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Normal differentiation and proliferation of cells are essential for maintaining homeostasis. Following the successful completion of whole genome sequencing, protein modification has been attracted increasing attention in order to understand the roles of protein diversification in protein function and to elucidate molecular targets in mechanisms of signal transduction. Vitamin A is an essential nutrient for health maintenance. It is present as β-carotene in green and yellow vegetables and retinyl ester in animal products and absorbed into the body from the intestines. After ingestion, it is converted to retinol and oxidized in target cells to retinal, which plays critical roles in vision. It is then further oxidized to retinoic acid (RA), which exhibits a number of effects prior to being metabolized by cytochrome P450 and excreted from the body. Since RA exhibits cell differentiation-inducing actions, it is used as a therapeutic agent for patients with acute promyelocytic leukemia. The current paper describes: (1) HL60 cell differentiation and cell differentiation induction therapy by RA; (2) roles played by RA and retinal and their mechanisms of action; (3) retinoylation, post-translational protein-modified by RA, a novel non-genomic RA mechanism of action without RA receptor; (4) new actions of β-carotene and retinol in vivo and (5) potent anticancer effects of p-dodecylaminophenol (p-DDAP), a novel vitamin A derivative created from the RA derivative fenretinide. We propose that nutritional management of vitamin A can be effective at preventing and treating diseases, and that p-DDAP is a promising anticancer drug.

Emerging evidences suggest that gut microbiota-derived substances play a pivotal role in the regulation of host homeostasis including vascular function. Actually, these substances and/or their metabolites can be presented in circulation and local tissue and their levels are often abnormal in the pathophysiological states. Therefore, to determine the role of them in physiological function is important in human health. On the other hand, vascular dysfunction is a key event in the initiation and progression of systematic complications of cardiovascular, kidney and metabolic diseases including hypertension, dyslipidemia, diabetes, and atherosclerosis. Although abnormalities in endothelial and vascular smooth muscle cells play an important role on vascular dysfunction, emerging evidences has suggested that gut microbiota-derived substances can directly or indirectly affect these cellular functions. The present review will focus on the relationship between vascular function and indoxyl sulfate or trimethylamine-N-oxide (TMAO).

For various pollutants with different specific gravity values, adsorbents having similar specific gravity values as those of target pollutants are necessary to achieve enough contact time. The purpose of this work is to introduce a self-vertical migration system to adsorbents for wastewater treatment. An alginate hydrogel composite with controlled specific gravity and buoyancy was successfully developed, which can first sink to the bottom of water and then float up on the surface after the adsorption. The alginate hydrogel composite was prepared using an alginate solution containing both glucose and yeast. In an experiment, the obtained beads first sank. However, 30 min later, most of them floated up to the water surface, where carbon dioxide was generated in the fermentation process. This behavior was repeated several times during the fermentation process, causing the beads to float. To confirm the efficiency of this unique property, the removal of cesium ions was demonstrated in a water column using Prussian Blue modified alginate gel beads with a repeated vertical migration system. The adsorbent showed a faster removal of cesium than the other adsorbents without the proposed system. We believe that the proposed system can be applied to treat large volumes of wastewater that cannot be stirred or pumped. Therefore, the novel adsorbent developed in this study is expected to significantly contribute to environmental remediation.

Studies on functional molecules starting from syntheses of cysteine-containing peptides and protein are described. Starting from evaluation of a cysteine specific side-reaction, a specific reaction for disulfide-bond formation was developed. The reaction made it possible to independently construct a disulfide bridge without effecting the existing disulfide bonds, which resulted in a unique approach for the synthesis of human insulin by site-specific disulfide bond formation. In a series of studies on sulfur-containing amino acids, another cysteine related un-natural amino acid, α-methyl cysteine, was used for the total syntheses of natural products containing a unique thiazorine/thiazole ring system. Chloroimidazolidium coupling reagent developed by us was effective for the successive couplings of the α-methyl cysteine residues. Based on these synthetic studies, design and evaluation of protease inhibitors were then studied, since a stereo-specific synthesis of the key structure is crucial to make the inhibitor an effective functional molecule in the interactions with its target protease. As the target proteases, β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and chymotrypsin-like protease of severe acute respiratory syndrome (SARS 3CL protease) were selected: the former is a crucial enzyme for amyloid β production and the latter is an essential enzyme for the re-construction of SARS corona virus in host cells. Structure optimization procedure of the respective inhibitors are described based on X-ray crystal structure analyses of the inhibitor-protease complex.

