Toward global standardization of conducting fair investigations of allegations of research misconduct.

In the United States, through nation-wide discussions, the procedures for handling allegations of research misconduct are now well established. Procedures are geared toward carefully treating both complainants and respondents fairly in accordance with the US framework. Other countries, which have their own cultural and legal framework, also need fair and legally compatible procedures for conducting investigations of allegations of research misconduct. Given the rapid growth of international collaboration in research, it is desirable to have a global standard, or common ground, for misconduct investigations. Institutions need clear guidance on important subjects such as what information should be included in the investigation reports, how the investigation committee should be organized once research misconduct allegation has been received, how to conduct the investigation, how the data and information obtained should be taken as evidence for vs. against misconduct, and what policies the investigation committee should follow. We explore these issues from the viewpoint of members of committees investigating accusations of research misconduct (hereafter referred to as "investigation committees") as well as persons overseeing the committees in Japan. We hope to engender productive discussions among experts in misconduct investigations, leading to a formulation of international standards for such investigation.

Physiological essay on Gulliver's Travels: a correction after three centuries.

Gulliver's Travels by Jonathan Swift, published in 1726, was analyzed from the viewpoint of scaling in comparative physiology. According to the original text, the foods of 1724 Lilliputians, tiny human creatures, are needed for Gulliver, but the author found that those of 42 Lilliputians and of 1/42 Brobdingnagians (gigantic human creatures) are enough to support the energy of Gulliver. The author further estimated their heartbeats, respiration rates, life spans and blood pressure. These calculations were made by the use of three equations, i.e., body mass index (BMI = W/H2) and quarter-power laws (E∝W3/4 and T∝W1/4), where W, H, E, and T denote body weight, height, energy and time, respectively. Their blood pressures were estimated with reference to that of the giraffe and barosaurus, a long-neck dinosaur. Based on the above findings, the food requirement of Gulliver in the original text should be corrected after almost three centuries.

New Classification of Research Misconduct from the Viewpoint of Truth, Trust, and Risk.

Fabrication, Falsification and Plagiarism (FFP) and Questionable Research Practice (QRP) have been used worldwide in the classification of research misconduct. However, FFP comprises two distinct categories of misconduct: FF is extreme research misconduct that betrays truth, while P undermines trust of science community. Irreproducibility and inadequate practice of research also betray trust. Research misconduct has the potential to cause serious risk of safety in daily life. The proposed classification system is outlined as follows: Class I misconduct: Betrayal of the truth: (1) Fabrication and (2) Falsification. Class II misconduct: Betrayal of trust: (1) Plagiarism of text ; Irreproducibility; and (3) Inadequate research practice. Class III misconduct: Risk to safety of health and industrial products: (1) Risk to safety of health and (2) Risk to safety of industrial products. The proposed classification reflects deeper values of truth, trust, and risk more directly than the previous classification and elucidates issues about nature and significance of misconduct.

Repeating probability of authors with retracted scientific publications.

Both the scientific community and the general public have expressed concern over scientific misconduct. The number of retracted articles has increased dramatically over the past 20 years and now comprises about .02% of the 2 million articles published each year. Retraction of publications available in large public databases can be analyzed as an objective measure for scientific misconduct and errors. In this project, we analyzed retractions of scientific publications using the Web of Science (WoS) and PubMed databases. We found that a power law is applicable to distributions of retracting authors and retracted publications with exponents of about -.6 and -3.0, respectively. Application of a power-law model for retracted publications implies that retraction is not a random event. Analysis of the retraction distributions suggests that a small fraction (1-2%) of retracting authors with ≧5 retractions are responsible for around 10% of retraction. The probabilities for their repeating retraction are calculated using a statistical model: 3-5% likelihood of repeat retraction for authors with a single retraction at five years after the latest retraction and 26-37% for authors with five retractions at five years after the latest retraction. By focusing on those with repeated retractions, this analysis could contribute to identification of measures to reduce such repetition of retractions.

Protein kinase C delta and eta differently regulate the expression of loricrin and Jun family proteins in human keratinocytes.

