Methyl 2-[3-(4-hydroxyphenyl)prop-2-enoylamino]-3-phenylpropanoate Is a Potent Cell-Permeable Anti-Cytokine Compound To Inhibit Inflammatory Cytokines in Monocyte/Macrophage-Like Cells.

Cytokines are signaling molecules involved in inflammation process. Interleukin (IL)-6 is one of pivotal inflammatory cytokines associated with many human diseases. Therefore, there are on-going efforts to find a therapeutic to inhibit IL-6 and other cytokines. Methyl 2-[3-(4-hydroxyphenyl)prop-2-enoylamino]-3-phenylpropanoate (MHPAP) is a phenolic amide ester, transported better than its non-ester form (NEF) in monocyte/macrophage-like cells. However, there is no information about the effects of their cell permeability on cytokines. Therefore, the effects of MHPAP and NEF on cytokines were investigated in lipopolysaccharide (LPS)-stimulated THP-1 and human peripheral blood mononuclear cells (PBMCs). In the THP-1 cells, MHPAP significantly inhibited IL-6, IL-1beta, IL-8, and tumor necrosis factor (TNF)-alpha (P < 0.05), but NEF showed no effects. MHPAP also inhibited nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 phosphorylation in the THP-1 cells (P < 0.05), without significant effects on c-FOS, ATF-2, and JUN phosphorylations. Because NF-κB p65 is phosphorylated by IκB kinase (IKK), in silico analysis was performed on IKK. MHPAP was found to bind to IKK better than an IKK inhibitor ((E)-2-fluoro-4'-methoxystilbene). Furthermore, MHPAP inhibited the luminescence increased in the LPS-stimulated NF-κB-Luc2 THP-1 cells. As anticipated, MHPAP was also found to inhibit IL-6, IL-1beta, IL-8, and TNF-alpha significantly in LPS-stimulated PBMCs (P < 0.05). Especially, MHPAP inhibited IL-6 and IL-1beta with an IC50 of 0.85 and 0.87 µM, better than IL-8 (1.58 µM) and TNF-alpha (1.22 µM) in the cells. Altogether, the data suggest that cell permeability may have a significant impact on MHPAP's ability to inhibit cytokines and MHPAP may be used as a potent cell-permeable compound to inhibit inflammatory cytokines in monocyte/macrophage-like cells. SIGNIFICANCE STATEMENT: Potential effects of MHPAP and NEF on inflammatory cytokines (IL-6, IL-8, IL-1beta, and TNF-alpha) were investigated in LPS-stimulated THP-1 and PBMCs. Cell transport had a great impact on cytokine inhibition in the cells. MHPAP was also found to inhibit NF-κB pathway, which was supported by in silico and NF-κB reporter (Luc)-THP-1 data. Also, in LPS-stimulated PBMCs, MHPAP significantly inhibited IL-6, IL-1beta, IL-8, and TNF-alpha, suggesting that MHPAP may be a potent cell-permeable compound to inhibit inflammatory cytokines in monocyte/macrophage-like cells.

Evaluation of Selected Markers in Kidneys of Cynomolgus Monkey (Macaca fascicularis) with Induced Diabetes during Renal Ischemia-reperfusion Injury.

