Intraradical colonization by arbuscular mycorrhizal fungi triggers induction of a lipochitooligosaccharide receptor.

Functional divergence of paralogs following gene duplication is one of the mechanisms leading to evolution of novel pathways and traits. Here we show that divergence of Lys11 and Nfr5 LysM receptor kinase paralogs of Lotus japonicus has affected their specificity for lipochitooligosaccharides (LCOs) decorations, while the innate capacity to recognize and induce a downstream signalling after perception of rhizobial LCOs (Nod factors) was maintained. Regardless of this conserved ability, Lys11 was found neither expressed, nor essential during nitrogen-fixing symbiosis, providing an explanation for the determinant role of Nfr5 gene during Lotus-rhizobia interaction. Lys11 was expressed in root cortex cells associated with intraradical colonizing arbuscular mycorrhizal fungi. Detailed analyses of lys11 single and nfr1nfr5lys11 triple mutants revealed a functional arbuscular mycorrhizal symbiosis, indicating that Lys11 alone, or its possible shared function with the Nod factor receptors is not essential for the presymbiotic phases of AM symbiosis. Hence, both subfunctionalization and specialization appear to have shaped the function of these paralogs where Lys11 acts as an AM-inducible gene, possibly to fine-tune later stages of this interaction.

[Socioeconomic costs due to tobacco smoking]. / Tobaksrygningens samfundsøkonomiske omkostninger.

The objective of the study was to calculate costs attributable to smoking from both a societal and a public finance perspective. The Cost-of-Illness analysis was based on incidence data from 1995 and 1996, estimated with the attributable fraction, based on English and Danish RR-estimates respectively. The indirect costs are calculated with both the friction and the human capital method. In 1995, smoking attributable costs in Denmark amounted to 4100 million DKK with the friction method and based on Danish RR-estimates, including 3600 million in direct costs and 500 million in indirect costs. A public cash flow analysis showed a net revenue of about 3900 to 5600 million DKK. Compared with previous results for Denmark (1983), the annual costs to society increased by about 118%. It is suggested that similar Cost-of-Illness analyses are carried out at regular intervals to monitor the economic consequences of smoking in society.

Detection of Streptococcus suis by in situ hybridization, indirect immunofluorescence, and peroxidase-antiperoxidase assays in formalin-fixed, paraffin-embedded tissue sections from pigs.

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1-31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.

Associations of Streptococcus suis serotype 2 ribotype profiles with clinical disease and antimicrobial resistance.

A total of 122 Streptococcus suis serotype 2 strains were characterized thoroughly by comparing clinical and pathological observations, ribotype profiles, and antimicrobial resistance. Twenty-one different ribotype profiles were found and compared by cluster analysis, resulting in the identification of three ribotype clusters. A total of 58% of all strains investigated were of two ribotypes belonging to different ribotype clusters. A remarkable relationship existed between the observed ribotype profiles and the clinical-pathological observations because strains of one of the two dominant ribotypes were almost exclusively isolated from pigs with meningitis, while strains of the other dominant ribotype were never associated with meningitis. This second ribotype was isolated only from pigs with pneumonia, endocarditis, pericarditis, or septicemia. Cluster analysis revealed that strains belonging to the same ribotype cluster as one of the dominant ribotypes came from pigs that showed clinical signs similar to those of pigs infected with strains with the respective dominant ribotype profiles. Furthermore, strains belonging to different ribotype clusters had totally different patterns of resistance to antibiotics because strains isolated from pigs with meningitis were resistant to sulfamethazoxazole and strains isolated from pigs with pneumonia, endocarditis, pericarditis, or septicemia were resistant to tetracycline.

Trends in the resistance to antimicrobial agents of Streptococcus suis isolates from Denmark and Sweden.

This study was conducted to determine the MIC values of historical and contemporary Streptoccocus suis (serotypes 2 and 7) from Denmark and S. suis (serotype 2) from Sweden. A total of 52 isolates originating from 1967 through 1981 and 156 isolates from 1992 through 1997 in Denmark and 13 isolates from Sweden were examined for their MICs against 20 different antimicrobial agents. Most antimicrobials were active against most isolates. A frequent occurrence of resistance to sulphamethoxazole was observed, with most resistance among historic isolates of serotype 7 and least resistance among isolates from Sweden. A large number of the isolates was resistant to macrolides. However, all historic serotype 2 isolates from Denmark were susceptible, whereas 20.4% of the contemporary isolates were resistant. Among serotype 7 isolates 23.3% of the historic isolates were resistant to macrolides, whereas resistance was found in 44.8% of the contemporary isolates. All isolates from Sweden were susceptible to macrolides. Time-associated frequency of resistance to tetracycline was also found. Only a single historic isolate of serotype 2 was resistant to tetracycline, whereas 43.9% of the contemporary serotype 2 isolates and 15.5% of the contemporary serotype 7 isolates were resistant. Only one (7.7%) of the isolates from Sweden was resistant. The differences in resistance between historic and contemporary isolates from Denmark were statistically significant. This study demonstrated a significant serotype-associated difference in the susceptibility to macrolides and tetracycline and demonstrated that an increase in resistance among S. suis isolates has taken place during the last 15 years to the two most commonly used antimicrobial agents (tylosin and tetracycline) in pig production in Denmark.