Barrier function of the epidermis is maintained by precise expression of keratinocyte-specific structural proteins to form the cornified cell envelope (CE). Loricrin, a major component of the CE, is expressed at the late stage of keratinocyte differentiation. In this study, we reveal the isoform-specific function of protein kinase C (PKC) in the regulation of loricrin expression. Both PKCdelta and PKCeta have been recognized as differentiation-promoting isoforms. However, loricrin expression was inversely controlled by PKCdelta and PKCeta in cultured keratinocytes and 3D skin culture; i.e. loricrin expression was decreased by PKCdelta and increased by PKCeta. To clarify the mechanisms that PKCdelta and PKCeta oppositely regulate the loricrin expression, we examined the expression of activator protein-1 (AP-1) family proteins, which modulate the transcription of loricrin and are downstream molecules of PKC. PKCdelta decreased c-Jun expression, whereas PKCeta increased JunD, which are positive regulators of loricrin transcription. These findings suggest that inverse effects of PKCdelta and PKCeta on loricrin expression attributes to the expression of c-Jun and JunD.

Discovery of novel motilin antagonists: Conversion of tetrapeptide leads to orally available peptidomimetics.

We successfully discovered peptidomimetic motilin antagonists (17c and 17d) through the improvement of physicochemical properties of a tetrapeptide antagonist (2). Furthermore, with oral administration and based on motilin antagonistic activity, both compounds suppressed motilin-induced colonic and gastric motility in conscious dogs.

UV irradiation increases ROS production via PKCdelta signaling in primary murine fibroblasts.

Ultraviolet (UV) irradiation is a major environmental factor responsible for a high incidence of premature skin aging, referred to as photoaging, as well as skin cancer and melanoma. UVA irradiation represents 90% of the solar UV light reaching the earth's surface, and yet the mechanisms by which it exerts its biological effects are not clear. UVA penetrates into the skin tissue, reaching the basal layers of the active dividing cells and, therefore, the contribution of UVA to skin damage may be significant. The majority of UVA energy is absorbed by unidentified photosensitizers in the cells which are postulated to generate reactive oxygen species (ROS). It has been believed that both chronological aging and photoaging share the same molecular features and, as such, it is very common to utilize UV irradiation for induction of skin aging. To determine the involvement of protein kinase isoforms in chronological aging and photoaging, we utilized in vitro aging model systems of primary murine fibroblasts and primary fibroblasts isolated from PKC null mice. We show for the first time distinct involvement of PKC isoforms PKCdelta and PKCalpha in photoaging versus cellular senescence. While chronological aging is accompanied by overexpression and activation of PKCalpha, UV irradiation and ROS production are associated with photoaging accompanied by PKCdelta downregulation and nuclear translocation.

Mutant epidermal growth factor receptor undergoes less protein degradation due to diminished binding to c-Cbl.

Gefitinib (Iressa) sensitivity in non-small cell lung cancer (NSCLC) is associated with activating mutations in epidermal growth factor receptor (EGFR). It was reported that autophosphorylation of the mutant EGFR is prolonged compared with wild-type EGFR. To explore the mechanism of sustained autophosphorylation, the mutant and wild-type EGFR degradation activities were examined in NSCLC cell lines. EGFR degradation activity was measured by 125I-EGF. The degradation rate of EGFR was lower in the PC-9 NSCLC cell line, which expressed 15-bp deletion mutant EGFR, compared with that in the PC-14 NSCLC (wild-type EGFR). To clarify the mechanism, the stable transfected cell lines, 293_pEGFR and 293_pdelta15, expressing wild-type and mutant EGFR, respectively, were used. In 293_pdelta15, EGFR degradation and binding of c-Cbl ubiquitin ligase to this receptor were reduced compared with 293_pEGFR. Based on these results, we conclude that the mutant EGFR underwent less protein degradation due to diminished binding to c-Cbl.

Effectiveness of cervical cancer screening over cervical cancer mortality among Japanese women.

