Broth disk elution method for anaerobic bacteria: a collaborative study to assess its reliability for clinical purposes

A collaborative study involving seven laboratories was undertaken to evaluate the reproducibility and the reliability of the broth disk elution test against anaerobic bacteria by comparing with the reference agar dilution method. A two breakpoint broth test was also evaluated. Assays were performed using the same testing conditions (i.e. medium, temperature, atmosphere and incubation time). One hundred Gram-negative and Gram-positive clinical isolates were initially studied. Overall agreement of 98.5% and 97.5%, were found for disk elution and the two breakpoint tests, respectively. In order to assess the reliability of the disk elution test, two different lots (LOT1 and LOT2) of disks of piperacillin and clindamycin were selected, to obtain two final concentrations after dilution (10 and 60 mg/mL; 1 and 4 mg/mL, respectively). Two hundred and eighty assays were performed against one strain of both Bacteroides fragilis(piperacillin MIC, 8.0 mg/mL; clindamycin MIC, <0.5 mg/mL) and Bacteroides thetaiotaomicron(piperacillin MIC, 16.0 mg/mL; clindamycin MIC, <0.5 mg/mL). With LOT 1, considering both species and both antibiotics, the agreement among six laboratories ranged from 85% to 100% (P > 0.05) with the higher concentration. Overall agreement among all laboratories was 91%. No optimal agreement (>90%) for clindamycin-Bacteroides thetaiotaomicron using the LOT1 (77%) was found. Since this finding was not observed with LOT2 (100% agreement), discrepancies were attributed to variation between lots. Overall agreement with LOT2 was 100% for all centres. The present study indicates that the broth disk elution method proved to be a reliable and suitable alternative for routine susceptibility testing for anaerobic bacteria, as a resistance screening method for clinical purposes.     

Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae

A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae. Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBA1 coding for yeast aldolase. The primary structure of the FBA1 gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39,608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5' end and the 90 base pairs at the 3' end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBA1 gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase allele fba1::URA3 lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.     

Cloning of dehydrin coding sequences from Brassica juncea and Brassica napus and their low temperature-inducible expression in germinating seeds.

A novel subclass of dehydrin genes, homologous to the Raphanus sativus late embryogenesis-abundant (LEA) protein (RsLEA2) and the Arabidopsis thaliana dehydrin, was isolated from Brassica juncea and Brassica napus, here designated BjDHN1 and BnDHN1, respectively. The cDNA of BjDHN1 and BnDHN1 genes share 100% nucleotide identity. The encoded protein is predicted to consist of 183 amino acid residues (molecular mass of 19.2 kDa and pI of 7.0). It shares 85.3% and 65.4% amino acid sequence identity with the RsLEA2 and Arabidopsis dehydrin, respectively. This Brassica dehydrin also features a "Y(3)SK(2)" plant dehydrin structure. Expression analysis indicated that the Brassica dehydrin gene is expressed at the late stages of developing siliques, suggesting that the gene expression may be inducible by water-deficit. Analysis of gene expression also indicated that in germinating seeds the gene expression was inducible by low temperature. Seed germination under low temperature was compared between B. juncea and B. napus. The results showed that B. juncea seeds germinated faster than B. napus seeds. Expression of Brassica dehydrin gene was also examined as a function of seed germination under low temperature.     

Naturally occurring human autoantibodies recognize a fetal brain antigen identified as microtubulus associated protein 1B

We have recently reported naturally occurring autoantibodies against a large fetal brain antigen (FBA). Now we describe the process of purification and identification of this particular FBA. The brains of newborn rabbits were solubilized and purified with preparative gel electrophoresis. The protein fractions were concentrated and desalted and the fractions were tested by a known positive serum. On membrane digestion of the FBA-band gave a twelve amino acid sequence that resulted in best identity score for mouse, rat and human microtubule-associated protein (MAP) 1B: a member of the microtubule-associated protein family. Monoclonal anti-MAP1B recognized a band in immunoblots of the brain homogenate and of the partially purified fractions with the same electrophoretic mobility as that recognized by a known anti-FBA positive serum. When adult rabbit brain was used as an antigen, the anti-MAP1B failed to recognize any bands on immunoblots. MAP lB has not been previously known as an autoantigen, even though many structural proteins of the neuronal cytoskeleton are known to be targets of naturally occurring autoantibodies. MAP 1B is a functionally important regulatory protein in the developing brain; thus autoantibodies against MAP1B may affect the normal development.     

