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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.

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.

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) β 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.

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.

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-Guerin (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.

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.

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.

Phosphatidic acid (PA) is one of the phospholipids composing the plasma membrane and acts as a second messenger to regulate a wide variety of important cellular events, including mitogenesis, migration and differentiation. PA consists of various molecular species with different acyl chains at the sn-1 and sn-2 positions. However, it has been poorly understood what PA molecular species are produced during such cellular events. Here we identified the PA molecular species generated during retinoic acid (RA)-induced neuroblastoma cell differentiation using a newly established liquid chromatography/mass spectrometry (LC/MS) method. Intriguingly, the amount of 32:0-PA species was dramatically and transiently increased in Neuro-2a neuroblastoma cells 24–48 h after RA-treatment. In addition, 30:0- and 34:0-PA species were also moderately increased. Moreover, similar results were obtained when Neuro-2a cells were differentiated for 24 h by serum starvation. MS/MS analysis revealed that 32:0-PA species contains two palmitic acids (16:0 s). RT-PCR analysis showed that diacylglycerol kinase (DGK) δ and DGKζ were highly expressed in Neuro-2a cells. The silencing of DGKζ expression significantly decreased the production of 32:0-PA species, whereas DGKδ-siRNA did not. Moreover, neurite outgrowth was also markedly attenuated by the deficiency of DGKζ. Taken together, these results indicate that DGKζ exclusively generates very restricted PA species, 16:0/16:0-PA, and up-regulates neurite outgrowth during the initial/early stage of neuroblastoma cell differentiation., Graphical abstract fx1, Highlights • We analyzed phosphatidic acid (PA) molecular species in neuroblastoma cells. • We identified a marked increase of 32:0-PA during neuroblastoma cell differentiation. • 32:0-PA species contained two palmitic acids (16:0 s). • Diacylglycerol kinase (DGK) ζ-siRNA decreased 16:0/16:0-PA production. • Neurite outgrowth was markedly attenuated by deficiency of DGKζ.

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, β and γ), type II (δ, η and κ) and type III (e) DGKs have 7.9-19.2% 2-MGK activity compared to their DGK activities, whereas their 1-MGK activities were

A variety of diacylglycerol (DG) molecular species are produced in stimulated cells. Conventional (α, βII and γ) and novel (δ, ε, η and θ) protein kinase C (PKC) isoforms are known to be activated by DG. However, a comprehensive analysis has not been performed. In this study, we analyzed activation of the PKC isozymes in the presence of 2–2000 mmol% 16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- or 18:0/22:6-DG species. PKCα activity was strongly increased by DG and exhibited less of a preference for 18:0/22:6-DG at 2 mmol%. PKCβII activity was moderately increased by DG and did not have significant preference for DG species. PKCγ activity was moderately increased by DG and exhibited a moderate preference for 18:0/22:6-DG at 2 mmol%. PKCδ activity was moderately increased by DG and exhibited a preference for 18:0/22:6-DG at 20 and 200 mmol%. PKCε activity moderately increased by DG and showed a moderate preference for 18:0/22:6-DG at 2000 mmol%. PKCη was not markedly activated by DG. PKCθ activity was the most strongly increased by DG and exhibited a preference for 18:0/22:6-DG at 2 and 20 mmol% DG. These results indicate that conventional and novel PKCs have different sensitivities and dependences on DG and a distinct preference for shorter and saturated fatty acid-containing and longer and polyunsaturated fatty acid-containing DG species, respectively. This differential regulation would be important for their physiological functions., Highlights • We comprehensively analyzed activation of c/nPKC isozymes by different DG species. • c/nPKCs have different sensitivities and dependences on DG. • c/nPKCs have a distinct preference for different fatty acid-containing DG species. • This differential regulation would be important for PKCs' physiological functions.

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.

