Author Correction: A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins.

It is critical for development of high-quality antibodies in research and diagnostics to predict accurately their cross-reactivities with "off-target" molecules, which potentially induce false results. Herein, we report a good example of such a cross-reactivity for an off-target due to a stereochemical environment of epitopes, which does not simply depend on amino acid sequences. We found that significant subpopulation of a polyclonal peptide antibody against Bcnt (Bucentaur) (anti-BCNT-C antibody) cross-reacted with a completely different protein, glutamine synthetase (GS), and identified four amino acids, GYFE, in its C-terminal region as the core amino acids for the cross-reaction. Consistent with this finding, the anti-BCNT-C antibody strongly recognized endogenously and exogenously expressed GS in tissues and cultured cells by Western blotting and immunohistochemistry. Furthermore, we elucidated that the cross-reaction is caused by a spatial similarity between the stereochemical environments formed by amino acid residues, including the GYFE of GS and the GYIE of Bcnt, rather than by their primary sequences. These results suggest it is critical to comprehensively analyze antibody interactions with target molecules including off-targets with special attention to the physicochemical environments of epitope-paratope interfaces to decrease the risk of false interpretations of results using antibodies in science and clinical applications.

KLF15 Enables Rapid Switching between Lipogenesis and Gluconeogenesis during Fasting.

Hepatic lipogenesis is nutritionally regulated (i.e., downregulated during fasting and upregulated during the postprandial state) as an adaptation to the nutritional environment. While alterations in the expression level of the transcription factor SREBP-1c are known to be critical for nutritionally regulated lipogenesis, upstream mechanisms governing Srebf1 expression remain unclear. Here, we show that the fasting-induced transcription factor KLF15, a key regulator of gluconeogenesis, forms a complex with LXR/RXR, specifically on the Srebf1 promoter. This complex recruits the corepressor RIP140 instead of the coactivator SRC1, resulting in reduced Srebf1 and thus downstream lipogenic enzyme expression during the early and euglycemic period of fasting prior to hypoglycemia and PKA activation. Through this mechanism, KLF15 overexpression specifically ameliorates hypertriglyceridemia without affecting LXR-mediated cholesterol metabolism. These findings reveal a key molecular link between glucose and lipid metabolism and have therapeutic implications for the treatment of hyperlipidemia.

Hepatic CREB3L3 controls whole-body energy homeostasis and improves obesity and diabetes.

Transcriptional regulation of metabolic genes in the liver is the key to maintaining systemic energy homeostasis during starvation. The membrane-bound transcription factor cAMP-responsive element-binding protein 3-like 3 (CREB3L3) has been reported to be activated during fasting and to regulate triglyceride metabolism. Here, we show that CREB3L3 confers a wide spectrum of metabolic responses to starvation in vivo. Adenoviral and transgenic overexpression of nuclear CREB3L3 induced systemic lipolysis, hepatic ketogenesis, and insulin sensitivity with increased energy expenditure, leading to marked reduction in body weight, plasma lipid levels, and glucose levels. CREB3L3 overexpression activated gene expression levels and plasma levels of antidiabetic hormones, including fibroblast growth factor 21 and IGF-binding protein 2. Amelioration of diabetes by hepatic activation of CREB3L3 was also observed in several types of diabetic obese mice. Nuclear CREB3L3 mutually activates the peroxisome proliferator-activated receptor (PPAR) α promoter in an autoloop fashion and is crucial for the ligand transactivation of PPARα by interacting with its transcriptional regulator, peroxisome proliferator-activated receptor gamma coactivator-1α. CREB3L3 directly and indirectly controls fibroblast growth factor 21 expression and its plasma level, which contributes at least partially to the catabolic effects of CREB3L3 on systemic energy homeostasis in the entire body. Therefore, CREB3L3 is a therapeutic target for obesity and diabetes.

TFE3 controls lipid metabolism in adipose tissue of male mice by suppressing lipolysis and thermogenesis.

