The recombinant Ca2+-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of a fine-grained polymer or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with a biotin residue on the 3'-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide, was used as a DNA template. The probe in the hybridization complex was labeled by elongation of the chain using Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, ensures a high sensitivity of the assay (no less than 10(-15) mol of DNA template), and enables quantitative determination of the amount of DNA template in the tested sample.
A Gram-reaction-negative, rod-shaped, yellow-pigmented, motile by gliding bacterial strain, designated RU-4-M-4(T), was isolated from intertidal sediment of Sakhalin Island in Russia. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RU-4-M-4(T) was related to the genus Algibacter and had highest 16S rRNA gene sequence similarity with Algibacter pectinivorans KACC 14153(T) (97.2%). The major cellular fatty acids were iso-C15 : 0 3-OH, C15: 0 and iso-C15 : 1 G. The predominant menaquinone was MK-6. The polar lipid profile contained phosphatidylethanolamine, three unidentified aminolipids and two unidentified lipids. The genomic DNA G+C content of strain RU-4-M-4(T) was 36.4 mol%. Combined data from phenotypic, phylogenetic and DNA-DNA relatedness studies demonstrated that strain RU-4-M-4(T) is a representative of a novel species of the genus Algibacter , for which we propose the name Algibacter amylolyticus sp. nov. (type strain RU-4-M-4(T)?=LMG 28383(T)?=DSM 29199(T)).
The taxonomic position of 12 isolates tentatively assigned to the genus Bifidobacterium on the basis of a limited phenotypic characterization was examined. The isolates were collected between 1978 and 2005 in Belgium, Sweden and Norway, and originated from various human clinical samples, including urine, blood, urethra, oral cavity, tonsil, and abscesses of lung and aortic valve. On the basis of band number and clustering analysis, repetitive DNA element-based PCR fingerprinting using the BOXA1R and (GTG)(5) primers indicated that the clinical isolates represented a taxon probably not belonging to the genus Bifidobacterium. Analysis of 16S rRNA gene sequence similarities revealed that the isolates were most closely affiliated to Parascardovia denticolens LMG 18312(T) (93.0-93.2 %), Scardovia inopinata LMG 18313(T) (92.9-93.1 %) and other members of the Bifidobacteriaceae, indicating that the isolates belong to a novel genus within that family. This observation was further substantiated by the results of partial sequencing of the heat-shock protein 60 gene (hsp60) and determination of the DNA G+C contents (47.3-48.3 mol%). Members of the novel taxon can be phenotypically distinguished from S. inopinata, P. denticolens and Gardnerella vaginalis by the ability to grow on agar under aerobic conditions and on the basis of positive reactions for acid production from L-arabinose, raffinose, salicin and D-xylose. Unambiguous phenotypic differentiation from Aeriscardovia aeriphila and Bifidobacterium species may be difficult, so phenotypic analyses should be complemented by molecular methods. The values for DNA-DNA binding among four members of the novel genus were in the range of 89-100 %, indicating that the strains should be considered as a single novel species of a novel genus, for which the name Alloscardovia omnicolens gen. nov., sp. nov. is proposed. The type strain of Alloscardovia omnicolens is CCUG 31649(T) (=LMG 23792(T)).
This molecular study analyzed the microbiota of primary root canal infections from adult Norwegian patients.
Samples were taken from the necrotic root canals of teeth with symptomatic (n = 13) or asymptomatic (n = 21) apical periodontitis and chronic apical abscesses (n = 9). DNA was extracted from samples, and bacterial identifications were performed by a closed-ended reverse-capture checkerboard approach targeting 50 candidate endodontic pathogens.
Bacterial DNA was detected in all cases. In teeth with asymptomatic apical periodontitis, the most frequent taxa were Dialister invisus (71%), Fusobacterium nucleatum (62%), and Porphyromonas endodontalis (62%). In chronic apical abscesses, the most prevalent taxa were P. endodontalis (100%), D. invisus (89%), Parvimonas micra (78%), and Solobacterium moorei (78%). In teeth with symptomatic apical periodontitis, the most prevalent taxa were D. invisus, P. endodontalis, S. moorei, Propionibacterium acnes, and Streptococcus species (all in 69%). None of the targeted taxa were significantly associated with either sinus tract or pain (P > .05), except for Selenomonas sputigena, which was more frequently found in painful cases (P = .04). No taxa were found in significantly higher levels in any conditions (P > .05). Cluster analyses revealed bacterial groupings that differed between cases with and without pain.
Although basically the same species were highly prevalent in the different conditions examined and none of the most prevalent taxa were positively associated with symptoms, results revealed that species formed different partnerships and associations in samples from teeth with or without pain. Therefore, it is possible that more virulent multispecies communities can form as a result of overall bacterial combinations and give rise to acute inflammation.
