Kegg Pathway: Biosynthesis of ansamycins

Sheng Wu Gong Cheng Xue Bao. 2009 Jun; 25(6): 847-53
Ni S, Zhang K, Wang Y, He W, Wang Y, He J, Wu L

ansamycins, such as rifamycin and ansamitocin, usually consist of a group of structural similar components. Geldanamycin, a benzenic ansamycin, has been found to consist of four structural similar components. We analyzed the geldanamycin (GDM) preparation from Streptomyces hygroscopicus 17997 by LC-ESI(+)-MS/MS, and discovered five novel and one known GDM analogues in trace amounts. Based on the ESI(+)-MS/MS spectra of these GDM analogues, and the present understanding of GDM Biosynthesis, we proposed the possible chemical structures of these GDM analogues. Three novel GDM analogues, all having the same molecular formula of C29H42N2O10, were GDM biosynthetic derivatives with one of the three C-C double bonds between C2-C3, C4-C5 and C8-C9 in GDM changed to mono-hydroxylated C-C single bond. The other two novel GDM analogues, having the same molecular formula of C28H38N2O8, were 17(or 12, or 4)-desmethoxylgeldanamycin and 4,5-dihydro-10,11-dehydrate-17-desmethyl-17-hydroxylgeldanamycin, respectively. The known GDM analogue, having the molecular formula of C29H42N2O9, was 4, 5-dihydrogeldanamycin, an intermediate in GDM Biosynthesis. The discovery of novel GDM analogues provided us new insights in understanding the biosynthetic details of GDM, and clues of obtaining GDM derivatives by gene-disruption and combinatorial Biosynthesis.

J Immunol. 2009 Oct 1; 183(7): 4385-94
Fionda C, Soriani A, Malgarini G, Iannitto ML, Santoni A, Cippitelli M

Modulation of the host immune system represents a promising therapeutic approach against cancer, including multiple myeloma. Recent findings indicate that the NK group 2D (NKG2D)- and DNAX accessory molecule-1 (DNAM-1)-activating receptors play a prominent role in tumor recognition and elimination by cytotoxic lymphocytes, suggesting that the levels of NKG2D and DNAM-1 ligand expression on tumor cells may be a critical factor to improve the immune response against cancer. In this study, we tested the effect of 17-allylaminogeldanamycin and radicicol, drugs targeting the heat shock protein-90 (HSP-90) chaperone protein and displaying antimyeloma activity, on the expression of NKG2D and DNAM-1 ligands in human myeloma cell lines. We demonstrate that HSP-90 inhibitors are able to up-regulate both MHC class I chain-related (MIC) A and MICB protein surface and mRNA expression in human myeloma cell lines, without any significant effect on the basal expression of the DNAM-1 ligand poliovirus receptor CD155, or induction of nectin-2 and UL16-binding proteins. Activation of the transcription factor heat shock factor-1 by HSP-90 inhibitors is essential for the up-regulation of MICA/MICB expression and knockdown of heat shock factor-1 using small hairpin RNA interference blocks this effect. Moreover, in vitro and in vivo binding of heat shock factor-1 to MICA and MICB promoters indicates that it may enhance NKG2D ligand expression at the transcriptional level. Finally, exposure to HSP-90 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and a blocking Ab specific for NKG2D significantly reduces this effect. Thus, these results provide evidence that targeting NKG2D ligands expression may be an additional mechanism supporting the antimyeloma activity of HSP-90 inhibitors and suggest their possible immunotherapeutic value.

Biomed Environ Sci. 2009 Jun; 22(3): 253-8
Sun AH, Fan XL, Li LW, Wang LF, Ans WY, Yan J

OBJECTIVE: To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray. METHODS: Four wild-type and 8 mutant probes were used to detect rifampin resistant strains. Target DNA of M. tuberculosis was amplified by PCR, hybridized and scanned. Direct sequencing was performed to verify the results of oligonucleotide microarray. RESULTS: Of the 102 rifampin-resistant strains 98 (96.1%) had mutations in the rpoB genes. CONCLUSION: Oligonucleotide microarray with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of rifampin resistance in M. tuberculosis isolates.