We previously reported that induced type 1 diabetes mellitus (DM) increases the susceptibility of acute kidney injury in- duced by ischemia-reperfusion injury (IRI) in cynomolgus monkeys. In this follow-up study, we compared the expression of selected markers in the renal tissues of monkeys subjected to bilateral renal IRI with and without diabetes. All tissues were obtained from the original study. Renal biopsies were obtained before and 24 and 48 h after ischemia and were examined for expression of KI-67 (tubular proliferation), Na+ /K+ ATPase (sodium-potassium pump), TNF-α(tumor necrosis factor-α, inflammation), CD31 (microvessels), CD3 (T-cells), 2 fibrotic markers (fibroblast specific protein-1, FSP-1;α-smooth muscle actin,α -SMA), and cleaved caspase 3 (apoptosis). Generally, the expression of these markers differed in monkeys with and without DM. As compared with non-DM monkeys, DM monkeys had more cells that expressed KI-67 during progression of acute kidney injury (AKI). Na+ /K+ ATPase expression was clearly present at baseline in the basolateral tubular areas only in the non-DM monkeys. At 48 h, its expression in the basolateral area was not visible in DM monkeys, but was still present in intercellular junctions of non-DM monkeys. The expression of TNF-αwas higher in DM before and 48 h after ischemia. Before and 24 h after ischemia, the number of CD31-positive capillaries was not different between 2 groups, although more collapsed vessels were found at in DM at 24 h. At 48 h, the number of capillaries was less in DM compared with those from non-DM animals. DM monkeys had more interstitial CD3-positive cells than did non-DM monkeys at 24 and 48 h after ischemia. Finally, FSP-1-stained cells were more abundant in DM than non-DM at 24 and 48 h. Our results show that DM aggravates the recovery of renal ischemia/reperfusion injury by affecting tubular proliferation, capillary density, T cell infil- tration and by altering protein and mRNA expression of various genes involved in ion channel, inflammation, and fibrotic change. The results from this observational study demonstrate that DM aggravates the recovery of renal ischemia/reperfusion injury by affecting multiple events including tubular necrosis, proliferation, function, inflammation and by inducing capillary rarefaction in cynomolgus monkeys.

Impact of roasting on javamide-I/-II in Arabica and Robusta coffee beans.

Javamide-I/-II are anti-inflammatory compounds found in coffee beans. However, potential effects of roasting on javamide-I/-II in coffee beans are currently unknown. Therefore, in this paper, the effects of roasting on javamide-I/-II were investigated in Arabica and Robusta beans. Coffee beans were roasted light, medium and dark, and the amounts of javamide-I/-II in the beans were quantified by a HPLC method. The data showed the different amounts of javamide-I/-II in the beans; not detected and ≤ 3.1 mg in Arabica beans, and 0.5-3.7 mg and 1.0-13.8 mg in Robusta beans, respectively. Furthermore, the data showed that roasting process significantly reduced the amounts of javamide-I/-II in both Arabica and Robusta beans (p < 0.05). These data were also confirmed by multivariate analyses. Additionally, these differences were validated in light, medium and dark roast coffee products in the market. Altogether, roasting can have a significant impact on javamide-I/-II amounts in coffee beans.

Finding a cell-permeable compound to inhibit inflammatory cytokines: Uptake, biotransformation, and anti-cytokine activity of javamide-I/-II esters.

AIM: Currently, there is limited information available about cell-permeability and anti-cytokine activity of javamide-I/-II esters in monocyte/macrophage-like cells. Therefore, the aim of this study was to investigate their cell-permeability and anti-cytokine activity in the cells. MATERIALS AND METHODS: The uptake of javamide-I/-II and esters was studied in THP-1 cells and PBMCs. Also, kinetic and inhibition studies were conducted using THP-1 cells. Western Blot was performed to determine the level of ATF-2 phosphorylation in THP-1 cells, and ELISA assays were carried out to measure TNF-alpha, MCP-1, IL-1beta and IL-8 levels in PBMCs. KEY FINDINGS: In THP-1 cells, the uptake of javamide-I/-II esters was significantly higher than javamide-I/-II (P < 0.001), and the Km for javamide-I ester was 27 µM. Also, the uptake of the esters was inhibited by PepT2 substrate/blocker. In THP-1 cells, javamide-I/-II esters were also biotransformed into javamide-I/-II. Furthermore, javamide-I ester could inhibit ATF-2 phosphorylation better than javamide-I in the cells, suggesting that the ester could be transported inside the cells better than javamide-I. Similarly, javamide-I/-II esters were found to be transported and biotransformed in PBMCs involved in inflammation processes. As anticipated, the esters were found to inhibit TNF-alpha and MCP-1 significantly in PBMCs (P < 0.005). Especially, javamide-I ester inhibited TNF-alpha, MCP-1, IL-1beta and IL-8 with IC50 values of 1.79, 0.88, 0.91 and 2.57 µM in PBMCs. SIGNIFICANCE: Javamide-I/-II esters can be transported, biotransformed and inhibit inflammatory cytokines significantly in monocyte/macrophage-like cells, suggesting that they may be utilized as a potent cell-permeable carrier to inhibit inflammatory cytokines in the cells. CHEMICAL COMPOUNDS: Javamide-I, javamide-I-O-methyl ester, javamide-II, javamide-II-O-methyl ester, tryptophan, coumaric acid, caffeic acid, GlySar, enalapril.

