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New photochemically removable protecting groups have been developed based on classical nitrobenzyl compounds modified by the inclusion of a pentadienyl group. It serves to trap through an internal Diels-Alder reaction the nitroso group produced as part of the photochemical deprotection process, preventing its further photochemistry or chemical reactions with nucleophiles.

Background Skeletal muscle atrophy, whether caused by chronic disease, acute critical illness, disuse or aging, is characterized by tissue-specific decrease in oxidative capacity and broad alterations in metabolism that contribute to functional decline. However, the underlying mechanisms responsible for these metabolic changes are largely unknown. One of the most highly upregulated genes in atrophic muscle is AMP deaminase 3 (AMPD3: AMP → IMP + NH3), which controls the content of intracellular adenine nucleotides (AdN; ATP + ADP + AMP). Given the central role of AdN in signaling mitochondrial gene expression and directly regulating metabolism, we hypothesized that overexpressing AMPD3 in muscle cells would be sufficient to alter their metabolic phenotype similar to that of atrophic muscle. Methods AMPD3 and GFP (control) were overexpressed in mouse tibialis anterior (TA) muscles via plasmid electroporation and in C2C12 myotubes using adenovirus vectors. TA muscles were excised one week later, and AdN were quantified by UPLC. In myotubes, targeted measures of AdN, AMPK/PGC-1α/mitochondrial protein synthesis rates, unbiased metabolomics, and transcriptomics by RNA sequencing were measured after 24 h of AMPD3 overexpression. Media metabolites were measured as an indicator of net metabolic flux. At 48 h, the AMPK/PGC-1α/mitochondrial protein synthesis rates, and myotube respiratory function/capacity were measured. Results TA muscles overexpressing AMPD3 had significantly less ATP than contralateral controls (−25%). In myotubes, increasing AMPD3 expression for 24 h was sufficient to significantly decrease ATP concentrations (−16%), increase IMP, and increase efflux of IMP catabolites into the culture media, without decreasing the ATP/ADP or ATP/AMP ratios. When myotubes were treated with dinitrophenol (mitochondrial uncoupler), AMPD3 overexpression blunted decreases in ATP/ADP and ATP/AMP ratios but exacerbated AdN degradation. As such, pAMPK/AMPK, pACC/ACC, and phosphorylation of AMPK substrates, were unchanged by AMPD3 at this timepoint. AMPD3 significantly altered 191 out of 639 detected intracellular metabolites, but only 30 transcripts, none of which encoded metabolic enzymes. The most altered metabolites were those within purine nucleotide, BCAA, glycolysis, and ceramide metabolic pathways. After 48 h, AMPD3 overexpression significantly reduced pAMPK/AMPK (−24%), phosphorylation of AMPK substrates (−14%), and PGC-1α protein (−22%). Moreover, AMPD3 significantly reduced myotube mitochondrial protein synthesis rates (−55%), basal ATP synthase-dependent (−13%), and maximal uncoupled oxygen consumption (−15%). Conclusions Increased expression of AMPD3 significantly decreased mitochondrial protein synthesis rates and broadly altered cellular metabolites in a manner similar to that of atrophic muscle. Importantly, the changes in metabolites occurred prior to reductions in AMPK signaling, gene expression, and mitochondrial protein synthesis, suggesting metabolism is not dependent on reductions in oxidative capacity, but may be consequence of increased AMP deamination. Therefore, AMP deamination in skeletal muscle may be a mechanism that alters the metabolic phenotype of skeletal muscle during atrophy and could be a target to improve muscle function during muscle wasting.