Comparative Analysis of Mitochondrial Genomes in <Emphasis Type="Italic">Bombina</Emphasis> (Anura; Bombinatoridae)

The complete mitochondrial genomes of two basal anurans, Bombina bombina and B. variegata (Anura; Bombinatoridae), were sequenced. The gene order of their mitochondrial DNA (mtDNA) is identical to that of canonical vertebrate mtDNA. In contrast, we show that there are structural differences in regulatory regions and protein coding genes between the mtDNA of these two closely related species. Corrected sequence divergence between the mtDNA of B. bombina and B. variegata amounts to 8.7% (2.3% divergence in amino acids). Comparisons with two East Asian congeners show that the control region contains two repeat regions, LV1 and LV2, present in all species except for B. bombina, in which LV2 has been secondarily lost. The rRNAs and tRNAs are characterized by low nucleotide divergence. The protein coding genes are considerably more disparate, although functional constraint is high but variable among genes, as evidenced by dN/dS ratios. A mtDNA phylogeny established the distribution of autapomorphic nonsynonomous substitutions in the mitogenomes of B. bombina and B. variegata. Nine of 98 nonsynonomous substitutions led to radical amino acid replacements that may alter mitochondrial protein function. Most radical substitutions were found in ND2, ND4, or ND5, encoding mitochondrial subunits of complex I of the electron transport system. The extensive divergence between the mitogenomes of B. bombina and B. variegata is discussed in terms of its possible role in impeding gene flow in natural hybrid zones between these two species.     

Physical Linkage of the B29/Ig-β (CD79B) Gene to the Skeletal Muscle, Sodium-Channel, and Growth Hormone Genes in Rat and Human

In the region between the polyadenylation site of the rat skeletal muscle (SkM) Na-channel gene and the 5′ end of the growth hormone (GH) gene, a gene coding for B-cell-specific membrane protein B29/Ig-β was found and noted to have the same orientation as the Na-channel and GH genes. Rat B29/Ig-β gene was 3.1 kb in length with six exons and was separated by 3.3 and 9.3 kb from Na-channel and GH genes, respectively. Rat B29/Ig-β protein comprised 228 amino acids, and its amino acid sequence was 85 and 69% identical with the mouse and human counterparts, respectively. With the long-area PCR method, genomic DNA connecting human SkM Na-channel (SCN4A) and B29/Ig-β (CD79B) genes and CD79B and GH (GH1) genes was amplified, and the physical linkage of SCN4A/CD79B/GH1 genes in the human genome was established. The human CD79B gene was separated by 6.3 and 10.5 kb from the SCN4A and GH1 genes, respectively.     

Genome-Scale Metabolic Network Validation of Shewanella oneidensis Using Transposon Insertion Frequency Analysis

Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA). TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1) previous genome-wide direct gene-essentiality assignments; and, 2) flux balance analysis (FBA) predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions.     

Gluconeogenic mutations in Pseudomonas aeruginosa: genetic linkage between fructose-bisphosphate aldolase and phosphoglycerate kinase

Mutants of mucoid Pseudomonas aeruginosa defective in fructose-bisphosphate aldolase (FBA), NADP-linked glyceraldehyde-3-phosphate dehydrogenase (GAP) or 3-phosphoglycerate kinase (PGK) were unable to grow on gluconeogenic precursors like glutamate, succinate or lactate. The gap and pgk mutants could grow on glucose, gluconate or glycerol, but fba mutants could not. This suggests that the metabolism of glucose or gluconate does not require either PGK or NADP-linked GAP but does require the operation of the aldolase-catalysed step. For gluconeogenesis, however, all three steps are essential. Recombinant plasmids carrying genes for FBA, PGK, GAP or phospho-2-keto-3-deoxygluconate aldolase (EDA) activities were constructed from a genomic library of mucoid P. aeruginosa selecting for complementation of deficiency mutations. Analysis of their complementation profile indicated that one group of plasmids carried fba and pgk genes, while another group carried eda, 6-phosphogluconate dehydratase (edd) and glucose-6-phosphate dehydrogenase (zwf) genes. The gap gene was not linked to any of these markers. Partial restoration of FBA activity in spontaneous revertants of Fba- mutants was accompanied by a concomitant loss of PGK activity. These experiments indicate a linkage between the fba and pgk genes on the P. aeruginosa chromosome.     

Mapping and association studies of diabetes related genes in the pig

The mitogen-activated protein kinase 8 (MAPK8), resistin (RETN), 11 beta hydroxysteroid dehydrogenase isoform 1 (HSD11B1) and protein kinase B Akt2 (AKT2) genes are all genes known to affect insulin signalling and have been implicated in the progression of obesity and type 2 diabetes in humans. In this study, polymorphisms in the porcine diabetes related MAPK8, RETN, HSD11B1 and AKT2 genes were identified, mapped and their associations with phenotypic measurements in swine were analysed. Polymorphisms detected in the MAPK8, RETN and HSD11B1 loci were used to genotype a Berkshire-Yorkshire pig breed reference family. Using linkage analysis, RETN, HSD11B1 and MAPK8 genes were mapped to pig chromosomes 2, 9 and 14, respectively, while the AKT2 gene was physically mapped to pig chromosome 6q21. Results presented here suggest associations between the polymorphisms in the MAPK8, RETN and HSD11B1 genes with several phenotypic measurements, including fat deposition traits in the pig. Because these genes have been implicated in obesity and diabetes in humans, and this study suggests associations with fat related traits, further research on these genes in swine may provide useful information on genetic factors underlying lean pork production.