In Vivo Effects of Coffee Containing Javamide-I/-II on Body Weight, LDL, HDL, Total Cholesterol, Triglycerides, Leptin, Adiponectin, C-Reactive Protein, sE-Selectin, TNF-α, and MCP-1.

BACKGROUND: Diet plays an unequivocal role in the development of obesity. Interestingly, recent studies have demonstrated that coffee products containing javamide-I/-II may be commonly found in the market. However, there is no information about in vivo effects of coffee containing javamide-I/-II (CCJ12) on obesity-related metabolic factors (body weight, LDL, HDL, total cholesterols, triglycerides, adiponectin, and leptin) in nonobese people. OBJECTIVES: The objective of this study was to investigate in vivo effects of CCJ12 on these metabolic factors as well as inflammatory/cardiovascular disease risk factors [C-reactive protein (CRP), soluble E-selectin (sE-selectin), TNF-α, monocyte chemoattractant protein-1 (MCP-1)] in a nonobese model. METHODS: Sprague-Dawley male rats were fed a complete diet for 20 wk with either drinking water containing CCJ12 [coffee containing javamide-I/-II group (CG), n = 10] or unsupplemented drinking water [water control group (NCG), n = 10]. The amounts of javamide-I/-II in CCJ12 were quantified by HPLC. Water/food consumption and body weight were monitored weekly, and the concentrations of metabolic/inflammatory/cardiovascular disease risk factors were measured by ELISA. RESULTS: There was no significant difference in water/food consumption between the NCG and CG during the study. Also, no significant difference was found in average body weights between the groups either. In addition, after 20 wk, both groups did not show any significant difference in plasma LDL, HDL, and total cholesterol concentrations. Likewise, adiponectin and leptin concentrations were not significantly different between the groups. As expected, the 2 groups did not show any significant difference in plasma concentrations of CRP and sE-selectin. Furthermore, there was no significant difference in plasma concentrations of TNF-α and MCP-1 between the groups. CONCLUSIONS: The data suggest that CCJ12 may not have significant effects on the metabolic/inflammatory/cardiovascular disease risk factors in the CG, compared with the NCG.

Soybean Callus-A Potential Source of Tocopherols.

In vitro cultures have been used as an effective means to achieve a high level of secondary metabolites in various plants, including soy. In this study, the contents of α-, γ-, and δ- tocopherol were quantified in soybean callus, and their amounts were compared to those of soybeans cultivated using the conventional tillage system with three weed controls (respectively without herbicide and with two variants of herbicide). Soybean callus was produced using Murashige and Skoog 1962 (MS) medium supplemented with 0.1 mg/L 6-Benzylaminopurine (BAP) and 0. 1 mg/L Thidiazuron (TDZ). The highest amount of fresh callus was obtained from soybeans from the conventional tillage system with second weed control (S-metolachlor 960 g/L, imazamox 40 g/L, and propaquizafop 100 g/L) respectively 13,652.4 ± 1177.62 mg. The analyzed tocopherols were in much higher content in soy dry callus than the soybean seeds (5.63 µg/g compared with the 0.35 α-toco in soybean, 47.57 µg/g compared with 18.71 µg/g γ-toco or, 5.56 µg/g compared with 1.74 µg/g ß-toco). The highest content of the three analyzed tocopherols was γ -tocopherol, both in callus and soybeans. Furthermore, the data showed that herbicides used in soybean culture significantly influenced both the in vitro callus production and the tocopherol callus content (p ˂ 0.05). Altogether, soybean callus can be an important source of tocopherols, and herbicides significantly influence in vitro callus production and the tocopherol callus content.

Kahweol Found in Coffee Inhibits IL-2 Production Via Suppressing the Phosphorylations of ERK and c-Fos in Lymphocytic Jurkat Cells.

