The Induction of Antigen 85B-Specific CD8+ T Cells by Recombinant BCG Protects against Mycobacterial Infection in Mice.

Mycobacterium tuberculosis (Mtb) infection remains a major health problem worldwide. Although the Bacillus Calmette-Guérin (BCG) vaccine is the most widely used vaccination for preventing tuberculosis (TB), its efficacy is limited. We previously developed a new recombinant BCG (rBCG)-based vaccine encoding the Ag85B protein of M. kansasii (Mkan85B), termed rBCG-Mkan85B, and its administration is followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). Previously, we identified MHC-I (H2-Kd)-restricted epitopes that highly cross-react with those of Mtb in BALB/c (H2d) and CB6F1 (H2b/d) mice. We also reported that the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination protocol protected CB6F1 mice against M. kansasii infection. In this study, to investigate the protective effect of our novel rBCG against Mtb infection, CB6F1 mice were either left unimmunized or immunized with the BCG, rBCG-Mkan85B, or rBCG-Mkan85B/DNA-Mkan85B vaccine for 10 weeks prior to inhalation exposure to the virulent Mtb Erdman strain for another 6 weeks. Compared with the BCG and rBCG-Mkan85B vaccinations, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination protocol significantly reduced the numbers of pulmonary colony-forming units (CFUs). Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination induced antigen-specific polyfunctional CD4+ and CD8+ T cells. These results suggest that CD8+ T-cell immunity to immunodominant epitopes of Mtb is enhanced by rBCG vector-based immunization. Thus, rBCG vector-based vaccinations may overcome the limited ability of the current BCG vaccine to elicit TB immunity.

SOCS1 Antagonist-Expressing Recombinant Bacillus Calmette-Guérin Enhances Antituberculosis Protection in a Mouse Model.

Suppressor of cytokine signaling 1 (SOCS1) plays a key role in the negative regulation of JAK/STAT signaling, which is involved in innate immunity and subsequent adaptive immunity. Bacillus Calmette-Guérin (BCG) induces upregulation of SOCS1 expression in host cells, which may lead to the suppression of immune responses by BCG via inhibition of the JAK/STAT signaling pathway. This might cause A reduction in the protective effect of a BCG vaccine. In the current study, we assessed the immune responses to and the protective efficacy of a recombinant BCG secreting a dominant negative mutant of the SOCS1 molecule (rBCG-SOCS1DN). C57BL/6 mice were immunized with rBCG-SOCS1DN or parental BCG Tokyo vaccine strain harboring an empty plasmid vector (rBCG-pSO). rBCG-SOCS1DN enhanced the activation of bone marrow-derived dendritic cells and the activation of T cells compared with those with rBCG-pSO. The amounts of IFN-γ, TNF-α, and IL-6 produced by splenocytes of rBCG-SOCS1DN-immunized mice were larger than those produced by splenocytes of rBCG-pSO-immunized mice. Moreover, the rBCG-SOCS1DN-immunized mice showed a substantial reduction in the number of CFU of Mycobacterium tuberculosis in the lungs and spleens compared with that in control BCG-immunized mice when the immunized mice were infected with a highly pathogenic M. tuberculosis strain by inhalation. These findings provide evidence for the possibility of rBCG-SOCS1DN being an effective M. tuberculosis vaccine with a novel concept of rBCG as a tool for immunomodulation in host cells.

MHC-restricted Ag85B-specific CD8+ T cells are enhanced by recombinant BCG prime and DNA boost immunization in mice.

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8+ T cell response to Bacillus Calmette-Guérin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8+ T cells with an rBCG-based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG-Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). We identified two MHC-I (H2-Kd )-restricted epitopes that induce cross-reactive responses to Mtb and other related mycobacteria in both BALB/c (H2d ) and CB6F1 (H2b/d ) mice. The H2-Kd -restricted peptide epitopes elicited polyfunctional CD8+ T cell responses that were also highly cross-reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2-Kd -restricted epitopes elicit distinct CD8+ T cell populations, a result explained by the X-ray structure of the two peptide/H2-Kd complexes. These results suggest that rBCG-Mkan85B vector-based immunization and DNA-Mkan85B boost may enhance CD8+ T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.

Diacylglycerol kinase α-selective inhibitors induce apoptosis and reduce viability of melanoma and several other cancer cell lines.

