Use of Etest to assess synergy of antibiotic combinations against clinical isolates of Pseudomonas spp.
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Anti-Bacterial Agents↗
Biomedical subjects
E Fodor
Publications and source records attributed to E Fodor.
Orthomyxovirus replication, transcription, and polyadenylation.
Efficient in vitro and in vivo systems are now in place to study the role of viral proteins in replication and/or transcription, the regulation of these processes, polyadenylation of viral mRNAs, the viral promoter structures, or the significance of noncoding regions for virus replication. In this chapter, we review the status of current knowledge of the orthomyxovirus RNA synthesis.
DNA-Directed RNA Polymerases↗
Alternative base pairs attenuate influenza A virus when introduced into the duplex region of the conserved viral RNA promoter of either the NS or the PA gene.
The development of plasmid-based rescue systems for influenza virus has allowed previous studies of the neuraminidase (NA) virion RNA (vRNA) promoter to be extended, in order to test the hypothesis that alternative base pairs in the conserved influenza virus vRNA promoter cause attenuation when introduced into other gene segments. Influenza A/WSN/33 viruses with alternative base pairs in the duplex region of the vRNA promoter of either the polymerase acidic (PA) or the NS (non-structural 1, NS1, and nuclear export, NEP, -encoding) gene have been rescued. Virus growth in MDBK cells demonstrated that one of the mutations, the D2 mutation (U-A replacing G-C at nucleotide positions 12'-11), caused significant virus attenuation when introduced into either the PA or the NS gene. The D2 mutation resulted in the reduction of PA- or NS-specific vRNA and mRNA levels in PA- or NS-recombinant viruses, respectively. Since the D2 mutation attenuates influenza virus when introduced into either the PA or the NS gene segments, or the NA gene segment, as demonstrated previously, this suggests that this mutation will lead to virus attenuation when introduced into any of the eight gene segments. Such a mutation may be useful in the production of live-attenuated viruses.
Animals↗
Isolation and characterization of an imipenem-resistant Bacteroides fragilis strain from a prostate abscess in a dog.
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Abscess↗
The predicted antigenicity of the haemagglutinin of the 1918 Spanish influenza pandemic suggests an avian origin.
In 1982 we characterized the antigenic sites of the haemagglutinin of influenza A/PR/8/34, which is an influenza strain of the H1 subtype that was isolated from humans in 1934, by studying mutants which escaped neutralization by antibody. Four antigenic sites, namely Cb, Sa, Sb and Ca, were found to be located near the tip of the trimeric haemagglutinin spike. Based on the sequence of the haemagglutinin of the 1918 Spanish influenza, we can now specify the extent of divergence of antigenic sites of the haemagglutinin during the antigenic drift of the virus between 1918 and 1934. This divergence was much more extensive (40%) than the divergence (20%) in predicted antigenic sites between the 1918 Spanish influenza and an avian H1 subtype consensus sequence. These results support the hypothesis that the human 1918 pandemic originated from an avian virus of the H1 subtype that crossed the species barrier from birds to humans and adapted to humans, presumably by mutation and/or reassortment, shortly before 1918.
Animals↗
A clinical isolate of Bacteroides fragilis from Hungary with high-level resistance to imipenem.
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Adult↗
Occurrence of metronidazole and imipenem resistance among Bacteroides fragilis group clinical isolates in Hungary.
During the period between 1987 and 1997, various surveillances of the antibiotic resistance of B. fragilis group isolates revealed that practically all the isolates tested were susceptible to imipenem, metronidazole and chloramphenicol; very few isolates (2.5%) exhibited resistance to amoxicillin/clavulanic acid. However, similarly as in some southern European countries, the percentages of the isolates that were resistant to ampicillin, tetracycline and clindamycin were high throughout this period, and the resistance to cefoxitin increased from 6% to 16%. In 2000, isolates with intermediate or high resistance to imipenem and isolates with increased MICs to metronidazole were emerging among the clinical isolates of B. fragilis. The presence of the cfiA gene was demonstrated by PCR in 7 of 242 isolates (2.9%); 2 of them with high MICs to carbapenems harboured the IS942 element immediately upstream of the resistance genes. In the 2 B. fragilis isolates with increased MICs to metronidazole, the nim gene could be detected by PCR. The IS1186 element was found in these isolates upregulating the metronidazole resistance gene.
Anti-Bacterial Agents↗
Docosahexaenoic acid-containing phospholipid molecular species in brains of vertebrates.