The number of patients with type 2 diabetes mellitus (T2DM) has rapidly increased, especially in East and Southeast Asia. In these areas, in general, people have especially vulnerable β-cells and insulin secretion deficiency and reduced β-cell mass are the primary cause of T2DM. Therefore, the alleviation of such β-cell dysfunction would provide therapeutic approaches to prevent the development of T2DM. Nobiletin, a polymethoxylated flavonoid found in citrus fruits, has been shown to improve obesity and insulin resistance in T2DM model mice. We focused on β-cells and investigated the effects of nobiletin on insulin secretion and β-cell apoptosis. In β-cell line INS-1, nobiletin increased glucose-induced insulin secretion (GSIS) in a concentration-dependent manner, which was inhibited by an Epac inhibitor. In addition, nobiletin at 10 μM inhibited thapsigargin-induced apoptosis, which was inhibited by a PKA inhibitor. Nobiletin also suppressed thapsigargin-induced increases in cleaved caspase-3 and phosphorylated JNK. Thus, nobiletin is suggested to promote GSIS and prevent ER stress-induced β-cell apoptosis, which are mediated via Epac and PKA-dependent pathways, respectively. In summary, nobiletin is suggested to exhibit insulinotropic and anti-apoptotic effects on β-cells, which are one of the causes of its anti-diabetic effect. Moreover, nobiletin seems to be able to alleviate the development of T2DM by protecting β-cells from apoptosis.

Biomolecule-derived peptides are attractive research resources to develop drugs and elucidate the basic mechanisms of life phenomena. This review article focuses on two biomolecules called "neuromedin U (NMU)" and "myostatin" that are deeply involved in obesity and muscle weakness caused by modern lifestyles and aging. A structure-activity relationship (SAR) study based on a biomolecule reveals the structural features required for the biological activity and gives clues leading the drug discovery process. NMU activates two types of receptors (NMUR1 and NMUR2). NMU, which is an attractive candidate for treating obesity, displays a variety of physiological actions in addition to appetite suppression. The discovery of useful receptor-selective agonists helps in elucidating the detailed roles of the respective receptors for each action and in developing therapeutic drugs based on receptor function. Hence, SAR studies focused on the amidated C-terminal heptapeptide of NMU were carried out to obtain selective agonists. Consequently, the respective hexapeptidic NMUR1 and NMUR2 agonists CPN-267 and CPN-116 were discovered. Myostatin, an endogenous negative regulator of skeletal muscle mass, is a promising target for treating muscle atrophy disorders. Focused on the inactivation mechanism of mature myostatin by the myostatin precursor-derived prodomain, a core peptide (23-mer) for effective myostatin inhibition was identified from the mouse myostatin prodomain sequence. The SAR study based on this core peptide afforded a 25-fold more potent derivative (16-mer), which increased skeletal muscle mass and hindlimb grip strength. Therefore, this derivative could be a novel platform for a peptidic drug useful in the treatment of muscle atrophy.

