Biomedical subjects
Publications and source records attributed to J K Andersen.
A specific PCR assay was used to detect Salmonella in enriched broths of 48 natural samples of minced pork and 48 natural samples of minced beef. By comparison with a routine culture method, the sensitivity of the PCR method was estimated to be 92% and the specificity of the PCR method was estimated to be 99%. The sensitivity of the culture method was estimated to be 50% in this study.
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To examine whether expressing high levels of monoamine oxidase (MAO-B) activity abberently in neurons results in increased sensitivity of dopaminergic neurons to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 8-week-old transgenic mice expressing high neuronal levels of MAO-B were compared with age-matched nontransgenic littermates following i.p. injections of 30 mg/kg body weight of the protoxin. Levels of striatal dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), as well as tyrosine hydroxylase (TH)-immunopositive cell numbers in the substantia nigra (SN) were compared 1 week later between transgenics and controls. No difference was found in any of these parameters, indicating that high neuronal MAO-B levels does not cause increased sensitivity to MPTP, and therefore neither conversion of MPTP to its active form, 1-methyl-4-phenyl pyridium (MPP+) by MAO-B nor MPP+ uptake by the dopaminergic transporter are likely to be the rate-limiting step in the toxicity of this compound.
To achieve gene delivery to sensory neurons of the trigeminal ganglion, thymidine kinase-negative (TK-) herpes simplex viruses (HSV) containing the reporter gene lacZ (the gene for E. coli beta-galactosidase) downstream of viral (in vectors RH116 and tkLTRZ1) or mammalian (in vector NSE-lacZ-tk) promoters were inoculated onto mouse cornea and snout. Trigeminal ganglia were removed 4, 14, 30, and 60 days after inoculation with vectors and histochemically processed with 5-bromo-4-chloro-3 indolyl-beta-galactoside (X-Gal). With vector tkLTRZ1, large numbers of labeled neurons were observed in rostromedial and central trigeminal ganglion at 4 days after inoculation. A gradual decline in the number of labeled neurons was observed with this vector at subsequent time points. With vectors RH116 and NSE-lacZ-tk, smaller numbers of labeled neurons were seen at 4 days following inoculation than were observed with vector tkLTRZ1. No labeled neurons could be observed at 14 days after inoculation with vectors RH116 and NSE-lacZ-tk. Immunocytochemistry for E. coli beta-galactosidase and in situ hybridization to HSV latency-associated transcripts revealed labeled neurons in regions of the trigeminal ganglion similar to that observed with X-Gal staining. A comparable distribution of labeled neurons in trigeminal ganglion was also observed after application of the retrograde tracer Fluoro-Gold to mouse cornea and snout. These data provide evidence that retrogradely transported tk- herpes virus vectors can be used to deliver a functional gene to sensory neurons in vivo in an anatomically predictable fashion.
A pair of polymerase chain reaction (PCR) primers, YC1 and YC2, selected from the sequence of the invasin locus (inv) of Y. enterocolitica, has been evaluated for specific detection of pathogenic Y. enterocolitica by PCR. The primers were hybridized at high stringency conditions to DNA from 65 pathogenic Y. enterocolitica, 16 non-pathogenic Y. enterocolitica, 18 other Yersinia and 124 non-Yersinia strains. YC2 hybridized to the pathogenic Y. enterocolitica only, while YC1 hybridized weakly to nine non-Yersinia as well. In a PCR with annealing at 64 degrees C all Y. enterocolitica, pathogenic and non-pathogenic, were positive. However, DNA from 60 non-Y. enterocolitica was amplified. With annealing at 72 degrees C, 10 non-pathogenic Y. enterocolitica and 41 non-Y. enterocolitica were positive. When a two-step PCR assay with annealing at 72 degrees C, hot-start and 1% dimethylsulphoxide (DMSO) were used, only DNA from the pathogenic Y. enterocolitica were amplified. The limit of detection was shown for four different strains to be less than 10 cells per PCR tube.