The fatty acid composition of phospholipids and the contents of docosahexaenoic acid (DHA)-containing diacyl phosphatidylcholine and diacyl phosphatidylethanolamine molecular species were determined from brains of five fresh-water fish species from a boreal region adapted to 5 degrees C, five fresh-water fish species from a temperate region acclimated to 5 degrees C, five fresh-water fish species from a temperate region acclimated to 20 degrees C, and three fresh water fish species from a subtropic region adapted to 25-26 degrees C, as well as six mammalian species and seven bird species. There was little difference in DHA levels of fish brains from the different thermal environments; mammalian and bird brain phospholipids contained a few percentage points less DHA than those of the fish investigated. Molecular species of 22:6/22:6, 22:6/20:5, 22:6/20:4, 16:0/22:6, 18:0/22:6, and 18:1/22:6 were identified from all brain probes, and 16:0/22:6, 18:0/22:6, and 18:1/22:6 were the dominating species. Cold-water fish brains were rich in 18:1/22:6 diacyl phosphatidylethanolamine (and, to a lesser degree, in diacyl phosphatidylcholine), and its level decreased with increasing environmental/body temperature. The ratio of 18:0/22:6 to 16:0/22:6 phosphatidylcholine and phosphatidylethanolamine was inversely related to body temperature. Phospholipid vesicles from brains of cold-acclimated fish were more fluid, as assessed by using a 1, 6-diphenyl-1,3,5-hexatriene fluorescent probe, than those from bird brains, but the fluidities were almost equal at the respective body temperatures. It is concluded that the relative amounts of these molecular species and their ratios to each other are the major factors contributing to the maintenance of proper fluidity relationships throughout the evolutionary chain as well as helping to maintain important brain functions such as signal transduction and membrane permeability.
Animals↗
DNA sequence and functional characterization of the human and rat epidermal growth factor promoter: regulation by cell growth.
Epidermal growth factor (EGF) regulates cell growth and differentiation through intracellular transduction networks activated by its tyrosine kinase receptor, EGFR. In this report we describe the structure and DNA sequence of transcriptional control regions from both human and Wistar-rat single copy EGF genes and their functional analysis in epithelial cell cultures. By sequence comparison we show these proximal gene regions have remained conserved in evolution to -640 (relative to the rodent mRNA initiation site), where similarity is interrupted by a rodent interspersed-repeat element (SINE). Transcript mapping reveals complexity in EGF initiation site selection: whereas a single rat liver initiation site (+1) appears 30bp 3' to the TTTAA element, an additional upstream site is detected in kidney RNA at -14. In contrast, in human RNA a single initiation is observed, which is displaced 12bp 3' to the rodent RNA terminus. Both promoters were defined in transient expression assays. Our results show the human promoter to be at least 20-fold more active than the equivalent rodent sequence, although both are activated during cell proliferation and negatively regulated in contact inhibited and quiescent cultures. The results indicate EGF gene expression and cell division are temporally linked, suggesting its promoter comprises a growth responsive regulatory domain.
3T3 Cells↗
Adaptation of composition and biophysical properties of phospholipids to temperature by the Crustacean, Gammarus spp.
The compositions of lipid classes as well as the molecular species composition of subclasses (diacyl, alkylacyl, and alkenylacyl forms) of choline and ethanolamine phosphoglycerides in marine amphipod crustaceans, Gammarus spp., collected in the Baltic Sea at 8 and 15 degrees C, were studied in relation to environmental temperature. The structural order of phospholipid multibilayers was also determined. Environmental temperature had little effect on fatty acid composition. The level of some polyunsaturated fatty acids, such as 20:4, even increased in choline and ethanolamine phosphoglycerides at 15 degrees C. Ethanolamine phosphoglycerides were rich in alkenylacyl forms, especially in crustaceans collected at 15 degrees C. The accumulation of sn-1 monoenic, sn-2 polyenic diacyl, alkyl, and alkenylacyl phosphatidylethanolamines and diacyl phosphatidylcholines was observed at 8 degrees C. The phospholipid vesicles of crustaceans collected at 8 degrees C were more disordered than expected compared to those obtained from animals collected at 15 degrees C. It was concluded that, in addition to variations in the levels of sn-1 monoenic and sn-2 polyenic phospholipid molecular species with temperature, ethanolamine plasmalogens may play a role in controlling membrane biophysical properties in marine amphipod crustaceans.
Animals↗
Messenger RNAs that are not synthesized by RNA polymerase II can be 3' end cleaved and polyadenylated.