The prodrugs cyclophosphamide (CP) and ifosfamide (IF) each metabolize to an active alkylating agent through a cytochrome P450-mediated oxidation at the C-4 position. Competing with this activation pathway are enzymatic oxidations at the exocyclic α and α' carbons, which result in dechloroethylation of CP and IF. The incidence of oxidation at one position relative to another is believed to be at least one factor underlying the high degree of interpatient variability in both CP and IF pharmacokinetics. As standards for the mass spectrometry quantification of dechloroethylation, the following were synthesized: (1) [4,4,5,5-(2) H4 ]-2-dechloroethylcyclophosphamide (equivalent to [4,4,5,5-(2) H4 ]-3-dechloroethylifosfamide); (2) [α,α,4,4,5,5-(2) H6 ]-2-dechloroethylcyclophosphamide (equivalent to [α,α,4,4,5,5-(2) H6 ]-3-dechloroethylifosfamide); and (3) [α,α,4,4,5,5-(2) H6 ]-2-dechloroethylifosfamide. The common precursor to all of the target compounds was [2,2,3,3-(2) H4 ]-3-aminopropanol. A one-pot reaction of this compound with POCl3 and unlabeled or labeled 2-chloroethylamine hydrochloride gave the d4 and d6 labeled 2-dechloroethylcyclophosphamides. The construction of the 2-dechloroethylifosfamide from the aminopropanol required five discreet steps. Optimization of the synthetic pathways and stability studies are discussed.

Effects of neurodegeneration have been linked to inefficient detoxification of free radicals due to lowered concentrations of antioxidants, especially glutathione, in the brain. In the biosynthesis of glutathione, cysteine concentration is generally the limiting factor. Glutathione and cysteine administrations are not effective treatments for neurodegeneration because glutathione inefficiently crosses cell membranes and cysteine is neurotoxic at high concentrations. Prodrugs of glutathione and cysteine may have more favorable uptake and/or toxicity profiles. Three such prodrugs were synthesized with a 13C-label such that in vivo uptake of each and conversion to glutathione in the brain could be monitored by magnetic resonance imaging. L-[3-13C]-Cysteine was treated with sodium acetate trihydrate and acetic anhydride to give 2(R)-N-acetyl-[3-13C]-cysteine ([13C]-NAC; 96%). Addition of triphosgene to L-[3-13C]-cysteine provided 4(R)-[5-13C]-2-oxothiazolidine-4-carboxylic acid ([13C]-OTZ; 65%). A four-step pathway was used to synthesize ethyl γ-L-glutamyl-[3-13C]-L-cysteinate ([13C]-GCEE). L-[3-13C]-Cysteine was esterified (100% yield) and then cyclized with acetaldehyde to give ethyl 2(R,S)-methyl-[5-13C]-thiazolidine-4(R)-carboxylate (73%) as a mixture of two diastereomers (65:35). The thiazolidine was silylated (bis(trimethylsilyl)trifluoroacetamide) and reacted with N-phthaloyl-L-glutamic anhydride. Treatment with hydrazine afforded ethyl N-[γ-4′(S)-glutamyl]-2(R,S)-methyl-[5-13C]-thiazolidine-4(R)-carboxylate (48%; 73:27 mixture of diastereomers). This was converted to the desired product, [13C]-GCEE (49%), using mercury (II) acetate and hydrogen sulfide.

A variety of deuterated 3-amino-1-propanols were made by the LiAlD 4 or AlD 3 reduction of nitrile or ester precursors. The labeled aminopropanols and/or deuterated bis(2-chloroethyl)amines were used to synthesize [4,4- 2 H 2 ]-, [6,6- 2 H 2 ]-, [α,α.α',α- 2 H 4 ]-, and [4,4,β,β,β',β'- 2 H 6 ]-cyclophosphamide. The labeled aminopropanols were also used to synthesize deuterated 3-(2'-benzyloxyethylamino)-1-propanols [BnOCH 2 CH 2 NH(CH 2 ) 3 OH] which, along with labeled and unlabeled 2-chloroethylamines, led to [4,4- 2 H 2 ]-, [6,6- 2 H 2 ]-, [α,α- 2 H 2 ]-, [α',α'- 2 H 2 ]-, [α,α.α',α'- 2 H 4 ]-, and [6,6,α,α,α',α'- 2 H 6] -ifosfamide.

