Comparative genome analysis of mycobacteria focusing on tRNA and non-coding RNA.

BACKGROUND: The Mycobacterium genus encompasses at least 192 named species, many of which cause severe diseases such as tuberculosis. Non-tuberculosis mycobacteria (NTM) can also infect humans and animals. Some are of emerging concern because they show high resistance to commonly used antibiotics while others are used and evaluated in bioremediation or included in anticancer vaccines. RESULTS: We provide the genome sequences for 114 mycobacterial type strains and together with 130 available mycobacterial genomes we generated a phylogenetic tree based on 387 core genes and supported by average nucleotide identity (ANI) data. The 244 genome sequences cover most of the species constituting the Mycobacterium genus. The genome sizes ranged from 3.2 to 8.1 Mb with an average of 5.7 Mb, and we identified 14 new plasmids. Moreover, mycobacterial genomes consisted of phage-like sequences ranging between 0 and 4.64% dependent on mycobacteria while the number of IS elements varied between 1 and 290. Our data also revealed that, depending on the mycobacteria, the number of tRNA and non-coding (nc) RNA genes differ and that their positions on the chromosome varied. We identified a conserved core set of 12 ncRNAs, 43 tRNAs and 18 aminoacyl-tRNA synthetases among mycobacteria. CONCLUSIONS: Phages, IS elements, tRNA and ncRNAs appear to have contributed to the evolution of the Mycobacterium genus where several tRNA and ncRNA genes have been horizontally transferred. On the basis of our phylogenetic analysis, we identified several isolates of unnamed species as new mycobacterial species or strains of known mycobacteria. The predicted number of coding sequences correlates with genome size while the number of tRNA, rRNA and ncRNA genes does not. Together these findings expand our insight into the evolution of the Mycobacterium genus and as such they establish a platform to understand mycobacterial pathogenicity, their evolution, antibiotic resistance/tolerance as well as the function and evolution of ncRNA among mycobacteria.

Comparative genomics of Mycobacterium mucogenicum and Mycobacterium neoaurum clade members emphasizing tRNA and non-coding RNA.

BACKGROUND: Mycobacteria occupy various ecological niches and can be isolated from soil, tap water and ground water. Several cause diseases in humans and animals. To get deeper insight into our understanding of mycobacterial evolution focusing on tRNA and non-coding (nc)RNA, we conducted a comparative genome analysis of Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clade members. RESULTS: Genome sizes for Mmuc- and Mneo-clade members vary between 5.4 and 6.5 Mbps with the complete MmucT (type strain) genome encompassing 6.1 Mbp. The number of tRNA genes range between 46 and 79 (including one pseudo tRNA gene) with 39 tRNA genes common among the members of these clades, while additional tRNA genes were probably acquired through horizontal gene transfer. Selected tRNAs and ncRNAs (RNase P RNA, tmRNA, 4.5S RNA, Ms1 RNA and 6C RNA) are expressed, and the levels for several of these are higher in stationary phase compared to exponentially growing cells. The rare tRNAIleTAT isoacceptor and two for mycobacteria novel ncRNAs: the Lactobacillales-derived GOLLD RNA and a homolog to the antisense Salmonella typhimurium phage Sar RNA, were shown to be present and expressed in certain Mmuc-clade members. CONCLUSIONS: Phages, IS elements, horizontally transferred tRNA gene clusters, and phage-derived ncRNAs appears to have influenced the evolution of the Mmuc- and Mneo-clades. While the number of predicted coding sequences correlates with genome size, the number of tRNA coding genes does not. The majority of the tRNA genes in mycobacteria are transcribed mainly from single genes and the levels of certain ncRNAs, including RNase P RNA (essential for the processing of tRNAs), are higher at stationary phase compared to exponentially growing cells. We provide supporting evidence that Ms1 RNA represents a mycobacterial 6S RNA variant. The evolutionary routes for the ncRNAs RNase P RNA, tmRNA and Ms1 RNA are different from that of the core genes.

Asymmetric growth and division in Mycobacterium spp.: compensatory mechanisms for non-medial septa.