Interleukin-2 (IL-2) is a cytokine involved in the development and maturation of the subsets of T cells, critically associated with the progression of several immune-related diseases (e.g. liver disease, bowel disease). Interestingly, a recent study suggests that coffee may contain several compounds to inhibit IL-2 expression in activated T-lymphocytic cells. However, there is little information about the potential effects of several coffee compounds (e.g. kahweol, cafestol, trigonelline, niacin and chlorogenic acids) on IL-2 expression in activated T-lymphocytic cells. Therefore, in this paper, their effects on IL-2 expression were evaluated in PHA/PMA-activated lymphocytic Jurkat cells. Among the tested compounds, only kahweol and cafestol were able to reduce IL-2 production significantly in the cells (p < 0.05). However, the inhibition of kahweol was a bit stronger than cafestol. Therefore, the molecular mechanism underlying the IL-2 inhibition was investigated using kahweol. Kahweol (≤ 20 µM) was able to inhibit the phosphorylations of ERK and c-Fos (p < 0.05) with little effects on p38 and JNK phosphorylations in the Jurkat cells. Subsequently, the inhibition of ERK/c-Fos led to the reduction of IL-2 mRNA expression in the Jurkat cells. In summary, the data suggest that kahweol may be a potential coffee compound to reduce IL-2 production via inhibiting the phosphorylations of ERK/c-Fos in PHA/PMA-activated lymphocytic Jurkat cells.

Javamide-II Inhibits IL-6 without Significant Impact on TNF-alpha and IL-1beta in Macrophage-Like Cells.

The main aim of this study is to find a therapeutic compound to inhibit IL-6, not TNF-alpha and IL-1beta, in macrophage-like cells, because the high-levels of IL-6 production by macrophages are reported to cause unfavorable outcomes under several disease conditions (e.g., autoimmune diseases, and acute viral infections, including COVID-19). In this study, the potential effects of javamide-II on IL-6, IL-1beta and TNF-alpha productions were determined using their ELISA kits in macrophage-like THP-1 cells. Western blots were also performed using the same cells, to determine its effects on signaling pathways (ERK, p38, JNK, c-Fos, ATF-2, c-Jun and NF-κB p65). At concentrations of 0.2-40 µM, javamide-II inhibited IL-6 production significantly in the THP-1 cells (IC50 of 0.8 µM) (P < 0.02). However, javamide-II did not inhibit IL-1beta or TNF-alpha productions much at the same concentrations. In addition, the treatment of javamide-II decreased the phosphorylation of p38 without significant effects on ERK and JNK phosphorylations in the THP-1 cells. Furthermore, the p38 inhibition, followed by the reduction of ATF-2 phosphorylation (not c-Fos, c-Jun or NF-κB p65), led to the suppression of IL-6 mRNA expression in the cells (P < 0.02). The data indicate that javamide-II may be a potent compound to inhibit IL-6 production via suppressing the p38 signal pathway, without significant effects on the productions of TNF-alpha and IL-1beta in macrophage-like THP-1 cells.

Javamide-II Found in Coffee Is Better than Caffeine at Suppressing TNF-α Production in PMA/PHA-Treated Lymphocytic Jurkat Cells.

Recent studies have suggested positive benefits of coffee consumption on inflammation-related diseases, such as liver diseases and diabetes, where activated lymphocytes and TNF-α are critically implicated. Interestingly, some reports suggested that javamide-II found in coffee may have anti-inflammatory activity greater than that of caffeine, but there is limited information about its effect on TNF-α production by activated lymphocytes. Therefore, the inhibitory effect of javamide-II on TNF-α was investigated in PMA/PHA-treated lymphocytic Jurkat cells. At 5 µM, javamide-II, not caffeine, inhibited TNF-α production in the cells (45 ± 4%, P < 0.001). To elucidate the underlying mechanism, the phosphorylation of MAP kinases (ERK, p38, and JNK) was investigated in the Jurkat cells. Javamide-II had little effect on JNK or p38 phosphorylation, but javamide-II (<20 µM) decreased ERK phosphorylation, consequently reducing TNF-α mRNA expression in the cells ( P < 0.001). The involvement of ERK phosphorylation was also confirmed by an ERK1/2 inhibitor (SCH772984). Furthermore, javamide-II was also found to inhibit IL-2 production, which is up-regulated by ERK phosphorylation in cells ( P < 0.001). These data suggested that javamide-II may be a potent compound to suppress TNF-α production more efficiently than caffeine by inhibiting ERK phosphorylation in Jurkat cells.