An experimental rat model to study acute cytomegalovirus infections is described. Eight-week old male Brown Norway rats, immunosuppressed by total body irradiation, were infected with rat cytomegalovirus (RCMV). The effects of infection were determined by survival rates and the presence of virus or viral components in different organs was assayed by plaque test, immunoperoxidase staining, dot-blot DNA hybridization and in situ DNA hybridization. At days 10-post infection nearly 90% of the animals had died. Spleen, liver and bone marrow were heavily infected. Interstitial pneumonia was observed. Pathological findings strongly resembled the full scale of lesions in human CMV infections. Anti-RCMV hyperimmune serum was effective against mortality from RCMV infection and viral spread to lungs and liver was prevented. This model is appropriate for studies on the pathogenesis and antiviral therapy of CMV infections in the immunocompromised host.
After infection with the neurotropic CAM/RBH measles virus (MV) strain, newborn Lewis rats succumb to an acute necrotizing encephalopathy. Passive transfer of neutralizing monoclonal antibodies directed against MV hemagglutinin prevented this disease process. Instead, either an antibody-induced acute or subacute measles encephalitis developed after a prolonged incubation period with a restricted expression of MV structural proteins. The molecular biological analysis of MV gene expression in brain tissue of rats treated with MV-neutralizing antibodies revealed a transcriptional restriction of viral mRNAs, particularly for the envelope proteins, leading to a steep expression gradient. Based on in situ hybridization, it was concluded that the efficiency of transcription of viral genes at the single-cell level is reduced compared with that of controls. Passive immunization with monoclonal antibodies directed against other MV structural proteins proved to be ineffective. Similar results were obtained in MV-infected weanling Brown Norway rats. These rats developed a clinically silent encephalitis in the presence of high titers of neutralizing antibodies. In such animals, a pronounced attenuation of the viral gene transcription was observed. These findings indicated that neutralizing antibodies directed against a restricted set of specific antigenic sites on the viral hemagglutinin protein expressed on cell membranes exert a modulating effect on the viral gene expression at the level of transcription. This phenomenon contributes to the switch from the acute cytopathic effect to a persistent infection in the central nervous system.
An underlying cause of type III hyperlipoproteinemia is the presence of variant forms of apolipoprotein (apo) E that are defective in binding to apo B,E low density lipoprotein receptors. This disorder is associated almost exclusively with the apo E2/2 phenotype. However, structural and functional heterogeneity have been demonstrated within this phenotype. The apo E2(Arg158----Cys) variant, displaying 1% of normal apo E3 binding activity, is the most defective known form. In this study, we describe a method in which a pair of 19-mer synthetic oligonucleotide probes were used to distinguish between DNA coding for arginine or cysteine at position 158 in apo E. The specificity of the probes was demonstrated by using DNA from subjects whose apo E protein sequence or phenotype was known. The probes were used to screen a French-Canadian population of 34 apo E2/2 subjects to determine the frequency of the apo E2(Arg158----Cys) variant. All 34 subjects, most of whom displayed clinical or biochemical features of type III hyperlipoproteinemia, were found to be homozygous for apo E2(Arg158----Cys), strongly suggesting that this variant is the most common form of apo E2 within this ethnic and clinical population. In addition, the utility of this approach in detecting new apo E mutants was demonstrated when DNA from one of the apo E3/3 control subjects, whose family has a history of hyperlipidemia and coronary artery disease, reacted with both probes. This result suggests that this subject is heterozygous for normal apo E3 and a new apo E3 variant that is likely to be functionally equivalent to apo E2(Arg158----Cys).
Four bacterial strains, LFT 1.7T, LT2C 2.5, LT4C 2.8 and TM 4.6, were isolated from great scallop (Pecten maximus) larvae and tank seawater in a Norwegian hatchery and characterized by a polyphasic approach including determination of phenotypic, chemotaxonomic and genomic traits. All were Gram-stain-negative, motile rods, oxidase- and catalase-positive and required sea salts for growth. Major fatty acids present were summed feature 3 (C16?:?1?7c/C16?:?1?6c), summed feature 8 (C18?:?1?7c or C18?:?1?6c), C16?:?0, C14?:?0, summed feature 2 (C14?:?0 3-OH/iso-C16?:?1 I), C12?:?0 3-OH and C12?:?0. Strain LFT 1.7T contained menaquinone MK-6 as the sole respiratory quinone. Phylogenetic analysis based on 16S rRNA gene sequences indicated that all strains formed a distinct lineage within the genus Arcobacter with a low similarity to known species (94.77-95.32?%). The DNA G+C content was 28.7?mol%. Results of in silico DNA-DNA hybridization and average nucleotide identity confirmed that the isolates constitute a novel species of Arcobacter, for which the name Arcobacter lekithochrous sp. nov. is proposed. The type strain is LFT 1.7T (=CECT 8942T=DSM 100870T).
Biopsy specimens from Alaskan Native patients with nasopharyngeal carcinoma (NPC) and from other patients seen on the otolaryngology service were tested for Epstein-Barr virus-specific DNA and nuclear antigen (EBNA). Serum samples from both groups were tested for various EBV-related antibodies. EBV DNA and EBNA results were in agreement in 29 of 31 tissue specimens tested by the two methods. Ten of 11 biopsies containing NPC cells were positive for EBV DNA. Two NPC patients had biopsies that showed only atypical epithelium but were also positive for EBV DNA or EBNA. The other tissue specimens were negative except for biopsies from two patients: one with a parotid gland lymphoepithelial lesion; another with undifferentiated carcinoma of salivary gland origin.