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Biomedical subjects

W M Johnson

Publications and source records attributed to W M Johnson.

At least 19 recordsLinked to original sources
PubMed2006

Comparative vaccine efficacy of different isoforms of recombinant protective antigen against Bacillus anthracis spore challenge in rabbits.

The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyl-transferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90-100% of rabbits challenged parenterally with 129 LD50 of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine.

Animals↗
PubMed2001

Structure-based design of inhibitors of the rice blast fungal enzyme trihydroxynaphthalene reductase.

Rice Blast Disease, caused by the fungus Pyricularia oryzae, is one of the most important diseases of rice. Several enzymes in the melanin biosynthetic pathway have proven to be valuable targets for development of rice blast fungicides. In particular, inhibitors of trihydroxynaphthalene reductase (3HNR), which catalyzes the conversion of trihydroxynaphthalene to vermelone, have yielded commercially useful rice fungicides. The X-ray structure of 3HNR has been published recently, presenting an opportunity to use this information in the de novo design of novel 3HNR inhibitors that may exhibit useful rice blast activity. We used the LeapFrog program to develop a docking model for interaction of ligands with the active site of THNR. The final model gave a good correlation between calculated binding energy and log Ki and was used to design novel ligands and score compounds for synthesis. Using this as a tool, we synthesized inhibitors in the nanomolar range and also developed several inhibitors that did not conform to the properties of the THNR active site. Leapfrog was able to locate a previously unrecognized binding pocket that could accommodate these otherwise anomalous regions of structure.

Ascomycota↗
PubMed2001

Biomechanical evaluation of occipitocervical fixation devices.

Human cadaveric occipitocervical specimens were implanted with three types of instrumentation. The devices were tested biomechanically under three modes of loading to determine the stiffness of spinal constructs and the failure mechanisms of the constructs under extreme flexion. The devices tested were the AXIS Fixation System (with custom plate), the Y-Plate, and the Luque rectangle. No significant differences in stiffness among the devices were found under compression and flexion. The stiffnesses of the plate systems were statistically higher than the Luque rectangle in extension and torsion. In extreme flexion, the plate systems failed by fracture of the C2 pedicles. Modern plate systems, for occipitocervical fixation, provide more stiffness and stability than traditional wiring techniques. This study provides surgeons with information on the relative merits of modern plate and screw systems compared with traditional rod and wire constructs.

Biomechanical Phenomena↗
PubMed2001

Phospholipase region of Mycobacterium tuberculosis is a preferential locus for IS6110 transposition.

Enzymes with phospholipase C activity in Mycobacterium tuberculosis have been recently described. The three genes encoding these proteins, plcA, plcB, and plcC, are located at position 2351 of the genomic map of M. tuberculosis H37Rv and are arranged in tandem. We have previously described the presence of variations in the restriction fragment length polymorphism patterns of the plcA and plcB genes in M. tuberculosis clinical isolates. In the present work we investigated the origin of this polymorphism by sequence analysis of the phospholipase-encoding regions of 11 polymorphic M. tuberculosis clinical isolates. To do so, a long-PCR assay was used to amplify a 5,131-bp fragment that contains the plcA and plcB genes and part of the plcC gene. In the M. tuberculosis strains studied the production of an amplicon approximately 1,400 bp larger than anticipated was observed. Sequence analysis of the PCR products indicated the presence of a foreign sequence that corresponded to an IS6110 element. We observed insertion elements in the plcA, plcB, and plcC genes. One site in plcB had the highest incidence of transposition (5 out of 11 strains). In two strains the insertion element was found in plcA in the same nucleotide position. In all the cases, IS6110 was transposed in the same direction. The high level of transposition in the phospholipase region can lead to the excision of fragments of genomic DNA by recombination of neighboring IS6110 elements, as demonstrated by finding the deletion, in two strains, of a 2,837-bp fragment that included plcA and most of plcB. This can explain the negative results obtained by some authors when detecting the mtp40 sequence (plcA) by PCR. Given the high polymorphism in this region, the use of the mtp40 sequence as a genetic marker for M. tuberculosis sensu stricto is very restricted.