Transcription factor E3 (TFE3) is a transcription factor that binds to E-box motifs and promotes energy metabolism-related genes. We previously reported that TFE3 directly binds to the insulin receptor substrate-2 promoter in the liver, resulting in increased insulin response. However, the role of TFE3 in other tissues remains unclear. In this study, we generated adipose-specific TFE3 transgenic (aP2-TFE3 Tg) mice. These mice had a higher weight of white adipose tissue (WAT) and brown adipose tissue than wild-type (WT) mice under fasting conditions. Lipase activity in the WAT in these mice was lower than that in the WT mice. The mRNA level of adipose triglyceride lipase (ATGL), the rate-limiting enzyme for adipocyte lipolysis, was significantly decreased in aP2-TFE3 Tg mice. The expression of Foxo1, which directly activates ATGL expression, was also suppressed in transgenic mice. Promoter analysis confirmed that TFE3 suppressed promoter activities of the ATGL gene. In contrast, G0S2 and Perilipin1, which attenuate ATGL activity, were higher in transgenic mice than in WT mice. These results indicated that the decrease in lipase activity in adipose tissues was due to a decrease in ATGL expression and suppression of ATGL activity. We also showed that thermogenesis was suppressed in aP2-TFE3 Tg mice. The decrease in lipolysis in WAT of aP2-TFE3 Tg mice inhibited the supply of fatty acids to brown adipose tissue, resulting in the inhibition of the expression of thermogenesis-related genes such as UCP1. Our data provide new evidence that TFE3 regulates lipid metabolism by controlling the gene expression related to lipolysis and thermogenesis in adipose tissue.

Tickling stimulation causes the up-regulation of the kallikrein family in the submandibular gland of the rat.

We recently showed that tactile stimulation (tickling) accompanied by positive emotion altered the expression of many genes in the rat hypothalamus (Hori et al., 2009 [15]). In this study, the effect of repeated tickling on gene expressions of the rat salivary gland was examined. After 4-week stimulation, several genes of the kallikrein (Klk) family were remarkably up-regulated and the alpha-amylase (amylase) gene was down-regulated in DNA microarray analysis. In quantitative analysis using real-time PCR of the submandibular gland of the rats tickled for 2 weeks, mRNAs of Klk1, Klk2 (Klk1c2, Tonin), Klk7 (Klk1l), Klk1b3 (Nerve growth factor, gamma), Klk1c10, Klks3 (Klk1c9) and GK11 were significantly 2-5-fold increased among 18 members of the Klk gene family examined and the submandibular amylase was decreased compared with the lightly touched and untouched control rats. In immunoblot analysis the increase in Klk7 protein was observed in the whole cell lysate fraction of the submandibular gland. In immunohistochemical analysis with anti-Klk7 polyclonal antibody, the immunostain was increased in duct cells of the submandibular gland of the tickled rat when compared with the lightly touched and untouched control rats. These results suggest that tactile sensory processing in the central nervous system affects the gene expression in the peripheral tissue probably via hormonal and/or autonomic neural activities. Submandibular Klks may be biochemical markers indicating positive emotional states.

Dicer has a crucial role in the early stage of adipocyte differentiation, but not in lipid synthesis, in 3T3-L1 cells.

Dicer is a rate-limiting enzyme for microRNA (miRNA) synthesis. To determine the effects of Dicer on adipogenesis, we performed stage-specific knockdown of Dicer using adenovirus encoding short-hairpin RNAi against Dicer in 3T3-L1 cells. When cells were infected with the adenovirus before induction of adipocyte differentiation, Dicer RNAi suppressed the gene expression of inducers of adipocyte differentiation such as PPARγ, C/EBPα, and FAS in 3T3-L1 cells during adipocyte differentiation. Concurrently, both adipocyte differentiation and cellular lipid accumulation were cancelled by Dicer RNAi when compared with control RNAi. Meanwhile, we addressed the roles of Dicer in lipid synthesis and accumulation in the final stages of differentiation. When the differentiated cells at day 4 after induction of differentiation were infected with adenovirus Dicer RNAi, cellular lipid accumulation was unchanged. Consistent with this, Dicer RNAi had no effects on the expression of genes related to cellular lipid accumulation, including PPARγ and FAS. Thus, Dicer controls proadipogenic genes such as C/EBPα and PPARγ in the early, but not in the late, stage of adipogenesis via regulation of miRNA synthesis.

Neurogenesis in the dentate gyrus of the rat hippocampus enhanced by tickling stimulation with positive emotion.

Hippocampal neurogenesis is influenced by many factors. In this study, we examined the effect of tactile stimulation (tickling), which induced positive emotion, on neurogenesis in the dentate gyrus (DG) of the hippocampus. Four week-old rats were tickled for 5 min/day on 5 consecutive days and received 5-bromo-2'-deoxyuridine (BrdU) administration for 4 days from the second tickling day. Then they were allowed to survive for 18 h or 3 weeks after the end of BrdU treatment. Neurogenesis in the DG was compared between the tickled and untickled rats by using immunohistochemistry with anti-BrdU antibody. The result showed that the number of BrdU- and NeuN (neural cell marker)-double positive neurons on 18h as well as 3 weeks of the survival periods was significantly increased in the tickled group as compared with the untickled group. The expression of mRNA of brain-derived neurotrophic factor (BDNF) in the hippocampus of the tickled rats was not altered when compared with the control rats. In conclusion, tickling stimulation which induces positive emotion may affect the generation and survival of new neurons of the DG through the BDNF-independent pathway.