J Bacteriol. 2009 Nov; 191(21): 6584-91
Brzostek A, Pawelczyk J, Rumijowska-Galewicz A, Dziadek B, Dziadek J

It is expected that the obligatory human pathogen Mycobacterium tuberculosis must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that M. tuberculosis grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.

Radiat Res. 2009 Sep; 172(3): 321-30
Kiang JG, Smith JT, Agravante NG

Inducible nitric oxide synthase (iNOS) expression and NO production increase after radiation exposure. We showed previously that inhibiting iNOS expression prevents hemorrhage injury; we therefore investigated whether inhibiting iNOS expression also limits radiation injury. Human Jurkat T cells were exposed to gamma radiation (2, 4, 6 or 8 Gy), and cell lysates were collected for analysis at selected times afterward. Radiation exposure increased iNOS expression within 4 h postirradiation by increasing the levels of the iNOS transcription factors NF-kappaB and KLF6. By 24 h postirradiation cell viability was reduced. In these cells, NO production, lipid peroxidation, protein nitration, apoptosomes (formed by cytochrome c, caspase 9 and Apaf-1), and caspase 3 activity were significantly elevated, suggesting that the iNOS pathway had been activated. Treatment with the iNOS inhibitors 17-DMAG or L-NIL-6 24 h prior to irradiation limited these changes, as did treatment with iNOS siRNA to silence the iNOS gene. These results suggest radiation injury involves the iNOS pathway, and iNOS-mediated NO produced endogenously in the T cell alters overall T-cell function and results in apoptosis and cell lethality. Control of iNOS expression may represent a useful approach for protecting T cells from radiation injury.

Antivir Ther. 2009; 14(5): 687-95
Cohen K, Grant A, Dandara C, McIlleron H, Pemba L, Fielding K, Charalombous S, Churchyard G, Smith P, Maartens G

BACKGROUND: Rifampicin induces expression of the cytochrome P450 isoenzyme 2B6 (CYP2B6), which metabolizes efavirenz. The CYP2B6 516G>T polymorphism impairs efavirenz metabolism and occurs more commonly in Africans than in Caucasians. We explored the effect of rifampicin-based antitubercular therapy and the 516G>T polymorphism on efavirenz concentrations in HIV-infected patients in South Africa. METHODS: Between-patient and within-patient comparisons were made of mid-dosing interval efavirenz plasma concentrations in adults on antiretroviral therapy including efavirenz 600 mg daily, with and without antitubercular therapy. RESULTS: There were 142 participants (40 were on antitubercular therapy and 102 were controls), the mean weight was 66 kg. Median efavirenz concentration was 2.4 mg/l (interquartile range [IQR] 1.3-3.1) and 1.8 mg/l (IQR 1.4-4.4) in participants on antitubercular therapy and controls, respectively (P=0.734). Paired efavirenz concentrations during and after antitubercular therapy in 17 participants were also similar (P=0.113). Genotyping results were 60 (49%) G/G homozygotes, 46 (38%) G/T heterozygotes and 16 (13%) T/T homozygotes. In a multivariate logistic regression model adjusted for sex, weight and concomitant antitubercular therapy, the 516G>T polymorphism was strongly associated with high (>4 mg/l) efavirenz concentrations: odds ratio (OR) 4.4 (95% confidence interval [CI] 1.3-14.9) for G/T versus G/G and 31.1 (95% CI 6.6-146.6) for T/T versus G/G. High efavirenz concentrations were associated with severe sleep disturbance (P=0.048). Low (<1 mg/l) efavirenz concentrations were associated with virological failure (OR 12.5, 95% CI 2.7-57.3). CONCLUSIONS: Efavirenz can be used together with rifampicin-based antitubercular therapy without dose adjustment in this population. The 516G>T polymorphism occurred commonly and was associated with high efavirenz concentrations.