Mycobacterium spp., rod-shaped cells belonging to the phylum Actinomycetes, lack the Min- and Noc/Slm systems responsible for preventing the placement of division sites at the poles or over the nucleoids to ensure septal assembly at mid-cell. We show that the position for establishment of the FtsZ-ring in exponentially growing Mycobacterium marinum and Mycobacterium smegmatis cells is nearly random, and that the cells often divide non-medially, producing two unequal but viable daughters. Septal sites and cellular growth disclosed by staining with the membrane-specific dye FM4-64 and fluorescent antibiotic vancomycin (FL-Vanco), respectively, showed that many division sites were off-centre, often over the nucleoids, and that apical cell growth was frequently unequal at the two poles. DNA transfer through the division septum was detected, and translocation activity was supported by the presence of a putative mycobacterial DNA translocase (MSMEG2690) at the majority of the division sites. Time-lapse imaging of single live cells through several generations confirmed both acentric division site placement and unequal polar growth in mycobacteria. Our evidence suggests that post-septal DNA transport and unequal polar growth may compensate for the non-medial division site placement in Mycobacterium spp.

Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.

RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.

tRNA accumulation and suppression of the bldA phenotype during development in Streptomyces coelicolor.

Streptomyces coelicolor undergoes distinct morphological changes as it grows on solid media where spores differentiate into vegetative and aerial mycelium that is followed by the production of spores. Deletion of bldA, encoding the rare tRNA(Leu) UAA, blocks development at the stage of vegetative mycelium formation. From previous data it appears that tRNA(Leu) UAA accumulates relatively late during growth while two other tRNAs do not. Here, we studied the expression of 17 different tRNAs including bldA tRNA, and the RNA subunit of the tRNA processing endoribonuclease RNase P. Our results showed that all selected tRNAs and RNase P RNA increased with time during development. However, accumulation of bldA tRNA and another rare tRNA(Leu) isoacceptor started at an earlier stage compared with the other tRNAs. We also introduced the bldA tRNA anticodon (UAA) into other tRNAs and introduced these into a bldA deletion strain. In particular, one such mutant tRNA derived from the tRNA(Leu) CAA isoacceptor suppressed the bldA phenotype. Thus, the bldA tRNA scaffold is not critical for function as a regulator of S. coelicolor cell differentiation. Further substitution experiments, in which the 5'- and 3'-flanking regions of the suppressor tRNA were changed, indicated that these regions were important for the suppression.

Sporulation in mycobacteria.

Mycobacteria owe their success as pathogens to their ability to persist for long periods within host cells in asymptomatic, latent forms before they opportunistically switch to the virulent state. The molecular mechanisms underlying the transition into dormancy and emergence from it are not clear. Here we show that old cultures of Mycobacterium marinum contained spores that, upon exposure to fresh medium, germinated into vegetative cells and reappeared again in stationary phase via endospore formation. They showed many of the usual characteristics of well-known endospores. Homologues of well-known sporulation genes of Bacillus subtilis and Streptomyces coelicolor were detected in mycobacteria genomes, some of which were verified to be transcribed during appropriate life-cycle stages. We also provide data indicating that it is likely that old Mycobacterium bovis bacillus Calmette-Guérin cultures form spores. Together, our data show sporulation as a lifestyle adapted by mycobacteria under stress and tempt us to suggest this as a possible mechanism for dormancy and/or persistent infection. If so, this might lead to new prophylactic strategies.

Intracellular localization of the mycobacterial stressosome complex.

Microorganisms survive stresses by alternating the expression of genes suitable for surviving the immediate and present danger and eventually adapt to new conditions. Many bacteria have evolved a multiprotein "molecular machinery" designated the "Stressosome" that integrates different stress signals and activates alternative sigma factors for appropriate downstream responses. We and others have identified orthologs of some of the Bacillus subtilis stressosome components, RsbR, RsbS, RsbT and RsbUVW in several mycobacteria and we have previously reported mutual interactions among the stressosome components RsbR, RsbS, RsbT and RsbUVW from Mycobacterium marinum. Here we provide evidence that "STAS" domains of both RsbR and RsbS are important for establishing the interaction and thus critical for stressosome assembly. Fluorescence microscopy further suggested co-localization of RsbR and RsbS in multiprotein complexes visible as co-localized fluorescent foci distributed at scattered locations in the M. marinum cytoplasm; the number, intensity and distribution of such foci changed in cells under stressed conditions. Finally, we provide bioinformatics data that 17 (of 244) mycobacteria, which lack the RsbRST genes, carry homologs of Bacillus cereus genes rsbK and rsbM indicating the existence of alternative σF activation pathways among mycobacteria.

Author Correction: Extensive genomic diversity among Mycobacterium marinum strains revealed by whole genome sequencing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Insight into the biology of Mycobacterium mucogenicum and Mycobacterium neoaurum clade members.