In Silico Screening and In Vitro Activity Measurement of Javamide Analogues as Potential p38 MAPK Inhibitors.

p38 Mitogen-activated protein kinase (p38 MAPK) is a protein kinase critically involved in the progress of inflammation/stress-associated diseases. Our data suggested that javamide analogues may contain strong anti-inflammation activities, but there is little information about their effects on p38 MAPK. Therefore, in this paper, the effects of thirty javamide analogues on p38 MAPK were investigated using in silico screening and in vitro p38 MAPK assay methods. The javamide analogues were synthesized and their chemical structures were confirmed using nuclear magnetic resonance (NMR) spectroscopic methods. Then, the javamide analogues were screened using an in silico modeling program. The screened analogues demonstrated a wide range of binding energy (ΔE; -20 to -39) and several analogues with ΔE; -34 to -39 showed strong binding affinity to p38 MAPK. In vitro p38 MAPK assay, the kinase was significantly inhibited by the analogues with great binding energy (ΔE; -34 to -39) and in silico scores (Avg. score; -27.5 to -29.3). Furthermore, the comparative analysis of both assays showed a positive correlation between the in silico scores and p38 MAPK inhibition. In fact, the javamide analogues with top five in silico scores (Avg. score; -27.5 to -29.3) were found to inhibit p38 MAPK by 27-31% (p < 0.05) better than those with less scores (ΔE < -27.0). Especially, javamide-II-O-ethyl ester with relatively high in silico score (Avg. score; -29.2) inhibited p38 MAPK (IC50 = 9.9 µM) a little better than its methyl ester with best in silico score (Avg. score; -29.3). To support the ability to inhibit p38 MAPK, the treatment of javamide-II-ethyl and -methyl esters could suppress the production of IL-8 and MCP-1 protein significantly by 22-73% (p < 0.05) in the differentiated THP-1 cells, and the inhibition was slightly stronger by the ethyl ester than the methyl ester. Altogether, this study suggests that javamide-II-O-ethyl ester may be a most potent p38 MAPK inhibitor among the tested compounds and the combining in silico and in vitro assay approach may be a useful and efficient solution as a functional screening approach in searching new lead compounds for targeted molecules.

NMR Confirmation and HPLC Quantification of Javamide-I and Javamide-II in Green Coffee Extract Products Available in the Market.

Javamide-I/javamide-II are phenolic amides found in coffee. Recent reports suggested that they may contain several biological activities related to human health. Therefore, there is emergent interest about their quantities in coffee-related products. Green coffee extract is a powder extract made of unroasted green coffee beans, available as a dietary supplement. However, there is little information about the amounts of javamide-I/javamide-II in green coffee extract products in the market. Therefore, in this paper, javamide-I/javamide-II were extracted from green coffee extract products and their identifications were confirmed by NMR. After that, the amounts of javamide-I/javamide-II were individually quantified from seven different green coffee extract samples using the HPLC method coupled to an electrochemical detector. The HPLC method provided accurate and reliable measurement of javamide-I/javamide-II with excellent peak resolution and low detection limit. In all seven green coffee extract samples, javamide-II was found to be between 0.28 and 2.96 mg/g, but javamide-I was detected in only five samples in the concentration levels of 0.15-0.52 mg/g, suggesting that green coffee extract products contain different amounts of javamide-I/javamide-II. In summary, javamide-I/javamide-II can be found in green coffee extract products sold in the market, but their amounts are likely to be comparatively different in between green coffee extract brands.

A Simple Way to Eradicate Methicillin-Resistant Staphylococcus aureus in Cynomolgus Macaques (Macaca fascicularis).

Our investigation of indoor-housed cynomolgus macaques (Macaca fascicularis) by using automated identification followed by antibiotic susceptibility testing revealed 1 of 7 immunocompetent animals and 2 of 9 immunosuppressed monkeys as carriers of methicillin-resistant Staphylococcus aureus (MRSA). Follow-up management involving mupirocin treatment resulted in the conversion of the 3 MRSA carriers into MRSA-negative cases. Prospective assessment of newly imported monkeys involving 24-h culture of nasal swabs on chromogenic agar revealed that 22% (18 of 82 animals) were MRSA-positive. Mupirocin treatment successfully converted all of the MRSA-positive macaques into non-carriers, suggesting the feasibility of this simple, one-step screening procedure for rapidly identifying MRSA carriers in large cohorts. In addition, 8 animals that had been diagnosed MRSA-positive and subsequently treated with mupirocin demonstrated no recolonization during follow-up, even under immunosuppressive conditions. We propose rapid screening using chromogenic agar followed by mupirocin treatment as a time- and cost-effective regimen for managing MRSA in cynomolgus monkeys.