Reduction of diethyliminodiacetate with [ 3 H]-LiAlH 4 and then reaction with SOCl 2 gave bis(2-chloro-2-[ 3 H] ethyl)amine hydrochloride. This compound, together with [ 33 P]-phosphorus oxychloride, provided for the synthesis of [ 3 H, 33 P]-phosphoramide mustard (as its cyclohexylammonium salt) in three steps over 2 days. Similarly, 2-[ 3 H]-ethanolamine was reacted with SOCl 2 to give 2-chloro-2-[ 3 H]-ethylamine hydrochloride which, along with [ 33 P]-POCl 3 , was used to synthesize [ 3 H, 33 P]-isophosphoramide mustard in two steps over 1 day. 1 H NMR studies were carried out to determine optimal times for in situ formation and storage of chloroethylaziridine and aziridine. A solution of 10 mM bis(2-chloroethyl)amine hydrochloride in 0.1M phosphate/D 2 O, pD 7.9 at 37°C for 3h gave chloroethylaziridine without contamination by starting material or hydrolysis products. For aziridine, the disappearance of 0.2 M 2-chloroethylamine hydrochloride in 2 M NaOD/D 2 O at pD 14, 37°C, gave κ = 0.00455 min -1 (R 2 = 1.000) and τ 1/2 = 2.55 h; no hydrolysis product was observed over the course of the NMR experiment (4 h). It was concluded that ∼ 13 h (5 half-lives) of reaction time would yield a solution of aziridine which was relatively free of contaminants. Using these reaction conditions, 3 H-labeled chloroethylamines were used to synthesize 1-(2-chloro-2-[ 3 H]-ethyl)-2-[ 3 H]-aziridine and 2-[ 3 H]-aziridine in situ.

O6-benzylguanine (BG) is a unique purine analog that has been shown to influence nitrogen mustard activity and increase cytotoxicity. Ifosfamide is a nitrogen mustard with growing clinical applications; effective modulation may lead to improved efficacy. We thus undertook a preliminary investigation of BG's effects on ifosfamide and ifosfamide derivatives in vitro.BG's effect on ifosfamide toxicity was studied in CHO cells transfected with O6-alkylguanine-DNA alkyltransferase (AGT) (CHOwtAGT) or control plasmid pcDNA3 (CHOpcDNA) using five ifosfamide derivatives and two control compounds: 4-hydroperoxyifosfamide (4HI), isophosphoramide mustard (IPM), phenylketoifosfamide (PKIF), 4-hydroperoxydidechloroifosfamide (4HDI), chloroacetaldehyde (CAA), didechloroisophosphoramide mustard (d-IPM), didechlorophenylketoifosfamide (d-PKIF). To further explore the mechanism of interaction, BG's effect on apoptosis (annexin V-FITC) and cell cycle distribution in cells exposed to ifosfamide was also analyzed.BG substantially enhanced cytotoxicity induced only by agents that produce IPM (4HI, IPM, PKIF) in both CHOwtAGT and CHOpcDNA cell lines. BG did not modulate 4HDI or CAA cytotoxicity. The addition of BG to IPM in CHO cells increased the percentage of apoptotic cells from 5.5% to 28.9% at 72 h after treatment. Cell cycle analysis showed that BG exposure was associated with G1 arrest. At 16 h following treatment with IPM, PKIF, or phosphoramide mustard (PM), BG increased the percentage of cells in G1 from 16-20% to 29-64%.BG's ability to increase 4HI-, IPM-, and PKIF-mediated cytotoxicity in cells devoid of AGT activity suggests a novel AGT-independent mode of action that is associated with increased apoptosis and may involve G1 arrest. BG selectively enhanced IPM toxicity without enhancement of acrolein and CAA toxicity. The data strongly support further investigation into combinations of BG and nitrogen mustards.

