[Interaction of Salmonella with Macrophages-Critical Roles of Salmonella SEp22, a Pathogenicity-related Protein, and Macrophage Reactive-Oxygen Intermediate Species (ROIs) on the Infection and Survival of Salmonella].

Salmonella is a Gram-negative [Gram(-)] bacteria, distributed widely in such natural environments as soil, dust, or river water, causing food poisoning as well as oral infections such as Typhi or Paratyphi. Salmonella is highly tissue invasive, easily spreading throughout the whole body after initial growth in the phagocytic vesicles of macrophages as an intra-cellular parasite. Because there remain many unknown elements in the Salmonella-macrophage interaction, I started my study by focusing on the molecules and mechanisms underlying the interaction; for example, how Salmonella escapes natural biodefense systems armed by macrophages, and how macrophages surround and inactivate Salmonella. In addition, I developed insight into Salmonella survival in the face of both environmental stresses and immunological stresses, including attacks from macrophages, based on the idea that "pathogenicity" is not limited simply to an attack, but to both the attack and defense against hazards. In this study, I found a novel pathogenicity-related protein of Salmonella, SEp22, an iron-chelating protein of MW 18.7 kDa, to cope with reactive-oxygen intermediates (ROIs) generated by activated macrophages pre-treated with lipopolysaccharides (LPS), one of the major components of Salmonella outer membrane. We also showed that Salmonella attacks macrophages by a novel mechanism through the induction of apoptosis with large amounts of LPS and protein synthesis inhibition, in addition to the well-known mechanisms of type-three secretion system (TTSS)-induced cell damage, including InvA, an attacking, virulent factor of Salmonella. We showed that macrophages could escape from this type of cell death by LPS-induced macrophage activation and LPS-tolerance.

Sodium bicarbonate regulates nitric oxide production in mouse macrophage cell lines stimulated with lipopolysaccharide and interferon γ.

Macrophages are known to play pivotal roles in host-defense through inflammation via both innate and acquired immune systems, and so on. In an earlier paper, we showed the influence of the type of culture medium, Ham's F-12 or DMEM, on activated macrophage phenotypes induced by LPS and IFNγ. The production of nitric oxide (NO), pro-inflammatory cytokines such as TNFα and IL-1ß, as well as the induction of superoxide-generating activity of J774.1/JA-4 cells was different depending on the type of culture medium. In this present study, we showed that sodium bicarbonate concentrations in these culture media, 14 mM in Ham's F-12 and 44 mM in DMEM, were crucial to explaining the differences in the induction of activated macrophage phenotypes, especially in that of iNOS. A concentration-dependent change in pH did not result in any remarkable difference in iNOS expression or NO production. Moreover, high sodium bicarbonate in culture medium increased not only NO production but also TNFα production in the activated macrophages. These results suggest that sodium bicarbonate would be a regulatory factor of NO and TNFα production in macrophages and that its concentration has a crucial role in macrophage activation.

Induction of different activated phenotypes of mouse peritoneal macrophages grown in different tissue culture media.

The role of activated macrophages in the host defense against pathogens or tumor cells has been investigated extensively. Many researchers have been using various culture media in in vitro experiments using macrophages. We previously reported that J774.1/JA-4 macrophage-like cells showed great differences in their activated macrophage phenotypes, such as production of reactive oxygen, nitric oxide (NO) or cytokines depending on the culture medium used, either F-12 (Ham's F-12 nutrient mixture) or Dulbecco modified Eagle's medium (DMEM). To examine whether a difference in the culture medium would influence the functions of primary macrophages, we used BALB/c mouse peritoneal macrophages in this study. Among the activated macrophage phenotypes, the expression of inducible NO synthase in LPS- and/or IFN-γ-treated peritoneal macrophages showed the most remarkable differences between F-12 and DMEM; i.e., NO production by LPS- and/or IFN-γ-treated cells was far lower in DMEM than in F-12. Similar results were obtained with C57BL mouse peritoneal macrophages. Besides, dilution of F-12 medium with saline resulted in a slight decrease in NO production, whereas that of DMEM with saline resulted in a significant increase, suggesting the possibility that DMEM contained some inhibitory factor(s) for NO production. However, such a difference in NO production was not observed when macrophage-like cell lines were examined. These results suggest that phenotypes of primary macrophages could be changed significantly with respect to host inflammatory responses by the surrounding environment including nutritional factors and that these altered macrophage phenotypes might influence the biological host defense.