Diacylglycerol (DG) kinase (DGK), which phosphorylates DG to generate phosphatidic acid (PA), consists of ten isozymes (α-к). Recently, we identified a novel small molecule inhibitor, CU-3, that selectively inhibits the activity of the α isozyme. In addition, we newly obtained Compound A, which selectively and strongly inhibits type I DGKs (α, ß, and γ). In the present study, we demonstrated that both CU-3 and Compound A induced apoptosis (caspase 3/7 activity and DNA fragmentation) and viability reduction of AKI melanoma cells. Liquid chromatography-mass spectrometry revealed that the production of 32:0- and 34:0-PA species was commonly attenuated by CU-3 and Compound A, suggesting that lower levels of these PA molecular species are involved in the apoptosis induction and viability reduction of AKI cells. We determined the effects of the DGKα inhibitors on several other cancer cell lines derived from refractory cancers. In addition to melanoma, the DGKα inhibitors enhanced caspase 3/7 activity and reduced the viability of hepatocellular carcinoma, glioblastoma, and pancreatic cancer cells, but not breast adenocarcinoma cells. Interestingly, Western blot analysis indicated that the DGKα expression levels were positively correlated with the sensitivity to the DGK inhibitors. Because both CU-3 and Compound A induced interleukin-2 production by T cells, it is believed that these two compounds can enhance cancer immunity. Taken together, our results suggest that DGKα inhibitors are promising anticancer drugs.

Primary Role of Suppressor of Cytokine Signaling 1 in Mycobacterium bovis BCG Infection.

Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of JAK/STAT signaling and is induced by mycobacterial infection. To understand the major function of SOCS1 during infection, we established a novel system in which recombinant Mycobacterium bovis bacillus Calmette-Guérin expressed dominant-negative SOCS1 (rBCG-SOCS1DN) because it would not affect the function of SOCS1 in uninfected cells. When C57BL/6 mice and RAG1-/- mice were intratracheally inoculated with rBCG-SOCS1DN, the amount of rBCG-SOCS1DN in the lungs was significantly reduced compared to that in the lungs of mice inoculated with a vector control counterpart and wild-type BCG. However, these significant differences were not observed in NOS2-/- mice and RAG1-/- NOS2-/- double-knockout mice. These findings demonstrated that SOCS1 inhibits nitric oxide (NO) production to establish mycobacterial infection and that rBCG-SOCS1DN has the potential to be a powerful tool for studying the primary function of SOCS1 in mycobacterial infection.

Distinct 1-monoacylglycerol and 2-monoacylglycerol kinase activities of diacylglycerol kinase isozymes.

Diacylglycerol kinase (DGK) consists of ten isozymes and is involved in a wide variety of patho-physiological events. However, the enzymological properties of DGKs have not been fully understood. In this study, we performed a comprehensive analysis on the 1-monoacylglycerol kinase (MGK) and 2-MGK activities of ten DGK isozymes. We revealed that type I (α, ß and γ), type II (δ, η and κ) and type III (ε) DGKs have 7.9-19.2% 2-MGK activity compared to their DGK activities, whereas their 1-MGK activities were <3.0%. Both the 1-MGK and 2-MGK activities of the type IV DGKs (ζ and ι) were <1% relative to their DGK activities. Intriguingly, type V DGKθ has approximately 6% 1-MGK activity and <2% 2-MGK activity compared to its DGK activity. Purified DGKθ exhibited the same results, indicating that its 1-MGK activity is intrinsic. Therefore, DGK isozymes are categorized into three types with respect to their 1-MGK and 2-MGK activities: those having (1) 2-MGK activity relatively stronger than their 1-MGK activity (types I-III), (2) only negligible 1-MGK and 2-MGK activities (type IV), and (3) 1-MGK activity stronger than its 2-MGK activity (type V). The 1-MGK activity of DGKθ and the 2-MGK activity of DGKα were stronger than those of the acylglycerol kinase reported as 1-MGK and 2-MGK to date. The presence or absence of 1-MGK and 2-MGK activities may be essential to the patho-physiological functions of each DGK isozyme.

Development of a liquid chromatography-mass spectrometry based enzyme activity assay for phosphatidylcholine-specific phospholipase C.

Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) hydrolyzes PC to generate the second messenger 1,2-diacylglycerol (DG) and phosphocholine. PC-PLC plays pivotal roles in inflammation, carcinogenesis, tumor progression, atherogenesis, and subarachnoid hemorrhage. Although the activity of PC-PLC in mammalian tissues was discovered approximately 40 years ago, neither the protein nor its gene has been identified. In the present study, we developed a non-radioactive enzyme activity assay for PC-PLC based on mass spectrometric detection of DG following HPLC separation. This new liquid chromatography-mass spectrometry (LC-MS) assay directly determines a specific reaction product, 1-palmitoyl-2-oleoyl-DG, that is generated from 1-palmitoyl-2-oleoyl-PC by purified Bacillus cereus PC-PLC. The LC-MS assay offers several advantages including a lower background (0.02% versus 91%), higher signal background ratio (4242 versus 1.06)/signal noise ratio (7494 versus 4.4), higher sensitivity (≥32-fold), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than a conventional fluorometric assay, which indirectly detects phosphocholine produced in the reaction. In addition to Bacillus cereus PC-PLC, the LC-MS assay was applicable to the measurement of mammalian PC-PLC prepared from the mouse brain. The radioisotope-free, highly sensitive and precise LC-MS assay for PC-PLC would be useful for the purification and identification of PC-PLC protein.

A novel diacylglycerol kinase α-selective inhibitor, CU-3, induces cancer cell apoptosis and enhances immune response.

Diacylglycerol kinase (DGK) consists of 10 isozymes. The α-isozyme enhances the proliferation of cancer cells. However, DGKα facilitates the nonresponsive state of immunity known as T-cell anergy; therefore, DGKα enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGKα activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl)amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGKα with an IC50 value of 0.6 µM. CU-3 targeted the catalytic region, but not the regulatory region, of DGKα. CU-3 competitively reduced the affinity of DGKα for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGKα and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.

Both the C1 domain and a basic amino acid cluster at the C-terminus are important for the neurite and branch induction ability of DGKß.

We previously reported that diacylglycerol kinase ß (DGKß) induces neurites and branches, contributing to higher brain function including emotion and memories. However, the detailed molecular mechanism of DGKß function remains unknown. Therefore, we constructed various mutants of DGKß and compared their enzyme activity, intracellular localization, and ability to induce neurites and branching in SH-SY5Y cells. Even when RVH-domain and EF-hand motif were deleted, the mutant showed similar plasma membrane localization and neurite induction compared to wild type (WT), although the kinase activity of the mutant was three times higher than that of WT. In contrast, further deletion of C1 domain reduced the activity to 50% and abolished plasma membrane localization and neurite induction ability. When 34 amino acids were deleted from C-terminus, the mutants completely lost enzyme activity, plasma membrane localization, and the ability to induce neurites. A kinase-negative mutant of DGKß retained plasma membrane localization and induced significant neurites and branches; however, the rate of induction was weaker than that of WT. Furthermore, C1A and C1B mutants, which have a mutation in a cysteine residue in the C1A or C1B domain, and the RK/E mutant, which has substitutions of arginine and lysine to glutamic acid in a cluster of basic amino acids at the C-terminus, lost their plasma membrane localization and neurite induction ability. These results indicate that in addition to kinase activity, plasma membrane localization via the C1 domain and basic amino acids at the C-terminus were indispensable for neurite induction by DGKß.

Solidification characteristics of solid biofuel densified by two-step torrefaction process.

Coal coke, which is used widely in industrial furnaces, emits large amounts of CO2. To utilize solid biofuels as alternatives to coal coke, the fuel ratio of the biofuels must be improved to generate functions, such as deoxidization, permeability, and carbon pickup. In this study, an innovative densification molding method is proposed; it uses a two-step torrefaction process with a high CO2 reduction effect. The molding method consists of the following two-step torrefaction process at torrefaction temperatures of 463-773 K: In the first step, raw biomass is torrefied to remove some of the volatile matter that inhibits densified molding. In the second step, the torrefied biomass is densified at the above temperature. The purpose of the second torrefaction step is to further enhance the fuel ratio due to the conversion of volatile matter to fixed carbon and to develop the thermal softening of lignin. Solid biofuel densified using a two-step torrefaction process was produced from a Japanese cedar sample, and it was found that its fuel ratio was significantly improved. Furthermore, the mechanism of the adhesive effect during carbonization was elucidated by analyzing the structure of the densified solid biofuel using Raman spectroscopy.

Culture filtrate proteins from BCG act as adjuvants for cytotoxic T lymphocyte induction.