The up-regulation of microRNA-335 is associated with lipid metabolism in liver and white adipose tissue of genetically obese mice.

MicroRNAs (miRNAs) are short non-coding RNA that post-transcriptionally regulates gene expression. Some miRNAs have been proposed to be associated with obesity. However, miRNAs, which are related to the development of obesity in vivo remains unknown. Here in, we found the up-regulation of miR-335 in obesity using microarray analysis for miRNA. The expressions of miR-335 in liver and white adipose tissue (WAT) were up-regulated in obese mice including ob/ob, db/db, and KKAy mice. Increased miR-335 expressions were associated with an elevated body, liver and WAT weight, and hepatic triglyceride and cholesterol. Furthermore, miR-335 levels were closely correlated with expression levels of adipocyte differentiation markers such as PPARgamma, aP2, and FAS in 3T3-L1 adipocyte. These findings provide the first evidence that the up-regulated expressions of miR-335 in liver and WAT of obese mice might contribute to the pathophysiology of obesity.

Positive emotion-specific changes in the gene expression profile of tickled rats.

The aim of this study was to investigate changes in gene expression after tactile stimulation (tickling) accompanied by positive emotion in the adolescent rat brain. We observed a positive emotional response (50-kHz ultrasonic vocalizations) after tickling using a modified version of the Panksepp method, and then comprehensively compared gene expression levels in the hypothalamus of the tickled rats and control rats using the microarray technique. After 4 weeks of stimulation, the expression levels of 321 of the 41,012 genes (including transcripts) were changed; 136 genes were up-regulated (>1.5-fold) and 185 were down-regulated (>0.67-fold) in the tickled rat group. Upon ontology analysis, the up-regulated genes were assigned to the following Gene Ontology (GO) terms: feeding behavior, neuropeptide signaling pathway, biogenic amine biosynthesis and catecholamine biosynthesis. Down-regulated genes were not assigned to any GO term categorized as a biological process. In conclusion, repeated tickling stimulation with positive emotion affected neuronal circuitry directly and/or indirectly, and altered the expression of genes related to the regulation of feeding in the adolescent rat hypothalamus.

Laughter modulates prorenin receptor gene expression in patients with type 2 diabetes.

OBJECTIVE: The purpose of this study was to assess whether laughter influences the expression of the receptor gene for prorenin that participates in the progression of diabetic nephropathy. METHODS: Sixteen normal subjects and 23 patients with type 2 diabetes [12 nephropathy (-) and 11 nephropathy (+)] were recruited to examine the effects of laughter on the modulation of prorenin receptor gene expression. After watching a comedy show, laughter-induced changes in the levels of blood prorenin and the expression of prorenin receptor gene were analyzed by an antibody-activating direct enzyme kinetic assay and by reverse transcriptase polymerase chain reaction, respectively. RESULTS: In diabetic patients, laughter decreased the level of blood prorenin [93.4-60.4 ng/l in nephropathy (-) patients, 196.6-166.7 ng/l in nephropathy (+) patients] and up-regulated the prorenin receptor gene [1.49-fold in nephropathy (-) patients, 1.46-fold in nephropathy (+) patients]. No significant changes in the expression of this gene were recognized in normal subjects. CONCLUSION: The beneficial effects of laughter on preventing the exacerbation of diabetic nephropathy are strongly suggested in terms of normalizing the expression of the prorenin receptor gene followed by reducing the level of blood prorenin.

[Investigation on the affinity between questions in the national examination for medical technologists and student level of education in the course of laboratory medicine--nine years history of the committee evaluating appropriate questions].

In order to evaluate the each question in the National Examination for Medical Technologist by comparison with the educated level in the course of laboratory medicine and the practical level of medical technologists, the investigation has been carried out by our committee established in the National University Association for Education of Laboratory Medicine during 9 years since 1997. The committee has asked each school of the 20 members of the Association to pick up good and/or inappropriate questions with the reasons why they are classified as good or improper ones. Some questions were considered as good ones by a large number of schools, while the others were considered improper. The reasons for the judgment have been sometimes very controversial and opinions uncommonly run counter to each other. It is suggested that a good question may become to be improper when the questionnaire is carelessly arranged even if the concept of the question is initially well considered. It is presumed that the annual reports of our committee may have played a role to gain common recognition that the numbers of the inappropriate questions have been decreasing in the recent National Examination for Medical Technologist.

RANTES production from mononuclear cells in response to the specific allergen in asthma patients.