Clin Ther. 2009 Jun; 31(6): 1256-63
Deng S, Chen XP, Cao D, Yin T, Dai ZY, Luo J, Tang L, Li YJ

BACKGROUND: Pravastatin is a potent cholesterol-lowering agent; ~34% of an oral dose of pravastatin is eliminated unchanged through biliary and urinary excretion. Rifampicin is an inducer of drug metabolism enzymes, and it affects the activities of transporters involved in pravastatin disposition. Drug-drug interaction between rifampicin and pravastatin is possible because of the effects of rifampicin on the activities of drug transporters. OBJECTIVE: This study was designed to investigate the effects of a single oral dose of rifampicin on the pharmacokinetics of pravastatin. METHODS: Healthy Chinese male volunteers were recruited for this 2-phase, single-blind, placebo-controlled, crossover study. The subjects were randomly divided into 2 groups to receive either rifam-picin or placebo concomitantly with pravastatin. All subjects received a 20-mg oral dose of pravastatin on days 1 and 9, separated by an 8-day washout period. Subjects in the rifampicin group received a single 600-mg oral dose of rifampicin on day 1 and placebo on day 9; those in the placebo group received placebo on day 1 and a single 600-mg oral dose of rifampicin on day 9. High-performance liquid chromatography-tandem mass spectrometry was used to determine plasma concentrations of pravastatin for up to 12 hours after administration. Results: Twelve volunteers participated in the study (6 per group). The mean (SD) age of the subjects was 20 (2) years (range, 18-25 years). The mean height of the subjects was 174 (4) cm (range, 168-180 cm), and the mean weight was 69.2 (3.7) kg (range, 65-77 kg). The mean pharmacokinetic parameters for pravastatin that changed significantly were as follows (rifampicin and placebo groups, respectively): C(max) (315.7 [227.2] and 115.8 [77.5] ng . mL(-1) [P = 0.009]); AUC(0-12) (604.8 [73.3] and 259.0 [133.4] ng . h . mL(-1) [P < 0.001]); AUC(0-infinity)) (623.3 [248.8] and 275.1 [58.5] ng . h . mL(-1) [P < 0.001]); and apparent oral clearance (CL/F) (0.52 [0.18] and 1.30 [0.58] L . h(-1) . kg(-1) [P < 0.001]). No significant changes in the T(max) or t((1/2)) of pravastatin were observed. All subjects tolerated pravastatin well during both phases of the study, with or without coadministration of rifampicin. None of the subjects withdrew from the study. CONCLUSION: Coadministration of a single oral dose of rifampicin significantly increased the plasma concentration of pravastatin in this group of healthy Chinese male subjects.

Cancer Res. 2009 Sep 1; 69(17): 6995-7003
Kawabe M, Mandic M, Taylor JL, Vasquez CA, Wesa AK, Neckers LM, Storkus WJ

EphA2, a member of the receptor tyrosine kinase family, is commonly expressed by a broad range of cancer types, where its level of (over)expression correlates with poor clinical outcome. Because tumor cell expressed EphA2 is a nonmutated "self" protein, specific CD8(+) T cells are subject to self-tolerance mechanisms and typically exhibit only moderate-to-low functional avidity, rendering them marginally competent to recognize EphA2(+) tumor cells in vitro or in vivo. We have recently reported that the ability of specific CD8(+) T cells to recognize EphA2(+) tumor cells can be augmented after the cancer cells are pretreated with EphA2 agonists that promote proteasomal degradation and up-regulated expression of EphA2/class I complexes on the tumor cell membrane. In the current study, we show that treatment of EphA2(+) tumor cells with the irreversible heat shock protein 90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), similarly enhances their recognition by EphA2-specific CD8(+) T-cell lines and clones in vitro via a mechanism that is dependent on proteasome and transporter-associated protein function as well as the retrotranslocation of EphA2 into the tumor cytoplasm. When 17-DMAG and agonist anti-EphA2 monoclonal antibodies are coapplied, T-cell recognition of tumor cells is further increased over that observed for either agent alone. These studies suggest that EphA2 represents a novel heat shock protein 90 client protein and that the treatment of cancer patients with 17-DMAG-based "pulse" therapy may improve the antitumor efficacy of CD8(+) T effector cells reactive against EphA2-derived epitopes.

