A Cross-Sectional Study Based on Forty Systematic Reviews of Foods with Function Claims (FFC) in Japan: Quality Assessment Using AMSTAR 2.

BACKGROUND: The Foods with Function Claims (FFC) was introduced in Japan in April 2015 to make more products available that are labeled with health functions. The products' functionality of function claims must be explained by scientific evidence presented in systematic reviews (SRs), but the quality of recent SRs is unclear. This study assessed the quality of SRs in the FFC registered on the Consumer Affairs Agency (CAA) website in Japan. METHODS: We searched the database from 1 April to 31 October 2022. Confidence in the methodological quality of each SR was evaluated by the AMSTAR 2 checklist. RESULTS: Forty SRs were randomly extracted on the basis of the eligibility criteria and recruitment procedures. Overall confidence was rated as "high" (N = 0, 0%), "moderate" (N = 0, 0%), "low" (N = 2, 5%), or "critically low" (N = 38, 95%). The mean AMSTAR 2 score was 51.1% (SD 12.1%; range 19-73%). Among the 40 SRs, the number of critical domain deficiencies was 4 in 7.5% of SRs, 3 in 52.5% of SRs, 2 in 35% of SRs, and 1 in 5% of SRs. Registering the review's protocol and comprehensive search strategies were particularly common deficiencies. Additionally, the risk of bias (RoB) was insufficiently considered. CONCLUSION: Overall, the methodological quality of the SRs based on the FFC, introduced eight years earlier, was very poor. This was especially true in the interpretation and discussion of critical domains, which had many deficiencies in terms of protocol registration, a comprehensive literature search strategy, and accounting for the RoB.

In Vitro and In Vivo Antiglycation Effects of Connarus ruber Extract.

Accumulation of advanced glycation end products (AGEs) of the Maillard reaction has been implicated in the pathogenesis of diabetes and its complications. Connarus ruber has been used as a folk remedy for several diseases, including diabetes; however, its underlying mechanism has not yet been investigated. This study investigated the effects of C. ruber extract against glycation on collagen-linked AGEs in vitro and streptozotocin-induced diabetic rats (STZ-DM rats) in vivo. The antiglycation activities of C. ruber extract and aminoguanidine (AG) were examined using a collagen glycation assay kit. Nonfluorescent AGE, Nε-carboxymethyl lysine (CML), Nω-carboxymethyl arginine, and Nε-carboxyethyl lysine levels were measured via electrospray ionization-liquid chromatography-tandem mass spectrometry. The effect of the extract on the cytotoxicity of methylglyoxal (MG), a precursor of AGEs, was examined in HL60 cells. STZ-DM rats were treated with the extract for 4 wk, and the effect was assessed using biochemical markers in the serum and CML-positive cells in renal tissues. C. ruber extract dose-dependently inhibited the glycation of collagen and formation of nonfluorescent AGEs, which was comparable to AG, and it significantly attenuated MG-induced cytotoxicity in HL60 cells. Furthermore, the glycated albumin levels in STZ-DM rats decreased, the increase in serum lipid levels was reversed, and immunohistochemistry demonstrated that CML deposition in the glomerulus of STZ-DM rats significantly decreased. Although further studies are needed, C. ruber could be a potential therapeutic for preventing and progressing many pathological conditions, including diabetes.

Quality of Systematic Reviews of the Foods with Function Claims in Japan: Comparative Before- and After-Evaluation of Verification Reports by the Consumer Affairs Agency.

Background: In Japan, a new type of foods with health claims, called Foods with Function Claims (FFC), was introduced in April 2015 in order to make more products available that are clearly labeled with certain health functions. Regarding substantiating product effectiveness, scientific evidence for the proposed function claims must be explained by systematic reviews (SRs), but the quality of SRs was not clear. The objectives of this review were to assess the quality of SRs based on the FFC registered on the Consumer Affairs Agency (CAA) website in Japan, and to determine whether the CAA's verification report in 2016 was associated with improvement in the quality of SRs. Methods: We evaluated the reporting quality of each SR by the AMSTAR checklist on methodological quality. We searched the database from 1 April to 31 October 2015 as the before-SR and from 1 July 2017 to 31 January 2018 as the after-SR. Results: Among the 104 SRs reviewed, 96 final products were included: 51 (53.1%) were supplements, 42 (43.8%) were processed foods without supplements, and 3 (3.1%) were fresh foods. Of the 104 SRs, 92 (88.5%) were qualitative reviews (i.e., without meta-analysis) and 12 (11.5%) performed a meta-analysis. The average quality score of before-SRs and after-SRs was 6.2 ± 1.8 and 5.0 ± 1.9, respectively, a statistically significant decrease (p < 0.001). Conclusion: Overall, the methodology and reporting quality of after-SRs based on the FFC were poorer than those of before-SRs. In particular, there were very poor descriptions and/or implementations of study selection and data extraction, search strategy, evaluation methods for risk of bias, assessment of publication bias, and formulating conclusions based on methodological rigor and scientific quality of the included studies.