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1. Herpesvirus infection with genetically engineered vectors is a way to deliver foreign gene products to various cell populations in culture and in vivo. Selective neuronal gene expression can be achieved using the neuron-specific enolase (NSE) promoter regulating expression of a transgene placed in and delivered by a herpesvirus vector. 2. We sought to determine the anatomical specificity and efficiency of herpesvirus-mediated gene transfer into the rat brain following placement of virus particles carrying a transgene (lacZ) under control of the NSE promoter. The virus utilized was thymidine kinase (TK) deficient and therefore replication deficient in the brain. 3. Infusion of 10(6) plaque-forming units of virus into the striatum caused a limited number of striatal neurons to express the lacZ transgene mRNA and protein product 7 days postinfection. In addition, small numbers of neurons expressing the transgene mRNA and protein were found ipsilateral to the viral injection in the frontal cortex, substantia nigra pars compacta, and thalamus. Neurons at these anatomic loci project directly to the striatal injection site. No other cells within the brains of injected animals expressed the lacZ gene. 4. While this herpesvirus NSE vector was capable of introducing novel functional genetic information into postmitotic neurons within defined neuroanatomic constraints, the numbers of neurons expressing detectable levels of beta-galactosidase was minimal. The calculated efficiency of delivery and transgene expression at 7 days postinfection was 1 transgenic neuron per 10(4) virus particles infused. 5. We conclude that NSE probably is not an optimal promoter for use in gene delivery to CNS neurons in herpesvirus vectors and that the efficacy of gene delivery using other neuron-specific promoters placed at various sites in the herpes viral genome needs to be explored.
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A herpes simplex virus (HSV) vector in which the mammalian promoter for neuron-specific enolase (NSE) controls expression of a marker gene was analyzed for its ability to drive expression of this foreign gene in culture and in vivo. In cultured cells, the vector appeared to give neuron-specific expression. Introduction of 10(6) pfu of the virus into the adult rat caudate nucleus by stereotactic injection was not toxic to the animals and yielded beta-galactosidase (beta-gal)-positive neurons for at least 30 days after viral inoculation. This recombinant herpes virus vector is the first described to use a mammalian promoter to yield extended expression of a foreign gene product in the adult mammalian central nervous system (CNS).
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Human monoamine oxidases A and B were expressed under the control of a galactose inducible promoter in Saccharomyces cerevisiae. The two MAO isoenzymes were found located in the yeast mitochondrial outer membrane, probably in different orientations as suggested by controlled proteolysis experiments. A high level of both human MAO-A or -B activities is measured in intact mitochondria without the need for any detergent solubilisation step. The substrate and inhibitor selectivities of the membrane-bound MAOs are highly similar to those of purified human enzymes. The level of MAO-B activity, however, is selectively lowered when bound to the membrane.
A review of works concerning different aspects of the epidemiology of human pathogenic Yersinia enterocolitica biogroup IV/serogroup O:3 (Y. enterocolitica O:3) is given. To investigate the epidemiology of Y. enterocolitica O:3 in Danish herds of pigs, tonsil swabs from 2218 freshly slaughtered pigs originating from 99 herds were examined. The organism was isolated from 25% of the pigs and from 82% of the herds. No herd management factor could be associated with the presence of the organism. The effect of slaughtering technique on surface contamination with Y. enterocolitica O:3 was investigated. 1256 pigs were slaughtered by different evisceration techniques. When a mechanical bung cutter was used instead of the traditional, manual evisceration the contamination was reduced markedly, especially when the rectum and anus were enclosed in a plastic bag prior to the removal of the gut. It was possible to reduce the rate of the surface contamination from 26% on the medial hind limb and 13% on the split sternum and surroundings to about 2% for both sampling sites. An investigation of the presence of Y. enterocolitica O:3 in meat and meat products in retail butcher's shops was performed. The organism was detected in 10 of 33 samples of minced pork and in three of 24 samples of minced beef, but in none of 32 samples of sliced vacuum packed, low to medium salt meat products. The positive minced beef samples were collected at butcher's shops from which positive samples of minced pork were found as well. It is concluded that Y. enterocolitica O:3 is common in pork with a risk of cross-contamination to other products for example ready-to-eat meat products that might be a source of human infection.
Intra-species restriction fragment length polymorphisms (RFLPs) of Yersinia enterocolitica were detected in assays with a cloned DNA fragment from Legionella pneumophila that included the 16S and 23S rRNA genes. By use of this method it was possible to identify different RFLP types within biogroups/serogroups which were indistinguishable by other means. Thus the 37 biogroup IV/serogroup O3 strains isolated worldwide from pig carcasses, pork and human patients, were subdivided into five different RFLP-types. Typing based on RFLP analysis was sensitive and independent of phenotypic characters. The method will be of value for the identification and evaluation of possible reservoirs and routes of infection.
We have cloned and characterized two variants of PC12 cells. MPT1 cells were selected by their resistance to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and variant 2068 was isolated nonselectively as a large, flat-cell variant commonly occurring in PC12 cultures. Variant 2068 cells also exhibit resistance to MPTP. Karyotype analysis revealed that these variants are true derivatives of wild-type PC12 cells; however, each variant is tetraploid, whereas the wild-type parent is diploid. The two variants contain an altered level and composition of lactate dehydrogenase isoenzymes, which could account for a previously described difference in lactate metabolism. Both variants exhibit a partial loss of transformed phenotype in culture in that they are nonrefractile, grow in monolayers, and fail to multiply in soft agar. We suggest that this alteration in transformed phenotype may result in altered mitochondria and lactate dehydrogenase and thus account for their resistance to MPTP. Compared with wild-type PC12 cells, MPT1 cells have a decreased level of fos mRNA and an increased level of myc mRNA; the latter results from an increased level of transcription of exon 1 of the myc gene. Studies with hybrid cells obtained by fusing MPT1 cells with wild-type-like cells show that most, but not all, of the parameters of the MPT1 phenotype predominate.