Base Sequence↗
PubMed2000

Differentiation of clinical Helicobacter pullorum isolates from related Helicobacter and Campylobacter species.

BACKGROUND: Helicobacter pullorum, first detected in the liver and intestinal contents of poultry, was defined as a new species in 1994. This organism has since been isolated from humans with gastroenteritis. Phenotypic as well as genotypic methods have been used to identify H. pullorum associated with cases of human disease. MATERIALS AND METHODS: Clinical isolates were submitted for identification to the National Laboratory for Enteric Pathogens by Provincial Public Health Laboratories within Canada. Phenotypic characterization was conducted using a variety of growth and biochemical tests including oxidase, catalase, indoxyl acetate, H2S production in triple sugar iron (TSI) agar, antimicrobial susceptibility testing, and fatty acid analysis. Genotypic identification was performed using a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis of a 1-kb fragment of the Helicobacter 16S rRNA gene. RESULTS: During the last 7 years (1993-1999) a total of 11 isolates of H. pullorum were detected from patients with gastroenteritis for inclusion in this study. Typically, these isolates were oxidase and catalase positive, produced optimal growth at 42 degrees C, and produced H2S in TSI. Of these 11 isolates, 1 showed DNase activity, while another did not produce H2S in TSI, and only 2 showed tolerance to 1% bile. Antimicrobial susceptibility assays indicated that 6 of the 11 strains were resistant to nalidixic acid. The fatty acid profiles of the isolates were similar to each other and provided a distinguishing profile from the other related species. Genetically identical and distinct species-specific restriction fragment-length polymorphism (RFLP) patterns were produced using the restriction enzymes Bsr I and Dde I. CONCLUSION: Phenotypic and genotypic procedures were used to identify H. pullorum. Interspecies phenotypic variability was apparent and supported the use of a polyphasic approach for identification. Similarities to the more prominent human pathogens Campylobacter coli and C. lari were also noted. The use of a combination of phenotypic and, in particular, genotypic markers for H. pullorum should prove valuable both for epidemiological investigations and for the diagnosis of disease related to this emerging human pathogen.

Campylobacter↗
PubMed2000

Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin resistance.

A multiplex PCR assay for detection of genes for staphylococcal enterotoxins A to E (entA, entB, entC, entD, and entE), toxic shock syndrome toxin 1 (tst), exfoliative toxins A and B (etaA and etaB), and intrinsic methicillin resistance (mecA) was developed. Detection of femA was used as an internal positive control. The multiplex PCR assay combined the primers for sea to see and femA in one set and those for eta, etb, tst, mecA, and femA in the other set. Validation of the assay was performed using 176 human isolates of Staphylococcus aureus. This assay offers a very specific, quick, reliable, and inexpensive alternative to conventional PCR assays used in clinical laboratories to identify various staphylococcal toxin genes.

Bacterial Toxins↗
PubMed2000

Molecular characterization of Campylobacter jejuni from patients with Guillain-Barré and Miller Fisher syndromes.

Campylobacter jejuni has been identified as the predominant cause of antecedent infection in Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS). The risk of developing GBS or MFS may be higher after infection with specific C. jejuni types. To investigate the putative clonality, 18 GBS- or MFS-related C. jejuni strains from The Netherlands and Belgium and 17 control strains were analyzed by serotyping (Penner and Lior), restriction fragment length polymorphism analysis of PCR products of the flaA gene, amplified fragment length polymorphism analysis, pulsed-field gel electrophoresis, and randomly amplified polymorphic DNA analysis. Serotyping revealed 10 different O serotypes and 7 different Lior serotypes, thereby indicating a lack of serotype clustering. Two new O serotypes, O:35 and O:13/65, not previously associated with GBS or MFS were found. Serotype O:19 was encountered in 2 of 18 strains, and none was of serotype O:41. The results of all genotypic methods also demonstrated substantial heterogeneity. No clustering of GBS- or MFS-related strains occurred and no molecular marker capable of separating pathogenic GBS or MFS from non-GBS- or non-MFS-related enteritis strains could be identified in this study. Sialic-acid-containing lipopolysaccharides (LPS) are thought to be involved in the triggering of GBS or MFS through molecular mimicry with gangliosides in human peripheral nerves. Therefore, further characterization of GBS- or MFS-related C. jejuni should target the genes involved in the synthesis of LPS and the incorporation of sialic acid.

