesis to alter each codon individually and in combination. Residues 75 and 237, in combination, appeared to be the major E1 determinants affecting pathogenesis. In addition, the effect of directly combining independently attenuating E1 and E2 mutations in the same virus was examined. The attenuating E1 sequences characterized in this study were coupled to a previously characterized attenuating mutation at E2 residue 114. The resulting recombinant virus, constructed in vitro, exhibited an increased attenuation of neurovirulence as compared with recombinant viruses containing either of the attenuating elements alone.

Animals↗

PubMed1989

In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant.

A molecular clone of Venezuelan equine encephalitis virus (VEE) was constructed from four cDNAs that were synthesized using the viral RNA genome as template. Together, these cDNAs are believed to represent all but the nine 5'-terminal nucleotides of the VEE genome sequence. A T7 promoter, followed by a single intervening G residue, and the exact 5'-terminus of VEE were added to the 5'-most clone using in vitro mutagenesis. Appropriate restriction fragments isolated from the cloned cDNAs were joined to form a candidate full-length VEE cDNA clone. RNA transcripts synthesized in vitro from the cDNA clone were able to initiate a productive infection in DEAE-dextran-treated chicken embryo fibroblasts (CEF). VEE antigens were demonstrated in RNA-transfected cells, and supernatants from transfected cultures contained infectious virus particles. The candidate full-length cDNA clone lacked 102 nucleotides of the VEE genome sequence. The deletion, which also was present in the genomes of progeny virions derived from the clone, did not appear to affect growth in cultured CEF, baby hamster kidney cells, or Vero cells. The site of the deletion was mapped to the 3'-end of the nsP3 gene by comparison to other alphavirus sequences. In this region, the VEE genome sequence includes two tandem 102-nucleotide repeats which can be arranged in a stable stem and loop structure. The sequence remaining in the deleted clone retains one copy of the duplicated sequence and, in addition, faithfully preserves a portion of the predicted stem.

Antigens, Viral↗

PubMed1989

Generation and characterization of monoclonal antibodies reactive with the 49-kDa proteinase of tobacco etch virus.

Monoclonal antibodies (McAbs) were generated against two tobacco etch virus (TEV)-encoded nonstructural proteins, the 49-kilodalton (kDa) proteinase and the 58-kDa putative RNA-dependent RNA polymerase. This process was facilitated by the fact that these two TEV nonstructural proteins cocrystallize in the nuclei of virus-infected cells to form nuclear inclusion (NI) bodies which can be purified readily. The anti-NI McAbs were shown by Western blot analysis to be specific for either the TEV 49-kDa or the 58-kDa protein. Those McAbs reactive with the 49-kDa proteinase were characterized further with respect to the 49-kDa domain with which they reacted and with respect to their ability to inhibit the autocatalytic or self-processing activity of the 49-kDa proteinase. The 49-kDa antigens were synthesized from a TEV cDNA sequence using cell-free transcription and translation systems. Each anti-49-kDa McAb was used in immunoprecipitation studies with a series of 49-kDa antigens which represented a nested set of 49-kDa proteins with common amino termini but varying in length. Immunoprecipitation results showed that all of the anti-49-kDa proteinase McAbs reacted with one of five binding regions, designated A through E from the carboxy terminus of the proteinase, which were 77, 38, 81, 18, and 61 amino acids long, respectively. The 38-amino-acid binding region B contained the proposed catalytic cysteine 339 residue and was recognized by only one McAb, 4911. McAb 4911 was the only anti-49-kDa McAb capable of inhibiting the self-processing reaction in which the 49-kDa proteinase is released from its 75-kDa polyprotein precursor.

Antibodies, Monoclonal↗

PubMed1989

Sindbis virus mutations which coordinately affect glycoprotein processing, penetration, and virulence in mice.