BACKGROUND: Various studies have revealed that cervical cancer (CC) screening significantly reduces both CC incidence and mortality in developed countries. Although Japan introduced a nationwide government funded annual CC screening for the women aged 30+ in 1982, the effectiveness of CC screening on CC mortality has not yet been evaluated by any prospective cohort study. Therefore, the present study evaluated the association of CC mortality with self-reported CC screening and some other factors by a nationwide cohort study. METHODS: Baseline survey of the Japan Collaborative Cohort Study for the enrollment of subjects was completed during 1988-90 and followed until 2003. This study only analyzed 63,541 women, aged 30-79 years, who were free from any cancer history at enrollment. RESULTS: During the follow-up period, 38 CC deaths were identified. The mean age at mortality was 67.0 years, with a mortality rate of 4.2 per 100,000 person-years. Participation rate in CC screening was 46.9%. Age-adjusted Cox model indicated significantly lower CC mortality [hazard ratio (HR) = 0.30, 95% confidence interval (CI) = 0.12-0.74] due to CC screening. Protectiveness remained almost the same (HR = 0.30, 95% CI = 0.12-0.76) when adjusted for age, body mass index and number of deliveries. The results also revealed that CC screening could reduce at least 50% of CC deaths even after excluding the effect of possible self-selection bias. CONCLUSIONS: CC screening in Japan may reduce CC mortality significantly for women aged 30-79 years. However, further studies with more CC deaths and increased statistical power are needed to validate the findings.

The role of protein kinase C delta activation and STAT3 Ser727 phosphorylation in insulin-induced keratinocyte proliferation.

Activation of the STAT family of transcription factors is regulated by cytokines and growth factors. STAT tyrosine and serine phosphorylation are linked to the transcriptional activation and function of STAT. We have previously described a unique pathway inducing keratinocyte proliferation, which is mediated by insulin stimulation and depends on protein kinase C delta (PKCdelta). In this study, we assessed STAT3 activation downstream of this pathway and characterized the role of PKCdelta activation in STAT3 tyrosine and serine phosphorylation and keratinocyte proliferation. Following insulin stimulation, STAT3 interacted with PKCdelta but not with any other PKC isoform expressed in skin. Activated forms of PKCdelta and STAT3 were essential for insulin-induced PKCdelta-STAT3 activation in keratinocyte proliferation. Abrogation of PKCdelta activity inhibited insulin-induced STAT3 phosphorylation, PKCdelta-STAT3 association and nuclear translocation. In addition, overexpression of STAT3 tyrosine mutant eliminated insulin-induced PKCdelta activation and keratinocyte proliferation. Finally, overexpression of a STAT3 serine mutant abrogated insulin-induced STAT3 serine phosphorylation and STAT3-induced keratinocyte proliferation, whereas STAT3 tyrosine phosphorylation was induced and nuclear localization remained intact. This study indicates that PKCdelta activation is a primary regulator of STAT3 serine phosphorylation and that PKCdelta is essential in directing insulin-induced signaling in keratinocyte proliferation.

Enhancement of sensitivity to tumor necrosis factor alpha in non-small cell lung cancer cells with acquired resistance to gefitinib.

Tumor cells that have acquired resistance to gefitinib through continuous drug administration may complicate future treatment. To investigate the mechanisms of acquired resistance, we established PC-9/ZD2001, a non-small-cell lung cancer cell line resistant to gefitinib, by continuous exposure of the parental cell line PC-9 to gefitinib. After 6 months of culture in gefitinib-free conditions, PC-9/ZD2001 cells reacquired sensitivity to gefitinib and were established as a revertant cell line, PC-9/ZD2001R. PC-9/ZD2001 cells showed collateral sensitivity to several anticancer drugs (vinorelbine, paclitaxel, camptothecin, and 5-fluorouracil) and to tumor necrosis factor alpha (TNF-alpha). Compared with PC-9 cells, PC-9/ZD2001 cells were 67-fold more sensitive to TNF-alpha and PC-9/ZD2001R cells were 1.3-fold more sensitive. Therefore, collateral sensitivity to TNF-alpha was correlated with gefitinib resistance. PC-9/ZD2001 cells expressed a lower level of epidermal growth factor receptor (EGFR) than did PC-9 cells; this down-regulation was partially reversed in PC-9/ZD2001R cells. TNF-alpha-induced autophosphorylation of EGFR (cross-talk signaling) was detected in all three cell lines. However, TNF-alpha-induced Akt phosphorylation and IkappaB degradation were observed much less often in PC-9/ZD2001 cells than in PC-9 cells or PC-9/ZD2001R cells. Expression of the inhibitor of apoptosis proteins c-IAP1 and c-IAP2 was induced by TNF-alpha in PC-9 and PC-9/ZD2001R cells but not in PC-9/ZD2001 cells. This weak effect of EGFR on Akt pathway might contribute to the TNF-alpha sensitivity of PC-9/ZD2001 cells. These results suggest that therapy with TNF-alpha would be effective in some cases of non-small-cell lung cancer that have acquired resistance to gefitinib.