Mycobacterium bovis bacilli Calmette-Guerin (BCG) is a licensed vaccine against tuberculosis. It requires attenuated live bacteria to be effective, possibly because actively secreted proteins play a critical role in inducing anti-tuberculosis immunity. BCG also functions as an effective adjuvant. Moreover, the effects of BCG components as adjuvants are not important as those of attenuated live BCG, which is used in cancer immunotherapy. However, the BCG secreted proteins have not been paid attention in anticancer immunity. To understand mycobacterial secreted proteins' function, we investigate immune responses to BCG culture filtrate proteins (CFP). Here, CFP strongly induce both antigen-specific CD4+ T cells and specific CD8+ T cells, which may be functional cytotoxic T lymphocytes (CTLs). In this study, we clearly demonstrate that CFP acts as an adjuvant for CTL induction against specific co-administered proteins and propose CFP as a new protein adjuvant. The CTL response shows potent anticancer effects in mice. These findings could provide insight into the contribution of mycobacterial secreted proteins in both anticancer and antimycobacterial immunity.

Mycobacterial mycolic acids trigger inhibitory receptor Clec12A to suppress host immune responses.

Mycobacteria often cause chronic infection. To establish persistence in the host, mycobacteria need to evade host immune responses. However, the molecular mechanisms underlying the evasion strategy are not fully understood. Here, we demonstrate that mycobacterial cell wall lipids trigger an inhibitory receptor to suppress host immune responses. Mycolic acids are major cell wall components and are essential for survival of mycobacteria. By screening inhibitory receptors that react with mycobacterial lipids, we found that mycolic acids from various mycobacterial species bind to mouse Clec12A, and more potently to human Clec12A. Clec12A is a conserved inhibitory C-type lectin receptor containing immunoreceptor tyrosine-based inhibitory motif (ITIM). Innate immune responses, such as MCP-1 production, and PPD-specific recall T cell responses were augmented in Clec12A-deficient mice after infection. In contrast, human Clec12A transgenic mice were susceptible to infection with M. tuberculosis. These results suggest that mycobacteria dampen host immune responses by hijacking an inhibitory host receptor through their specific and essential lipids, mycolic acids. The blockade of this interaction might provide a therapeutic option for the treatment or prevention of mycobacterial infection.

Mycobacteria exploit host hyaluronan for efficient extracellular replication.

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.

Recombinant BCG-Prime and DNA-Boost Immunization Confers Mice with Enhanced Protection against Mycobacterium kansasii.

The incidence of infections with nontuberculous mycobacteria (NTM) has been increasing worldwide. The emergence of multidrug-resistant NTM is a serious clinical concern, and a vaccine for NTM has not yet been developed. We previously developed a new recombinant Bacillus Calmette-Guérin (rBCG) vaccine encoding the antigen 85B (Ag85B) protein of Mycobacterium kansasii-termed rBCG-Mkan85B-which was used together with a booster immunization with plasmid DNA expressing the same M. kansasii Ag85B gene (DNA-Mkan85B). We reported that rBCG-Mkan85B/DNA-Mkan85B prime-boost immunization elicited various NTM strain-specific CD4+ and CD8+ T cells and induced Mycobacterium tuberculosis-specific immunity. In this study, to investigate the protective effect against M. kansasii infection, we challenged mice vaccinated with a rBCG-Mkan85B or rBCG-Mkan85B/DNA-Mkan85B prime-boost strategy with virulent M. kansasii. Although BCG and rBCG-Mkan85B immunization each suppressed the growth of M. kansasii in the mouse lungs, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination reduced the bacterial burden more significantly. Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination induced antigen-specific CD4+ and CD8+ T cells. Our data suggest that rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination effectively enhances antigen-specific T cells. Our novel rBCG could be a potential alternative to clinical BCG for preventing various NTM infections.

Characterization of α-synuclein N-terminal domain as a novel cellular phosphatidic acid sensor.

Tracking the localization and dynamics of the intracellular bioactive lipid phosphatidic acid (PA) is important for understanding diverse biological phenomena. Although several PA sensors have been developed, better ones are still needed for comprehensive PA detection in cells. We recently found that α-synuclein (α-Syn) selectively and strongly bound to PA in vitro. Here, we revealed that the N-terminal region of α-Syn (α-Syn-N) specifically bound to PA, with a dissociation constant of 6.6 µm. α-Syn-N colocalized with PA-producing enzymes, diacylglycerol kinase (DGK) ß at the plasma membrane (PM), myristoylated DGKζ at the Golgi apparatus, phorbol ester-stimulated DGKγ at the PM, and phospholipase D2 at the PM and Golgi but not with the phosphatidylinositol-4,5-bisphosphate-producing enzyme in COS-7 cells. However, α-Syn-N failed to colocalize with them in the presence of their inhibitors and/or their inactive mutants. These results indicate that α-Syn-N specifically binds to cellular PA and can be applied as an excellent PA sensor.