16S rDNA sequence variations of some Streptococcus suis serotypes.

Streptococcus suis 16S rDNA from selected serotypes has been sequenced and compared with the 16S rDNA sequences from serotypes 1 and 2 present in Genbank. After alignment the sequenced serotypes show clusters of variation. Based on these clusters, a limited phylogenetic tree showing the relationships of all of the serotypes was constructed.

[Specific serotonin uptake inhibitors in patients referred to a psychiatrist]. / Specifikke serotoningenoptagelseshaemmere hos patienter henvist til psykiatrisk speciallaege.

During a twelve month period (1995), 261 patients were referred to a psychiatrist (the author) by their general practitioners. Forty-eight patients (18%) were being treated with antidepressive medicine when first seen. Only seven patients were being treated with the traditional tricyclic antidepressive medicine, while 39 received specific serotonin reuptake inhibitors (SSRI) and two patients received other kinds of antidepressants. The SSRIs seem to be prescribed to a wide range of patients providing an unclear clinical picture, and it is assumed that the patients' desire to try the famous and popular pills is crucial when the therapeutic decision is made. The weakening of the ontological stase of endogenous depression should also not be underestimated.

Detection of immobilized amplicons by ELISA-like techniques.

The NucleoLink surface is a physically modified, thermostable, optically clear resin. It allows the covalent binding of 5'-phosphorylated oligonucleotides. Target DNA amplification by polymerase chain reaction (PCR) is accomplished by asymmetric amplification on the covalently immobilized primer that develops into immobilized amplicons. A DNA fragment of bovine leukemia virus is used as a model system for the detection of immobilized amplicons by ELISA-like techniques. Covalently bound oligonucleotides are also utilized as capture probe in the hybridization-based signal amplification for detection of an infectious organism.

Combined polymerase chain reaction-hybridization microplate assay used to detect bovine leukemia virus and Salmonella.

Here we describe the use of an assay that integrates the polymerase chain reaction (PCR) with hybridization of the amplified product for detection in the same microwell. Traditional PCR requires transportation of the amplified product to another system for characterization of samples. Transportation means time-consuming manipulation and risk of contaminating the laboratory with amplified product. Integration of amplification and specific product detection greatly reduces sample manipulations and the risk of contamination. We used the assay for detection of bovine leukemia virus and Salmonella. The results were identical with those produced by two traditional PCR methods. This assay could easily be adapted for other organisms, simply by using other primers and probes.

Fluorescent detection-polymerase chain reaction (FD-PCR) assay on microwell plates as a screening test for salmonellas in foods.

This study evaluates a polymerase chain reaction assay coupled with a fluorescent detection in microwell plates for salmonellas in food samples. Chelex 100-extracted cultures and bulk and processed food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 206 bp segment of IS200. The PCR products were then denatured by heat and transferred to CovaLink NH plates (Nunc) to which capture oligonucleotides were covalently bound. Hybridization was performed for 1 h at 55 degrees C, the microwells were washed and an alkaline phosphatase-labelled probe, complementary of an internal sequence of the PCR product, was added. After stringent washes, 100 microliters of 1 mmol 1(-1) AttoPhos (JBL Scientific) was then added to the wells and the fluorescence measurement system (Millipore). The level of detection of the assay was as low as 1-10 cfu. A total of 172 food samples were tested, both by culture and FD-PCR. Of these 53 were culture positive and 119 culture negative. The sensitivity of the FD-PCR assay was 100% and the specificity was 90.1%. Positive and negative predictive values were 82.8 and 100%, respectively. Based on the results obtained in this study it appears that the FD-PCR assay described here can be useful to screen a large number of food samples for contamination by salmonellas.

PCR product quantification by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells.

Oligonucleotides derived from IS6110, an insertion sequence from Mycobacterium tuberculosis, have been covalently immobilized on polystyrene Covalink NH microwells to develop a sandwich and a competitive non-radioactive hybridization assay for the quantitative determination of the DNA fragments obtained by polymerase chain reaction (PCR). Using the appropriate standard DNA, the method can be employed for the quantitative analysis of PCR fragments. The sandwich assay can detect as little as 3 fmol of target DNA per well and the standard curve may be used with quantities ranging from 3 to 300 fmol per well. The competitive hybridization assay is less sensitive since it is quantitative between 100 and 8000 fmol per well. We show here that both kinds of assays can be used to identify M. tuberculosis strains isolated from clinical samples. The non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells involve few and simple operations, and are thus suitable for routine diagnosis. Moreover, when stored at 5 degrees C, precoated strips can still be used for hybridization up to at least 10 months.

Covalent immobilization of DNA onto polystyrene microwells: the molecules are only bound at the 5' end.

Carbodiimide-mediated condensation of DNA onto microwells is investigated. DNA is bound onto the microwells by formation of a phosphoramidate bond between the 5' terminal phosphate group and the microwells. Immobilization of 25 to 30 ng DNA per well is obtained. DNA molecules bound covalently at only the 5' end are, ideally, perfect for hybridization. The practicability of DNA molecules bound to microwells for hybridization is investigated.