Curr Med Chem. 2009; 16(24): 3081-92
Hwang M, Moretti L, Lu B

With the rapid rise of tumor resistance, combinatorial anticancer therapies have gained favor over single-molecule inhibition to maximize the suppression of oncogenic pathways. In this regard, HSP90 inhibitors have rapidly emerged as a class of promising drugs that can target multiple oncogenic pathways simultaneously. HSP90 is a highly conserved protein chaperone involved in essential cellular functions such as protein folding and cell signaling in both stressed and unstressed cells. In the last decade, a large number of oncogenic client proteins have been identified to associate with HSP90 and contribute to malignant transformation. Development of HSP90 inhibitors, derived from the natural compound geldanamycin that mimics the ATP binding site of HSP90, was designed to target HSP90 and allow degradation of these client proteins. Preclinical and clinical data with HSP90 inhibitors in various cancer models are promising, and evidences also hint at the potential for tumor-selective cytotoxicity as well as enhanced sensitization to chemo- and radiotherapy. This review will discuss the effects of HSP90 inhibition in cancer, the known mechanistic basis for the oncogenic toxicity and selectivity, as well as the current progress on single or combinatorial therapies with HSP90 inhibitors.

Biochim Biophys Acta. 2009 Oct; 1793(10): 1597-603
Banz VM, Medová M, Keogh A, Furer C, Zimmer Y, Candinas D, Stroka D

N-myc downstream-regulated gene 1 (NRDG1) is a stress-induced protein whose putative function is suppression of tumor metastasis. A recent proteonomic study showed NDRG1 interacts with the molecular chaperone heat shock protein 90 (Hsp90). From their reported association, we investigated if NDRG1 is dependent on Hsp90 for its stability and is therefore a yet unidentified Hsp90 client protein. Here, we demonstrate that endogenous NDRG1 and Hsp90 physically associate in hepatocellular cancer cell lines. However, geldanamycin (GA)-mediated inhibition of Hsp90 did not disrupt their interaction or result in NDRG1 protein destabilization. On the contrary, inhibition of Hsp90 led to a transcriptional increase of NDRG1 protein which was associated with cell growth arrest. We also observed that GA inhibited the phosphorylation of NDRG1 by targeting its regulating kinases, serum- and glucocorticoid-induced kinase 1 (SGK1) and glycogen synthase kinase 3 beta (GSK3beta). We demonstrate that in the presence of GA, GSK3beta protein and activity were decreased thus indicating that Hsp90 is necessary for GSK3beta stability. Taken together, our data demonstrate that NDRG1 is not a classic client protein but interacts with Hsp90 and is still dually regulated by Hsp90 at a transcriptional and post-translational level. Finally, we suggest for the first time GSK3beta as a new client protein of Hsp90.

Am J Trop Med Hyg. 2009 Aug; 81(2): 322-9
Barroso EC, Pinheiro VG, Façanha MC, Carvalho MR, Moura ME, Campelo CL, Peloquin CA, Guerrant RL, Lima AA

This study evaluates the serum concentrations of rifampin (RMP), isoniazid (INH), and intestinal barrier function in patients with multidrug-resistant tuberculosis (MDR-TB), drug susceptible tuberculosis (DS-TB), and health volunteers (HC; controls). Peak serum concentrations of RMP were significantly lower in MDR-TB and DS-TB as compared with HC (odds ratio [OR] = 3.125, confidence interval [CI] [1.037-9.418] and OR = 4.025, CI [1.207-13.418], respectively). The INH peak serum concentration was not significantly different between MDR-TB versus DS-TB or DS-TB versus HC. The percent of mannitol excretion was significantly lower in the MDR-TB group compared with DS-TB (13.18 versus 16.03, analysis of covariance [ANCOVA], P = 0.0369) and compared with HC (13.18 versus 16.61, ANCOVA, P = 0.0291) the other study groups. These data suggested a lower peak serum concentration of RMP for both MDR-TB and DS-TB as compared with the HC group. The data also showed a lower intestinal area of absorption in patients with tuberculosis and even worse in MDR-TB.