Establishment of a guinea pig model of latent tuberculosis with GFP-introduced Mycobacterium tuberculosis.

There exists latent tuberculosis, in which small numbers of tubercle bacilli remain viable in the host without visible granulomatous lesions. As few data exist on the mechanisms of latent tuberculosis, it is important to examine latent tuberculosis in terms of pathogenesis and efficacy of chemotherapy. As a first step, we used green fluorescent protein (GFP)-introduced H37Rv Mycobacterium tuberculosis to establish latent tuberculosis in the guinea pig that provides one of the best animal models of tuberculosis. We inoculated the guinea pigs subcutaneously with 100 or 1,000 colony-forming unit (CFU) of tubercle bacilli. During the 300-day follow-up period after infection, there were no clinical signs of disease, suggesting a lack of visible granulomatous lesions. In fact, upon necropsy, no macroscopic tuberculous lesions were recognized, but histopathological examination of the lung, spleen and liver revealed microgranulomas consisting of epithelioid macrophages and lymphocytes without central necrosis. Importantly, photon imaging visualized granulomatous lesions corresponding to these histologically apparent microgranulomas. Tuberculin skin testing of infected guinea pigs showed strong positivity (> or = 10 mm induration) until the end of the experiments. Real-time PCR analysis showed a significant increase in the expression levels of interferon-gamma, tumor necrosis factor-alpha, interleukin-12, and inducible nitric oxide synthase mRNAs in infected lung tissues after 300 days (P < 0.01). As human samples are hardly available to study latent tuberculosis, our guinea pig model would be useful for examining the pathogenesis and molecular mechanisms of latent tuberculosis as well as for monitoring the results of chemotherapy with green fluorescence emission of tubercle bacilli.

Higher susceptibility of type 1 diabetic rats to Mycobacterium tuberculosis infection.

An association between diabetes mellitus and tuberculosis has been implicated for a long time. We have previously reported that Goto Kakizaki type 2 diabetic rats are highly susceptible to Mycobacterium (M.) tuberculosis infection. As a next step, we attempted to clarify whether type 1 diabetic rats are more susceptible to M. tuberculosis than non-diabetic wild-type (WT) rats. Here, we used the Komeda diabetes-prone (KDP) rat, as a model of type 1 diabetes mellitus. The infected KDP rats developed large granulomas without central necrosis in their lungs, liver or spleen. This was consistent with a significant increase in the number of colony-forming units (cfu) of M. tuberculosis in the lungs and spleen (p < 0.01). Insulin treatment resulted in significant reduction of tubercle bacilli in the infected KDP rats (p < 0.01). Pulmonary levels of interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta mRNAs were higher in the infected diabetic rats than in WT rats. Alveolar macrophages from KDP rats were not fully activated by M. tuberculosis infection because the macrophages did not secrete nitric oxide (NO) that can kill M. tuberculosis (p < 0.01), but no significant difference in phagocytosis of tubercle bacilli by alveolar macrophages was observed between KDP and WT rats. Taken together, our findings indicate that type 1 diabetic rats are more susceptible to M. tuberculosis that WT rats.