Nontuberculous mycobacteria, NTM, are of growing concern and among these members of the Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clades can cause infections in humans and they are resistant to first-line anti-tuberculosis drugs. They can be isolated from different ecological niches such as soil, tap water and ground water. Mycobacteria, such as Mmuc and Mneo, are classified as rapid growing mycobacteria, RGM, while the most familiar, Mycobacterium tuberculosis, belongs to the slow growing mycobacteria, SGM. Modern "omics" approaches have provided new insights into our understanding of the biology and evolution of this group of bacteria. Here we present comparative genomics data for seventeen NTM of which sixteen belong to the Mmuc- and Mneo-clades. Focusing on virulence genes, including genes encoding sigma/anti-sigma factors, serine threonine protein kinases (STPK), type VII (ESX genes) secretion systems and mammalian cell entry (Mce) factors we provide insight into their presence as well as phylogenetic relationship in the case of the sigma/anti-sigma factors and STPKs. Our data further suggest that these NTM lack ESX-5 and Mce2 genes, which are known to affect virulence. In this context, Mmuc- and Mneo-clade members lack several of the genes in the glycopeptidolipid (GLP) locus, which have roles in colony morphotype appearance and virulence. For the M. mucogenicum type strain, MmucT, we provide RNASeq data focusing on mRNA levels for sigma factors, STPK, ESX proteins and Mce proteins. These data are discussed and compared to in particular the SGM and fish pathogen Mycobacterium marinum. Finally, we provide insight into as to why members of the Mmuc- and Mneo-clades show resistance to rifampin and isoniazid, and why MmucT forms a rough colony morphotype.

Extended insight into the Mycobacterium chelonae-abscessus complex through whole genome sequencing of Mycobacterium salmoniphilum outbreak and Mycobacterium salmoniphilum-like strains.

Members of the Mycobacterium chelonae-abscessus complex (MCAC) are close to the mycobacterial ancestor and includes both human, animal and fish pathogens. We present the genomes of 14 members of this complex: the complete genomes of Mycobacterium salmoniphilum and Mycobacterium chelonae type strains, seven M. salmoniphilum isolates, and five M. salmoniphilum-like strains including strains isolated during an outbreak in an animal facility at Uppsala University. Average nucleotide identity (ANI) analysis and core gene phylogeny revealed that the M. salmoniphilum-like strains are variants of the human pathogen Mycobacterium franklinii and phylogenetically close to Mycobacterium abscessus. Our data further suggested that M. salmoniphilum separates into three branches named group I, II and III with the M. salmoniphilum type strain belonging to group II. Among predicted virulence factors, the presence of phospholipase C (plcC), which is a major virulence factor that makes M. abscessus highly cytotoxic to mouse macrophages, and that M. franklinii originally was isolated from infected humans make it plausible that the outbreak in the animal facility was caused by a M. salmoniphilum-like strain. Interestingly, M. salmoniphilum-like was isolated from tap water suggesting that it can be present in the environment. Moreover, we predicted the presence of mutational hotspots in the M. salmoniphilum isolates and 26% of these hotspots overlap with genes categorized as having roles in virulence, disease and defense. We also provide data about key genes involved in transcription and translation such as sigma factor, ribosomal protein and tRNA genes.

Extensive genomic diversity among Mycobacterium marinum strains revealed by whole genome sequencing.

Mycobacterium marinum is the causative agent for the tuberculosis-like disease mycobacteriosis in fish and skin lesions in humans. Ubiquitous in its geographical distribution, M. marinum is known to occupy diverse fish as hosts. However, information about its genomic diversity is limited. Here, we provide the genome sequences for 15 M. marinum strains isolated from infected humans and fish. Comparative genomic analysis of these and four available genomes of the M. marinum strains M, E11, MB2 and Europe reveal high genomic diversity among the strains, leading to the conclusion that M. marinum should be divided into two different clusters, the "M"- and the "Aronson"-type. We suggest that these two clusters should be considered to represent two M. marinum subspecies. Our data also show that the M. marinum pan-genome for both groups is open and expanding and we provide data showing high number of mutational hotspots in M. marinum relative to other mycobacteria such as Mycobacterium tuberculosis. This high genomic diversity might be related to the ability of M. marinum to occupy different ecological niches.

The length of the 5' leader of Escherichia coli tRNA precursors influences bacterial growth.