Iron is the most abundant transition metal in our body and plays various pivotal roles in our lives including oxygen transport, energy production, and metabolic reactions. At the same time, an excess amount of iron may cause cellular damages through undesired oxidative reactions due to the high redox activity of Fe ion. We have developed the several fluorescent probes which can detect Fe(II) ion selectively with fluorescence enhancement to understand both the physiological and pathological contributions of Fe ion in living systems. These fluorescent probes worked in an aqueous buffer, living cells, and histochemical-stained samples (Chem Sci. 2013;4:1250, Chem Sci. 2017;8:4858, Free Radic Res. 2014;48:990, Sci Rep. 2017;7:10621). We established a color series of Fe(II)-selective fluorescent probes from blue to deep-red, which were applied to organelle-targeted fluorescent probes for mitochondria, lysosome, and endoplasmic reticulum. Herein, I would like to focus on fluorescence imaging study about the alteration of labile Fe(II) level in each organelle during ferroptosis, iron-dependent cell death, by using the various organelle-targeted fluorescent probes of Fe(II).

Neglected tropical diseases (NTDs) are an extremely important issue facing global health care. To improve "access to health" where people are unable to access adequate medical care due to poverty and weak healthcare systems, we have established two consortiums: the NTD drug discovery research consortium, and the pediatric praziquantel consortium. The NTD drug discovery research consortium, which involves six institutions from industry, government, and academia, as well as an international non-profit organization, is committed to developing anti-protozoan active compounds for three NTDs (Leishmaniasis, Chagas disease, and African sleeping sickness). Each participating institute will contribute their efforts to accomplish the following: selection of drug targets based on information technology, and drug discovery by three different approaches (in silico drug discovery, "fragment evolution" which is a unique drug designing method of Astellas Pharma, and phenotypic screening with Astellas' compound library). The consortium has established a brand new database (Integrated Neglected Tropical Disease Database; iNTRODB), and has selected target proteins for the in silico and fragment evolution drug discovery approaches. Thus far, we have identified a number of promising compounds that inhibit the target protein, and we are currently trying to improve the anti-protozoan activity of these compounds. The pediatric praziquantel consortium was founded in July 2012 to develop and register a new praziquantel pediatric formulation for the treatment of schistosomiasis. Astellas Pharma has been a core member in this consortium since its establishment, and has provided expertise and technology in the area of pediatric formulation development and clinical development.

A decrease in nitric oxide (NO) production may induce pathological conditions associated with endothelial dysfunction and diabetes. Although a decrease in NO production caused by impaired Akt/endothelial nitric oxide synthesis (eNOS) signaling has been demonstrated at the aorta in the presence of diabetic vascular complications, little is known regarding the details of the mechanism. We identified G-protein-coupled receptor kinase 2 (GRK2) as a critical factor in diabetic endothelial dysfunction. GRK2 plays a role in many physiological functions including regulation of G-protein-coupled receptors (GPCRs). We found that the vasculature affected by type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction caused by impaired Akt/eNOS signaling. GRK2 activation also induces changes in the subcellular localization of GRK2 and β-arrestin 2, a downstream protein, from the cytosol to membrane. In mouse aorta GRK2 may be, on translocation, a key negative regulator and an important regulator of β-arrestin 2/Akt/eNOS signaling, which has been implicated in diabetic endothelial dysfunction. Furthermore, in the aortic membrane of type 2 diabetic model mice under insulin stimulation, the impaired Akt/eNOS signaling was improved by a selective GRK2 inhibitor. These results suggest that in diabetes the GRK2 inhibitor ameliorates vascular endothelial dysfunction via Akt/eNOS signaling by inhibiting GRK2 activity and enhancing β-arrestin 2 translocation to the membrane under GPCR or non-GPCR stimulation, thereby contributing to blood pressure- and blood glucose-lowering effects. We propose that the GRK2 inhibitor may be a promising therapeutic target for cardiovascular complications in type 2 diabetes.