BACKGROUND: Eosinophils are considered to be the major inflammatory cells in asthma. Since regulated on activation, normal T expressed and secreted (RANTES) is a potent chemoattractant for various important inflammatory cells such as eosinophils as well as memory T cells potentially recruiting these cells to an inflamed focus, RANTES has been considered to play a key role in various allergic disorders such as asthma. METHODS: To extend our understanding of the participation of eosinophils and T cells in relation to the production of RANTES in response to the specific allergen in asthma, we examined the production of RANTES from peripheral blood mononuclear cells cultured with specific allergen in atopic asthma patients by a sandwich enzyme-linked immunosorbent assay. RESULTS: It was revealed that mononuclear cells produced RANTES but not eotaxin in response to the specific allergen in asthma. RANTES production from mononuclear cells of asthma patients with eosinophilia was greater than that of asthma patients without eosinophilia. Moreover, in this study, no differences in RANTES production between CD4 negative cells and CD8 negative cells were observed. CONCLUSIONS: Taken together, these findings may suggest that mononuclear cells play a crucial role in the pathogenesis, particular in eosinophil and T lymphocyte recruitment into the inflamed focus of asthma through RANTES production in response to the specific allergen.

Laughter regulates gene expression in patients with type 2 diabetes.

BACKGROUND: Positive emotions influence endocrinological and immunological response. This study examined the effect of laughter,as an expression of positive emotion, in terms of gene expression changes. METHODS: Using a microarray technique, we analyzed the changes in expression of 18,716 genes from peripheral blood leukocytes in patients with type 2 diabetes, which were induced by laughter. RESULTS: Of the 18,716 genes, 23 genes showed significantly different expression changes after listening to the comic story compared to the lecture. Eight were relatively upregulated and 15 were downregulated 1.5 h after the laughing episode. However, these genes did not include genes that are directly involved in blood glucose metabolism. Among the 23 genes discriminated, all 4 genes encoding proteins involved in the immune response and all 4 signal transduction genes were downregulated. Moreover, it is noteworthy that 5 of the 8 relatively upregulated genes were related to the cell cycle, apoptosis, and cell adhesion. CONCLUSIONS: We demonstrated that laughter, which is an expression of positive emotion, is linked to gene expression. However, the finding of this study does not allow reasonable interpretation for the regulation of gene expression by laughter. A more focused study is needed that may identify the candidate genes for the association between physical condition and positive emotion.

Laughter therapy modulates the parameters of renin-angiotensin system in patients with type 2 diabetes.

The effect of laughter therapy on the plasma levels of renin, angiotensinogen, and prorenin was investigated in patients with type 2 diabetes. In the diabetic patients, the mean plasma renin concentrations were 24.6+/-12.1 ng/ml/h in the first observation (at the beginning of laughter therapy), 8.2+/-3.4 ng/ml/h in the second observation (three months after the beginning of laughter therapy) and 7.7+/-1.7 ng/ml/h in the third observation (six months after the beginning of laughter therapy). The mean plasma angiotensinogen concentrations in the 1st, 2nd and 3rd observations were 0.19+/-0.08, 0.47+/-0.12, 0.42+/-0.14 microg/ml, respectively. The mean plasma prorenin concentrations in the 1st, 2nd and 3rd observations during the laughter therapy were 195.1+/-66.2, 193.4+/-88.2 and 170.7+/-52.5 pg/ml, respectively. Plasma renin concentrations were significantly decreased (p<0.05) by the therapy. Subnormal concentrations of plasma angiotensinogen were found in the 1st observation and increased significantly (p<0.05) to the normal range after the therapy. Plasma prorenin concentration only slightly changed during the laughter therapy. Other biochemical parameters remained unchanged during the laughter therapy. These results indicated that a long-term laughter therapy changed the plasma components of renin-angiotensin system in patients with diabetes. Thus, laughter therapy can be used as non-pharmacological treatment for the prevention of diabetic microvascular complications.

[Reference value and cut-off value for diagnosis of insulin resistance in type 2 diabetes mellitus].

The homeostasis model assessment-insulin resistance(HOMA-IR) index has been proposed to assess insulin resistance, which plays an important role in the pathogenesis of type 2 diabetes mellitus. However, it has not generally introduced into the outpatient care in Japan, because of lack in definite reference values. In this paper we studied the precision in the assay of immuno-reactive insulin and then the HOMA-IR index was determined in 90 healthy individuals and 281 type 2 diabetic patients. The reference value was calculated to be 1.77 +/- 0.44(mean +/- SD) in healthy individuals without obesity. Receiver operating characteristic curve analysis gave the cut-off values discriminating between healthy and diabetic subjects to be 2.53, 3.50 and 3.00 in non-obese, obese and total individuals, respectively. These values should be available for the diagnosis of insulin resistance in type 2 diabetes mellitus.