Culinary continuity in central Japan across the transition to agriculture.

Rice and millet arrived in Western Japan from Korea around 3,000 years ago and spread eastwards across the archipelago in the next 700 years. However, the extent to which agriculture transformed traditional Jomon hunter-gatherer-fisher communities is debated. Central Japan is a key area of study as remodelling of radiocarbon dates shows a slowdown in the dispersal rate of rice agriculture in this area. Here, we examine and compare the use of pottery by Final Jomon and Early to Middle Yayoi communities in the Tokai and the Central Highland regions of central Japan, using lipid residue analysis. Although the identification of specific biomarkers for rice remains elusive, an increase in the ratio of E/H C18 APAAs with the arrival of rice and millet indicates a potential change in plant processing and consumption. We were also able to identify biomarkers for broomcorn millet (miliacin) in both Final Jomon and Yayoi pottery. However, evidence for millet consumption is sparse and in all cases was likely mixed with wild hunted and foraged foods. We conclude therefore that, despite the introduction of rice and millet agriculture in central Japan, pre-existing diets and culinary habits of Jomon hunter-gatherers remain important. Supplementary Information: The online version contains supplementary material available at 10.1007/s12520-024-01992-9.

Role of tyrosine residues in modulation of claudin-4 by the C-terminal fragment of Clostridium perfringens enterotoxin.

The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) modulates the barrier function of claudin-4 via its C-terminal 16 amino acids. In the current study, we investigated the roles of tyrosine residues (Y306, Y310 and Y312) in this region in the modulation of TJs by C-CPE. Single mutations of Y306, Y310 and Y312 to alanine resulted in partial reduction of claudin-4 binding. We also prepared double mutants of C-CPE to further evaluate the roles of these tyrosine residues. Replacement of Y310 and Y312 with alanine (Y310A/Y312A) partly reduced the ability of C-CPE to bind to claudin-4. Double mutants Y306A/Y310A and Y306A/Y312A, however, lost the ability to bind to claudin-4 and to modulate the TJ barrier. We also found that a triple mutant (Y306A/Y310A/Y312A) lost the ability to bind claudin-4, modulate the TJ barrier, and enhance jejunal absorption in rats. These results indicate that tyrosines 306, 310, and 312 are critical for the interaction of C-CPE with claudin-4 and for the modulation of TJ barrier function by C-CPE. This study provides information that should help in the development of claudin modulators based on C-CPE.

Role of Tyr306 in the C-terminal fragment of Clostridium perfringens enterotoxin for modulation of tight junction.

We previously reported that the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a novel type of absorption enhancer that interacts with claudin-4 and that Tyr306 of C-CPE plays a role in ability of C-CPE to modulate barrier of tight junctions. In the current study, to investigate effects of Tyr306 on the C-CPE activity, we prepared some C-CPE mutants substituted Tyr306 with Trp (Y306W), Phe (Y306F) and Lys (Y306K). We found that Y306W and Y306F mutants of C-CPE had claudin-4 binding affinities and effects on the barrier function of tight junctions, whereas both of these properties were greatly reduced with the Y306K mutant. Finally, the Y306K but not the Y306F and Y306W mutants had reduced abilities to enhance absorption in rat jejunum. These results indicate that aromatic and hydrophobic properties, not hydrogen bonding potential, of Tyr306 are involved in the interaction of C-CPE with claudin-4 and in the modulation of the tight junction barrier function by C-CPE.

Preparation of a claudin-targeting molecule using a C-terminal fragment of Clostridium perfringens enterotoxin.