The poly(A) tail of influenza virus mRNAs is synthesized by the viral RNA polymerase by reiterative copying of a U5-7 sequence near the 5' end of the viral RNA (vRNA) template. We have engineered a vRNA molecule by replacing its viral U6 poly(A) site with a negative-sense eukaryotic polyadenylation signal. The vRNA was transcribed by the viral RNA polymerase and the transcription product was processed by the cellular 3' end processing machinery in vivo. According to the current model, 3' end processing of eukaryotic pre-mRNAs is coupled to cellular RNA polymerase II (pol II) transcription; thus only RNAs synthesized by pol III are believed to be polyadenylated efficiently. Our results show that the cellular polyadenylation machinery is nevertheless able to recognize and process RNA transcripts that are not synthesized by pol II, indicating that synthesis by pol II is not an absolute requirement for 3' end processing in vivo.
Base Sequence↗
Prevalence of the carbapenemase gene (cfiA) among clinical and normal flora isolates of Bacteroides species in Hungary.
The carbapenemase gene (cfiA) was detected in 4 (5.7%) of 70 clinical isolates of Bacteroides fragilis from different parts of Hungary. Among 24 other Bacteroides species isolated from infectious processes or from normal faecal flora, none was cfiA-positive. The MIC of imipenem and meropenem for all cfiA-positive B. fragilis isolates was < or =0.25 mg/L, but 17% of the B. fragilis and 46% of the non-fragilis Bacteroides isolates exhibited reduced susceptibility to imipenem (MICs 0.5-2 mg/L). Only one of these isolates produced increased levels of beta-lactamase. No difference was observed in the outer-membrane proteins of B. fragilis isolates that harboured the cfiA gene and those with reduced susceptibility to imipenem.
Bacterial Proteins↗
Reduced levels of neuraminidase of influenza A viruses correlate with attenuated phenotypes in mice.
We have previously obtained four transfectant influenza A viruses containing neuraminidase (NA) genes with mutated base pairs in the conserved double-stranded RNA region of the viral promoter by using a ribonucleoprotein transfection system. Two mutant viruses (D2 and D1/2) which share a C-G-->A-U mutation at positions 11 and 12 of the 3' and 5' ends, respectively, of the NA gene, showed an approximate 10-fold reduction of NA-specific mRNA and protein levels (Fodor et al., Journal of Virology 72, 6283-6290, 1998). These viruses have now allowed us to determine the effects of decreased NA levels on virus pathogenicity. Both D2 and D1/2 viruses were highly attenuated in mice, and their replication in mouse lungs was highly compromised as compared with wild-type influenza A/WSN/33 virus. The results highlight the importance of the level of NA activity in the biological cycle and virulence of influenza viruses. Importantly, mice immunized by a single intranasal administration of 10(3) infectious units of D2 or D1/2 viruses were protected against challenge with a lethal dose of wild-type influenza virus. Attenuation of influenza viruses by mutations resulting in the decreased expression of a viral protein represents a novel strategy which could be considered for the generation of live attenuated influenza virus vaccines.
Animals↗
Polyuridylated mRNA synthesized by a recombinant influenza virus is defective in nuclear export.
The poly(A) tail of influenza virus mRNA is synthesized by reiterative copying of a U track near the 5' end of the virion RNA (vRNA) template by the viral RNA polymerase. We have engineered a novel influenza A/WSN/33 virus which contains a neuraminidase (NA) vRNA with its U track mutated into an A track. Instead of synthesizing poly(A)-tailed NA mRNA, this novel virus synthesizes poly(U)-tailed NA mRNA. In infected cells, most poly(U)-tailed NA mRNA was retained in the nucleus, while most control polyadenylated NA mRNA was transported to the cytoplasm. These results suggest that the poly(A) tail is important for efficient nuclear export of NA mRNA. The mutant virus produced a reduced amount of NA and showed an attenuated phenotype, suggesting that poly(A) signal mutants of this type might be useful as potential live attenuated virus vaccines. In addition, this virus mutant might provide a useful model to further elucidate the basic mechanisms of mRNA nuclear export.
Animals↗
Expression of epidermal growth factor and its receptor in cirrhotic liver disease.
Polypeptide growth factors, including epidermal growth factor (EGF), play a central role in regulating hepatocyte growth both in vivo and in primary culture. To characterize EGF gene expression in the pathogenesis of regenerative cirrhotic fibrosis, we employed biotinylated antisense oligonucleotide probes to localize hepatic mRNA transcripts in situ. In control tissue and regenerative hepatic nodules, EGF receptor (EGFR) mRNA transcripts were expressed constitutively. In contrast, oligonucleotide probes targeting the human EGF coding region showed that EGF transcription was extremely low in control liver but was highly elevated and localized to regenerative hepatic nodules and bile duct epithelia of cirrhotic liver. To determine whether EGF mRNA accumulation accompanied a comparable increase in the EGF peptide, we performed immunohistochemistry using an antibody specific for the nonprocessed peptide aminoterminus. We observed that positive localized EGF staining paralleled its mRNA transcript. These results indicate that EGF upregulation is a characteristic of cirrhotic liver disease and suggest that persistent de novo ligand synthesis and its signaling contribute to an autocrine-mediated hepatocyte proliferation within the regenerative nodule.