Bacterial Typing Techniques↗
PubMed2000

Epidemiologic typing of Salmonella enterica serotype enteritidis in a Canada-wide outbreak of gastroenteritis due to contaminated cheese.

A major Canada-wide outbreak of gastroenteritis due to Salmonella enterica serotype Enteritidis phage type (PT) 8 occurred in 1998, and this was traced to contaminated cheese in a commercial lunch pack product. Phage typing and pulsed-field gel electrophoresis linked the clinical and cheese isolates of serotype Enteritidis but failed to differentiate outbreak from nonoutbreak PT 8 strains. Further differentiation was made by biotyping based on melibiose fermentation.

Bacterial Typing Techniques↗
PubMed1999

Rapid identification of Campylobacter, Arcobacter, and Helicobacter isolates by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene.

A rapid two-step identification scheme based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S rRNA gene was developed in order to differentiate isolates belonging to the Campylobacter, Arcobacter, and Helicobacter genera. For 158 isolates (26 reference cultures and 132 clinical isolates), specific RFLP patterns were obtained and species were successfully identified by this assay.

Arcobacter↗
PubMed1999

Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus.

An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3'-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N. brasiliensis strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces. All of the N. brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.

Actinomycetales↗
PubMed1999

Comparison of molecular methods for typing Vibrio parahaemolyticus.

An outbreak of Vibrio parahaemolyticus gastroenteritis on Canada's west coast in 1997 emphasized the need to develop molecular methods for differentiation and typing of these organisms. Isolates were analyzed by enterobacterial repetitive intergenic consensus sequence (ERIC) PCR, detection of restriction fragment length polymorphisms (RFLP) in rRNA genes (ribotyping), pulsed-field gel electrophoresis (PFGE), and RFLP analysis of the genetic locus encoding the polar flagellum (Fla locus RFLP analysis). ERIC PCR and ribotyping were the most informative typing methods, especially when used together, while Fla locus RFLP analysis was the least discriminatory. PFGE exhibited good discrimination but suffered from a high incidence of DNA degradation. ERIC PCR and ribotyping will be useful for the evaluation of genetic and epidemiological relationships among V. parahaemolyticus strains.

Bacterial Typing Techniques↗
PubMed1998

Microbiological and epidemiological investigation of cholera epidemic in Ukraine during 1994 and 1995.

The Ukraine cholera epidemic of 1994 and 1995 was caused by Vibrio cholerae O1, serotype Ogawa, biotype El Tor. This epidemic was centred in the area around Respublika Krim (Crimea) and Mykolajiv, and spread to include parts of southern Ukraine. Cases of cholera occurred between September and November of 1994 and between June and October of 1995. The 32 fatalities among 1370 recorded cases (case fatality ratio, 2.3%) occurred throughout the course of the epidemic. V. cholerae from patients with cholera produced cholera toxin and were resistant to multiple antibiotics, though no resistance plasmids were found. Conjugation experiments suggested that resistance to multiple antibiotics may be present on a self-transmissible genetic element. Environmental sources of V. cholerae O1 El Tor included sewage, sea and surface water, and fresh water and marine fish. All but one of the environmental V. cholerae isolated during the epidemic were very similar to selected isolates from patients at the same time, supporting the role of these environmental sources in the spread of disease.