Rapid penetration of baby hamster kidney cells was used as a selective pressure for the isolation of pathogenesis mutants of the S.A.AR86 strain of Sindbis virus. Unlike most Sindbis virus strains, S.A.AR86 is virulent in adult as well as neonatal mice. Two classes of mutants were defined. One class was attenuated in adult mice inoculated intracerebrally as well as in neonatal mice inoculated either intracerebrally or subcutaneously. Sequence analysis of the glycoprotein genes of the parent virus and three such mutant strains revealed a single point mutation which resulted in an amino acid change at position 1 in the E2 glycoprotein. The change from a serine in S.A.AR86 to an asparagine in the mutants created a new site for N-linked glycosylation which appeared to be utilized. This mutation did not retard release of infectious particles; however, mutant virions contained the E2 precursor protein (PE2) rather than the E2 glycoprotein itself. The mutants also lost the ability to bind two E2-specific monoclonal antibodies, R6 and R13. A second class of mutants was attenuated in neonatal mice upon subcutaneous inoculation but remained virulent in adults and in neonates when inoculated intracerebrally. Sequence analysis of three such strains revealed the substitution of an arginine residue for a serine at position 114 in the E2 glycoprotein. Reactivity with monoclonal antibodies R6 and R13 was reduced, yet members of this mutant class were more susceptible than S.A.AR86 to neutralization by these antibodies.

Amino Acid Sequence↗

PubMed1988

Acceleration of reproductive development in female Djungarian hamsters by adult males.

Housing young female Djungarian hamsters (Phodopus campbelli) with an adult male accelerates uterine and ovarian development and there is a strong relationship between uterine weight and ovarian measures (e.g., follicular size). Uterine weights of females housed with an adult male for 10 days following weaning are comparable to values from females housed alone for 25 days. Removal of endogenous androgens by castration eliminated the capacity of adult males to accelerate reproductive development in young females and treatment of castrated males with exogenous androgens maintained the production of the acceleratory chemosignal. When adult male urine and ventral gland sebum were examined as possible sources for the acceleratory chemosignal, only male urine had an acceleratory effect on reproductive development. Thus, female Djungarian hamsters respond with accelerated reproductive development to androgen-dependent chemosignals in the urine of adult males. These mechanisms are similar to those found in several other rodents but contrast with the lack of such effects in the golden hamster (Mesocricetus auratus).

Animals↗

PubMed1988

Antibody-mediated activation of Sindbis virus.

The biological activity of an anti-Sindbis monoclonal antibody (MCAB 49) has been explored. The antibody recognizes an epitope on the E2 glycoprotein of Sindbis virus and, in the presence of complement (C'), neutralizes virus infectivity. In the absence of C', reaction of the antibody with our laboratory strain of Sindbis, SB, increased the number of plaque-forming units (PFU) detected on baby hamster kidney (BHK) cells rather than neutralizing virus infectivity. The elevated titers of SB approached, but never exceeded, the number of virions calculated from the particle:PFU ratio, indicating that the additional PFU might have resulted from activation of normally noninfectious particles. The apparent activation could not be attributed to disaggregation of SB by MCAB 49 as shown by ultraviolet inactivation experiments with antibody-treated and untreated virus preparations. Fc receptors did not appear to be involved in the antibody-mediated activation. Fab' and F(ab')2 fragments of MCAB 49 also increased the number of observed PFU of SB. Control monoclonal antibodies of the same isotype, but specific for the tobacco etch virus capsid protein, were unable to compete for cellular binding sites with the SB/MCAB 49 complex. Rather, the SB/MCAB 49 complex appeared to utilize the same receptor(s) as SB in that SB and the SB/MCAB 49 complexes competed with each other for binding sites on BHK cells. Binding studies with 32P-labeled SB showed that a higher proportion of MCAB 49 activated virions than untreated virions associated with BHK cells. Moreover, activated virions were much less susceptible to elution. These results suggest that reaction which MCAB 49 may facilitate successful attachment of SB to its receptor, or receptors, on BHK cells.

Animals↗