Comparison of the effects of pachymic acid, moronic acid and hydrocortisone on the polysome loading of RNAs in lipopolysaccharide-treated THP-1 macrophages.

We have proposed that analysis of ribosome-loaded mRNAs (i.e., the translatome) is useful for elucidation of pharmacological effects of phytocompounds in immune cells, regarding the involvement of post-transcriptional regulation mechanisms. In the present study, we compared the effects of pachymic acid from Poria cocos fungus and moronic acid from propolis with those of hydrocortisone on the translatomes of THP-1 macrophages exposed to bacterial lipopolysaccharide (LPS) to find clues to their biological effects. Polysome-associated RNAs collected from cells treated for 3 h with LPS plus each of the compounds were analyzed by DNA microarray followed by analyses of pathways/gene ontologies (GO). Upregulated mRNAs in enriched pathways that were found to contain AUUUA (AU)-rich motifs were checked by real-time PCR, and expression of candidate RNA-binding proteins stabilizing/destabilizing such AU-rich mRNAs was checked by Western blotting. The numbers of upregulated and downregulated genes (fold-changes ± 2.0 versus vehicle-control) were, respectively, 209 and 125 for moronic acid, 23 and 2 for pachymic acid, and 214 and 59 for hydrocortisone treatment. Overlapping with hydrocortisone treatment for upregulation were 158 genes in moronic acid and 17 in pachymic acid treatment; of these, 16 overlapped within all treatments (C-X-C motif chemokine ligands, interferon-induced protein with tetratricopeptide repeats, etc.). Pathway analyses showed GO enrichments such as 'immune response', 'receptor binding', 'extracellular space' etc. The pachymic acid-upregulated mRNAs (highly overlapped with the other 2 treatments) showed the presence of signal peptides and AU-rich motifs, suggesting regulation by AU-rich element (ARE)-binding proteins. The expression of ARE-binding protein HuR/ELAV-1 was increased by the 3 compounds, and AUF1/hnRNP D was decreased by pachymic acid. These results suggested that pachymic acid and moronic acid effects may involve as yet unknown post-transcriptional modulation via ARE-binding proteins resembling that of glucocorticoids.

Shikonin changes the lipopolysaccharide-induced expression of inflammation-related genes in macrophages.

We aimed to find candidate molecules possibly involved in the anti-inflammatory activity of shikonin (active compound of "Shikon") by analyzing its effects on gene expression of lipopolysaccharide (LPS)-treated THP-1 macrophages. Polysome-associated mRNAs (those expected to be under translation: translatome) from cells treated with LPS alone (LPS: 5 µg/mL), shikonin alone (S: 100 nM), or LPS plus shikonin (LPS&S) for 3 h were analyzed by DNA microarray followed by detection of enriched pathways/gene ontologies using the tools of the STRING database. Candidate genes in enriched pathways in the comparison of LPS&S cells vs. LPS cells were analyzed by reverse-transcription quantitative real-time PCR (RT-qPCR; 1, 2, and 3 h). DNA microarray showed shikonin significantly influences gene expression. Gene expression changes between LPS&S cells and LPS cells were compared to detect relevant proteins and/or mRNAs underlying its anti-inflammatory effects: shikonin downregulated pathways which were upregulated in LPS cells, for example, 'innate immune response'. Within changed pathways, three genes were selected for RT-qPCR analyses as key candidates influencing inflammatory responses: CYBA (component of the superoxide-generating Nox2 enzyme), GSK3B (controller of cell responses after toll-like receptor stimulation), and EIF4E (a key factor of the eukaryotic translation initiation factor 4F complex that regulates abundance of other proteins involved in immune functions). All three mRNAs were decreased at 2 h, and CYBA continued low at 3 h relative to LPS cells. Given that shikonin decreased the expression of CYBA gene of Nox2, in addition to the direct inhibition of the Nox2 activity that we have previously shown, it is suggested that one of its anti-inflammatory mechanisms could be attenuation of oxidative stress.