Cholera↗
PubMed1998

Investigation of the 1994-5 Ukrainian Vibrio cholerae epidemic using molecular methods.

Thirty-seven Vibrio cholerae and four non-cholera Vibrio isolates from Ukraine, including strains from the epidemic of 1994-5, were analysed by molecular methods. Results from PFGE and ribotyping indicated that all Ukrainian toxigenic V. cholerae were closely related to each other and to an isolate from a patient from Pakistan. A non-toxigenic river water strain obtained during the height of the epidemic was more distantly related to these V. cholerae strains, while the Vibrio parahaemolyticus isolates and Vibrio alginolyticus isolate were not closely related to V. cholerae or each other. ERIC- and REP-PCR allowed the differentiation of strains identical by other methods. The results obtained confirm that the epidemic Ukrainian strains are most closely related to seventh pandemic strains from Asia and support a hypothesis that the Ukrainian epidemic of 1994-5 was caused by toxigenic environmental strains surviving since the time of the 1991 Ukrainian epidemic or before.

Cholera↗
PubMed1998

Serogroup B, electrophoretic type 15 Neisseria meningitidis in Canada.

Invasive meningococcal disease is nationally reportable in Canada. In recent years, a serogroup C genotype, designated electrophoretic type 15 (ET15), has been the most frequently isolated meningococcal genotype in Canada and has caused epidemics across the country. Between August 1993 and September 1995, there were 9 cases of invasive meningococcal disease caused by a variant of this genotype, expressing group B capsular polysaccharide. The appearance of serogroup B:ET15 was related temporally and geographically to mass immunization campaigns designed to control serogroup C meningococcal disease in Canada. Since there is no vaccine available to control serogroup B meningococcal disease, the appearance of this variant may have public-health significance if it demonstrates the same epidemic potential as its serogroup C counterpart.

Adult↗
PubMed1998

Combined use of bacteriophage typing and pulsed-field gel electrophoresis in the epidemiological analysis of Japanese isolates of enterohemorrhagic Escherichia coli O157:H7.

A total of 236 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates in Japan were investigated by bacteriophage typing, and the results were compared with those of pulsed-field gel electrophoresis (PFGE). Seven phage types (PTs) were observed in 71 isolates which were derived from 22 outbreaks. All of the isolates from ten outbreaks in the Kinki region (midwestern part of Japan) in July-August 1996 were grouped into the same PFGE type (IIa) and PT 32, while among total isolates, there were such varieties as PFGE type IIa containing five phage types and PT32 containing two PFGE types. These results suggest that the ten outbreaks should be considered to be a single outbreak, and show that the combined use of bacteriophage typing and PFGE enhances reliability in epidemiological surveys.

Bacteremia↗
PubMed1998

Double jeopardy: lung cancer after cardiac transplantation.

Two heart transplant patients were referred on the same day for evaluation of new chest radiograph abnormalities. Each proved to have advanced stage bronchogenic carcinoma. Review of the recent medical literature reveals that the combination of profound immunosuppression and a heavy smoking history puts cardiac transplant recipients at increased risk for the development of aggressive lung cancers.

Carcinoma, Bronchogenic↗
PubMed1998

Isolation of Arcobacter butzleri in raw water and drinking water treatment plants in Germany.

A total of 147 "Campylobacter-like" strains were isolated from six drinking water treatment plants within a two year investigation period. The strains were characterized by bio-, and serotyping according the scheme for Arcobacter butzleri. The result was that 100 strains were typed as Arcobacter butzleri, 17 strains as Arcobacter butzleri-like and 6 strains as Campylobacter jejuni/coli. 24 strains were typed as Arcobacter spp. The strains isolated from the treatment plants showed the same serotypes as described for human isolates. Therefore the spread of Arcobacter organisms via the drinking water path must be suspected.

Bacterial Typing Techniques↗