Indian J Exp Biol. 2009 Jun; 47(6): 469-74
Yadav AB, Sharma R, Muttil P, Singh AK, Verma RK, Mohan M, Patel SK, Misra A

Macrophage responses to infection with Mycobacterium tuberculosis (MTB) and treatment with soluble isoniazid (INH) plus rifabutin (RFB) versus microparticles containing equivalent amounts of drugs were compared. It was investigated whether macrophages driven to alternative activation upon infection with MTB could be rescued to display the classical activation phenotype. It was established that microparticles sustain high levels of drugs in cytosol of macrophages for longer period as compared to soluble drugs. Microparticles co-localized with intracellular bacteria, and induced a variety of innate bactericidal responses, including induction of free radicals, alteration of mitochondrial membrane potential and apoptosis. The data strongly suggest that additional benefit may be derived from the nature of the drug delivery system, which fulfils Koch's dictum 'stimulate the phagocyte' for curing tuberculosis.

J Mol Biol. 2009 Oct 2; 392(4): 923-36
Nilapwar S, Williams E, Fu C, Prodromou C, Pearl LH, Williams MA, Ladbury JE

Despite its importance as a target in anti-cancer therapeutics and the numerous rational-based inhibitor design efforts aimed at it, there are only limited data available on structural-thermodynamic relationships of interactions of the N-terminal ATP-binding domain of Hsp90 (N-Hsp90). Here, we redress this by presenting an investigation of binding of nucleotides and ansamycin compounds to this domain. Interactions of nucleotides with N-Hsp90 are relatively weak (>10 microM) and are strongly enthalpy driven over the temperature range 10-25 degrees C. Geldanamycin (GA) and its analogues 17-AAG [17-(allylamino)-17-demethoxy-GA] and 17-DMAG (17-N,N-dimethylaminoethylamino-17-demethoxy-GA) bind more strongly and have a dominant favourable enthalpic contribution over the temperature range investigated. We investigated the temperature dependence of the enthalpic contribution to binding. We found that while the ansamycin compounds have the commonly observed negative value, the nucleotides show a negligible or even a positive DeltaC(p) of binding. These data represent the first observation of a single binding site for which interactions with different ligands result in both negative and positive DeltaC(p) values. By addressing the likely impact of the potential contributions from protein-ligand interactions, we are able to attribute the anomalous DeltaC(p) for the nucleotides largely to a change in the conformation of the domain structure and local motion in the lid region of N-Hsp90 with the concomitant exposure of hydrophobic amino acid side chains.

Am J Respir Crit Care Med. 2009 Sep 15; 180(6): 553-7
Ibrahim M, Truffot-Pernot C, Andries K, Jarlier V, Veziris N

RATIONALE: The diarylquinoline R207910 (TMC207) has potent bactericidal activity in a murine model of tuberculosis (TB), but its sterilizing activity has not been determined. OBJECTIVES: To evaluate the sterilizing activity of R207910-containing combinations in the murine model of TB. METHODS: Swiss mice were intravenously inoculated with 6 log(10) of Mycobacterium tuberculosis strain H37Rv, treated with R207910-containing regimens, and followed for 3 months to determine relapse rates (modified Cornell model). MEASUREMENTS AND MAIN RESULTS: Quantitative lung and spleen colony-forming unit counts and bacteriological relapse rates 3 months after the end of therapy were compared for the following regimens: 2, 3, or 4 months of R207910 (J) and pyrazinamide (Z) combined with rifampin (R) or isoniazid (H) or both and 3 or 4 months of a moxifloxacin (M)-containing regimen and 6 months of the standard WHO regimen RHZ. All J-treated mice were culture negative after 4 months of therapy. The relapse rate in the group treated with 4 months of JHRZ was similar to that of mice treated for 6 months with the RHZ regimen (6 vs. 17%; P = 0.54) and lower than that of RMZ (6 vs. 42%; P = 0,03), a moxifloxacin-containing regimen that was the most active in mice on once-daily basis. CONCLUSIONS: Four months of treatment with some J-containing regimens was as effective as the 6-month standard regimen and more effective than 4 months of treatment with M-containing regimens. Supplementation of standard regimen (RHZ) with J or substitution of J for H may shorten the treatment duration needed to cure TB in patients.