Where do substrates of diacylglycerol kinases come from? Diacylglycerol kinases utilize diacylglycerol species supplied from phosphatidylinositol turnover-independent pathways.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprises ten isozymes (α-κ) and regulates a wide variety of physiological and pathological events, such as cancer, type II diabetes, neuronal disorders and immune responses. DG and PA consist of various molecular species that have different acyl chains at the sn-1 and sn-2 positions, and consequently, mammalian cells contain at least 50 structurally distinct DG/PA species. Because DGK is one of the components of phosphatidylinositol (PI) turnover, the generally accepted dogma is that all DGK isozymes utilize 18:0/20:4-DG derived from PI turnover. We recently established a specific liquid chromatography-mass spectrometry method to analyze which PA species were generated by DGK isozymes in a cell stimulation-dependent manner. Interestingly, we determined that DGKδ, which is closely related to the pathogenesis of type II diabetes, preferentially utilized 14:0/16:0-, 14:0/16:1-, 16:0/16:0-, 16:0/16:1-, 16:0/18:0- and 16:0/18:1-DG species (X:Y = the total number of carbon atoms: the total number of double bonds) supplied from the phosphatidylcholine-specific phospholipase C pathway, but not 18:0/20:4-DG, in high glucose-stimulated C2C12 myoblasts. Moreover, DGKα mainly consumed 14:0/16:0-, 16:0/18:1-, 18:0/18:1- and 18:1/18:1-DG species during cell proliferation in AKI melanoma cells. Furthermore, we found that 16:0/16:0-PA was specifically produced by DGKζ in Neuro-2a cells during retinoic acid- and serum starvation-induced neuronal differentiation. These results indicate that DGK isozymes utilize a variety of DG molecular species derived from PI turnover-independent pathways as substrates in different stimuli and cells. DGK isozymes phosphorylate various DG species to generate various PA species. It was revealed that the modes of activation of conventional and novel protein kinase isoforms by DG molecular species varied considerably. However, PA species-selective binding proteins have not been found to date. Therefore, we next attempted to identify PA species-selective binding proteins from the mouse brain and identified α-synuclein, which has causal links to Parkinson's disease. Intriguingly, we determined that among phospholipids, including several PA species (16:0/16:0-PA, 16:0/18:1-PA, 18:1/18:1-PA, 18:0/18:0-PA and 18:0/20:4-PA); 18:1/18:1-PA was the most strongly bound PA to α-synuclein. Moreover, 18:1/18:1-PA strongly enhanced secondary structural changes from the random coil form to the α-helix form and generated a multimeric and proteinase K-resistant α-synuclein protein. In contrast with the dogma described above, our recent studies strongly suggest that PI turnover-derived DG species and also various DG species derived from PI turnover-independent pathways are utilized by DGK isozymes. DG species supplied from distinct pathways may be utilized by DGK isozymes based on different stimuli present in different types of cells, and individual PA molecular species would have specific targets and exert their own physiological functions.

Retinoic acid therapy attenuates the severity of tuberculosis while altering lymphocyte and macrophage numbers and cytokine expression in rats infected with Mycobacterium tuberculosis.

Because retinoic acid (RA) exerts a stimulatory effect on macrophages and tubercle bacilli target alveolar macrophages, the therapeutic potential of RA was examined in rats with tuberculosis. In the main study, 15 rats were randomized to treatment with oil (control) or RA, 100 microg/100 g body weight per dose, given 3 times weekly for 3 and 5 wk after infection with Mycobacterium tuberculosis strain H37Rv. There was a significant difference in the severity of tuberculosis histopathology between control and RA-treated rats, and oral administration of RA decreased the number of colony-forming units (CFU) in both lung and spleen at 3 and 5 wk after H37Rv infection (P < 0.005). CD4-positive and CD8-positive T cells, natural killer cells, and CD163-positive macrophages increased (P < 0.05) in the infected lung tissues of RA-treated rats. Expression of IFNgamma and inducible nitric oxide synthetase messenger RNA (mRNA) was higher in the infected lung tissues of RA-treated rats than in control rats. Alveolar macrophages from rats treated in vivo with RA and infected in vitro with M. tuberculosis showed significantly higher expression of TNFalpha and IL-1beta mRNA than macrophages in control rats. To our knowledge, this is the first reported study to demonstrate that orally administered RA significantly inhibits the in vivo growth of M. tuberculosis and the development of tuberculosis.

Dioleoyl-phosphatidic acid selectively binds to α-synuclein and strongly induces its aggregation.

α-Synuclein (α-syn), which causally links to Parkinson's disease, binds to vesicles containing phosphatidic acid (PA). However, the effects of the fatty acyl chains of PA on its ability to bind to α-syn protein remain unclear. Intriguingly, we reveal that among several PA species, 18:1/18:1-PA is the most strongly bound PA to the α-syn protein. Moreover, 18:1/18:1-PA more strongly enhances secondary structural changes from the random coil form to the α-helical form than 16:0/18:1-PA. Furthermore, 18:1/18:1-PA more markedly accelerates generation of multimeric and proteinase K-resistant α-syn protein compared to 16:0/18:1-PA. These results indicate that among phospholipids examined so far, 18:1/18:1-PA demonstrates the strongest binding to α-syn, as well as the most effective enhancement of its secondary structural changes and aggregation formation.