In vitro activity of the chelating agents nitroxoline and oxine against Mycobacterium bovis BCG.

The chelating antibiotic nitroxoline (5-nitro-8-hydroxyquinoline) showed a bacteriostatic effect at a concentration of 10 microM for Mycobacterium bovis BCG. At higher concentrations the compound showed moderate cidal activity against growing bacilli. In contrast, its non-nitrated derivative oxine (8-hydroxyquinoline, MIC=2 microM) reduced the viability of a growing culture rapidly, 5000-fold at a concentration where the nitroxoline was merely bacteriostatic. Both compounds showed appreciable cidal activity against bacilli in their hypoxic dormant state.

Bactericidal activity of nitrofurans against growing and dormant Mycobacterium bovis BCG.

Depletion of oxygen triggers the shift-down of Mycobacterium bovis BCG to a state of dormancy. Bacilli in their dormant state are resistant to standard anti-mycobacterials. The nitroimidazole metronidazole was the first compound identified to show bactericidal activity against dormant tubercle bacilli. In contrast to metronidazole's selective toxicity for dormant bacilli, we report here that the nitrofurans nitrofurantoin, furaltadone and nitrofurazone showed bactericidal activity against dormant and growing bacteria. Importantly, the bactericidal effect of nitrofurans on dormant bacilli was 35- to 250-fold higher compared with metronidazole.

The effect of a single fraction compared to multiple fractions on painful bone metastases: a global analysis of the Dutch Bone Metastasis Study.

PURPOSE: To answer the question whether a single fraction of radiotherapy that is considered more convenient to the patient is as effective as a dose of multiple fractions for palliation of painful bone metastases. PATIENTS: 1171 patients were randomised to receive either 8 Gy x 1 (n = 585) or 4 Gy x 6 (n = 586). The primary tumour was in the breast in 39% of the patients, in the prostate in 23%, in the lung in 25% and in other locations in 13%. Bone metastases were located in the spine (30%), pelvis (36%), femur (10%), ribs (8%), humerus (6%) and other sites (10%). METHOD: Questionnaires were mailed to collect information on pain, analgesics consumption, quality of life and side effects during treatment. The main endpoint was pain measured on a pain scale from 0 (no pain at all) to 10 (worst imaginable pain). Costs per treatment schedule were estimated. RESULTS: On average, patients participated in the study for 4 months. Median survival was 7 months. Response was defined as a decrease of at least two points as compared to the initial pain score. The difference in response between the two treatment groups proved not significant and stayed well within the margin of 10%. Overall, 71% experienced a response at some time during the first year. An analysis of repeated measures confirmed that the two treatment schedules were equivalent in terms of palliation. With regard to pain medication, quality of life and side effects no differences between the two treatment groups were found. The total number of retreatments was 188 (16%). This number was 147 (25%) in the 8 Gy x 1 irradiation group and 41 (7%) in the 4 Gy x 6 group. It was shown that the level of pain was an important reason to retreat. There were also indications that doctors were more willing to retreat patients in the single fraction group because time to retreatment was substantially shorter in this group and the preceding pain score was lower. Unexpectedly, more pathological fractures were observed in the single fraction group, but the absolute percentage was low. In a cost-analysis, the costs of the 4 Gy x 6 and the 8 Gy x 1 treatment schedules were calculated at 2305 and 1734 Euro respectively. Including the costs of retreatment reduced this 25% cost difference to only 8%. The saving of radiotherapy capacity, however, was considered the major economic advantage of the single dose schedule. CONCLUSION: The global analysis of the Dutch study indicates the equality of a single fraction as compared to a 6 fraction treatment in patients with painful bone metastases provided that 4 times more retreatments are accepted in the single dose group. This equality is also shown in long term survivors. A more detailed analysis of the study is in progress.

Oxygen depletion-induced dormancy in Mycobacterium bovis BCG.

Gradual depletion of oxygen causes the shift-down of aerobic growing Mycobacterium bovis BCG to an anaerobic synchronized state of nonreplicating persistence. The persistent culture shows induction of glycine dehydrogenase and alpha-crystallin-like protein and is sensitive to metronidazole.

Upregulation of stress response genes and ABC transporters in anaerobic stationary-phase Mycobacterium smegmatis.

Oxygen starvation triggers an adaptive stationary-phase response in Mycobacterium smegmatis. During this anaerobic stationary phase, RNA synthesis continues at a low but significant level. Employing a modified expressed-sequence-tag (EST) approach, in combination with the M. tuberculosis genome data and comparative Northern analysis, we have identified the first genes that show an increase in transcription in M. smegmatis cells that have entered anaerobic stationary phase. One gene encodes the counterpart of the M. tuberculosis NifS-like protein Rv1464. Two genes are homologues of M. tuberculosis Rv1460 and Rv3368c, of unknown function. Strikingly, several genes induced by oxygen starvation encode putative stress protection proteins (counterparts of M. tuberculosis DnaK, Rv0350; betaine-aldehyde dehydrogenase, Rv0768; thioredoxin reductase, Rv3913) and ABC transporters (counterparts of M. tuberculosis Rv1463, Rv1473, Rv3197). We conclude that development of general stress resistance and certain active transport processes might play a role in the survival of oxygen-starved M. smegmatis.

Oxygen depletion induced dormancy in Mycobacterium smegmatis.