Quality of systematic reviews of the Foods with Function Claims registered at the Consumer Affairs Agency Web site in Japan: a prospective systematic review.

The objective of this review was to assess the quality of systematic reviews (SRs) based on the Foods with Function Claims (FFC) registered at the Consumer Affairs Agency (CAA) Web site in Japan by AMSTAR checklist. Study design was a prospective SR of SRs based on the FFC registered at the CAA Web site in Japan. We searched the database from 1 April 2015 (starting date) through 27 October 2015 on the CAA Web site. A full quality appraisal of identified articles was made using the combined tool based on the AMSTAR checklist developed to assess the methodological quality of SRs. Each item was scored as "present" (yes), "absent" (no), "unclear or inadequately described" (cannot answer), or "not applicable" (N/A). Forty-nine SRs met inclusion criteria. The quality of the articles was in the poor description category (mean±SD; 6.2±1.8 points, range; 2-11 points). Especially, there were very poor descriptions and/or implementations regarding the registration (2%), evaluation of publication bias (12%), and appropriate conclusions based on scientific quality of the included studies (27%). As a whole, the quality of SRs based on the FFC was poor in methodology and reporting. To develop SRs of the FFC and healthy foods, it will be important for future research to introduce and use (1) the AMSTAR checklist (ie, a tool to assess the methodological quality of SRs), (2) the PRISMA (ie, a checklist in the general description of SRs) and PRISMA-NMA checklists (ie, a checklist in the specific description of SRs with meta-analysis), (3) many English databases, (4) development of the original checklist for the FFC and healthy foods, and (5) notification documents (including SR) of the FFC in English.

Medicine reclassification processes and regulations for proper use of over-the-counter self-care medicines in Japan.

OBJECTIVES: Japan has actively reclassified substances ranging from prescription drugs to over-the-counter (OTC) drugs in recent years. The sale of most OTC drugs was deregulated several times and pharmacists' supervision was deemed no longer mandatory. Japan established a new OTC evaluation system in 2015 to hear opinions from various stakeholders regarding medicine types to be reclassified. This study aimed to examine the new framework to identify candidate substances for reclassification. Moreover, we examined how to manage the safe, self-care use of OTC drugs in Japan. METHODS: The necessary regulatory information on OTC approvals as of January 2015 was collected using an Internet search and relevant databases. To highlight the characteristics of OTC drugs in Japan, the UK was selected as a comparison country because it too was actively promoting the reclassification of medicines from prescription to nonprescription status, and because of economic similarity. RESULTS: Japan and the UK have a risk-based classification for nonprescription medicines. Japan has made OTC drugs available with mandatory pharmacists' supervision, face-to-face with pharmacists, or online instruction, which is similar to the "pharmacy medicine" practiced in the UK. Japan recently reformed the reclassification process to involve physicians and the public in the process; some interactions were back to "prescription-only medicine" in the UK. CONCLUSION: It is expected that the opinion of marketers, medical professionals, and the public will improve the discussion that will greatly contribute to the safe use of drugs. Monitoring the new system will be noteworthy to ensure that OTC drug users are managing their self-care properly and visiting a doctor only when necessary. The supply methods are similar in Japan and the UK; however, the expected growth in the Japanese OTC market by the Cabinet and the industry is still uncertain.

Inhibition of neutrophil superoxide generation by shikonin is associated with suppression of cellular Ca(2+) fluxes.