Effects of 5-fluorouracil, adriamycin and irinotecan on HSC-39, a human scirrhous gastric cancer cell line.

Therapies for patients with scirrhous gastric cancer remain ineffective. Current treatments for gastric cancer based on systemic therapy, such as the combination of S-1 with cisplatin or docetaxel, show good clinical response rates. S-1 plus cisplatin is the standard treatment for HER2-negative advanced scirrhous gastric cancer in Japan. In spite of recent advances in the treatment of gastric cancer, a standard chemotherapy regimen is yet to be established for scirrhous gastric cancer. To develop new therapeutic approaches based on characteristic biological features of cancer cells, we examined the mechanisms underlying the cytotoxicity of anticancer drugs and reactive oxygen species (ROS) toward a human scirrhous cancer cell line, HSC-39, in vitro. Anticancer drugs such as 5-fluorouracil (5-FU), adriamycin (ADR) and irinotecan (CPT-11), as well as ROS, were previously shown to have important cytotoxic effects on these tumor cells. We demonstrated that 5-FU effectively induced apoptosis in HSC-39 cells in a dose­dependent manner, while ADR and CPT-11 induced necrosis and/or aponecrosis. 5-FU effectively inhibited WST-1 decrease in the MTT viability assay, even at low doses where little LDH release was observed, while ADR and CPT-11 only inhibited WST-1 decrease at high doses where LDH release was induced. Moreover, HSC-39 cells showed high sensitivity to H2O2 and NOC-18, but less sensitivity to other ROS, suggesting a link between cell damage and membrane permeability changes induced by H2O2 and NOC-18 or related oxygen radical species such as OH· or ·O2. These results suggest that combination treatment of chemotherapeutics with a fluoropyrimidine such as 5-FU is effective chemotherapy for scirrhous gastric cancer.

Induction of Viable but Nonculturable Salmonella in Exponentially Grown Cells by Exposure to a Low-Humidity Environment and Their Resuscitation by Catalase.

Salmonella is a major cause of foodborne disease that sometimes occurs in massive outbreaks around the world. This pathogen is tolerant of low-humidity conditions. We previously described a method for induction of viable but nonculturable (VBNC) Salmonella enterica serovar Enteritidis by treatment with hydrogen peroxide (H2O2) and subsequent resuscitation with 0.3 mM sodium pyruvate. Here, we report a new method for the induction of the VBNC state in Salmonella Enteritidis cells, one involving dehydration. Exposure of Salmonella Enteritidis cells to dehydration stress under poor nutritional conditions (0.9% [wt/vol] NaCl) and 10 to 20% relative humidity at room temperature decreased the presence of culturable population to 0.0067%, but respiratory and glucose uptake active populations were maintained at 0.46 and 1.12%, respectively, meaning that approximately 1% may have entered the VBNC state. Furthermore, these VBNC cells could be resuscitated to acquire culturability by incubation with catalase in M9 minimal medium without glucose in a manner dependent on the dose of catalase but not sodium pyruvate. These results suggest that a low-humidity environment could cause Salmonella Enteritidis cells to enter the VBNC state and the cells could then be resuscitated for growth by treatment with catalase, suggesting a potential risk of Salmonella Enteritidis to survive in low water activity foods in the VBNC state and to start regrowth for foodborne illness.

Simultaneous Addition of Shikonin and Its Derivatives with Lipopolysaccharide Induces Rapid Macrophage Death.

Macrophages play pivotal roles in inflammatory responses. Previous studies showed that various natural products exert antiinflammatory effects by regulating macrophage activation. Recent studies have shown that shikonin (SHK) and its derivatives (ß-hydroxyisovalerylshikonin, acetylshikonin, and isobutylshikonin), which are 1,4-naphthoquinone pigments extracted from the roots of Lithospermum erythrorhizon, have various pharmacological, including antiinflammatory and antitumor, effects. Even though there have been many studies on the antiinflammatory activities of SHK derivatives, only a few have described their direct effects on macrophages. We investigated the effects of SHK derivatives on lipopolysaccharide (LPS)-treated macrophages. Low doses of SHK derivatives induced significant macrophage cytotoxicity (mouse macrophage-like J774.1/JA-4 cells and mouse peritoneal macrophages) in the presence of LPS. SHK activated caspases-3 and -7, which led to DNA fragmentation, but this cytotoxicity was prevented through a pan-caspase inhibitor in LPS-treated JA-4 cells. Maximal cytotoxic effects were achieved when SHK was added immediately before LPS addition. These results indicate that SHK derivatives induce caspase-dependent apoptotic cell death of LPS-treated macrophages and suggest that SHK acts during an early stage of LPS signaling.

