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Biomedical subjects

D M Johnston

Publications and source records attributed to D M Johnston.

At least 19 recordsLinked to original sources
PubMed2001

Correlates of hazard education programs for youth.

Virtually no research has examined the hypothesized benefits of hazard education programs for youth in helping to increase community resilience. This exploratory study examined the role of these programs in helping to increase child and family resilience to a range of future hazards. Various aspects of hazards programs were examined in relation to a wide range of child- and parent-reported hazard adjustments in a sample of 560 schoolchildren. Additional factors assessed included childrens' risk perceptions, knowledge of response-related protective activities, and hazard-related emotional factors. Overall, the results supported the role of hazards education programs in increasing hazard adjustments in the home. The findings also supported various aspects of education program involvement as being related to more realistic risk perceptions, increased knowledge, and increased interaction with caregivers. Analyses identified the following features of these programs as being particularly important: provision of specific knowledge (e.g., an emergency management perspective); multiple program involvement over time; and, importantly, promotion of increased interaction between children and parents. Overall, findings supported the idea that hazards education programs for youth provide one gateway through which communities can increase their resilience to the effects of a major hazardous event. Findings also provided an initial foundation for further research in this emerging area.

Adolescent↗
PubMed1999

Construction of mutant strains of Neisseria gonorrhoeae lacking new antibiotic resistance markers using a two gene cassette with positive and negative selection.

The pathogenesis of infections caused by Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can be studied using experimental infection of human male volunteers. The desire to avoid introducing new antibiotic resistance markers into strains to be used in human experimental infection has complicated the construction of genetically defined mutants in which expression of potential virulence factors is inactivated. To facilitate construction of such mutants, we have used a two-step mutagenesis strategy that allows for gene replacements without introducing new selectable markers into the final strain. The method uses a two-gene cassette containing both a selectable marker (ermC') and a counterselectable marker (rpsL). The cassette is cloned into the gene of interest and used to replace the wild-type gene on the chromosome by allelic exchange. A second transformation replaces the cassette-containing version of the gene with an engineered version with an unmarked deletion or other mutation. The rpsL gene of Escherichia coli functioned for the counterselection in the gonococcus, albeit with low efficiency. To improve the efficiency of the counterselection, we cloned the gonococcal rpsL gene and incorporated it into the cassette. This technique has been successful in creating defined mutants for human challenge, and also circumvents the limitation in the number of different selectable markers that are useful in Neisseria species.

Anti-Infective Agents↗
PubMed1999

Genotypic resistance and the treatment of HIV-1 infection in Espírito Santo, Brazil.

Before December 1997, in Espírito Santo, Brazil, combination antiretroviral therapy was used without routine virologic or immunologic monitoring. To examine consequences of therapy in this setting, clinical information, human immunodeficiency virus type 1 (HIV-1) RNA levels, CD4 cell counts, and protease and reverse transcriptase sequences were determined for consecutive HIV-1-infected outpatients. Of 48 treatment-naive individuals, 11 were started on therapy for HIV-related symptoms; however, 44 (92%) had an RNA level >20,000 copies/mL, a CD4 cell count <500/mm3, or symptoms. Eighteen (51%) of 35 patients on therapy had an RNA level >20,000 copies/mL. Nucleoside-resistance mutations were observed in 21 (68%) of 31 nucleoside-experienced subjects. Protease mutations necessary for high-level protease inhibitor (PI) resistance were present together with permissive mutations in 3 of 10 PI-experienced patients. Inability to identify high-risk individuals and to detect virologic failure may limit the effectiveness of antiretroviral drug programs and may promote the spread of drug resistance where virologic and immunologic monitoring are not available.

Adult↗
PubMed1999

A Neisseria gonorrhoeae immunoglobulin A1 protease mutant is infectious in the human challenge model of urethral infection.

Many mucosal pathogens, including Neisseria gonorrhoeae, produce proteases that cleave immunoglobulin A (IgA), the predominant immunoglobulin class produced at mucosal surfaces. While considerable circumstantial evidence suggests that IgA1 protease contributes to gonococcal virulence, there is no direct evidence that N. gonorrhoeae requires IgA1 protease activity to infect a human host. We constructed a N. gonorrhoeae iga mutant without introducing new antibiotic resistance markers into the final mutant strain and used human experimental infection to test the ability of the mutant to colonize the male urethra and to cause gonococcal urethritis. Four of the five male volunteers inoculated with the Iga- mutant became infected. In every respect-clinical signs and symptoms, incubation period between inoculation and infection, and the proportion of volunteers infected-the outcome of human experimental infection with FA1090iga was indistinguishable from that previously reported for a variant of parent strain FA1090 matching the mutant in expression of Opa proteins, lipooligosaccharide, and pilin. These results indicate that N. gonorrhoeae does not require IgA1 protease production to cause experimental urethritis in males.