The testicular fatty acid binding protein PERF15 regulates the fate of germ cells in PERF15 transgenic mice.

The quality control of sperm is critical for efficient reproduction. In germ cells, cell death involves different processes to those in somatic cells, and in many cases, the trigger to induce cell death in deficient germ cells is still unclear. It is known that the fatty acid composition of sperm is related to fertility. Composition of the fatty acid of germ cells changes dynamically during spermatogenesis, and fatty acid binding protein (FABP) may be involved in these changes. In this study, we developed transgenic mice with a testicular germ-cell-specific FABP (PERF15) transgene, whose expression was controlled by the Cre-LoxP site-specific recombination system. We also developed transgenic mice with the Cre gene under the control of the spermatocyte specific Pgk2 promoter. In double transgenic mice, following Cre-mediated recombination of the PERF15 containing transgene, PERF15 was strongly overexpressed. Its overexpression induced multinucleate symplasts to form, indicating programmed germ cell death occurred at the elongated spermatid stage. As a result, sperm harboring the transgene were significantly decreased, but the surviving sperm demonstrated higher fertility than natural sperm. Therefore, we conclude that PERF15 associate with the direction of germ cell fates and preserve the quality of sperm.

Protein kinase Calpha is implicated in cholecystokinin-induced activation of 70-kd S6 kinase in AR42J cells.

OBJECTIVES: We showed previously that cholecystokinin (CCK)-induced 70-kd S6 kinase activation is partly mediated by protein kinase C (PKC) in pancreatic acinar AR42J cells. Here, we examined which isoform of PKC is involved in this process. METHODS: AR42J cells were infected with adenovirus vectors carrying the kinase-deficient alpha, delta, and epsilon isoforms of PKC, the dominant-negative form of the 85-kd regulatory subunit of phosphatidylinositol (PI) 3-kinase, and the dominant-negative form of Sos. CCK-induced p70 S6 kinase activation was determined in AR42J cells infected with these adenovirus vectors. RESULTS: CCK-induced p70 S6 kinase activity was significantly reduced in cells overexpressing the dominant-negative p85 subunit of PI 3-kinase but not in cells overexpressing dominant-negative Sos or beta-galactosidase. CCK-induced p70 S6 kinase activity was inhibited in parallel with the expression levels of kinase-deficient PKCalpha, whereas it was unaffected by the expression of kinase-deficient PKCdelta or PKCepsilon. CONCLUSION: PKCalpha is implicated in CCK-induced activation of p70 S6 kinase in AR42J cells.

PKC-delta-dependent activation of oxidative stress in adipocytes of obese and insulin-resistant mice: role for NADPH oxidase.

Oxidative stress is thought to be one of the causative factors contributing to insulin resistance and type 2 diabetes. Previously, we showed that reactive oxygen species (ROS) production is significantly increased in adipocytes from high-fat diet-induced obese and insulin-resistant mice (HF). ROS production was also associated with the increased activity of PKC-delta. In the present studies, we hypothesized that PKC-delta contributes to ROS generation and determined their intracellular source. NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) reduced ROS levels by 50% in HF adipocytes, and inhibitors of NO synthase (L-NAME, 1 mM), xanthine oxidase (allopurinol, 100 microM), AGE formation (aminoguanidine, 10 microM), or the mitochondrial uncoupler (FCCP, 10 microM) had no effect. Rottlerin, a selective PKC-delta inhibitor, suppressed ROS levels by approximately 50%. However, neither GO-6976 nor LY-333531, effective inhibitors toward conventional PKC or PKC-beta, respectively, significantly altered ROS levels in HF adipocytes. Subsequently, adenoviral-mediated expression of wild-type PKC-delta or its dominant negative mutant (DN-PKC-delta) in HF adipocytes resulted in either a twofold increase in ROS levels or their suppression by 20%, respectively. In addition, both ROS levels and PKC-delta activity were sharply reduced by glucose depletion. Taken together, these results suggest that PKC-delta is responsible for elevated intracellular ROS production in HF adipocytes, and this is mediated by high glucose and NADPH oxidase.

Transcriptional and post-transcriptional regulation of the PKC delta gene by etoposide in L1210 murine leukemia cells: implication of PKC delta autoregulation.