Although most malignant tumors are epithelia-derived carcinomas, methods for specific and effective delivery of antitumor agents to carcinomas have not been developed. Recent reports indicate that epithelia overexpress claudin-3 and -4, which are integral membrane proteins of epithelial tight junctions. This suggests that claudins can be targeted for tumor therapy, but there is not currently a method for delivering drugs to claudin-expressing cells. In the present study, we evaluated whether a potent claudin-4-binding C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) would allow targeting to claudin-4-expressing cells. We fused C-CPE to the protein synthesis inhibitory factor (PSIF), which lacks the cell binding domain of Pseudomonas exotoxin. This fusion protein, C-CPE-PSIF, was cytotoxic to MCF-7 human breast cancer cells, which express endogenous claudin-4, but it was not toxic to mouse fibroblast L cells, which lack endogenous claudin-4. The cytotoxicity of C-CPE-PSIF was attenuated by pretreating the MCF-7 cells with C-CPE but not bovine serum albumin. Also, deletion of the claudin-4-binding region of C-CPE reduced the cytotoxicity of C-CPE-PSIF. Finally, we found that C-CPE-PSIF is toxic to L cells expressing claudin-4 but not to normal L cells or cells expressing claudin-1, -2, or -5. These results indicate that use of the C-CPE peptide may provide a novel way to target drugs to claudin-expressing cells.

Induction of apoptosis by new ent-kaurene-type diterpenoids isolated from the New Zealand liverwort Jungermannia species.

Some diterpenoids show various biological activities, including anti-inflammatory, anti-HIV and anti-tumor activity. Previously, we have focused our research on the apoptosis-inducing properties of diterpenoids and found that some ent-kaurene-type diterpenoids induced apoptosis in human leukemia HL-60 cells. In this study, we have investigated the induction of apoptosis in HL-60 cells by the novel ent-kaurene-type diterpenoids, jungermannenones A (JA), B (JB), C (JC) and D (JD), isolated from the New Zealand liverwort Jungermannia species. Treatment of the cells with each compound for 12 h resulted in cytotoxicity (IC (50) values: A, 1.3; B, 5.3; C, 7.8; D, 2.7 microM) and caused DNA fragmentation and nuclear condensation, both biochemical markers of the induction of apoptosis. Treatment with the compounds resulted in activation of caspases, including caspase-3 and caspase-8. A broad-spectrum inhibitor of caspases, Z-Asp-CH (2)-DCB, attenuated the cytotoxicity induced by these compounds, suggesting that JA, JB, JC and JD induced apoptosis through a caspase-dependent pathway. JA and JD inhibited the activity of nuclear factor-kappaB, which is a transcriptional factor of anti-apoptotic factors. Thus, some of these new ent-kaurene-type diterpenoids may be promising candidates for anti-tumor agents.

Role of N-terminal amino acids in the absorption-enhancing effects of the c-terminal fragment of Clostridium perfringens enterotoxin.

We recently found that a polypeptide, the C-terminal of Clostridium perfringens enterotoxin (C-CPE), was a novel type of drug absorption enhancer. The C-terminal of C-CPE is thought to play a role in the binding of C-CPE to its receptor, claudin-4; however, the function of the N-terminal of C-CPE is unclear. In the present study, we evaluated the role of the N-terminal domain of C-CPE in jejunal absorption and claudin-4 binding. The treatment of rat jejunum with C-CPE resulted in enhanced absorption of dextran, with a molecular weight of 4000 Da. However, treatment with C-CPE220, which lacks the 36 N-terminal amino acids of C-CPE, did not enhance jejunal absorption. C-CPE had affinity for claudin-4 in rat jejunum lysates and Caco-2 lysates, but C-CPE220 did not. Interaction of C-CPE with the recombinant extracellular domain 2 of human claudin-4 (EC2hCld-4), which is the putative binding site for C-CPE, was observed, but C-CPE220 had no affinity for EC2hCld-4. To investigate the effect of C-CPE220 on the barrier function of tight junctions, we measured transepithelial electric resistance (TER) in C-CPE- or C-CPE220-treated Caco-2 monolayer cells. Although C-CPE decreased TER in Caco-2 monolayer cells, C-CPE220 did not disrupt the barrier function of tight junctions. Together, these results indicate that the 36 N-terminal amino acids of C-CPE may be necessary for the enhanced absorption mediated by C-CPE and play a partial role in binding to claudin-4.