We report here that the physiological behaviour of the fast growing saprophytic Mycobacterium smegmatis under in vitro oxygen-depletion and reactivation conditions is strikingly similar to the characteristics shown by the slow growing pathogenic M. tuberculosis. M. smegmatis died rapidly when shifted abruptly from aerobic to anaerobic conditions. In contrast to the lethal shock of abrupt oxygen depletion, the slow depletion through a self generated oxygen gradient permitted an adaptation to a persistent state which showed increased resistance against the bactericidal effects of anaerobiosis. The anaerobic persistent culture did not synthesise DNA and showed synchronised division upon reactivation in oxygen rich medium, indicating that the persistent bacilli are uniformly arrested at a defined stage of the cell cycle. Upon reactivation the persistent culture started synthesising DNA only after the first cell division, suggesting that the persistent cells contain two chromosomes. Furthermore, the persistent culture developed sensitivity to metronidazole and resistance against ofloxacin. These results suggest that M. smegmatis might be useful as a fast growing non-pathogenic model for comparative molecular analyses of mycobacterial dormancy.

Inscuteable and numb mediate asymmetric muscle progenitor cell divisions during Drosophila myogenesis.

Each larval hemisegment comprises approximately 30 uniquely specified somatic muscles. These derive from muscle founders that arise as distinct sibling pairs from the division of muscle progenitor cells. We have analyzed the progenitor cell divisions of three mesodermal lineages that generate muscle (and pericardial cell) founders. Our results show that Inscuteable and Numb proteins are localized as cortical crescents on opposite sides of dividing progenitor cells. Asymmetric segregation of Numb into one of the sibling myoblasts depends on inscuteable and is essential for the specification of distinct sibling cell fates. Loss of numb or inscuteable results in opposite cell fate transformations-both prevent sibling myoblasts from adopting distinct identities, resulting in duplicated or deleted mesodermal structures. Our results indicate that the muscle progenitor cell divisions are intrinsically asymmetric; moreover, the involvement of both inscuteable and numb/N suggests that the specification of the distinct cell fates of sibling myoblasts requires intrinsic and extrinsic cues.

Upregulation of a histone-like protein in dormant Mycobacterium smegmatis.

The aerobic saprophyte Mycobacterium smegmatis, like its pathogenic counterpart M. tuberculosis, has the ability to adapt to anaerobiosis by shifting down to a dormant state. Here, we report the identification and molecular genetic characterisation of the first dormancy-induced protein in M. smegmatis. Comparative SDS-polyacrylamide gel electrophoresis of protein extracts of aerobically growing and dormant anaerobic M. smegmatis cultures revealed the upregulation of a 27-kDa protein in the dormant state. Peptide sequencing showed that the induced protein is a homologue of the histone-like protein H1p, predicted by the M. tuberculosis genome project. The corresponding hlp gene was cloned from M. smegmatis and sequenced. Disruption of the hlp gene eliminated the histone-like protein but did not affect the viability of the dormant culture.

Mutations in masquerade, a novel serine-protease-like molecule, affect axonal guidance and taste behavior in Drosophila.

The masquerade (mas) locus encodes an extracellular molecule with a striking similarity to serine proteases. The serine residue, which is essential for enzymatic activity, has been substituted by glycine, suggesting that MAS could serve to antagonize serine protease activity [Murugasu-Oei et al. (1995), Genes Dev. 9, 139-154]. We describe the expression pattern of mas mRNA and protein in the developing embryonic, larval and pupal nervous system and in the epidermis. Total loss of mas function is lethal and results in aberrations in the embryonic central and peripheral nervous systems, consistent with a role in axonal guidance. The possibility that the observed deficits in taste behavior, exhibited by animals with partial loss of mas function, are a result of defects in the adult brain are discussed.

Masquerade: a novel secreted serine protease-like molecule is required for somatic muscle attachment in the Drosophila embryo.

Diverse developmental processes, such as neuronal growth cone migration and cell shape changes, are mediated by the interactions of cells with the extracellular matrix. We describe here a secreted molecule encoded by the Drosophila masquerade (mas) gene. Total loss of mas function causes defective muscle attachment. This mutant phenotype suggests that mas normally acts to stabilize cell-matrix interaction and represents a novel functional and limiting component in the adhesion process. mas encodes a 1047-amino-acid preproprotein that is further processed by proteolytic cleavage to generate two polypeptides. The carboxy-terminal polypeptide is highly similar to serine proteases and has an extracellular localization; however, it is unlikely to possess proteolytic activity, because the catalytic site serine has been substituted by a glycine residue. During embryonic development, the mas amino- and carboxy-terminal polypeptides are differentially localized. The mas carboxy-terminal polypeptide accumulates at all somatic muscle attachment sites, which corresponds well with the morphological defect seen in the mas mutants. Our findings demonstrate the involvement of an extracellular component in somatic muscle attachment. We propose that mas acts via its modified serine protease motif, either as a novel adhesion molecule and/or as a competitive antagonist of serine proteases, to stabilize muscle attachment.

Monoclonal antibodies to blood group antigens.

To obtain haemagglutinating monoclonal antibodies as potential blood typing reagents 10 fusions were performed between mouse myeloma cells and spleen cells from mice immunised with either blood group substances or human red blood cells. Nineteen hybridomas with anti-A specificity and 10 with anti-B specificity were generated, Three were selected for further investigations. Some of these hybridoma clones produce and secrete both IgM and IgG.