Toxicol Appl Pharmacol. 2009 Oct 1; 240(1): 26-36
Chen X, Zhang C, Wang H, Xu J, Duan ZH, Zhang Y, Yu T, Wei W, Xu DX, Xu JM

Rifampicin is a well-known hepatotoxicant, but little is known about the mechanism of rifampicin-induced hepatotoxicity. The aim of this study was to characterize the expression and localization of hepatocyte tight junctions in rifampicin-induced cholestasis in mice. Cholestasis was induced by administration of rifampicin (200 mg/kg) for 7 consecutive days or treatment with a single dose of rifampicin (200 mg/kg) by gastric intubation. The expression of mRNA for hepatic zonula occludens (ZO)-1, ZO-2, ZO-3, occludin and claudin-1 was determined using RT-PCR. Localization of ZO-1 and occludin was detected using immunofluorescence. Results showed that there was an 82-fold increase in the conjugated bilirubin in serum in rifampicin-treated mice. In addition, an 8-fold increase in total bile acid in serum was observed after a seven-day administration of rifampicin. The integrity of hepatocyte ZO-1 and occludin was altered by a seven-day administration of rifampicin. Importantly, the integrity and intensity of hepatocyte tight junctions were altered as early as 30 min after a single dose of rifampicin. The expression of hepatic ZO-1 and ZO-2 mRNA was significantly decreased, beginning as early as 30 min and remaining a lower level 12 h after a single dose of rifampicin. Taken together, these results suggest that the altered integrity and internalization of hepatocyte tight junctions are associated with rifampicin-induced cholestasis.

Med Clin North Am. 2009 Jul; 93(4): 819-36, vii
Sundaram V, Shaikh OS

Hepatic encephalopathy is characterized by neuropsychiatric abnormalities in patients with liver failure. Severe hepatic encephalopathy is an indication for liver transplantation as it portends poor outcome. Treatment of hepatic encephalopathy involves correction of precipitating factors such as sepsis, gastrointestinal bleeding, medications, and electrolyte imbalance. Effective therapies include lactulose and antibiotics such as neomycin, metronidazole, and rifaximin.

Respir Res. 2009; 10: 60
Lopez JP, Vigerust DJ, Shepherd VL

BACKGROUND: Surfactant protein A (SP-A) is a C-type lectin involved in surfactant homeostasis as well as host defense in the lung. We have recently demonstrated that SP-A enhances the killing of bacillus Calmette-Guerin (BCG) by rat macrophages through a nitric oxide-dependent pathway. In the current study we have investigated the role of tyrosine kinases and the downstream mitogen-activated protein kinase (MAPK) family, and the transcription factor NFkappaB in mediating the enhanced signaling in response to BCG in the presence of SP-A. METHODS: Human SP-A was prepared from alveolar proteinosis fluid, and primary macrophages were obtained by maturation of cells from whole rat bone marrow. BCG-SP-A complexes were routinely prepared by incubation of a ratio of 20 microg of SP-A to 5 x 105 BCG for 30 min at 37 degrees C. Cells were incubated with PBS, SP-A, BCG, or SP-A-BCG complexes for the times indicated. BCG killing was assessed using a 3H-uracil incorporation assay. Phosphorylated protein levels, enzyme assays, and secreted mediator assays were conducted using standard immunoblot and biochemical methods as outlined. RESULTS: Involvement of tyrosine kinases was demonstrated by herbimycin A-mediated inhibition of the SP-A-enhanced nitric oxide production and BCG killing. Following infection of macrophages with BCG, the MAPK family members ERK1 and ERK2 were activated as evidence by increased tyrosine phosphorylation and enzymatic activity, and this activation was enhanced when the BCG were opsonized with SP-A. An inhibitor of upstream kinases required for ERK activation inhibited BCG- and SP-A-BCG-enhanced production of nitric oxide by approximately 35%. Macrophages isolated from transgenic mice expressing a NFkappaB-responsive luciferase gene showed increased luciferase activity following infection with BCG, and this activity was enhanced two-fold in the presence of SP-A. Finally, lactacystin, an inhibitor of IkappaB degradation, reduced BCG- and SP-A-BCG-induced nitric oxide production by 60% and 80% respectively. CONCLUSION: These results demonstrate that BCG and SP-A-BCG ingestion by macrophages is accompanied by activation of signaling pathways involving the MAP kinase pathway and NFkappaB.