A number of investigators have observed that the use of 4-hydroperoxycyclophosphamide (4-HC) in multiwell plate cytotoxicity assays can be associated with toxicity to cells in wells that contain no drug. Previous reports have implicated diffusion of 4-HC decomposition products, and acrolein in particular, as the active species. Purpose: The purpose of this study was to elucidate the species responsible for the airborne cytotoxicity of 4-HC, and to devise ways to minimize such effects in chemosensitivity assays. Methods: To this end, analogues of 4-HC were synthesized to identify the contributions of individual cyclophosphamide metabolites to cytotoxicity. The analogues were then tested for activity against three human breast tumor cell lines (including a line resistant to 4-HC), and one non-small-cell lung carcinoma line. Cytotoxicity was evaluated by assays that quantitate cellular metabolism and nucleic acid content. Results: Didechloro-4-hydroperoxycyclophosphamide, a compound that generates acrolein and a nontoxic analogue of phosphoramide mustard, gave no cross-well toxicity. In contrast, a significant neighboring well effect was observed with phenylketophosphamide, a compound that generates phosphoramide mustard but not acrolein. Addition of authentic chloroethylaziridine reproduced the airborne toxicity patterns generated by 4-HC and phenylketophosphamide. Increasing the buffering capacity of the growth medium and sealing the microtiter plates prevented airborne cytotoxicity. Conclusions: Since it is unlikely that phosphoramide mustard is volatile, these findings implicate chloroethylaziridine rather than acrolein as the volatile metabolite of 4-HC that is responsible for airborne cytotoxicity. The fact that chloroethylaziridine is generated in amounts sufficient to volatilize, diffuse across wells and cause cytotoxicity indicates that it is an important component in the overall cytotoxicity of 4-HC in vitro. Furthermore, these findings suggest that chloroethylaziridine may also contribute to the toxicity of cyclophosphamide in vivo.

Previous investigations demonstrated three taxonomic groups among 22 clinical isolates of Mycobacterium smegmatis. These studies were expanded to 71 clinical isolates, of which 35 (49%) (group 1) were identical to five ATCC reference strains including the type strain ATCC 19420T. Twenty-eight isolates (39%) were group 2, and eight isolates (11%) were group 3. Isolates of groups 2 and 3 were most often associated with post-traumatic or post-surgical wound infections including osteomyelitis, were susceptible to sulfamethoxazole, amikacin, imipenem and the tetracyclines, variably resistan to clarithromycin, and susceptible (group 1), intermediately resistant (group 2) or resistant (group 3) to tobramycin. The three groups were similar by routine biochemical and growth characteristics, but had different mycolic acid dimethoxy-4-coumarinylmethyl ester elution patterns by HPLC and different PCR-restriction enzyme patterns of a 439 bp fragment of the hsp-65 gene. Group 3 isolates differed from group 1 by 18 bp by 16S rRNA sequencing and exhibited < 25% homology by DNA-DNA hybridization, being most closely related to Mycobacterium mageritense. The 16S rRNA of group 1 and group 2 isolates differed by only 3 bp, but by DNA-DNA hybridization they exhibited only 40% homology. The following names are proposed: Mycobacterium goodii sp. nov. for group 2 isolates (type strain ATCC 700504T = M069T), Mycobacterium wolinskyi sp. nov. for group 3 isolates (type strain ATCC 700010T = M0739T) and Mycobacterium smegmatis sensu stricto for group 1 isolates.

To investigate the mechanism(s) of bisalkylation by isophosphoramide mustard (IPM), IPM-beta,beta,beta',beta'-d(4) was synthesized and the products of its reaction with thiosulfate (at pD 7.0) were analyzed by NMR. By both (1)H and (13)C NMR, the distribution of deuterium in the products was consistent with bisalkylation through sequential aziridinyl intermediates [(NCH(2)CD(2)S):(NCD(2)CH(2)S) = 53:47]. Under the given reaction conditions, label scrambling as a result of thiosulfate acting as a leaving group was ruled out through control experiments. The data gave a calculated kinetic isotope effect of 0.97 per deuterium. For the initial aziridine species formed from IPM, ab initio quantum chemical calculations gave a hybridization value of sp(2.4)(-)(2.5) for each of the C-H bonds of the reaction centers, and this correlated with the observed inverse isotope effect. Other structure and bond order data were also determined for this aziridine intermediate and related compounds.