Development of durable commercial/heavy fuel cell (FC) vehicles is required to decrease carbon dioxide emission from commercial/heavy vehicles. The mileage of heavy vehicles is far higher than that of passenger vehicles. Because FC stacks in vehicles deteriorate during long-term operation, their lifetime should be maximized by increasing durability. To improve FC stack durability, we must avoid catalyst degradation. When the hydrogen supply on the anode catalyst surface is inadequate, carbon of the cathode catalyst is oxidized with water. This occurs where there is insufficient hydrogen, such as at anode outlet during cell starting or water flooding. It is therefore important to prevent deterioration of FC operation caused by inadequate hydrogen. In stacks where the anode gas is circulated, nitrogen permeates and remains at the anode until it is exhausted, which lowers hydrogen concentration. Moreover, hydrogen is consumed in the piling direction, so its concentration is lower downstream. This leads to degradation at the anode outlet, so a high hydrogen flow rate is required during operation. To optimize hydrogen supply through feedback control, it is beneficial to measure the local hydrogen partial pressure. Hydrogen distributions exist in the reactive plane, gas manifolds, and piping system. To avoid FC deterioration caused by insufficient hydrogen, hydrogen concentration needs to be measured in the in-plane direction. However, it is difficult to measure hydrogen distribution in the in-plane direction. Some commercial sensors may affect FC materials. General hydrogen sensors are unsuitable for hydrogen concentrations over 4% inside FC systems. Raman spectroscopy is a useful hydrogen detection method. Generally, leaked hydrogen gas is intermittently detected with Raman spectroscopy. The intensity of Raman scattering is about 10−4 times that of Rayleigh scattering, and low-density gas scattering is weak compared with that of a liquid or solid. However, the Raman shift of hydrogen vibration (4160 cm−1) is far larger than that of other materials and does not require high wavelength resolution. Moreover, the hydrogen partial pressure in FC systems is high and easily detected. We developed a new method to measure the hydrogen Raman scattering from the reacting surface of an operating FC. During operation, hydrogen is consumed in the anode channel and its partial pressure drops near the outlet. To confirm this phenomenon with Raman spectroscopy, we needed to detect scattering light from the anode channel during cell operation. This measurement also required optics able to resolve very weak hydrogen Raman scattering. For these reasons, we fabricated a visualization cell that transmitted pump and scattered light without gas leakage. Pump light was introduced into a flow field in a channel with a depth of 1 mm, so the channel worked as an optical path . The optical path was from a mirror on the cathode flow field toward the anode to avoid interference with sealing materials. Sapphire windows were included in the anode flow field to allow visualization of scattered light. Using this cell and an Intensified Charge-Coupled Device (ICCD ) camera, we vertically observed Raman scattering from hydrogen. We included a bandpass filter with a center wavelength of 420-nm and halfwidth of 10 nm inside the camera lens to capture images of 417-nm scattered light from hydrogen excited by 355-nm pump light through the filter. A 355-nm notch filter was also added to remove the optical noise from Rayleigh scattering, allowing visualization of only hydrogen. Before the measurement, scattering intensity was calibrated using 100% hydrogen and nitrogen to clarify the relation between scattering intensity and partial pressure. The flow field was filled with 100% hydrogen (100 kPa) and an intensity distribution was obtained. Pure nitrogen was also measured to evaluate the dark noise and the correlation between intensity and hydrogen partial pressure. Local partial pressure was determined from the intensity at a desired point. We then measured the scattered light during cell operation at 100 kPaG. Hydrogen with a stoichiometric ratio of 1.1 diluted 45% by nitrogen was supplied to the cell to mimic stack operation. The cell operated at 0.5 A/cm2. The scattering distribution was measured at 10 Hz and ten measurements were integrated to determine hydrogen partial pressure distribution from the scattered light intensity . Hydrogen partial pressure decreased from 80 to 40 kPa along the in-plane flow, consistent with our rough predictions of 90 and 38 kPa, respectively. This data was thought to be affected by the sensitization noise from the photomultiplier tube elements in the ICCD camera. Suppressing optical and electronic noise or conducting an imaging process with dispersion treatment will allow detection of various gases with this Raman method.

