The production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages helps to control infection by Mycobacterium tuberculosis (Mtb). However, the protection is imperfect and infection persists. To identify genes that Mtb requires to resist RNI, we screened 10,100 Mtb transposon mutants for hypersusceptibility to acidified nitrite. We found 12 mutants with insertions in seven genes representing six pathways, including the repair of DNA (uvrB) and the synthesis of a flavin cofactor (fbiC). Five mutants had insertions in proteasome-associated genes. An Mtb mutant deficient in a presumptive proteasomal adenosine triphosphatase was attenuated in mice, and exposure to proteasomal protease inhibitors markedly sensitized wild-type Mtb to RNI. Thus, the mycobacterial proteasome serves as a defense against oxidative or nitrosative stress.

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SummaryGenes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 ∞ C allowed formation of an a 7 b 7 b 7 a 7 , 750 kDa cylindrical stack of four rings in which all 14 b -subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete , Rhodococcus erythropolis , Mtb's b -chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg 2 + or Ca 2 + . Electron microscopy revealed what appeared to be obstructed a -rings in the Mtb 750 kDa particle. Deletion of the Nterminal octapeptide from Mtb's a -chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes , the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator.

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Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known . We have found that DNA synthesized during chemically induced differentiation of Friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of < 1 .6% in 5-methylcytosine content .Hypomethylated DNA can be isolated from Friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis-acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced . DNA isolated from cells of a dimethyl sulfoxideresistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of Friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of Friend erythroleukemia cells.

Intramyocardial injection of B cells into early post-ischemic myocardium preserved cardiac function by cardiomyocyte salvage. Other BM MNC subtypes were either ineffective or suppressed cardioprotection conferred by an enriched B cell population.

Hexamethylenebisacetamide (HMBA)-induced murine erythroleukemia (MEL) differentiation is a multistep process. Commitment is the capacity to express terminal cell division and characteristics of the differentiated phenotype even after the cells are removed from culture with inducer. Culture of MEL cell line 745A-DS19 (DS19) with HMBA causes commitment to terminal differentiation after a latent period of about 10-12 hr. Previous studies have shown that during this latent period, HMBA causes a number of metabolic changes, including modulation in expression of certain protooncogenes. We now report the development of a MEL cell line (designated V3-17) derived from DS19 that is resistant to vincristine and is (i) markedly more sensitive to HMBA, (ii) induced to commitment without a detectable latent period, and (iN) resistant to the effects of phorbol ester and dexamethasone, which are potent inhibitors of HMBAmediated DS19 differentiation. We suggest that this V3-17 MEL cell line may express a factor that circumvents HMBAmediated early events, which prepare the cells for commitment to terminal differentiation.Hexamethylenebisacetamide (HMBA)-mediated murine erythroleukemia (MEL) cell terminal differentiation is a multistep process (1, 2). Upon culture of MEL cell line 745A-DS19 (DS19) (3) with HMBA (4), there is a latent period of -10 to 12 hr during which commitment to terminal differentiation cannot be detected. Commitment is defined as the capacity to express characteristics of the erythroid differentiated phenotype, including loss of proliferative capacity, despite removal of the inducer (5, 6). This early, latent period is followed by a period during which an increasing proportion of the population expresses characteristics of terminal differentiation, including loss of proliferative capacity.During the latent period, the inducer initiates a number of metabolic changes that precede irreversible commitment to differentiation. Among these are alterations in membrane permeability, which involve Na', K+, and Ca2+ flux (7-9); changes in cell volume (10); a transient increase in cyclic AMP concentration (11); a prompt increase in membraneassociated protein kinase C activity (PKC); the appearance in the cytosol of a Ca2 + -and phospholipid-independent form of PKC, presumably generated by proteolytic cleavage of membrane-bound PKC (12); and the modulation in expression of a number of genes, including c-myb, c-myc, c-fos, and p53 (13)(14)(15)(16). Upon more prolonged culture with HMBA, DS19 cells become irreversibly committed (5, 6). Morphological and molecular changes occur that are similar to normal erythroid terminal cell differentiation, including the coordinated expression of genes for a'-and 83mj-globin, for the heme synthetic enzymes, and for erythroid-specific membrane proteins, as well as suppression of DNA replication and rRNA synthesis (1,17,18).In the present studies we describe the development of a MEL cell line derived from DS19 that is resistant to inhibition of cell growth by vincristine and is de...