A scotochromogenic acid-fast bacillus was isolated from a lymph node of a 2-year-old female. On the basis of conventional testing, the mycobacterium appeared to be Mycobacterium scrofulaceum. Its mycolic acid profile, however, was not identical to that of Mycobacterium scrofulaceum but was similar to that of Mycobacterium interjectum. Direct sequencing of the 16S rRNA gene revealed a unique nucleic acid sequence, suggesting that the isolate represents a previously undescribed pathogenic species.

In order to determine the reactive geometries of enone−TiCl4 complexes in Diels−Alder reactions compounds 1a·TiCl4 and 1b·TiCl4 were prepared. A crystal structure of 1a·TiCl4 confirmed that the titanium in this complex has the desired out-of-plane geometry, with a TiOCC dihedral angle of 57.6°, a value remarkably similar to dihedral angles found in simple carbocyclic compounds. Difference NOE studies support a similar geometry in solution. The magnitudes of key 1H and 13C NMR chemical shift changes upon complex formation indicate that the out-of-plane TiCl4 in 1a·TiCl4 is a stronger Lewis acid than the in-plane TiCl4 of 1e·TiCl4. These results are further supported by the changes in carbonyl stretching frequencies upon complex formation, with a ΔνCO for 1a·TiCl4 which is 24 cm-1 larger than that for 1e·TiCl4. Expectations of heightened reactivity for 1a·TiCl4 as compared to 1e·TiCl4 have been confirmed; the former complex undergoes Diels−Alder reactions with cyclopentadiene 15 times more rapidly than the ...

Reaction of diastereomeric β-benzyloxy enones with TiCl4 results in the cleavage of the benzyl ethers and yields substrates for Diels−Alder reactions which incorporate covalently attached Lewis acids. The axial CH2OTiCl3 group of 2a unambiguously directs complexation of the Lewis acidic titanium to the carbonyl oxygen anti to the enone carbon−carbon double bond in an out-of-plane coordination geometry, while the equatorial CH2OTiCl3 of 2e directs complexation to an anti in-plane coordination geometry. The mono-THF adduct of the out-of-plane complex, 2a·THF, is found to undergo cycloaddition reactions with cyclopentadiene 2.1−2.4 times more rapidly than the corresponding in-plane complex 2e·THF. These results suggest that out-of-plane coordination geometries may play a significant role in titanium-derived Lewis acid-mediated Diels−Alder reactions and provide confirmation of our previous rate studies with substrates in which TiCl4 coordination is directed by simple chelation effects.

The diastereo selcctivities of addition reactions of allyltrimethylsilane to an α-benzyloxyaldehyde were found to be relatively insensitive to the amount of added TiCl 4 , and to the absolute concentration of the 1:1 aldehyde/TiCl 4 , complex. In contrast, diastereoselectivities of similar additions to a structurally related β-benzyloxyaldehyde were highly dependent on these variables.

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.

The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-(13)C]-thiazolidine-4-carboxylate, (13)C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by (13)C magnetic resonance spectroscopy. Although uptake and clearance of (13)C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of (13)C-OTZ over 20 h, however, resulted in (13)C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.

New photochemically removable protecting groups have been developed based on classical nitrobenzyl compounds modified by the inclusion of a pentadienyl group. It serves to trap through an internal Diels-Alder reaction the nitroso group produced as part of the photochemical deprotection process, preventing its further photochemistry or chemical reactions with nucleophiles.

It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by (31)P NMR. In 0.33 M BisTris, pH 7.4, 37 degrees C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of delta 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of delta 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (delta 33.0) and reactivity (half-life 0.3 h at 25 degrees C) of the species attributed to 13 were consistent with the assigned structure. Resonances with (31)P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 degrees C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 microM, while 12 (500 microM) was mutagenic with 246 mutant colonies/10(6) surviving cells.