Genotype↗
PubMed1998

Resistance of HIV-1 to antiretroviral agents in blood and seminal plasma: implications for transmission.

OBJECTIVES: To evaluate blood and genital secretions from HIV-infected men for HIV-1 resistant to antiretroviral agents. DESIGN: A longitudinal study of 11 men with HIV infection and persistent detectable HIV RNA levels in blood and semen on antiretroviral therapy. METHODS: HIV-1 from the blood and seminal plasma, obtained before the initiation of a new therapeutic regimen and on therapy, were evaluated by population-based sequencing of reverse transcriptase (RT) and protease RNA for the development of resistance to antiretroviral therapy. The genetic relatedness of sequences over time was compared. RESULTS: RT genotypic resistance markers were present in seminal plasma at baseline in three out of six individuals with previous RT inhibitor experience. Eight out of 10 men, from whom the viral sequence was available on new therapy, demonstrated the evolution of new resistance mutations in the blood or seminal plasma, or both. The evolution of resistance mutations in blood and semen were frequently discordant, although over time similar patterns were seen. In two individuals, protease inhibitor resistance mutations evolved in the blood but not in the major variant in seminal plasma. Comparisons of the viral sequences between blood and seminal plasma from six men revealed two patterns. Three men showed a clustering of sequences from blood and semen. Three had sequences that appeared to evolve separately in the two compartments. CONCLUSIONS: HIV-1 variants with genotypic resistance markers are present in the male genital tract and evolve over time on incompletely suppressive antiretroviral therapy. The absence of genotypic changes consistent with protease inhibitor resistance in the semen, despite their presence in blood plasma, suggests the possibility of limited penetration of these agents into the male genital tract. Sexual transmission of resistant variants may have a negative impact on treatment outcome in newly infected individuals and on the spread of the diseases within a population. Therapeutic strategies that fully suppress HIV-1 in the genital tract should be a public health priority.

Amino Acid Sequence↗
PubMed1992

Transformation of Acetobacter xylinum with plasmid DNA by electroporation.

Genetic analysis of Acetobacter xylinum, a cellulose-synthesizing bacterium, has been limited by lack of a successful transformation method. Transformation of A. xylinum was attempted using two broad-host-range plasmids (pUCD2 and pRK248) and a variety of transformation methods. Methods using CaCl2, freeze/thaw treatments, and polyethylene glycol were unsuccessful. Transformation of a cellulose-negative strain of A. xylinum with plasmid DNA has been achieved with high-voltage electroporation. Electroporation conditions of 25 microF capacitance, 2.5 kV, 400 ohms resistance, and pulse lengths of 6-8 ms were applied to a cell/DNA mixture in a 0.2-cm cuvette. Plasmid pUCD2 transformed at an efficiency of 10(6)-10(7) transformants/micrograms DNA and pRK248 yielded 10(5) transformants/micrograms DNA. The frequency of transformation increased linearly with increasing DNA concentration, while transformation efficiency remained constant. pUCD2 was recovered from transformants following chloramphenicol amplification and observed by agarose gel electrophoresis. Both plasmids could be reisolated from Escherichia coli after back-transformation with alkaline lysis DNA preparations from Acetobacter transformants. Electro-transformation of A. xylinum with plasmid DNA suggests its potential use for analysis of the A. xylinum genome.

Cellulose↗
PubMed1985

Dictyate oocytes of a kangaroo (Macropus robustus) show paternal inactivation at the X-linked Gpd locus.

Purified samples of large numbers of dictyate oocytes from 13 M. robustus pouch young heterozygous for glucose-6-phosphate dehydrogenase type and six homozygous controls were examined electrophoretically to determine activity states at the Gpd locus. Like somatic cortical and medullary cells, oocytes expressed only the maternal phenotype irrespective of the direction of the cross. No evidence was found of reactivation of the inactive (paternal) allele or inactivation of both maternal and paternal alleles. It was therefore concluded that unlike eutherian dictyate oocytes, only a single (maternal) allele is active in each dictyate oocyte in M. robustus. The stage of reactivation of the paternal allele remains to be determined.

Animals↗
PubMed1984

Consumption of monoclonal anti-idiotypic antibody by neoplastic B lymphocytes: a guide for immunotherapy.

