A study of enteric viruses in raw and treated sewage from two secondary treatment plants, which received sewage from Oslo city (plant A) and small municipalities in Hedmark county in Norway (plant B), showed high levels of noro-, adeno-, and bocavirus throughout the year. A seasonal variation was observed for adeno- and GII norovirus with higher levels during winter and bocavirus that had more positive samples during winter. The virus concentrations in raw sewage were comparable in the two plants, with medians (log10 genome copies per liter) of 6.1, 6.3, 6.0, and 4.5 for noro GI, noro GII, adeno-, and bocavirus, respectively. The level of hepatitis E virus was not determined as it was below the limit of quantification. The mean log10 virus reduction was 0.55 (plant A) and 1.44 (plant B) with the highest reduction found in the plant with longer hydraulic retention time. The adenoviruses were dominantly serotype 41, while serotype 12 appeared sporadically. Of the 102 raw and treated sewage samples that were tested, eight were positive for hepatitis E virus of which four were from treated sewage. Two of the four obtained gene sequences from hepatitis E virus originated from the rural sewage samples and showed high similarity with a genotype 3 strain of hepatitis E virus detected in local piglets. Two other hepatitis E virus sequences obtained from urban sewage samples showed high similarities with genotype 3 strains isolated from urban sewage in Spain and a human genotype 1 isolate from India. The study gives information on the levels of noroviruses in raw and treated sewage, which is valuable to risk assessment, information indicating that some infections with hepatitis E viruses in Norway have a regional origin and that human bocavirus 2 and 3 are prevalent in the Norwegian population.
The trypanosomatid parasite Trypanosoma brucei synthesizes fatty acids in the mitochondrion using the type II fatty acid synthesis (FAS) machinery. When mitochondrial FAS was characterized in T. brucei, all of the enzymatic components were identified based on their homology to yeast mitochondrial FAS enzymes, except for 3-hydroxyacyl-ACP dehydratase. Here we describe the characterization of T. brucei mitochondrial 3-hydroxyacyl-ACP dehydratase (TbHTD2), which was identified by its similarity to the human mitochondrial dehydratase. TbHTD2 can rescue the respiratory deficient phenotype of the yeast knock-out strain and restore the lipoic acid content, is localized in the mitochondrion and exhibits hydratase 2 activity.
Haplotype analysis of the low density lipoprotein receptor (LDLR) gene was performed in Norwegian subjects heterozygous for familial hypercholesterolemia (FH). Southern blot analysis of genomic DNA, using an exon 18 specific probe and the restriction enzyme NcoI, showed that two out of 57 unrelated FH subjects had an abnormal 3.6 kb band. Further analyses revealed that this abnormal band was due to a 9.6 kb deletion that included exons 16 and 17. The 5' deletion breakpoint was after 245 bp of intron 15, and the 3' deletion breakpoint was in exon 18 after nucleotide 3390 of cDNA. Thus, both the membrane-spanning and cytoplasmatic domains of the receptor had been deleted. A polymerase chain reaction (PCR) method was developed to identify this deletion among other Norwegian FH subjects. As a result of this screening one additional subject was found out of 124 subjects screened. Thus, three out of 181 (1.7%) unrelated Norwegian FH subject possessed this deletion. The deletion was found on the same haplotype in the three unrelated subjects, suggesting a common mutagenic event. The deletion is identical to a deletion (FH-Helsinki) that is very common among Finnish FH subjects. However, it is not yet known whether the mutations evolved separately in the two countries.
Processing and metabolism of beta-amyloid precursor protein (APP) and generation of a variety of beta-amyloid (Abeta) peptides in the human brain is essentially associated with pathophysiology of Alzheimer's disease (AD). APP degradation activity of the 68 kDa serine protease, which was originally prepared from familial AD lymphoblastoid cells and harbors beta-secretase-like activity, was analyzed by Western blot using anti Abeta 1/40 antibody and anti APP cytoplasmic domain (CT) antibody. Native lymphocyte APP (LAPP) prepared from normal or AD-derived lymphoblastoid cells was degraded by the protease, generating a 16 kDa Abeta-bearing C-terminal fragment of APP. N-terminal amino acid sequencing of the fragment indicated that the protease cleaves LAPP at the Abeta-N-terminus. When the LAPP was treated with chondroitinase ABC prior to proteolysis, the activity to generate the fragment was inhibited, but pretreatment with heparitinase resulted in no effect. Native hippocampal APP prepared from normal brain, however, did not generate the 16 kDa peptide by the protease treatment. These results suggest that the process of APP degradation and Abeta-peptides generation, including beta-secretase activity, is associated with tissue specificity of both APP substrate and proteases. They also indicate that sulfated glycoconjugates attached to a portion of APP isoforms may play a role as a molecular determinant in the proteolysis.