Differentiation agents that induce neoplastic cells to regain a normal phenotype and/or cause growth arrest without significantly affecting normal cells represent an attractive option for cancer treatment. Analogues of short chain fatty acids, such as phenylbutyrate (PB), have been studied as clinically relevant agents. In an attempt to improve its pharmacokinetic profile, structural modifications of PB and other fatty acids have been studied. We hypothesize that strategic isotopic modification of PB would result in a longer half-life and thus translate into a more potent differentiation agent for clinical use. Using a colon cancer model, we demonstrated that 2,2,3,3-tetradeuterated PB (D4PB) significantly increased induction of apoptosis and inhibition of cell proliferation as compared with PB and butyrate. Difference in potency could not be explained by the effect of D4PB on the expression of specific regulatory proteins of the apoptotic cascade or from the inhibitory effect of D4PB on histone deacetylase activity. Interestingly, exposure of HT-29 colon cancer cells to D4PB resulted in a slowing of S transit, in contrast to butyrate and PB, which induced a G2/M cell cycle block. This difference in cell cycle effect may explain the differences seen in the potency of the phenotypic changes seen with treatment with D4PB. Further studies are needed to elucidate the mechanisms underlying effects of D4PB on the cell cycle. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1054–1064, 2002

The Mycobacterium chelonae-like organism (MCLO) is a recently described member of the Mycobacterium fortuitum complex which causes posttraumatic skin infections and catheter sepsis. This taxon is a distinct group biochemically and has a unique mycolic acid profile as determined by high-performance liquid chromatography. Its phylogenetic relationships to other mycobacteria, however, have not been studied previously. We sequenced 1,062 bp of the 16S rRNA genes from three MCLO strains obtained from the American Type Culture Collection and compared our results with the sequences of previously described taxa of rapidly growing and slowly growing mycobacteria. Two biochemically typical strains (ATCC 49650T [T = type strain] and ATCC 49651) had identical sequences, while the sequence of a biochemically atypical strain (ATCC 49649) differed by 4 bp from the sequence of the two typical strains. The Hamming distances between these MCLO strains and related rapidly growing mycobacteria are comparable to the Hamming distances among taxa of rapidly growing mycobacteria established as species by DNA-DNA hybridization. We propose the name Mycobacterium mucogenicum sp. nov. for this new taxon because of the highly mucoid nature of most isolates on solid media.

The Mycobacterium tuberculosis ahpC gene, encoding the mycobacterial orthologue of alkylhydroperoxide reductase, undergoes an unusual regulatory cycle. The levels of AhpC alternate between stages of expression silencing in virulent strains grown as aerated cultures, secondary to a natural loss of the regulatory oxyR function in all strains of the tubercle bacillus, and expression activation in static bacilli by a yet undefined mechanism. The reasons for this unorthodox regulatory cycle controlling expression of an antioxidant factor are currently not known. In this work, M. tuberculosis H37Rv and Mycobacterium smegmatis mc(2)155 ahpC knockout mutants were tested for sensitivity to reactive nitrogen intermediates, in particular peroxynitrite, a highly reactive combinatorial product of reactive nitrogen and oxygen species, and sensitivity to bactericidal mechanisms in resting and activated macrophages. Both M. tuberculosis ahpC::Km(r) and M. smegmatis ahpC::Km(r) showed increased susceptibility to peroxynitrite. In contrast, inactivation of ahpC in M. tuberculosis did not cause increased sensitivity to donors of NO alone. M. tuberculosis ahpC::Km(r) also showed decreased survival in unstimulated macrophages, but the effect was no longer detectable upon IFNgamma activation. These studies establish a specific role for ahpC in antioxidant defences involving peroxynitrite and most likely additional cidal mechanisms in macrophages, with the regulatory cycle likely contributing to survival upon coming out of the stationary phase during dormancy (latent infection) or upon transmission to a new host.