Shikonin, an anti-inflammatory compound of "Shikon", inhibits the neutrophil superoxide (O2 (•-)) generation by NADPH oxidase 2 (Nox2); however, the mechanisms of how shikonin affects Nox2 activity remained unclear. We aimed to elucidate the relationship between the inhibition of Nox2 activity and influences on intracellular Ca(2+) concentration ([Ca(2+)]i) by shikonin. For this purpose, we used a simultaneous monitoring system for detecting changes in [Ca(2+)]i (by fluorescence) and O2 (•-) generation (by chemiluminescence) and evaluated the effects of shikonin on neutrophil-like HL-60 cells stimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Since fMLP activates Nox2 by elevation in [Ca(2+)]i via fluxes such as inositol 1,4,5-trisphosphate-induced Ca(2+) release (IICR) and store-operated Ca(2+) entry (SOCE), we also evaluated the effects of shikonin on IICR and SOCE. Shikonin dose-dependently inhibited the fMLP-induced elevation in [Ca(2+)]i and O2 (•-) generation (IC50 values of 1.45 and 1.12 µM, respectively) in a synchronized manner. Analyses of specific Ca(2+) fluxes showed that shikonin inhibits IICR and IICR-linked O2 (•-) generation (IC50 values: 0.28 and 0.31 µM for [Ca(2+)]i and O2 (•-), respectively), as well as SOCE and SOCE-linked O2 (•-) generation (IC50 values: 0.39 and 0.25 µM for [Ca(2+)]i and O2 (•-), respectively). These results suggested that shikonin inhibits the O2 (•-) generation by Nox2 in fMLP-stimulated neutrophils by targeting Ca(2+) fluxes such as IICR and SOCE.

Localizations of γ-Actins in Skin, Hair, Vibrissa, Arrector Pili Muscle and Other Hair Appendages of Developing Rats.

Six isoforms of actins encoded by different genes have been identified in mammals including α-cardiac, α-skeletal, α-smooth muscle (α-SMA), ß-cytoplasmic, γ-smooth muscle (γ-SMA), and γ-cytoplasmic actins (γ-CYA). In a previous study we showed the localization of α-SMA and other cytoskeletal proteins in the hairs and their appendages of developing rats (Morioka K., et al. (2011) Acta Histochem. Cytochem. 44, 141-153), and herein we determined the localization of γ type actins in the same tissues and organs by immunohistochemical staining. Our results indicate that the expression of γ-SMA and γ-CYA is suggested to be poor in actively proliferating tissues such as the basal layer of the epidermis and the hair matrix in the hair bulb, and as well as in highly keratinized tissues such as the hair cortex and hair cuticle. In contrast, the expression of γ-actins were high in the spinous layer, granular layer, hair shaft, and inner root sheath, during their active differentiations. In particular, the localization of γ-SMA was very similar to that of α-SMA. It was located not only in the arrector pili muscles and muscles in the dermis, but also in the dermal sheath and in a limited area of the outer root sheath in both the hair and vibrissal follicles. The γ-CYA was suggested to be co-localized with γ-SMA in the dermal sheath, outer root sheath, and arrector pili muscles. Sparsely distributed dermal cells expressed both types of γ-actin. The expression of γ-actins is suggested to undergo dynamic changes according to the proliferation and differentiation of the skin and hair-related cells.

Morphological Modifications in Myofibrils by Suppressing Tropomyosin 4α in Chicken Cardiac Myocytes.

Tropomyosin (TPM) localizes along F-actin and, together with troponin T (TnT) and other components, controls calcium-sensitive muscle contraction. The role of the TPM isoform (TPM4α) that is expressed in embryonic and adult cardiac muscle cells in chicken is poorly understood. To analyze the function of TPM4α in myofibrils, the effects of TPM4α-suppression were examined in embryonic cardiomyocytes by small interference RNA transfection. Localization of myofibril proteins such as TPM, actin, TnT, α-actinin, myosin and connectin was examined by immunofluorescence microscopy on day 5 when almost complete TPM4α-suppression occurred in culture. A unique large structure was detected, consisting of an actin aggregate bulging from the actin bundle, and many curved filaments projecting from the aggregate. TPM, TnT and actin were detected on the large structure, but myosin, connectin, α-actinin and obvious myofibril striations were undetectable. It is possible that TPM4α-suppressed actin filaments are sorted and excluded at the place of the large structure. This suggests that TPM4α-suppression significantly affects actin filament, and that TPM4α plays an important role in constructing and maintaining sarcomeres and myofibrils in cardiac muscle.

[Development of an Informative System for Consumers and Pharmacists Aimed at the Proper Use of Kampo OTCs].