Methyl (E)-(3-(3,4-dihydroxyphenyl)acryloyl)tryptophanate can suppress MCP-1 expression by inhibiting p38 MAP kinase and NF-κB in LPS-stimulated differentiated THP-1 cells.

Methyl (E)-(3-(3,4-dihydroxyphenyl)acryloyl)tryptophanate (MHAT) is an O-methyl ester of javamide-II showing strong anti-inflammatory activity. Therefore, in this study, MHAT was chemically synthesized, and its effects on p38 MAP kinase, NF-κB, and monocyte chemotactic factor-1 (MCP-1) expression were investigated in LPS-stimulated differentiated THP-1 cells. MHAT inhibited p38 MAP kinase with an IC50 of 12µM, and the inhibition was supported by an in silico model showing that its binding to p38 MAP kinase was stronger than that of SB203580. At the concentration of 20µM, the p38 inhibition reduced ATF-2 phosphorylation by 55% (P < 0.05). Additionally, MHAT inhibited NF-κB (p65) phosphorylation by 30% (P < 0.05) at the same concentration, suggesting that MHAT was able to reduce NF-κB transcriptional activity. This supposition was confirmed by the NF-κB reporter assay, demonstrating that MHAT (20µM) could suppress NF-κB transcriptional activity by 29% (P < 0.05) in the NF-κB reporter (Luc)-HEK293 cell line. As expected, the treatment with MHAT (5-40µM) significantly inhibited MCP-1 mRNA expression by 9-73% (P < 0.05) and the production of MCP-1 protein by 10-70% (P < 0.05) in the THP-1 cells. Furthermore, MHAT was found to inhibit RANTES expression as well in the same THP-1 cells, supporting its purported inhibition of p38 MAP kinase and NF-κB. All these data suggest that MHAT is a potent compound that can inhibit MCP-1 production by suppressing p38 kinase/ATF-2 phosphorylation and NF-κB in the differentiated THP-1 cells.

N-Caffeoyltryptamine, a Potent Anti-Inflammatory Phenolic Amide, Suppressed MCP-1 Expression in LPS-stimulated THP-1 Cells and Rats Fed a High-Fat Diet.

Monocyte chemoattractant protein-1 (MCP-1) is a well-known chemokine critically involved in the pathophysiological progression of several inflammatory diseases including arthrosclerosis. N-caffeoyltryptamine is a phenolic amide with strong anti-inflammatory effects. Therefore, in this paper, the potential effect of N-caffeoyltryptamine on MCP-1 expression was investigated as a potential p38 mitogen-activated protein (MAP) kinase inhibitor in vitro and in vivo. At the concentration of 20 µM, N-caffeoyltryptamine significantly inhibited p38 MAP kinase α, ß, γ and δ by 15-50% (p < 0.05), particularly p38 MAP kinase α (IC50 = 16.7 µM) and ß (IC50 = 18.3 µM). Also, the pretreatment of the lipopolysaccharide (LPS)-stimulated THP-1 cells with N-caffeoyltryptamine (10, 20 and 40 µM) led to significant suppression of MCP-1 production by 10-45% (p < 0.05) in the cells. Additionally, N-caffeoyltryptamine was also able to significantly downregulate MCP-1 mRNA expression in the THP-1 cells (p < 0.05). On the basis of this strong inhibition in vitro, an animal study was conducted to confirm this inhibitory effect in vivo. Rats were divided into three groups (n = 8): a normal control diet (C), a high-fat diet (HF), or a high-fat diet supplemented with N-caffeoyltryptamine (2 mg per day) (HFS). After 16 weeks, blood samples were collected from the rats in each group, and MCP-1 levels were determined in plasma with other atherogenic markers (C-reactive protein and soluble E-selectin (sE-selectin)). As expected, the average MCP-1 levels of the HF group were found to be higher than those of the C group (p < 0.05). However, the MCP-1 levels of the HFS group were significantly lower than those of the HF group (p < 0.05), suggesting that N-caffeoyltryptamine could decrease MCP-1 expression in vivo. Related to other atherogenic markers such as C-reactive protein and sE-selectin, there was no significant difference in their levels between the HF and HFS groups. These data suggest that N-caffeoyltryptamine may specifically suppress MCP-1 expression in vitro and in vivo, possibly by inhibiting p38 MAP kinase.