Based on a computational analysis of the 5' regions of tRNA-encoding genes, the average length of the 5' leaders in tRNA precursors in Escherichia coli appears to be 17-18 residues long. An in vivo assay based on tRNA nonsense suppression was developed and used to investigate the function of the 5' leader of the tRNA precursors on tRNA processing and bacterial growth. Our data indicate that the 5' leader influences bacterial growth but is surprisingly not absolutely necessary for growth. These findings are consistent with previous in vitro data where it was demonstrated that the 5' leader plays a role in the interaction with RNase P, the endoribonuclease responsible for removing the 5' leader in the cell. We discuss the plausible role of the 5' leader in processing and tRNA gene expression.

On the paradoxically concentration-dependent metabolism of 6-mercaptopurine in WEHI-3b murine leukemia cells.

The intracellular metabolism of 6-mercaptopurine (6-MP) was studied in a murine leukemia cell line, WEHI-3b. Cells were incubated 3 to 24 h with 10 nM to 50 microM 6-MP. Nucleotides were extracted with perchloric acid, and the 6-thiopurine nucleotides were isolated on mercurial cellulose. The endogenous ribonucleotides in the perchloric acid extracts as well as 6-thiopurine nucleotides were separated and quantified with anion exchange high-performance liquid chromatography. The concentration of 6-thioinosinate (6-TIMP) and 6-thioxantinate (6-TXMP) increased with an increasing 6-MP dose. The concentration of the 6-thioguanosine nucleotides (6-TGN) increased with 6-MP concentrations between 10 nM and 1 microM. However, further increase in 6-MP concentration led to a decrease in the formation of 6-TGN. At 50 microM 6-MP, the concentration of 6-thioguanosine 5'-triphosphate was one fifth of that seen at 1 microM. The incorporation of 6-[35S]mercaptopurine into DNA was also slightly higher at 1 microM compared with 50 microM. The cytocidal effect on clonogenic cells was one log greater at 1 microM 6-MP compared with 50 microM 6-MP. The decrease of 6-TGN was accompanied not only by an increased 6-TIMP concentration but also by an inhibition of the purine de novo synthesis and consequently by a decrease of the cellular ATP concentration. The ATP concentration in the cells treated with 1 microM 6-MP could be reduced to the level seen in cells treated with 50 microM 6-MP by simultaneous incubation with 0.3 microM antimycin A. This decrease of ATP concentration was accompanied by a reduction of 6-TGN and to a lesser extent of 6-TXMP. These experiments suggest that the "self-limiting" phenomenon in the metabolism of 6-MP might be caused by a depletion of ATP by inhibition of purine de novo synthesis presumably by 6-TIMP.

The Mycobacterium phlei Genome: Expectations and Surprises.

Mycobacterium phlei, a nontuberculosis mycobacterial species, was first described in 1898-1899. We present the complete genome sequence for theM. phlei CCUG21000(T)type strain and the draft genomes for four additional strains. The genome size for all five is 5.3 Mb with 69.4% Guanine-Cytosine content. This is ≈0.35 Mbp smaller than the previously reported M. phlei RIVM draft genome. The size difference is attributed partly to large bacteriophage sequence fragments in theM. phlei RIVM genome. Comparative analysis revealed the following: 1) A CRISPR system similar to Type 1E (cas3) in M. phlei RIVM; 2) genes involved in polyamine metabolism and transport (potAD,potF) that are absent in other mycobacteria, and 3) strain-specific variations in the number of σ-factor genes. Moreover,M. phlei has as many as 82 mce(mammalian cell entry) homologs and many of the horizontally acquired genes in M. phlei are present in other environmental bacteria including mycobacteria that share similar habitat. Phylogenetic analysis based on 693 Mycobacterium core genes present in all complete mycobacterial genomes suggested that its closest neighbor is Mycobacterium smegmatis JS623 and Mycobacterium rhodesiae NBB3, while it is more distant toM. smegmatis mc2 155.

Flavoprotein-linked substrate oxidation in preparations of hamster brown adipocytes. A discrimination between internally and externally oxidized substrates.