Influence of the culture medium on the production of nitric oxide and expression of inducible nitric oxide synthase by activated macrophages in vitro.

Macrophages play an important role in immune and inflammatory responses, and have been extensively studied in vitro using culture media such as RPMI1640 medium, Dulbecco's modified Eagle medium (DMEM), and Ham's F-12 medium (F-12). We found that the activation phenotypes of a murine macrophage-like cell line, J774.1/JA-4, were obviously different in two distinct culture media (F-12 and DMEM), both of which were supplemented with 10% of the same fetal bovine serum (FBS). Among these phenotypes, nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression, induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), were remarkably different. iNOS expression was higher in the macrophages cultured in DMEM than in F-12 for 20 h, while no significant differences were shown in NO production between in F-12 and DMEM. It might be the reason why DMEM have reduced NO production by the induced iNOS. Besides, [Formula: see text]-generating activity, and production of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in the activated macrophages were also different between the cultures in F-12 and DMEM. These results suggest that F-12 and DMEM contain certain components responsible for modification of macrophage activation processes and/or macrophage functions. Our present results provide evidence that the choice of culture medium is important in the study and analysis of macrophage activation.

Use of Flow Cytometry for Quantitative Analysis of Metabolism of Viable but Non-culturable (VBNC) Salmonella.

A simple and rapid assay method for analysis of the metabolic activity of viable but non-culturable (VBNC) Salmonella was established. An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost its culturability during incubation with 1-10 mM H2O2 in Luria-Bertani (LB) medium. To assess the viability of the bacteria, we measured 3 different metabolic activities: Respiratory activity by 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) reduction, glucose uptake assessed with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and DNA synthesis activity evaluated by 5-ethynyl-2'-deoxyuridine (EdU) incorporation. These activities were analyzed by both confocal laser-scanning microscopy and flow cytometry, together with colony-formation assays on LB-agar plates. The results showed that some of the H2O2-treated SE cells were in the VBNC state and that the extent of H2O2-induced decrease in each metabolic activity varied according to the activity. That is, glucose-uptake activity was not markedly changed, being kept at the highest level; whereas the respiratory activity was less than that of the glucose-uptake, and DNA synthesis activity was the lowest among them. These results suggest that the VBNC state might be characterized by different metabolic activities that vary and correspond to the kind and strength of the stress, threatening bacterial survival in an adverse environment.

Nitric oxide is an important regulator of heme oxygenase-1 expression in the lipopolysaccharide and interferon-γ-treated murine macrophage-like cell line J774.1/JA-4.

Heme oxygenase-1 (HO-1) catabolizes the degradation of heme into bilirubin, carbon monoxide, and iron ions. The HO-1 products provide antioxidant cytoprotection in addition to having potent antiinflammatory and immunomodulatory functions. HO-1 is induced by its substrate heme and environmental factors including oxidative and heat stresses. Although previous studies reported that lipopolysaccharide (LPS) induced the expression of both the HO-1 gene and its protein in macrophages, the major regulators of HO-1 expression remain unknown. To identify these regulators, we used two types of cell, the murine macrophage-like cell line J774.1/JA-4 and its LPS-resistant mutant, LPS1916. Based on a comparison of the results obtained with these cells, we found that nitric oxide (NO) was closely linked to the induction of HO-1. Real-time polymerase chain reaction (PCR) showed that the time course for inducible HO-1 mRNA by LPS or LPS+interferon (IFN)-γ was similar to that for inducible NO synthase (iNOS) mRNA. Furthermore, the expression of iNOS mRNA and protein increased earlier than that of HO-1 mRNA and protein. N-Nitro-L-arginine methyl ester, an NO synthase inhibitor, reduced both HO-1 expression and NO production in LPS+IFN-γ-treated JA-4 cells. Furthermore, NOC-12, an NO donor, significantly induced HO-1 expression not only in JA-4 but also in LPS1916 cells. Reactive oxygen species (ROS) scavengers, such as superoxide dismutase and catalase, did not affect HO-1 protein expression in LPS+IFN-γ-treated JA-4 cells. These results suggest that, among ROS, NO plays an important role in HO-1 induction in activated macrophages treated with LPS+IFN-γ.