Antimicrob Agents Chemother. 2009 Sep; 53(9): 3675-82
Ramón-García S, Martín C, Thompson CJ, Aínsa JA

Bacterial efflux pumps have traditionally been studied as low-level drug resistance determinants. Recent insights have suggested that efflux systems are often involved with fundamental cellular physiological processes, suggesting that drug extrusion may be a secondary function. In Mycobacterium tuberculosis, little is known about the physiological or drug resistance roles of efflux pumps. Using Mycobacterium bovis BCG as a model system, we showed that deletion of the Rv1410c gene encoding the P55 efflux pump made the strain more susceptible to a range of toxic compounds, including rifampin (rifampicin) and clofazimine, which are first- and second-line antituberculosis drugs. The efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin inhibited the P55-determined drug resistance, suggesting the active export of the compounds by use of the transmembrane proton and electrochemical gradients as sources of energy. In addition, the P55 efflux pump mutant was more susceptible to redox compounds and displayed increased intracellular redox potential, suggesting an essential role of the efflux pump in detoxification processes coupled to oxidative balance within the cell. Finally, cells that lacked the p55 gene displayed smaller colony sizes and had a growth defect in liquid culture. This, together with an increased susceptibility to the cell wall-targeting compounds bacitracin and vancomycin, suggested that P55 is needed for proper cell wall assembly and normal growth in vitro. Thus, P55 plays a fundamental role in oxidative stress responses and in vitro cell growth, in addition to contributing to intrinsic antibiotic resistance. Inhibitors of the P55 efflux pump could help to improve current treatments for tuberculosis.

J Biol Chem. 2009 Aug 21; 284(34): 22898-904
Kohli RM, Abrams SR, Gajula KS, Maul RW, Gearhart PJ, Stivers JT

Enzymes of the AID/APOBEC family, characterized by the targeted deamination of cytosine to generate uracil within DNA, mediate numerous critical immune responses. One family member, activation-induced cytidine deaminase (AID), selectively introduces uracil into antibody variable and switch regions, promoting antibody diversity through somatic hypermutation or class switching. Other family members, including APOBEC3F and APOBEC3G, play an important role in retroviral defense by acting on viral reverse transcripts. These enzymes are distinguished from one another by targeting cytosine within different DNA sequence contexts; however, the reason for these differences is not known. Here, we report the identification of a recognition loop of 9-11 amino acids that contributes significantly to the distinct sequence motifs of individual family members. When this recognition loop is grafted from the donor APOBEC3F or 3G proteins into the acceptor scaffold of AID, the mutational signature of AID changes toward that of the donor proteins. These loop-graft mutants of AID provide useful tools for dissecting the biological impact of DNA sequence preferences upon generation of antibody diversity, and the results have implications for the evolution and specialization of the AID/APOBEC family.

Cell Immunol. 2009; 259(1): 49-55
Fang S, Fu J, Yuan X, Han C, Shi L, Xin Y, Luo L, Yin Z

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the conformational maturation and function of certain signaling proteins. Hsp90 inhibitors cause the inactivation, destabilization and eventual degradation of Hsp90 client proteins through occupying the ATP/ADP binding pocket of Hsp90. In the present study, we found that Hsp90 interacted with MEKK3 in HEK293 cells. Hsp90 inhibitors reduced the level of endogenous MEKK3 in time- and dose-dependent manners, and this decrease was reversed by Hsp90 overexpression. In addition, Hsp90 RNAi destabilized MEKK3. A selective inhibitor of Hsp90, geldanamycin (GA), shortened MEKK3 half-life, and induced ubiquitination and proteasomal degradation of MEKK3. These results strongly suggested that Hsp90 could work as the molecular chaperone of MEKK3.