Noradrenaline-stimulated oxidative metabolism in isolated hamster brown fat cells is very reproducible between different cell preparations, 565 +/- 81 (S.D.) nmol O/min per 10(6) cells (n = 25). In contrast, the oxygen consumption rate induced by the addition of succinate or sn-glycerol 3-phosphate strongly varies between different cell preparation, although these substances have been reported to be potent substrates for isolated hamster brown fat cells. By filtration and by successive washings we demonstrate that the flavoprotein-linked substrate oxidation is mainly dependent on extracellular succinate and sn-glycerol 3-phosphate-oxidizing enzymes. These enzymes originate from damaged and broken cells and are present in different amounts in different cell preparations. In discriminating between intra- and extracellular succinate oxidation 5,5'- dithiobis(2-nitrobenzoate) is used as an inhibitor of the extracellular portion. This application of 5,5'-dithiobis(2-nitrobenzoate) ought to be useful also in other cell or tissue preparations. Added succinate can, however, be oxidized by the intact brown adipocyte but at very low rate, probably as a result of a limited transport rate over the membrane(s). In the presence of noradrenaline, added succinate can potentiate the noradrenaline-inducible oxygen consumption by catalytically increasing the oxidative capacity of the citric acid cycle. Our conclusions is that the only effectors which significantly increase oxidative metabolism in intact isolated hamster brown fat cells are catecholamines and free fatty acids. Provided the cells are uncoupled, also pyruvate can function as substrate for these cells.

Importance of the +73/294 interaction in Escherichia coli RNase P RNA substrate complexes for cleavage and metal ion coordination.

We have studied an interaction, the "73/294-interaction", between residues 294 in M1 RNA (the catalytic subunit of Escherichia coli RNase P) and +73 in the tRNA precursor substrate. The 73/294-interaction is part of the "RCCA-RNase P RNA interaction", which anchors the 3' R(+73)CCA-motif of the substrate to M1 RNA (interacting residues underlined). Considering that in a large fraction of tRNA precursors residue +73 is base-paired to nucleotide -1 immediately 5' of the cleavage site, formation of the 73/294-interaction results in exposure of the cleavage site. We show that the nature/orientation of the 73/294-interaction is important for cleavage site recognition and cleavage efficiency. Our data further suggest that this interaction is part of a metal ion-binding site and that specific chemical groups are likely to act as ligands in binding of Mg(2+) or other divalent cations important for function. We argue that this Mg(2+) is involved in metal ion cooperativity in M1 RNA-mediated cleavage. Moreover, we suggest that the 73/294-interaction operates in concert with displacement of residue -1 in the substrate to ensure efficient and correct cleavage. The possibility that the residue at -1 binds to a specific binding surface/pocket in M1 RNA is discussed. Our data finally rationalize why the preferred residue at position 294 in M1 RNA is U.

Biochemical pharmacology and resistance to 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, a novel analogue of cladribine in human leukemic cells.

The objective of the present study was to investigate the biochemical pharmacology of 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA)--a fluorinated analogue of cladribine [2-chloro-2'-deoxyadenosine, Leustatin (CdA)] with improved acid and metabolic stability--in human leukemic cell lines and in mononuclear cells isolated from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). We have also made and characterized two cell lines that are not sensitive to the growth inhibitory and cytotoxic effects of CAFdA. Incubation of cells isolated from the blood of CLL and AML patients with various concentrations of CdA or of CAFdA accumulated CdA and CAFdA nucleotides in a dose-dependent manner. A significantly higher rate of phosphorylation to monophosphates was observed for CAFdA than for CdA in cells from CLL patients (n = 14; P = 0.04). The differences in the phosphorylation were even more pronounced for the respective triphosphates in both CLL (n = 14; P = 0.001) and AML (n = 4; P = 0.04) cells. Retention of CAFdA 5'-triphosphate (CAFdATP) was also longer than that for CdA 5'-triphosphate (CdATP) in cells from leukemic patients. The relative efficacy of CAFdA as a substrate for purified recombinant deoxycytidine kinase (dCK), the key enzyme in the activation of nucleoside analogues, was very high and exceeded that of CdA as well as the natural substrate, deoxycytidine, by a factor of 2 and 8, respectively. The Km for CAFdA with dCK was also lower than that for CdA, as measured in crude extracts from the human acute lymphoblastic leukemia cell line CCRF-CEM and the promyelocytic leukemia cell line HL60. Acquired resistance to CAFdA in HL60 and in CCRF-CEM cell lines was directly correlated to the decreased activity of the nucleoside phosphorylating enzyme, dCK. Resistant cells also showed a considerable degree of cross-resistance to analogues that were activated by dCK. These observations demonstrated that dCK phosphorylates CAFdA more efficiently than CdA. Furthermore, CAFdATP is apparently more stable than CdATP and the mechanisms of resistance to CAFdA are similar to those leading to CdA resistance. These results encourage studies on the clinical effect of CAFdA in lymphoproliferative diseases.

Pharmacokinetics of cladribine in plasma and its 5'-monophosphate and 5'-triphosphate in leukemic cells of patients with chronic lymphocytic leukemia.