Induction of viable but non-culturable (VBNC) state in Salmonella cultured in M9 minimal medium containing high glucose.

An environmental isolate of Salmonella enterica serovar Enteritidis (SE) clone, SE Cl#15-1, loses its culturability during 72-h culture in M9 minimal medium containing 0.8% glucose, a concentration twice higher than that in normal M9 medium, whereas the bacterium retains its culturability in normal M9 medium. Live/dead analysis using the 5-cyano-2,3-di(p-tolyl) tetrazolium chloride (CTC)-reduction assay revealed that SE cells cultured in M9 medium containing 0.8% glucose died with time when in the "viable but non-culturable" (VBNC) state. Assay of the culturability of SE cells in the used supernatant (0.4 spent M9 or 0.8 spent M9) also indicated that 0.8 spent M9 soon showed a lethal effect on intact SE cells. These results suggest that large amounts of glucose metabolites might have been responsible for the toxicity. Analysis of the 0.8 spent M9 revealed that formate rapidly accumulated in the medium. The pH of the medium rapidly dropped to 4.7, leading to conversion of formate to formic acid, which might have damaged the bacterial cell membrane. These results suggest that the excessive amount of glucose in the M9 medium might have injured SE cells in the VBNC state by being metabolized to formic acid and other acidic compounds.

The effect of capsaicin on circulating biomarkers, soluble tumor necrosis factor and soluble tumor necrosis factor-receptor-1 and -2 levels in vivo using lipopolysaccharide-treated mice.

The circulating soluble tumor necrosis factor (sTNF) and sTNF-receptor (R) 1 and -R2 have known as septic biomarker. The pungent component of capsicum, capsaicin (Cap), has several associated physiological activities, including anti-oxidant, anti-bacterial and anti-inflammatory effects. The aim of this study was to elucidate the effect of Cap on circulating sTNF and sTNF-R1 and -R2 in vivo using lipopolysaccharide (LPS)-treated mice. LPS (20 mg/kg, ip)-treated group was significantly increased circulating sTNF, sTNF-R1, and -R2 and TNF-α mRNA expression levels compared to the vehicle group. Treatment with LPS (20 mg/kg, ip) + Cap (4 mg/kg, sc)-treated group was significantly decreased both circulating sTNF levels (after 1 h only) and TNF-α mRNA expression (after 6 h) compared to the LPS-treated group. There is an early increase in circulating sTNF, sTNR-R1, and -R2 observed in the LPS-treated mice. Since Cap inhibits this initial increase as biomarkers, circulating sTNF, it is considered a potent treatment option for TNF-α-related diseases, such as septicemia. In conclusion, Cap interferes with TNF-α mRNA transcription and exerts an inhibiting effect on TNF-α release from macrophages in the early phase after LPS stimulation. Thus, Cap is considered a potent agent for the treatment of TNF-α-related diseases, such as septicemia.

Differential resuscitative effect of pyruvate and its analogues on VBNC (viable but non-culturable) Salmonella.

An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost culturability by the addition of 3 mM H2O2 to cultures grown in Luria-Bertani (LB) medium; however, some H2O2-treated bacteria regained their culturability in M9 minimal medium, if sodium pyruvate was present at at least 0.3 mM. In addition, most pyruvate analogues, such as bromopyruvate or phenylpyruvate, did not show restoration activity similar to that of pyruvate, except in the case of α-ketobutyrate. Further analysis of the mechanism underlying the resuscitation by pyruvate revealed that although many of the bacteria showed respiratory activity on CTC (5-cyano-2,3-di-(p-tolyl) tetrazolium chloride) reduction with or without pyruvate, the biosynthesis of DNA and protein synthesis were quite different in the presence or absence of pyruvate, i.e., pyruvate endowed the cells with the ability to incorporate much more radio-label into precursors during the resuscitation process. These results suggest that pyruvate is one of the key molecules working in the resuscitation process by taking bacteria from the non-culturable state to the growing and colony-forming state by triggering the synthesis of macromolecules such as DNA and protein.

Regulatory role of cardiolipin in the activity of an ATP-dependent protease, Lon, from Escherichia coli.