Enalaprilat (H2L), which is the active metabolite of the pro-drug enalapril, is an angiotensin-converting enzyme inhibitor. Some side effects such as neurodegeneration and taste disorder can be related to copper or zinc deficiency, which would be caused by the metal complex formation of dianionic elalaprilat (L(2-)). For a better understanding of this phenomenon, we investigated the solution species of enalaprilat in the presence of copper(II) or zinc(II) ions by pH titration analysis with I=0.10 M (NaCl) at 25℃. The 1:1 complex formation constants (KML=[ML]/[M(2+)][L(2-)] M(-1)) of 10(7.4) for CuL and 10(4.4) for ZnL complexes were evaluated, indicating the presence of those complexes at a physiological pH. Furthermore, partition experiments with a two-phase system of 1-butanol/water at 25℃ disclosed that copper(II) and zinc(II) complexes of enalaprilat were partially extracted into the organic layer. In the absence of those metal ions, enalaprilat was not soluble in the 1-butanol phase. The increase in lipophilicity of enalaprilat by metal complexation suggests that the long-term administration of enalapril could be a possible risk factor for the disrupted distribution of those metal ions in biological systems.

Recently, highly active anti-retroviral therapy (HAART), which involves a combinational use of reverse transcriptase inhibitors and HIV protease inhibitors, has brought us a great success in the clinical treatment of AIDS patients. However, HAART has several serious clinical problems. These drawbacks encouraged us to find novel drugs and increase repertoires of anti-HIV agents with various action mechanisms. The recent disclosing of the dynamic supramolecular mechanism in HIV-entry has provided potentials to find a new type of drugs. To date, we have synthesized HIV-entry inhibitors, especially coreceptor CXCR4 antagonists. In addition, CD4 mimics in consideration of synergic effects with other entry inhibitors or neutralizing antibodies have been developed. The development of the above anti-HIV agents is based on the concept of reverse chemical genomics, in which target molecules are fixed. On the other hand, based on the concept of forward chemical genomics, in which active compounds are searched according to the screening of random libraries, effective peptide leads such as integrase inhibitors derived from fragment peptides of HIV-1 Vpr have been discovered. As such, from a point of view on chemical biology, anti-HIV leads have been found utilizing reverse and forward chemical genomics. Furthermore, antibody-based therapy or AIDS vaccine is still thought to be a promising treatment. Thus, peptidic antigen molecules based on artificial remodeling of the dynamic structures of a surface protein gp41 in HIV fusion have been developed. The present chemical biology approaches would be essential for discovery of anti-HIV agents in consideration of cocktail therapy of AIDS.

The structure of tumor vasculature is crucial for the nanocarrier-mediated chemotherapy. Recently, transforming growth factor-β (TGF-β) inhibitor was reported to increase the tumor accumulation of nanocarriers by changing the structure of tumor vasculature. To identify the parameters of tumor vasculature function following TGF-β inhibitor (A-83-01) treatment, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed using Gd-DTPA and its liposomal formulation (Gd-L) as contrast agents. Observation of tumor MR image before, during, and after injection of contrast agent could calculate the parameters of vascular function, such as volume transfer constant between blood plasma and extracellular space (K(trans)) and fractional plasma volume (v(p)). A-83-01 treatment significantly increased these parameters within 24 h that was positively related to pericyte coverage and tumor cell proliferation. Furthermore, apparent diffusion coefficient (ADC) determined by diffusion-weighed imaging was decreased by A-83-01 treatment, suggesting the decrease of tumor interstitial fluid pressure. Vascular function of the tumor improved by A-83-01 treatment well assessed on post-Gd-L-enhanced MR images, which predicted delivery of liposomal drug to the tumor. These findings suggest that DCE-MRI and, in particular, K(trans) and v(p) quantitation, provide important additional information about tumor vasculature by A-83-01 treatment.