Biotin↗
Lipid compositional correlates of temperature-adaptive interspecific differences in membrane physical structure.
Teleost species from cold environments possess more disordered brain synaptic membranes than species from warm habitats, thereby providing equivalent physical structures at their respective habitat temperatures. We have related this adaptive interspecific biophysical response to the fatty acid composition of brain membranes from 17 teleost species obtained from Antarctic, temperate and semi-tropical waters, as well as from rat and turkey as representative homeotherms. Cold-adaptive increases in membrane disorder (determined by fluorescence anisotropy with diphenylhexatriene as probe) were correlated with large and linear increases in the proportion of unsaturated fatty acids, from 35 to 60 % in phosphatidylcholine (PtdCho) and from 55 to 85 % in phosphatidylethanolamine (PtdEth). For PtdCho, the cold-adaptive increase in unsaturation was associated almost entirely with increased proportions (from 7 to 40 %) of polyunsaturated fatty acids (PUFAs), with mono-unsaturates (MUFAs) providing an approximately constant proportion in all species. Exactly opposite effects were evident for phosphatidylethanolamine (PtdEth). Thus, the compositional adaptation for PtdCho occurred largely by exchange of polyunsaturated and mono-unsaturated fatty acid in the sn-2 position, whilst for PtdEth it involved exchanges between saturates and mono-unsaturates at the sn-1 position. This difference may be related to the different molecular shapes of the two phosphoglycerides and the need to maintain the balance between bilayer-stabilising and -destabilising tendencies. This comparative study provides a more comprehensive view of the compositional adjustments that accompany and perhaps account for temperature-adaptive interspecific differences in membrane physical structure.
Adaptation, Physiological↗
Transcription and replication of the influenza a virus genome.
The genome of influenza A virus consists of eight segments of negative-strand viral RNA (vRNA). During the replication cycle of the virus, the genomic vRNA is transcribed into positive-strand mRNA and complementary RNA (cRNA) in the cell nucleus. The promoter for the synthesis of mRNA molecules is located in a partially double-stranded RNA structure formed by the 5'- and 3'-terminal sequences of genomic vRNA segments. The virus encoded RNA-dependent RNA polymerase complex has to interact with both ends of the vRNA in order to generate capped RNA primers by endonucleolytic cleavage of cellular pre-mRNAs for the initiation of viral mRNA synthesis. Conserved sequence elements in the 5'-end, e.g. a polymerase binding site and a U(5-7) sequence are required for polyadenylation of virus-specific mRNAs. Polyadenylation occurs by reiterative copying of the U(5-7) sequence by the viral RNA polymerase, which is bound to the 5'end of the vRNA template. The U(5-7) sequence acts directly as a template for the poly(A)-tail. During the replication cycle of the virus, a "switch" from mRNA to cRNA synthesis occurs, but the mechanism by which this switch occurs remains unclear. The viral nucleoprotein and its interaction with the polymerase proteins and vRNA might play a role in this process. In contrast to transcription, the process of replication--the synthesis of cRNA and vRNA, which are known to occur in the absence of primers--is poorly understood.
Animals↗
Characterization of beta-lactam-resistant Bacteroides fragilis isolates by use of PCR fingerprinting.
PCR fingerprinting was used for characterization of 35 beta-lactam-resistant Bacteroides fragilis strains isolated in Sweden and Hungary. Ten B. fragilis strains showed unique PCR fingerprints by use of the M13 core primer. Their main product was a DNA fragment with a length of 2000-bp which was absent in the other 25 strains and the reference strain B. fragilis ATCC 25285. The 2000-bp fragment from four imipenem-resistant strains gave rise to positive reactions in a specific PCR for detection of ccrA. Printed by the T3B primer, five B. fragilis strains, including the imipenem-resistant strains showed unique PCR fingerprints. The investigated imipenem-resistant strains produced carbapenem-hydrolysing metallo-beta-lactamases. The study indicates that the unique PCR fingerprinting profiles shown in highly beta-lactam resistant B. fragilis strains are correlated to antimicrobial resistance. The PCR fingerprinting technique is a useful tool for differentiation of Bacteroides fragilis strains with high-level beta-lactam resistance.
Journal Article↗