Activation of p38 mitogen-activated protein kinase during ent-11alpha-hydroxy-16-kauren-15-one-induced apoptosis in human leukemia HL-60 cells.

Kaurene-type diterpenes possess various biological activities including antitumor and anti-inflammatory effects. Indeed, we have found that an ent-kaurene diterpene, ent-11alpha-hydroxy-16-kauren-15-one (KD), induced apoptosis via caspase-8 activation in human promyelocytic leukemia HL-60 cells. However, the mechanism of caspase-8 activation by KD is not clear. In this study, we investigated the involvement of p38 mitogen-activated protein kinase (p38 (MAPK)) in KD-induced apoptosis. p38 (MAPK) was activated by treatment with KD parallel to DNA ladder formation. Pretreatment with SB203580, a specific inhibitor of p38 (MAPK), attenuated induction of apoptosis by KD and inhibited activation of caspase-8. Cleavage of Bid, a typical substrate of caspase-8, was also inhibited by treatment with SB203580, suggesting that activation of p38 (MAPK) occurs upstream of caspase-8 during KD-induced apoptosis.

An ent-kaurene diterpene enhances apoptosis induced by tumor necrosis factor in human leukemia cells.

Some antitumor agents, including tumor necrosis factor-alpha (TNF-alpha) and camptothecin (CPT), often cause resistance of tumor cells to antitumor agents through activation of the nuclear factor-kappa B (NF-kappa B) pathway that leads to up-regulation of anti-apoptotic proteins. Therefore, co-treatment of an inhibitor of the NF-kappa B pathway with antitumor agents is a useful strategy for chemotherapy. Here we report that ent-11 alpha-hydroxy-16-kauren-15-one (KD) selectively inhibits NF-kappa B-dependent gene expression due to treatment with TNF-alpha. KD in combination with TNF-alpha caused a dramatic increase in apoptosis in human leukemia cells accompanied by activation of caspases. A broad-spectrum inhibitor of caspases decreased the apoptosis induced by treatment with KD and TNF-alpha. KD in combination with CPT also caused an increase in apoptosis. These results suggest that the apoptotic potency of co-treatment of KD with TNF-alpha or CPT is elicited through selective inhibition of NF-kappa B-dependent anti-apoptotic proteins and thus may provide a basis for the development of useful approaches to the treatment of leukemia.

Kaurene diterpene induces apoptosis in human leukemia cells partly through a caspase-8-dependent pathway.

Defects in apoptosis signaling pathways contribute to tumorigenesis and drug resistance, and these defects are often a cause of failure of chemotherapy. Thus, a major goal in chemotherapy is to find cytotoxic agents that restore the ability of tumor cells to undergo apoptosis. We previously found that an Ent-kaurene diterpene, Ent-11alpha-hydroxy-16-kauren-15-one (KD), induced apoptosis in human promyelocytic leukemia HL-60 cells. Here, we found that caspase-8, an apoptotic factor, is involved in KD-induced apoptosis. Although treatment of HL-60 cells with KD resulted in the activation of caspase-8 and -9, a caspase-8-specific inhibitor but not a caspase-9-specific inhibitor attenuated KD-induced apoptosis. Expression of a catalytically inactive caspase-8 partly attenuated KD-induced apoptosis. Treatment with KD led to a time-dependent cleavage of Bid, a substrate of caspase-8, as well as to the proteolytic processing of procaspase-8, indicating that KD treatment induces apoptosis through a caspase-8-dependent pathway. Moreover, overexpression of the drug resistance factor Bcl-2, which is frequently overexpressed in many tumors, failed to confer resistance to KD-induced cytotoxicity. Thus, KD may be a promising experimental cytotoxic agent that possibly points to new strategies to overcome a drug resistance.