Vascular tone is tightly regulated by endothelium-derived factors. These include relaxing factors (EDRFs) such as nitric oxide (NO), hyperpolarizing factors (EDHFs), and contracting factors (EDCFs). Although EDHF is a prominent vasodilator, particularly in smaller arteries, little attention has been paid to the potential role of EDHF responses in diabetes. EDHF signaling may involve various factors, including several diffusible factors and non-diffusible factors (e.g., gap junctions). It has been demonstrated that the alterations in EDHF relaxation seen in mesenteric arteries from diabetic rats may be attributable to an increase in phosphodiesterase 3 (PDE3) activity, leading to a reduction in the action of adenosine 3',5'-cyclic monophosphate (cAMP), and consequently the activity of protein kinase A (PKA) is decreased in such arteries. Moreover, it has been suggested that the imbalance between EDRFs and EDCFs is present in mesenteric arteries from type 2 diabetic rats and the EDHF relaxation can be partly reversed by suppression of EDCF signaling. Indeed, chronic treatment with metformin, eicosapentaenoic acid, or thromboxane synthase inhibitor can reduce EDCF signaling and normalize EDHF signaling in mesenteric arteries from type 2 diabetic rats. Although the improvement or restoration of EDHF responses has not been the direct subject of any pharmaceutical effort, increasing cAMP/PKA signaling (e.g., by inhibiting PDE3 activity) or reducing EDCFs signaling has potential as an interesting therapeutic target in diabetic vasculopathy.

Macro- and microvascular disease states currently represent the principal causes of morbidity and mortality in patients with type I or type II diabetes mellitus. Abnormal vasomotor responses and impaired endothelium-dependent vasodilation have been demonstrated in various beds in different animal models of diabetes and in humans with type I or type II diabetes. The principal mediators of diabetes-associated vascular dysfunction are increases in glucose, oxidized low-density lipoprotein, endothelin-1, angiotensin II, insulin, or growth factors. An accumulating body of evidence indicates that abnormal production of oxidative stress may be one of several factors contributing to vascular dysfunction in diabetes. It is possible that in diabetic states, hyperinsulinemia initiates oxidant stress, leading to vascular dysfunction at a later stage. We and others have demonstrated that in models of hyperinsulinemia and hyperglycemia, . NO production and/or . NO responsiveness are impaired in aortic strips. Several recent studies have shown that the formation of nitrotyrosine and/or peroxynitrite impairs vascular . NO responsiveness and . NO production. Our findings suggest that the coexistence of a high insulin level and an established diabetic state may lead to the excessive generation of peroxynitrite, and that this may in turn trigger an impairment of endothelium-dependent relaxation via a decrease in sarcoendo plasmic reticulum Ca(2+) ATPase function. This review summarizes the results of our recent studies on the involvement of insulin and oxidative stress in the blood vessels of diabetic animals.

The aim of the present study was to investigate the link between the changes in vascular responsiveness associated with hyperinsulinemia in established STZ-induced diabetes and the growth factors signal system. We have shown that in rats with established diabetes, high-insulin treatment can enhances NA-induced contractility. This enhancement probably results from an upregulation of the expression of the mRNA for the alpha 1B- or alpha 1D-adrenergic receptor that is secondary to the hyperinsulinemia. The above effects may be made possible as a result of the increase in IGF-1 receptors and the decreased IGFBPs expressions that occur in the aorta in long-term insulin deficiency. In contrast, those insulin treatments can normalise the impaired endothelium-dependent relaxation, probably by inducing an overexpression of eNOS and VEGF. Furthermore, the expression of the IGF-1 receptor was higher in the aorta in insulin-treated diabetic than in untreated diabetes. This presumably increased the expression of VEGF mRNA, and the increased VEGF presumably upregulated eNOS, thereby resulting in an amelioration in the endothelial dysfunction otherwise seen in diabetic rats. The downside is that such a perturbation of the activity in the IGF-1 system in diabetes could be a key event in the progress of arteriosclerosis and hypertension in syndromes involving hyperinsulinemia.