Type IV collagenase (gelatinase) is a 70,000 dalton neutral metalloproteinase that specifically cleaves type IV collagen in addition to degrading denatured collagen (gelatin). It is secreted in a latent proenzyme form that is converted proteolytically in the extracellular space to a 62,000 dalton active enzyme. The primary structure, enzymatic properties as well as gene structure, demonstrate that type IV collagenase is closely related with the other well characterized metalloproteinases, interstitial collagenase and stromelysin. However, the structure of type IV collagenase differs from the others in that it is larger and contains three internal repeats that resemble the type II domains of fibronectin. Also, initial characterization of the promoter region of the gene indicates that its regulation differs from the other proteinase genes. Type IV collagenase is presumably required for the normal turnover of basement membranes. Augmented activity is linked with the invasive potential of tumor cells and the enzyme is believed to play a major role in the penetration of basement membranes by metastatic cells. Measurements of enzyme activity and mRNA levels as well as immunostaining of a variety of tumor cells and tissues suggest that assays for the enzyme may have value in the follow-up of malignant growth.
Inherited retinal dystrophies represent the most important cause of vision impairment in adolescence, affecting approximately 1 out of 3000 individuals. Mutations of the photoreceptor-specific gene ABCA4 (ABCR) are a common cause of retinal dystrophy. A number of mutations have been repeatedly reported for this gene, notably the 2588G>C mutation which is frequent in both patients and controls. Here we ascertained the frequency of the 2588G>C mutation in a total of 2343 unrelated random control individuals from 11 European countries and 241 control individuals from the US, as well as in 614 patients with STGD both from Europe and the US. We found an overall carrier frequency of 1 out of 54 in Europe, compared with 1 out of 121 in the US, confirming that the 2588G>C ABCA4 mutation is one of the most frequent autosomal recessive mutations in the European population. Carrier frequencies show an increasing gradient in Europe from South-West to North-East. The lowest carrier frequency, 0 out of 199 (0%), was found in Portugal; the highest, 11 out of 197 (5.5%), was found in Sweden. Haplotype analysis in 16 families segregating the 2588G>C mutation showed four intragenic polymorphisms invariably present in all 16 disease chromosomes and sharing of the same allele for several markers flanking the ABCA4 locus in most of the disease chromosomes. These results indicate a single origin of the 2588G>C mutation which, to our best estimate, occurred between 2400 and 3000 years ago.
The Asian-specific 9-bp deletion between the genes for mitochondrial cytochrome oxidase II and lysine transfer RNA has been used to trace aboriginal human movements out of Southeast Asia and into portions of the South Pacific. Although it has been used to estimate the number of independent lineages that occur in the New World, it has not been studied in native peoples of the Beringian region. Thus, we have used PCR to amplify and compare the lengths of DNA segments surrounding this deletion in native peoples of Beringia and the adjacent regions, as well as natives of the Altai Mountains of Southwestern Siberia. Of the 176 individuals analyzed here, the deletion was found in only 3 of 25 individuals from the Ust-Kan region of the Altai Mountains. We comment on the distribution of this marker and on potential relationships between Beringians and other Native American groups in which this marker has been surveyed. One Chukchi possessed three copies of the 9-bp sequence, which suggests (1) that the number of copies of this sequence in humans may be more variable than had been believed and (2) that a mechanism of replication based on tandem duplication may be a potential explanation for the origin of this length mutation in humans.