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-K(d))-restricted epitopes which induce cross-reactive responses to Mtb and other related mycobacteria in both BALB/c (H2(d)) and CB6F1 (H2(b/d)) mice. The H2-K(d)-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-K(d)-restricted epitopes elicit distinct CD8(+) T cell populations, a result explained by the X-ray structure of the two peptide/H2-K(d) 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.

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.

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, β, 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.

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.

Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol to phosphatidic acid. Since both diacylglycerol and phosphatidic acid serve as signaling molecules, DGK is regarded as a hub between diacylglycerol-mediated and phosphatidic acid-mediated signaling. One of the 10 DGK isozymes, DGKα, is shown to be involved in T cell function. Transfection studies using tagged expression vectors revealed that DGKα localizes to the cytoplasm and nucleus and translocates to the plasma membrane in response to T cell receptor stimulation. However, a limited number of studies reported the localization of native protein of DGKα in tissues and cells. In this study, we immunized mice with recombinant DGKα and developed several anti-DGKα monoclonal antibodies (mAbs). One of the established anti-DGKα mAbs is a clone DaMab-2 (mouse IgG1, kappa). In enzyme-linked immunosorbent assay, DaMab-2 recognized only DGKα, and did not react with the other isozymes, such as DGKγ, DGKζ, DGKη, and DGKδ. Importantly, DaMab-2 is very useful in immunocytochemical analysis of human cultured cells, indicating that DaMab-2 is advantageous to analyze the localization and function of DGKα.

Although effective liquid chromatography (LC)/mass spectrometry (MS) methods enabling the separation of phospholipid molecular species have been developed, there are still problems with an intracellular signaling molecule, phosphatidic acid (PA). In this study, we optimized LC/MS conditions to improve the quantitative detection of PA molecular species from a cellular lipid mixture. Using the newly developed LC/MS method, we showed that stimulation of CTLL-2 murine T-lymphocytes by interleukin-2 (IL-2) induced a significant increase of 36:1-, 36:2-, 40:5- and 40:6-diacyl-PA. A diacylglycerol kinase (DGK) inhibitor, R59949, attenuated the increase of 36:1-, 40:5-, 40:6-diacyl-PA, suggesting that DGK IL-2-dependently and selectively generated these diacyl-PA species., Highlights ▸ We optimized LC/MS conditions to improve quantitative detection of PA species. ▸ IL-2 induced increase of 36:1-, 36:2-, 40:5- and 40:6-diacyl-PA. ▸ IL-2 stimulation caused DGK-dependent increases of limited molecular species of PA. ▸ The LC/MS method developed in this study will be advantageous for lipidomics studies.

The aim of the Japanese cabinet council's 'Biomass Nippon Strategy' is to increase the utilization of biomass as one of the ways to reduce the dependence on fossil resources. However, there are various problems associated with the use biomass, one of which is the cost associated with the collection and transportation of biomass. Biomass has high moisture content and low bulk density. The biomass solidification technology developed by Ida et al. partly aims to solve the transportability problem. This study shows physical properties of high density and hardness of the new briquette produced from herby biomass. The relationship between apparent density and maximum compressive strength and an effect on the maximum compressive strength by black colorization are considered. Also, an attempt is made to explain the effect on properties of the briquette by hemi-cellulose and lignin in the region before semi-carbonized region. As a result, the relation between the apparent density and the compressive strength shows that maximum value exists as characteristics of the herby briquette. The maximum apparent density for all processing conditions is 1.38 g/cm3 at the initial moisture 0.10 kg/kg-wet - processing temperature 473 K. The maximum compressive strength for all processing conditions is 127 MPa at the initial moisture 0.05 kg/kg-wet - processing temperature 453 K. Furthermore, the effect of black colorization indicates that when the area of black colorization is increased for the surface of herby biomass briquette by increased in the processing temperature, the maximum compressive strength is decreased due to reduction in the adhesion of lignin. Consequently, high-density and high-hardness of the new briquette can be controlled by the initial moisture and the processing temperature.