6-Acyldecahydro [1,6]naphthyridines were synthesized as derivatives of matrine-type and allomatrine-type alkaloids, and the structure-activity relations were examined by the acetic acid-induced abdominal contraction test. All synthesized derivatives produced the antinociception in mice. The antinociceptive potencies of 15a-c and 16a-c were lower than those of 17a-c, 18a-c, 19a-c and 20a-c. Furthermore, those of the matrine-type derivatives 17b and 17c are greater than other derivatives. These findings suggest that less hindered tertiary amine and highly lipophilic acyl group are better functional groups for the greater antinociceptive potencies. Furthermore, these findings suggest that A or B ring of 1 and 2 are not essential for the antinociceptive effect.

Infection with Helicobacter pylori(H. pylori) is thought to be an important factor in the pathogenesis of gastritis, peptic ulcer and gastric cancer, and the 13C-urea breath test (13C-UBT) is a convenient and non-invasive method for the detection of H. pylori in the stomach. We have examined the sensitivity, specificity and accuracy of 13C-UBT, comparing it with histology. The 13CO2/12CO2 ratio was measured by using infra-red spectroscopy(IR) and gas chromatography/mass spectrometry(GC-MS). The IR analyzer should be particularly useful for the diagnostic test of H. pylori infection using 13C-UBT.

Exploitation of saturated macrocyclic polyamines has led to the discovery of numerous novel functions and new molecules such as 1) unique proton sponge properties; 2) uptake of biological polyanions; 3) peptide-like metal uptake properties; 4) stabilization of unusual oxidation states of metal ions (e.g., CuIII, NiIII); 5) novel uptake and activation of O2 by new NiII-macrocyclic complexes; 6) a new synthetic pathway to functionalize macrocyclic polyamines; 7) the first gold(III) complex that is a new candidate for gold-plating agents; 8) intrinsic zinc(II) properties pertinent to zinc enzymes; 9) selective recognition of thymine by ZnII complexes; and 10) new cage supermolecules. These newly discovered molecules and properties have opened up a new field of supramolecular science.

We previously demonstrated that Amaryllidaceae alkaloids, lycorine and lycoricidinol, inhibit induction of apoptosis by calprotectin derived from neutrophils, and that the latter alkaloid showed suppression in rat adjuvant-induced arthritis model. These findings suggest that the alkaloids have a modulating activity against inflammatory reaction. To explore further the mechanism of the suppression for inflammation, we studied the effect of the alkaloids on macrophage tumor necrosis factor (TNF-alpha) production in vitro, since TNF-alpha is recognized as a pivotal cytokine to regulate inflammation. As a result of this study, lycorine and lycoricidinol inhibited TNF-alpha production of murine macrophages stimulated with lipopolysaccharide (ID50 were 0.2 microgram/ml and 0.002 microgram/ml, respectively). The inhibition was also observed in macrophages treated by Gram-positive bacteria, Enterococcus faecalis. Both lycorine and lycoricidinol reportedly have inhibitory activity for protein biosynthesis. Although the inhibition of TNF-alpha production by lycoricidinol was mainly due to the inhibition of protein biosynthesis, lycorine showed inhibition against TNF-alpha production at lower concentrations than the case that they inhibited 35S-Cysteine/35S-Methionine incorporation into macrophages. These facts suggest that the inhibition of TNF-alpha production is not due to the inhibitory activity against protein translation at least at lower concentrations. From these results, it was concluded that these alkaloids exert inhibitory effects not only on neutrophil apoptosis-inducing protein, calprotectin, but also on macrophage TNF-alpha production.

Blockade of the action of angiotensin II (AII) has long been a target for the development of novel antihypertensive agents. We recently discovered a novel class of potent nonpeptide AII receptor antagonists, benzimidazole-7-carboxylic acids including candesartan. Candesartan is a highly potent and insurmountable AII type-1 receptor (AT1)-selective antagonist. Structure-activity relationship (SAR) studies revealed that the adjacent arrangement of a lipophilic substituent, a tetrazolylbiphenylmethyl moiety and a carboxyl group was the important structural requirement for potent AII antagonistic activity. Especially, the presence of a carboxyl group at the 7-position was found to be essential for insurmountable antagonism. To improve bioavailability of candesartan, chemical modification was examined to yield candesartan cilexetil, a prodrug of candesartan. Candesartan cilexetil is a potent and long-acting blocker that, when given once a day to patients, provides effective 24 hr blood pressure control.