Javamide-I-O-methyl ester increases p53 acetylation and induces cell death via activating caspase 3/7 in monocytic THP-1 cells.

BACKGROUND: Javamide-I and-II are phenolic amide compounds found in Coffea sp. Previous study suggested that javamide-II may be a potent compound with Sirt1/2 inhibition activity. PURPOSE: However, the effects of javamide-I and the its O-methyl ester on Sirt inhibition, p53 acetylation and cell death have not been investigated. METHODS: The isolation and synthesis of javamide-I and its O-methyl ester analogue were confirmed by NMR. Their potential effects on Sirt1/2/3, p53 acetylation and cell death were examined using sirt assay, silico analysis, Western blot, caspase 3/7 and apoptotic assay methods. RESULTS: Javamide-I and its O-methyl ester demonstrated a similar inhibition pattern; Sirt1 (IC50 of 19µM) better than Sirt2 (IC50 of 104µM) and Sirt3 (IC50 of 160µM). However, javamide-I and its O-methyl ester were found to inhibit Sirt1 better than Sirt2, which is different from javamide-II able to inhibit Sirt2 stronger than Sirt1. In silico analysis, javamide-I and its O-methyl ester showed a competitive binding pattern against NAD+, which was also supported by the kinetic analysis with Ki=20.1µM for javamide-I and 19.5µM for the O-methyl ester. However, the O-methyl ester increased p53 acetylation better than javamide-I in monocytic THP-1 cells. Since caspase 3/7 activation is often followed by the p53 activation, we investigated their effects on caspase 3/7, and found that O-methyl ester again activated caspase 3/7 greater than javamide-I in the cells, eventually leading to apoptotic cell death. CONCLUSION: These data suggest that the O-methyl modification in javamide-I may play a critical role in increasing p53 acetylation, activating caspase, and eventually inducing the THP-1 cell death.

Finding Potent Sirt Inhibitor in Coffee: Isolation, Confirmation and Synthesis of Javamide-II (N-Caffeoyltryptophan) as Sirt1/2 Inhibitor.

Recent studies suggest that Sirt inhibition may have beneficial effects on several human diseases such as neurodegenerative diseases and cancer. Coffee is one of most popular beverages with several positive health effects. Therefore, in this paper, potential Sirt inhibitors were screened using coffee extract. First, HPLC was utilized to fractionate coffee extract, then screened using a Sirt1/2 inhibition assay. The screening led to the isolation of a potent Sirt1/2 inhibitor, whose structure was determined as javamide-II (N-caffeoyltryptophan) by NMR. For confirmation, the amide was chemically synthesized and its capacity of inhibiting Sirt1/2 was also compared with the isolated amide. Javamide-II inhibited Sirt2 (IC50; 8.7 µM) better than Sirt1(IC50; 34µM). Since javamide-II is a stronger inhibitor for Sirt2 than Sirt1. The kinetic study was performed against Sirt2. The amide exhibited noncompetitive Sirt2 inhibition against the NAD+ (Ki = 9.8 µM) and showed competitive inhibition against the peptide substrate (Ki = 5.3 µM). Also, a docking simulation showed stronger binding pose of javamide-II to Sirt2 than AGK2. In cellular levels, javamide-II was able to increase the acetylation of total lysine, cortactin and histone H3 in neuronal NG108-15 cells. In the same cells, the amide also increased the acetylation of lysine (K382) in p53, but not (K305). This study suggests that Javamide-II found in coffee may be a potent Sirt1/2 inhibitor, probably with potential use in some conditions of human diseases.

Bioavailability of Alfrutamide and Caffedymine and Their P-Selectin Suppression and Platelet-Leukocyte Aggregation Mechanisms in Mice.