The pharmacokinetic parameters of cladribine (CdA) in patient plasma and its intracellular nucleotides CdA 5'-monophosphate (CdAMP) and CdA 5'-triphosphate (CdATP) were delineated in circulating leukemia cells in 17 patients with chronic lymphocytic leukemia, after the last dose intake and up to 72 h thereafter. Patients were treated with 10 mg/m2 CdA p.o. on 3 consecutive days. A novel and specific ion-pair liquid chromatographic method, which separates the intracellular CdA nucleotides, was used. The area under the concentration versus time curve (AUC) of CdAMP in leukemia cells was generally higher (median, 47 micromol/liter x h) than the AUC of CdATP (median, 22 micromol/liter x h); however, in some patients (3 of 17), the reverse relationship was seen. The median ratio between the AUC values for CdATP and CdAMP was 0.60 (95% confidence interval, 0.4-1.0). The median half-life (t(1/2)) of CdAMP was 15 h, and that of CdATP was 10 h. The median terminal t(1/2) of CdA in plasma was 21 h. A significant correlation was found between the maximum plasma CdA and cellular CdAMP concentrations (r = 0.56, P = 0.02). There was no correlation between the AUC values of cellular CdAMP and CdATP (r = 0.224, P = 0.55). No correlation was found between deoxycytidine kinase activity and intracellular pharmacokinetic parameters of CdAMP or CdATP. The response to treatment was not significantly related to intracellular concentration of CdAMP or active metabolite CdATP. There is great heterogeneity among patients in terms of AUC and t(1/2) of CdAMP and CdATP. Furthermore, the results emphasize the differences between the pharmacokinetics of plasma CdA and those of the metabolites in circulating leukemic cells.

Noise exposure and subjective hearing symptoms among school children in Sweden.

OBJECTIVE: The aim of the present study was to evaluate factors of importance for the experience of temporary threshold shift (TTS), noise- induced tinnitus (NIT), spontaneous tinnitus (ST) in school children. SUBJECTS AND METHODS: A total of 671 students aged 13-16 years old were asked to fill in a questionnaire containing items concerning TTS, NIT, ST, hearing loss (HL), heredity for HL, noise exposure, history of otitis media, symptoms of anxiety and depression, psychosocial factors and habits, life satisfaction, chronic medical conditions, age, gender and height. The questionnaire was filled in during school hours. RESULTS: Correlations were found with exercise and eating habits, sleep disturbances, BMI, depressive and anxiety disorders, heredity for HL and noise exposure dosage. The risk for TTS was nine times higher in students who reported having a verified hearing loss than in subjects without subjective or verified complaints of hearing loss. The risk for NIT was approximately four times higher in the group who visited concerts 6-12 times per year as compared to those who never attended concerts. There was almost a threefold increase in the risk for ST in the group that sometimes experienced TTS, as compared to those without TTS, and a tenfold increase in risk for ST in those who reported having a verified hearing loss. CONCLUSION: In school children, exposure to leisure noise is correlated with tinnitus and the risk increases with increasing noise exposure. Sensitivity to subjective hearing loss has similar risk factors as seen for metabolic syndrome and we suggest that this sensitivity may be another side of metabolic syndrome.

Characterization of Three Mycobacterium spp. with Potential Use in Bioremediation by Genome Sequencing and Comparative Genomics.

We provide the genome sequences of the type strains of the polychlorophenol-degrading Mycobacterium chlorophenolicum (DSM43826), the degrader of chlorinated aliphatics Mycobacterium chubuense (DSM44219) and Mycobacterium obuense (DSM44075) that has been tested for use in cancer immunotherapy. The genome sizes of M. chlorophenolicum, M. chubuense, and M. obuense are 6.93, 5.95, and 5.58 Mb with GC-contents of 68.4%, 69.2%, and 67.9%, respectively. Comparative genomic analysis revealed that 3,254 genes are common and we predicted approximately 250 genes acquired through horizontal gene transfer from different sources including proteobacteria. The data also showed that the biodegrading Mycobacterium spp. NBB4, also referred to as M. chubuense NBB4, is distantly related to the M. chubuense type strain and should be considered as a separate species, we suggest it to be named Mycobacterium ethylenense NBB4. Among different categories we identified genes with potential roles in: biodegradation of aromatic compounds and copper homeostasis. These are the first nonpathogenic Mycobacterium spp. found harboring genes involved in copper homeostasis. These findings would therefore provide insight into the role of this group of Mycobacterium spp. in bioremediation as well as the evolution of copper homeostasis within the Mycobacterium genus.