This review summarizes our recent findings in the syntheses of drug metabolites. The metabolites of Grepafloxacin (1) and OPC-14117 (10) were prepared from the common intermediates (5) and (21), respectively. Moreover, treatment of 10 with a model P450 system led to a benzyl alcohol derivative (11) in one step. OPC-31260 (22) was efficiently N-dealkylated using several metalloporphyrins with oxidants to afford three metabolites (23-25). In addition, I succeeded in obtaining the metabolite (23) in high yield from N-oxide (26) not only as an oxygen donor but also as a substrate, there after, in the model P450 system. Optically active metabolites of OPC-29030 (27) were prepared by enzyme-catalyzed enantioselective transesterification of racemic sulfinyl metabolites. On the other hand, a chiral 1,1'-bi-2-naphthol derivative (38a) was found to be an efficient asymmetric acylating agent for a secondary alcohol (36) which is a valuable intermediate for preparing optically active metabolites of 22. Furthermore, metabolites (45) and (47) of OPC-21268 (44) were prepared using SmI2-induced cyclization and oxidative decarboxylation with Pb(OAc)4 as key steps, respectively.

Exploitation of saturated macrocyclic polyamines has led to a discovery of numerous novel functions and new molecules such as (1) unique proton sponge properties; (2) uptake of biological polyanions; (3) peptide-like metal uptake properties; (4) stabilization of unusual oxidation states of metal ions (e.g. CuIII, NiIII); (5) novel uptake and activation of O2 by a new NiII-macrocyclic complex; (6) a new synthetic pathway to functionalize polyamines; (7) the first gold (III) complex that is a new candidate for gold-plating agents; (8) intrinsic zinc (II) properties pertinent to zinc enzymes; and (9) selective recognition of thymine by ZnII complexes. These newly discovered properties have created a way to a new type of supramolecular science.

The tumor necrosis factor (TNF) was first discovered as a substance that induced necrosis of transplanted tumors. Recently, TNF has been recognized as an important and endogenous mediator in host defense mechanisms. To prove the fact that plant foods contain substances which activate the host defense mechanisms, we first examined if the administration of flavonoids could induce TNF production in mice. Some selected flavonoids such as naringin, apiin, poncirin and rutin were shown to amplify TNF release from murine macrophages in vivo in response to OK-432 as a second stimulus. However, their aglycone forms were not effective. The differences in the saccharide-chain of flavonoids induced the variety of TNF production.

TNF-alpha, a cytokine with tumor necrosis activity produced by many types of cells including macrophages, was recently recognized as an important host defense factor that affects not only malignant cells, but many kinds of normal cells. Clinical applications of TNF-alpha are now limited, but local injection or local production of TNF-alpha should be given closer consideration in antitumor trials. Here we described the TNF receptor family and TNF-alpha-driven inflammation, and also discussed the physiological roles of TNF-alpha.

Stable isotopically labeled steroids can, without radiation hazards, be safely used as biological internal standards to perform pharmacokinetic studies of steroids in man. This technique offers an advantage in that an endogenous steroid and its stable isotopically labeled analogue exogenously administered can be differentiated easily by employing mass spectrometry. For example, we can examine the pharmacokinetics of cortisol and investigate the metabolic conversion of cortisol to cortisone in man by administering deuterium-labeled cortisol to human subjects as a tracer. The number and the stability of the label are fundamental requisites in stable isotope methodology. Successful application of methodology with stable isotopes is then always dependent on the availability of compounds that are labeled at predesignated chemically inert positions.