A quantitative analysis in vitro of events which might occur on administration of mouse monoclonal anti-idiotypic antibody to a recipient with a B cell neoplasm has been made. The L2C leukaemic cells of guinea pigs, which closely resemble those of human lymphoma in expression and metabolism of immunoglobulin have been used as a model. Exposure of neoplastic B cells to antibody results in rapid binding of approximately 420,000 molecules of antibody per cell at saturation, and the amount consumed does not increase markedly over the next 4 h of exposure at 37 degrees C. This is in spite of the fact that secretion of idiotypic IgM continues unaffected by the presence of antibody, and reflects the fact that the amount of IgM secreted during this period is low compared to the amount displayed on the cell surface. If cells undergo lysis, however, the antibody consumed is approximately doubled: thus a recipient with an estimated tumour load of 10(12) cells would require 200 mg of monoclonal anti-idiotype for binding to surface and intracellular antigen. The effect of the soluble idiotypic IgM found in serum on the ability of antibody to bind target cells has been examined by means of the fluorescence activated cell sorter. Access of antibody to the cells is efficiently blocked by competing idiotypic IgM in the fluid phase, with no indication of preferential binding to cell surface idiotype. Immunotherapeutic doses should be designed therefore to overcome this additional antigenic load in secreting tumours, which form the majority of B cell neoplasms.

Animals↗
PubMed1976

Nerve fibre velocity and refractory period distributions in nerve trunks.

With the use of a double stimulus technique, nerve fibre velocity range measurements were performed over a single conduction distance in 13 normal subjects and over two conduction distances in another 12 normal subjects. The velocity ranges were found to be dependent upon the conduction distance, owing to unknown refractory period delays. Refractory period values were calculated for the 12 subjects and also direct refractory period distribution measurements were made on 15 normal subjects using a twin stimulus and automatic subtraction technique. Corrections to the velocity range measurements were made upon differing assumptions as to the correlation between refractory period and fibre conduction velocity. It was concluded that a single median value refractory period obtained from the distribution was the best correction to use, based upon the hypothesis that for group A fibres the random scatter of refractory period values is far greater than any variation due to a correlation between refractory period and fibre conduction velocity. It was found important to recognize that calculated values of velocity range are a function not only of the spread of fibre conduction velocities but also of refractory periods.

Adult↗
PubMed1975

Neurotrophic regulation of dynamic properties of skeletal muscle: effects of botulinum toxin and denervation.

In order to determine the role of acetylcholine (ACh) transmission in neurotrophic regulation of dynamic properties of muscle, the effects of botulinum toxin treatment were compared with those of denervation. The extensor digitorum longus (EDL) and soleus muscles of rats were either denervated or injected with botulinum toxin. At times up to 25 days the isometric properties of these muscles were determined. 2. Both botulinum treatment and denervation produced progressive slowing of the time to peak of the twitch (TPT) and half-relaxation time of the twitch (1/2 RT), which was more pronounced in the EDL than in the soleus. 3. Both treatments produced slowing of the relaxation curve following tetanic contraction, more marked in the EDL than in the soleus muscle. This indicates a slowing of relaxation, and suggests a prolongation of the active state of the muscle. 4. The maximum rate of rise of the tetanus did not change significantly in the EDL and soleus muscles after botulinum treatment or denervation. This suggests that there is no major change in the speed of contraction under conditions of botulinum treatment or denervation. 5. The changes produced by botulinum treatment and denervation were virtually identical in all parameters tested. This is interpreted meaning that cholinergic transmission (including muscle usage), or some other factor closely related to cholinergic transmission, accounts for the motor nerve's trophic influence in maintaining these dynamic properties of skeletal muscles.

Acetylcholine↗
PubMed1973

Development of a mammalian fast muscle: dynamic and biochemical properties correlated.

1. The isometric properties and related biochemical properties of a developing rat fast muscle (extensor digitorum longus, EDL) have been determined during the 21 days after birth.2. At birth the maximum rate of rise of tension in a tetanus, a measure of speed of contraction, is slow; it begins to increase only after the 5th day and reaches adult values by 21 days.3. The increase in actomyosin ATPase activity of the rat EDL correlates closely (P << 0.001) with the changes in the maximum rate of rise during the same period of development.4. The relaxation phase of a tetanic response begins to increase in speed promptly after birth, and reaches adult values by the 21st day. The rate of calcium accumulation by the isolated sarcoplasmic reticulum (SR) increases with a similar time course.5. The separate contributions of contraction and relaxation mechanisms to the changes in the more conventional isometric properties of maturing muscles have been analysed.

Actomyosin↗