Kampo medicines must be used according to an individual's physical characteristics and symptoms to avoid lack of efficacy, adverse reactions or interactions with other drugs. As category-2 over-the-counter drugs (OTCs), Kampo OTCs are not targets of active explanations by pharmacists, and consumers usually decide to use OTCs by themselves on the basis of drug label information. However, information on the label is occasionally brief. To promote the proper use of Kampo OTCs, we herein propose an informative tool based on the Australian Shelf-Talker (information on the proper use of OTCs with a self-check questionnaire for consumers), the Self-Check Card (SCC). We also prepared the informative material, Information to the Pharmacist (IP), directed at pharmacists regarding these Kampo OTCs. We created SCCs and IPs for 16 prescriptions in the 5 most demanded categories (cold, cough/sore throat, urination problems, women's diseases, and shoulder stiffness or joint/nerve pain). The SCC with questions in a simplified language specifies individuals who should avoid the drug, or those who should consult a pharmacist before purchase, according to the safety information on the respective drug labels. The IP provides information to pharmacists on safety issues and those concerning recognition of the consumer's symptoms and physical characteristics needed for the selection of appropriate Kampo OTCs. Such SCCs and IPs are now in use in 2 pharmacies to know pharmacists' suggestions about improvements and consumer's opinions and degree of satisfaction. We believe these risk-benefit communication tools, SCC and IP, will contribute to improve the proper use of Kampo OTCs.

Electron microscopic examination of podosomes induced by phorbol 12, 13 dibutyrate on the surface of A7r5 cells.

The role of myosin light chain kinase (MLCK) in inducing podosomes was examined by confocal and electron microscopy. Removal of myosin from the actin core of podosomes using blebbistatin, a myosin inhibitor, resulted in the formation of smaller podosomes. Downregulation of MLCK by the transfection of MLCK small interfering RNA (siRNA) led to the failure of podosome formation. However, ML-7, an inhibitor of the kinase activity of MLCK, failed to inhibit podosome formation. Based on our previous report (Thatcher et al. J.Pharm.Sci. 116 116-127, 2011), we outlined the important role of the actin-binding activity of MLCK in producing smaller podosomes.

Evaluation of radical scavenging properties of shikonin.

With the aim of developing effective anti-inflammatory drugs, we have been investigating the biochemical effects of shikonin of "Shikon" roots, which is a naphthoquinone with anti-inflammatory and antioxidative properties. Shikonin scavenged reactive oxygen species like hydroxyl radical, superoxide anion (O2 (•-)) and singlet oxygen in previous studies, but its reactivity with reactive oxygen species is not completely understood, and comparison with standard antioxidants is lacking. This study aimed elucidation of the reactivity of shikonin with nitric oxide radical and reactive oxygen species such as alkyl-oxy radical and O2 (•-). By using electron paramagnetic resonance spectrometry, shikonin was found unable of reacting with nitric oxide radical in a competition assay with oxyhemoglobin. However, shikonin scavenged alkyl-oxy radical from 2,2'-azobis(2-aminopropane) dihydrochloride with oxygen radical absorbance capacity, ORAC of 0.25 relative to Trolox, and showed a strong O2 (•-)-scavenging ability (42-fold of Trolox; estimated reaction rate constant: 1.7 × 10(5) M(-1)s(-1)) in electron paramagnetic resonance assays with CYPMPO as spin trap. Concerning another source of O2 (•-), the phagocyte NADPH oxidase (Nox2), shikonin inhibited the Nox2 activity by impairing catalysis when added before enzyme activation (IC50: 1.1 µM; NADPH oxidation assay). However, shikonin did not affect the preactivated Nox2 activity, although having potential to scavenge produced O2 (•-). In conclusion, shikonin scavenged O2 (•-) and alkyl-oxy radical, but not nitric oxide radical.

Down-regulation of myosin light chain kinase expression in vascular smooth muscle cells accelerates cell proliferation: requirement of its actin-binding domain for reversion to normal rates.

Myosin light-chain kinase (MLCK) is a multi-domain protein with kinase and actin-binding domains, among others. Deficiency of MLCK expression in GBaSM-4 vascular smooth muscle cells enhanced cell proliferation rate and shortened cell doubling time. Transient transfection of the MLCK-deficient cells with cDNA constructs of either wild-type MLCK or its mutant lacking the kinase activity reverted the cell proliferation rate to that of wild-type cells, whereas that of MLCK lacking the actin-binding domain maintained cell proliferation at an elevated rate similar to the MLCK-deficient cells. Thus, the actin-binding domain of MLCK seems to play a role in regulating cell proliferation.