Lon is an ATP-dependent serine protease that plays a significant role in the quality control of proteins in cells, degrading misfolded proteins and certain short-lived regulatory proteins under stresses as such heat-shock and UV irradiation. It is known that some polymers containing phosphate groups regulate enzymatic activity by binding with Lon. We focused on the phospholipids of biological membrane components such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and cardiolipin (CL), and examined whether or not liposomes containing these phospholipids regulate the enzymatic activity of Lon. CL-containing liposomes specifically inhibited both the proteolytic and ATPase activities of Lon in a dose-dependent manner. In addition, on pull-down assay, we found that CL-containing liposomes selectively bound to Lon. The interaction between CL-containing liposomes and Lon changed with the order of addition of Mg(2+)/ATP. When CL-containing liposomes were added after the addition of Mg(2+)/ATP to Lon, the binding of CL-containing liposomes to Lon was significantly decreased as compared with the reversed order. In fact, we found that CL-containing liposomes bound to Lon, resulting in inhibition of the enzymatic activity of Lon. These results suggest that Lon interacts with CL in biological membranes, which may regulate the functions of Lon as a protein-degrading centre in accordance with environmental changes inside cells.

Niacin promotes adipogenesis by reducing production of anti-adipogenic PGF2α through suppression of C/EBPß-activated COX-2 expression.

Niacin is converted to NAD and NADP in tissues, whose products are involved in a number of cellular processes; and it is associated with the regulation of adipogenesis. In this study, we identified the molecular mechanism by which niacin promotes the adipogenesis in mouse 3T3-L1 cells. When the cells were cultured with niacin, the expression of adipogenic peroxisome proliferator-activated receptor γ, CCAAT enhancer binding protein (C/EBP)α, and their target genes was enhanced concomitant with an increase in triglyceride storage. Moreover, niacin suppressed the expression of cyclooxygenase-2 and decreased the production of prostaglandin (PG) F(2α) in the early phase of adipogenesis, which PG suppresses the progression of adipogenesis via the PGF(2α) receptor. Furthermore, niacin decreased the C/EBPß level in the early phase of adipogenesis. These results indicate that niacin promoted adipogenesis by suppressing the production of the anti-adipogenic PGF(2α) through down-regulation of C/EBPß-activated cyclooxygenase-2 expression in adipocytes.

Forkhead transcription factor Foxa1 is a novel target gene of C/EBPß and suppresses the early phase of adipogenesis.

Forkhead/winged helix transcription factors (Foxs) regulate differentiation, metabolism, and development. Although Foxa1 is expressed in adipocytes, the roles and regulation of Foxa1 in them remain unclear. Here, we found that under the control of C/EBPß, Foxa1 suppressed lipid accumulation and concomitantly caused a decrease in adipogenic gene expression in adipocytes. Foxa1 was expressed in undifferentiated mouse 3T3-L1 cells and in the early phase of adipogenesis, with its highest expression at 3h after the initiation of adipogenesis, which was followed by a subsequent decrease. SiRNA-mediated suppression of Foxa1 expression activated the expression of adipogenic genes such as PPARγ. Moreover, siRNAs for C/EBPß, but not those for C/EBPδ, reduced Foxa1 mRNA and protein levels. The results of a promoter-reporter assay and chromatin immunoprecipitation assay demonstrated that C/EBPß bound to the C/EBP binding element at -529 of the mouse Foxa1 promoter. Furthermore, siRNA-mediated knockdown of C/EBPß decreased the promoter activity of mouse Foxa1 gene. These results suggest that Foxa1 plays a suppressive role in the early phase of adipogenesis, acting under the control of C/EBPß, and might be involved in the regulation of the rate of progression of the early phase of adipogenesis.

Desulforubrerythrin from Campylobacter jejuni, a novel multidomain protein.

A novel multidomain metalloprotein from Campylobacter jejuni was overexpressed in Escherichia coli, purified, and extensively characterized. This protein is isolated as a homotetramer of 24-kDa monomers. According to the amino acid sequence, each monomer was predicted to contain three structural domains: an N-terminal desulforedoxin-like domain, followed by a four-helix bundle domain harboring a non-sulfur µ-oxo diiron center, and a rubredoxin-like domain at the C-terminus. The three predicted iron sites were shown to be present and were studied by a combination of UV-vis, EPR, and resonance Raman spectroscopies, which allowed the determination of the electronic and redox properties of each site. The protein contains two FeCys(4) centers with reduction potentials of +240 mV (desulforedoxin-like center) and +185 mV (rubredoxin-like center). These centers are in the high-spin configuration in the as-isolated ferric form. The protein further accommodates a µ-oxo-bridged diiron site with reduction potentials of +270 and +235 mV for the two sequential redox transitions. The protein is rapidly reoxidized by hydrogen peroxide and has a significant NADH-linked hydrogen peroxide reductase activity of 1.8 µmol H(2)O(2) min(-1) mg(-1). Owing to its building blocks and its homology to the rubrerythrin family, the protein is named desulforubrerythrin. It represents a novel example of the large diversity of the organization of domains exhibited by this enzyme family.