BACKGROUND: Alfrutamide and caffedymine are phenolic amides found in plants, including garlic and cocoa. However, the bioavailability of alfrutamide and caffedymine and their effects on cardiovascular diseases (CVDs), particularly via effects on P-selectin expression(PSE) and platelet-leukocyte aggregation (PLA), are unknown. OBJECTIVE: The objective of this study was to investigate the bioavailability of alfrutamide and caffedymine and their effects on PSE and PLA, which are frequently involved in the progression of CVDs. METHODS: Cyclooxygenase (COX) I and COX-II activities and cAMP were determined by using COX and cAMP kits. Bioavailability was determined by HPLC analysis of plasma samples from Swiss Webster mice orally administered alfrutamide and caffedymine (10 µg each). PSE and PLA were also measured by flow cytometry using blood samples from the same mice. RESULTS: At 0.05 µmol/L, alfrutamide and caffedymine inhibited COX-I and COX-II by 20-40% (P < 0.05) and 16-33% (P < 0.05), respectively, compared with the control. At 0.1 µmol/L, the 2 compounds also inhibited platelet PSE by 28% (P < 0.05) and 35% (P < 0.05), respectively, compared with the control. The ß2-adrenoceptor antagonists ICI118551 and butoxamine partially suppressed the inhibition of PSE by caffedymine, suggesting that ß2 receptors are involved in inhibition by caffedymine but not by alfrutamide. At the same concentration (0.1 µmol/L), however, these 2 compounds inhibited PLA by 24-32% (P < 0.05) compared with the control. In addition, mice administered caffedymine and alfrutamide orally (10 µg/35 g body weight) exhibited maximum concentrations >0.6 µmol/L and significant inhibition of PSE by 23-34% (P < 0.05) and PLA by 20-27% (P < 0.05) compared with control mice. CONCLUSIONS: These data show the adequate bioavailability of alfrutamide and caffedymine and their different mechanisms of suppressing PSE and PLA: alfrutamide exerts its effects only via COX inhibition, whereas caffedymine works through both COX inhibition and cAMP amplification.

Becatamide Found in Houttuynia cordata Suppresses P-selectin Expression Via Inhibiting COX Enzyme, Not Increasing cAMP in Platelets.

Atherosclerosis is a well-known inflammatory cardiovascular disease. Recent studies suggested potential anti-atherosclerosis effects of becatamide found in Houttuynia cordata. Therefore, in this study, we investigated potential effect of becatamide (1) and its analogues (enferamide (2), veskamide (3), oretamide (4) and amkamide (5)) on cyclooxygenase (COX)-1 and -2 and the production of cyclic adenosine monophosphate (cAMP), which are critically involved in platelet activation. Among them, becatamide was the most potent compound able to inhibit COX-1 (IC50 = 0.27 µm) and -2 (IC50 = 0.78 µm) (p < 0.05). The decreasing order of COX-1 and -2 inhibition activity was becatamide > veskamide > enferamide > oretamide > amkamide. As a result of the inhibition, the production of thromboxane B2 and P-selectin expression were suppressed by 35% (p < 0.05) and 28% (p < 0.05), respectively, in mouse blood treated with becatamide (0.25 µm). However, becatamide did not increase intracellular cAMP in platelets. Therefore, the suppression of P-selectin expression was not blocked by beta 2-adrenoceptor antagonists, suggesting that the COX inhibition is likely an underlying mechanism for the P-selectin suppression. In summary, becatamide may be a potent compound to inhibit platelet activation by inhibiting COX enzymes, not by increasing cAMP. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Comparison of Methods for Determining ABO Blood Type in Cynomolgus Macaques (Macaca fascicularis).

Thorough examination of ABO blood type in cynomolgus monkeys is an essential experimental step to prevent humoral rejection during transplantation research. In the present study, we evaluated current methods of ABO blood-antigen typing in cynomolgus monkeys by comparing the outcomes obtained by reverse hemagglutination, single-nucleotide polymorphism (SNP) analysis, and buccal mucosal immunohistochemistry. Among 21 animals, 5 were type A regardless of the method. However, of 8 serologically type B animals, 3 had a heterozygous type AB SNP profile, among which 2 failed to express A antigen, as shown by immunohistochemical analysis. Among 8 serologically type AB animals, 2 appeared to be type A by SNP analysis and immunohistochemistry. None of the methods identified any type O subjects. We conclude that the expression of ABO blood-group antigens is regulated by an incompletely understood process and that using both SNP and immunohistochemistry might minimize the risk of incorrect results obtained from the conventional hemagglutination assay.