Down-regulation of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), an endogenous allosteric alpha7 nicotinic acetylcholine receptor modulator, in murine and human asthmatic conditions.

Whereas acetylcholine (ACh) acts as a bronchoconstrictor and stimulator of mucus secretion from bronchial epithelium, it acts via alpha7 nicotinic Ach receptors (nAChRs) on macrophages in the airways to exert anti-inflammatory effects by reducing synthesis of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). Moreover, the effects of ACh are modified by secreted ly-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of alpha7 nAChR signaling. Our aim was to explore the roles played by SLURP-1 in the pathophysiology of asthma by assessing SLURP-1 expression in the OVA-sensitized murine asthma model and in cultured human bronchial epithelial cells. Using real-time PCR we found that expression of SLURP-1 mRNA is down-regulated in the lungs of asthmatic model mice, as compared to healthy mice. In addition, immunohistochemical studies confirmed the diminished expression of SLURP-1 in the bronchioles of asthmatic mice, and showed it was due to extensive metaplasia of mucus-secreting cells and the concomitant loss of ciliated epithelial cells. Expression of SLURP-1 mRNA and protein was also significantly down-regulated in human epithelial cells stimulated with the pro-inflammatory cytokine interleukin-13 (IL-13), which is related to asthmatic condition. Thus SLURP-1 appears to be down-regulated in both an animal model of asthma and human epithelial cells treated with an inflammatory cytokine related to asthma. Those findings suggest that diminished expression of SLURP-1 in asthma attenuates its negative regulation of airway inflammation, and that perhaps changes in SLURP-1 expression could serve as a marker of airway damage in asthma.

Shikonin directly inhibits nitric oxide synthases: possible targets that affect thoracic aorta relaxation response and nitric oxide release from RAW 264.7 macrophages.

Recently, an isomeric mixture of herbal anti-inflammatory naphthoquinones shikonin and alkannin, and their derivatives, have been found to impair cellular responses involving nitric oxide (NO) and NO synthesis, like the acetylcholine-induced relaxation response of rat thoracic aorta and NO release from murine RAW 264.7 macrophages. However, the mechanisms of such effects, including whether NO synthase (NOS) activity is affected, remained unclear. We herein investigate possible targets of shikonin in these NOS-related events. Shikonin by itself dose-dependently inhibited the rat thoracic aorta relaxation in response to acetylcholine (pD'(2) value: 6.29). Its optical enantiomer, alkannin, was equally inhibitory in the aorta relaxation-response assay. In RAW 264.7 cells, shikonin inhibited the lipopolysaccharide-induced NO production by 82% at 1 microM. A cell-free assay to verify direct effects on NOS activity showed that shikonin inhibits all isoforms of NOS (IC(50)s, 4 - 7 microM), suggesting NOS as an inhibition target in both the events. Further possible targets of shikonin that might be involved in the inhibitions of the acetylcholine-induced aorta relaxation response and the NO generation by RAW 264.7 cells are also discussed. It is shown for the first time that shikonin inhibits NOS activity.

Expression of SLURP-1, an endogenous alpha7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cells.

Mammalian secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) is a positive allosteric ligand for alpha7 nicotinic acetylcholine (ACh) receptors (alpha7 nAChRs) that potentiates responses to ACh and elicits proapoptotic activity in human keratinocytes. Mutations in the gene encoding SLURP-1 have been detected in patients with Mal de Meleda, a rare autosomal recessive skin disorder characterized by transgressive palmoplantar keratoderma. On the basis of these findings, SLURP-1 is postulated to be involved in regulating tumor necrosis factor-alpha (TNF-alpha) release from keratinocytes and macrophages via alpha7 nAChR-mediated pathways. In the present study, we assessed SLURP-1 expression in lung tissue from C57BL/6J mice to investigate the functions of SLURP-1 in pulmonary physiology and pathology. Immunohistochemical and in situ hybridization analyses revealed expression of SLURP-1 protein and mRNA, respectively, exclusively in ciliated bronchial epithelial cells. This was supported by Western blotting showing the presence of the 9.5-kDa SLURP-1 protein in whole-lung tissue and trachea. In addition, high-affinity choline transporter (CHT1) was detected in apical regions of bronchial epithelial cells and in neurons located in the lamina propria of the bronchus, suggesting that bronchial epithelial cells are able to synthesize both SLURP-1 and ACh. We also observed direct contact between F4/80-positive macrophages and bronchial epithelial cells and the presence of invading macrophages in close proximity to CHT1-positive nerve elements. Collectively, these results suggest that SLURP-1 contributes to the maintenance of bronchial epithelial cell homeostasis and to the regulation of TNF-alpha release from macrophages in bronchial tissue.