Prostaglandin F(2alpha) suppresses early phase of adipogenesis, but is not associated with osteoblastogenesis in mouse mesenchymal stem cells.

In this study, we investigated the roles of prostaglandin (PG) F(2alpha) in the differentiation of mouse ST2 mesenchymal stem cells (MSC) into adipocytes and osteoblasts. PGF(2alpha) was not produced in the undifferentiated ST2 MSC, but its highest level of production was detected at 3h after the initiation of adipogenesis and then quickly decreased. On the contrary, apparent PGF(2alpha) production was not detected during the osteoblastogenesis of ST2 MSC. The PGF(2alpha) production pattern during adipogenesis well resembled the expression profiles of aldo-keto reductase (AKR) 1B3, which acted as the PGF(2alpha) synthase, and cyclooxygenase-2 genes; but the pattern showed a slight delay compared with these profiles. The siRNA for AKR1B3, but not that for AKR1B8 or 1B10, decreased PGF(2alpha) production and enhanced the expression of adipogenic genes, but did not affect the mRNA levels of osteoblastogenic genes, during the adipogenesis of ST2 MSC. The FP receptor was expressed during adipogenesis of ST2 MSC, and its agonist or antagonist suppressed or enhanced, respectively, the lipid accumulation and the adipogenic gene expression; but this receptor was not associated with the osteoblastogenesis. These results indicate that AKR1B3-mediated PGF(2alpha) suppressed the early phase of adipogenesis through FP receptors, but did not affect osteoblastogenesis in ST2 MSC. Therefore, PGF(2alpha) suppressed the progression of early phase adipogenesis after determination of the cell fate that causes MSC to differentiate into adipocytes.

Suppression of adipocyte differentiation by aldo-keto reductase 1B3 acting as prostaglandin F2alpha synthase.

Prostaglandin (PG) F(2alpha) suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor gamma. However, PGF(2alpha) synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF(2alpha), was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF(2alpha) production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor gamma, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF(2alpha). These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF(2alpha) suppressed adipocyte differentiation by acting through FP receptors.

Administration of tomato and paprika beverages modifies hepatic glucose and lipid metabolism in mice: a DNA microarray analysis.

To examine whether the expression of hepatic genes, including biomarkers, is affected by the ingestion of tomato or paprika, mice were given tomato beverage (TB), paprika beverage (PB), or water (control) ad libitum for 6 weeks. The body weights in the TB and PB groups were significantly lower than those in the control group. Administration of PB significantly increased the plasma high-density lipoprotein-cholesterol level. Hepatic gene expression was investigated using DNA microarrays. The ingestion of TB or PB up-regulated the expression of 687 and 1045 genes and down-regulated the expression of 841 and 653 genes, respectively (false discovery rate<0.05). These changes in gene expression suggest that TB ingestion promotes glycogen accumulation and stimulates some specific steps in fatty acid oxidation. PB ingestion promoted the entire glucose and fatty acid metabolic pathways to improve lipid profiles. These results provide useful genetic information about a variety of biochemical processes by which vegetables can contribute to good health.

Novel 3alpha-methoxyserrat-14-en-21beta-ol (PJ-1) and 3beta-methoxyserrat-14-en-21beta-ol (PJ-2)-curcumin, kojic acid, quercetin, and baicalein conjugates as HIV agents.

Sixteen novel compounds; 3alpha-methoxyserrat-14-en-21beta-ol (1) and 3beta-methoxyserrat-14-en-21beta-ol (2) and their curcumin, kojic acid, quercetin, and baicalein conjugates (3)-(18) were designed, synthesized, and evaluated for in vitro anti-HIV-1 reverse transcriptase (RT) activity in infected C8166-CCR5 cells, a human CD4(+) T-lymphocyte cell line. Among them, kojic acid derivatives, 9-12 showed significant biological activity. In particular, the compound 13, the conjugate of two molecules of 3alpha-methoxyserrat-14-en-21beta-ol (1) and one molecule of kojic acid, exerted significant anti-HIV activity with an EC50 value of 0.12microg/mL.