Evidence is presented for the extent of contamination of freshly slaughtered pig carscasses with human pathogenic Yersinia enterocolitica and shows the significance of faecal contamination as a source of infection. Swab samples collected from the rectum and the surface of a total of 1458 pig carcasses were examined for the presence of human pathogenic Y. enterocolitica. Y. enterocolitica, biovar IV, serogroup 0:3, were isolated from the rectum of 360 pigs (24.7%). The organism was isolated from carcass surfaces with varying frequencies depending on the evisceration technique. Manual evisceration was found to correspond with high frequencies of contamination: 26.3% on the medial hind limb and 12.9% on the split sternum. The use of a mechanised bung cutter was found to reduce the rate of contamination, especially when the bung cutter was used in connexion with enclosing the anus and rectum in a plastic bag to minimise faecal contamination. When carcasses were eviscerated in this way, it was possible to reduce carcass contamination to 1.9% on the medial hind limb, 1.0% in the pelvic duct, and 2.2% on the split sternum.
Twenty-six children with exogenous asthma were studied by case history (CH), skin tests (ST), allergen-specific serum IgE (RAST), basophil histamine release (HR), and bronchial provocation tests (BPT). Nine standardized allergens were used from the following groups: house dust mite, animal dander, pollen, and mould fungi. For each allergen, the same batch was used throughout for all in vivo and in vitro studies, and the tests were carried out in a controlled, double-blinded manner, independent of each other. Different allergen concentrations were used and the results graded on a semi-quantitative scale. Approximately 120 comparable investigations, using in vivo and in vitro tests were carried out, followed by calculations of the sensitivity and specificity for each test alone, as well as for various combinations of tests, at various allergen-concentrations, and compared with the result of BPT, i.e. the true diagnosis. In this way the results could be used to directly compare the diagnostic value of each test, and combinations of tests. In general, a careful CH combined with ST gave the most reliable results. RAST was the most unreliable single analysis. When pollen or mite allergy was suspected, a combination of ST with either RAST or HR gave a further diagnostic reliability. HR as a single analysis did not seem to offer advantages over existing methods. None of the tests, or combinations of tests offered 100% sensitivity and specificity. By altering the concentration of allergen, the sensitivity, or specificity could be increased, but only at the expense of a decrease in the other. BPT is still necessary in many instances when a specific diagnosis is required.
The wound-induced carboxypeptidase inhibitor in potato leaves was shown to be localized in the central vacuoles of the cells. The inhibitor was quantified by immunological assays (ELISA) in protoplasts and vacuoles isolated from upper unwounded leaves of 5- to 6-week old potato plants that had been wounded on their lower leaves 48 hours earlier to induce the accumulation of the carboxypeptidase inhibitor. The regulation of the synthesis and compartmentation of the inhibitor is similar to that of wound-induced serine proteinase Inhibitors I and II in potato and tomato leaves and appears to be part of an induced defense response against attacking pests.
Within 4 hr after wounding the lower leaves of young potato and tomato plants, a rapid and remarkable change is induced in the cells of upper undamaged leaves that results in extensive lysis of protoplasts during their isolation. Protoplast yields from unwounded upper leaves, 4 hr after wounding a lower leaf by crushing with a hemostat, decreased 25% below yields from leaves of unwounded plants. From 8 to >20 hr after wounding, protoplast yields were less than half of those from control plants. Multiple woundings decreased yields even further, as did chewing of the lower leaves by tobacco hornworms over a period of several minutes. In addition, within 4 hr of excising young tomato plants at their base with a razor blade, a 90% decrease in leaf protoplast yields was recorded. The major loss of protoplasts induced by wounding was primarily due to an increased cell lysis during protoplast isolation. Cell lysis was apparently due to a weakened cell membrane, because newly recovered protoplasts released from leaves of wounded plants were extremely fragile and exhibited 70% lysis during low speed centrifugation, compared to 20% lysis of protoplasts recovered from control plants. We conclude that a signal is released by wounding that is rapidly transmitted or transported through the plants to induce a profound change in the leaf cell membranes that renders them fragile during protoplast isolation. It is proposed that this signal may play a role in inducing cellular changes in the plant cells as part of their responses to environmental stress such as pest attacks.