Suppression of cardiac troponin T induces reduction of contractility and structural disorganization in chicken cardiomyocytes.

We herein examine the effect of cardiac troponin T (CTnT) suppression in cultured chicken cardiomyocytes derived from embryonic cardiac ventricular muscle. TnT is an important protein participating in regulation of striated muscle contraction, but it is not clear whether TnT contributes to the formation of sarcomere structure in myofibrils. Double-stranded RNA homologous to the nucleotide sequence of CTnT (CTnT-siRNA) was introduced into cultured muscle cells two days after plating. Transfection efficiency was above 80%. Immunoblot analyses suggested that the expression of CTnT progressively falls for the three consecutive days after transfection, but partly reappears on the fourth day. Maximum suppression occurs three days after transfection, with almost invisible CTnT protein on immunoblots in all the examined conditions: 0.5-2 nmol CTnT-siRNA towards 1-3 x 10(6) cells. The suppression was specific to CTnT, and the other myofibrillar proteins such as myosin, connectin/titin, tropomyosin, alpha-actinin, and troponin I were all present in transfected cells. The following functional and morphological changes were detected in CTnT-suppressed cells. The population of beating cells decreased significantly after transfection, when compared to control cells. A part of CTnT-suppressed cells showed two non-overlapping types of morphological changes: 1) myofibrils presenting unusually long Z-Z intervals; 2) myofibrils with irregular small striations in cells not connected at their adhesion interfaces of a jagged-appearance. Thus, our results reveal that CTnT is important for stable beating in cultured ventricular muscle cells, and also to some extent, for maintaining myofibrillar structure and cell-to-cell adhesion.

Utilisation of human pharmacology studies with biomarkers for new drug applications in Japan.

OBJECTIVE: This study evaluated the utilisation of human pharmacology studies with biomarkers for either efficacy or safety estimation conducted for new drug applications (NDAs) submitted to the Japanese regulatory authority, the Ministry of Health, Labour and Welfare (MHLW). METHODS: A total of 50 new chemical entities (NCEs) posted on the Websites, which were approved from June 2000 to November 2001, were evaluated by investigating their approval information. The utilisation of human pharmacology studies with biomarkers was evaluated by focusing on the classification referred to biomarkers for either efficacy or safety estimation and timing of studies. RESULTS: The human pharmacology studies with biomarkers for either efficacy or safety estimation were conducted in 20 compounds classified by utilising measures of either efficacy (17 compounds) or safety (seven compounds). In 4 of 17 NCEs, some of the biomarkers in human pharmacology studies were similar to the clinical endpoints for efficacy assessment in therapeutic exploratory and/or therapeutic confirmatory studies. For safety assessment in therapeutic exploratory and/or therapeutic confirmatory studies, clinical endpoints rather than biomarkers in human pharmacology studies were used in all seven NCEs. The timing of each type of clinical study could only be obtained for 15 NCEs. Of these 15 NCEs, human pharmacology studies with biomarkers for either efficacy or safety estimation were conducted on six compounds. There were only two compounds for which human pharmacology studies with biomarkers for efficacy estimation were conducted before pivotal studies such as a therapeutic exploratory study or a bridging study. CONCLUSION: Our survey suggests that with Japanese NDAs, human pharmacology studies with biomarkers for either efficacy or safety estimation do not play a key role in accelerating drug development and maximising the knowledge gained from confirmatory trials. The relationship between a biomarker and a clinical endpoint should be investigated appropriately for accelerating drug development. We think that the utilisation of human pharmacology studies with biomarkers for either efficacy or safety estimation in the regulatory review process for NDAs should be encouraged with the advancements of drug evaluation research using an appropriate biomarker based on clinical pharmacology.