Protein kinase C delta (PKC delta) plays an important role in the regulation of apoptosis in response to diverse anticancer agents. PKC delta is cleaved irreversibly to a catalytically active fragment in response to apoptotic stimuli; however, little information is available about the regulation of PKC delta gene expression. In this study, we found that the amount of steady-state PKC delta mRNA and protein was increased by etoposide in mouse L1210 leukemia cells. The transcriptional rate of the PKC delta gene and the stability of PKC delta mRNA were increased by treatment with etoposide, resulting in the accumulation of PKC delta protein. Rottlerin inhibited etoposide-induced PKC delta gene expression significantly, while Go6976, LY294002 and PD98059 had no effect. Further, both stable and adenovirus-mediated expression of a dominant negative PKC delta(KR) abrogated etoposide-induced PKC delta expression. Etoposide-stimulated PKC delta transcription but not PKC delta mRNA stability was blocked completely by pretreatment with rottlerin. Our data reveal a novel mechanism whereby PKC delta gene is regulated at the transcriptional and post-transcriptional level in the L1210 leukemia cell line.

PKCalpha mediates TGFbeta-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11.

Growth regulation of epithelial cells is of major concern because most human cancers arise from them. We demonstrated previously a novel signal pathway involving S100C/A11 for high Ca2+-induced growth inhibition of normal human keratinocytes (Sakaguchi, M., M. Miyazaki, M. Takaishi, Y. Sakaguchi, E. Makino, N. Kataoka, H. Yamada, M. Namba, and N.H. Huh. 2003. J. Cell Biol. 163:825-835). This paper addresses a question whether transforming growth factor beta (TGFbeta) shares the pathway with high Ca2+. On exposure of the cells to TGFbeta1, S100C/A11 was phosphorylated, bound to nucleolin, and transferred to the nucleus, resulting in induction of p21WAF1/CIP1 and p15INK4B through activation of Sp1. Protein kinase C alpha (PKCalpha) was shown to phosphorylate 10Thr of S100C/A11, which is a critical event for the signal transduction. The TGFbeta1-induced growth inhibition was almost completely mitigated when PKCalpha activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCalpha-S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFbeta1.

Differential regulation of alpha6beta4 integrin by PKC isoforms in murine skin keratinocytes.

In mammalian epidermis, alpha6beta4 integrin is expressed exclusively on the basal layer localized to the hemidesmosomes, where it interacts extracellularly with the laminin-5 ligand. During differentiation, loss of alpha6beta4 is associated with keratinocyte detachment from the basement membrane and upward migration. The protein kinase C (PKC) family of isoforms participates in regulation of integrin function and is linked to skin differentiation. Exposure of primary murine keratinocytes to PKC activators specifically downregulates alpha6beta4 expression. Utilizing recombinant adenoviruses, we selectively overexpressed skin PKC isoforms in primary keratinocytes. PKCdelta and PKCzeta induced downregulation of alpha6beta4 protein expression, leading to reduced keratinocyte attachment to laminin-5 and enhanced gradual detachment from the underlying matrix. In contrast, PKCalpha upregulated alpha6beta4 protein expression, leading to increased keratinocyte attachment to laminin-5 and to the underlying matrix. Altogether, these results suggest distinct roles for specific PKC isoforms in alpha6beta4 functional regulation during the early stages of skin differentiation.

PKCdelta inhibits PKCalpha-mediated activation of phospholipase D1 in a manner independent of its protein kinase activity.

The regulation of phospholipase D1 (PLD1) by protein kinase C (PKC) isoforms was analyzed in human melanoma cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced PLD1 activation was suppressed by the introduction of PKCdelta as well as its kinase-negative mutant in MeWo cells, which contain PKCalpha but lack PKCbeta. PLD activity was not affected by PKCdelta in G361 cells, which have PKCbeta but are deficient in PKCalpha. In MeWo cells introduced by PKCalpha and PLD1, the association of these proteins was observed, which was enhanced by the TPA treatment. In cells overexpressing PKCdelta in addition to PKCalpha and PLD1, TPA treatment increased the association of PKCdelta and PLD1, while it attenuated the association of PKCalpha and PLD1. These results indicate that PKCdelta inhibits TPA-induced PLD1 activation mediated by PKCalpha through the association with PLD1.