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Numeric abundance, identity and pH preferences of methanotrophic Gammaproteobacteria (type I methanotrophs) inhabiting the northern acidic wetlands were studied. The rates of methane oxidation by peat samples from six-wetlands of European Northern Russia (pH 3.9-4.7) varied from 0.04 to 0.60 µg CH4 g(-1) peat h(-1). The number of cells revealed by hybridization with fluorochrome-labeled probes M84 + M705 specific for type I methanotrophs was 0.05-2.16 x 10(5) cells g(-1) dry peat, i.e. 0.4-12.5% of the total number of methanotrophs and 0.004-0.39% of the total number of bacteria. Analysis of the fragments of the pmoA gene encoding particulate methane monooxygenase revealed predominance of the genus Methylocystis (92% of the clones) in the studied sample of acidic peat, while the proportion of the pmoA sequences of type I methanotrophs was insignificant (8%). PCR amplification of the 16S rRNA gene fragments of type I methanotrophs with TypeIF-Type IR primers had low specificity, since only three sequences out of 53 analyzed belonged to methanotrophs and exhibited 93-99% similarity to those of Methylovulum, Methylomonas, and Methylobacter species. Isolates of type I methanotrophs obtained from peat (strains SH10 and 83A5) were identified as members of the species Methylomonaspaludis and Methylovulum miyakonense, respectively. Only Methylomonaspaludum SH10 was capable of growth in acidic media (pH range for growth 3.8-7.2 with the optimum at pH 5.8-6.2), while Methylovulum miyakonense 83A5 exhibited the typical growth characteristics of neutrophilic methanotrophs (pH range for growth 5.5-8.0 with the optimum at pH 6.5-7.5).
Abyssivirga alkaniphila gen. nov., sp. nov., an alkane-degrading, anaerobic bacterium from a deep-sea hydrothermal vent system, and emended descriptions of Natranaerovirga pectinivora and Natranaerovirga hydrolytica.
A strictly anaerobic, mesophilic, syntrophic, alkane-degrading strain, L81T, was isolated from a biofilm sampled from a black smoker chimney at the Loki's Castle vent field. Cells were straight, rod-shaped, Gram-positive-staining and motile. Growth was observed at pH?6.2-9.5, 14-42?°C and 0.5-6?% (w/w) NaCl, with optima at pH?7.0-8.2, 37?°C and 3% (w/w) NaCl. Proteinaceous substrates, sugars, organic acids and hydrocarbons were utilized for growth. Thiosulfate was used as an external electron acceptor during growth on crude oil. Strain L81T was capable of syntrophic hydrocarbon degradation when co-cultured with a methanogenic archaeon, designated strain LG6, isolated from the same enrichment. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain L81T is affiliated with the family Lachnospiraceae, and is most closely related to the type strains of Natranaerovirga pectinivora (92?% sequence similarity) and Natranaerovirga hydrolytica (90%). The major cellular fatty acids of strain L81T were C15?:?0, anteiso-C15?:?0 and C16?:?0, and the profile was distinct from those of the species of the genus Natranaerovirga. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, three unidentified phospholipids, four unidentified glycolipids and two unidentified phosphoglycolipids. The G+C content of genomic DNA was determined to be 31.7?mol%. Based on our phenotypic, phylogenetic and chemotaxonomic results, strain L81T is considered to represent a novel species of a new genus of the family Lachnospiraceae, for which we propose the name Abyssivirga alkaniphila gen. nov., sp. nov. The type strain of Abyssivirga alkaniphila is L81T (=DSM 29592T=JCM 30920T). We also provide emended descriptions of Natranaerovirga pectinivora and Natranaerovirga hydrolytica.
Commercial inactivated parenteral influenza vaccines reduced febrile (> or = 38 degrees C) respiratory illness by 53% (95% CL: 41-63%) during a 3 week outbreak in 1998 when A/Sydney/5/97(H3N2)-like influenza viruses were shown to be the predominant etiological agents and an older antigenic variant, A/Nanchang/933/95, served as the vaccine virus. The calculatory efficacy for preventing virologically diagnosed influenza infections was 57% (95% CL: 40-68%). The study population consisted of 1374 young male military conscripts. Vaccination coverage on a voluntary basis was 67%. Vaccination was ineffective in preventing febrile illness during a second epidemic wave lasting 2 weeks when mainly adenoviruses were shown to have been circulating in the garrison. Out of the 36 nasopharyngeal aspirates positive for influenza A by antigen detection, 18 A/Sydney/5/97-like strains (10 from non-vaccinated and eight from vaccinated subjects) and two A/Nanchang/933/95-like strains (both from non-vaccinated subjects) were isolated in MDCK cell cultures. Intraepidemic variation was detected among the A/Sydney/5/97-like field strains in their HA1 sequences and reactivity in HI tests, but no evidence was obtained that this variation would have been of significance to the virus in breaking through the vaccination-induced immunity.