Research perspectives on the regulation and physiological functions of FGF21 and its association with NAFLD

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Takeshi eInagaki

2015-09-01

Full Text Available Fibroblast growth factor 21 (FGF21 is a metabolic hormone primarily secreted from the liver and functions in multiple tissues. Various transcription factors induce FGF21 expression in the liver, which indicates that FGF21 is a mediator of multiple environmental cues. FGF21 alters metabolism under starvation conditions, protects the body from energy depletion, and extends life span. Pharmacological administration of FGF21 alleviates dyslipidemia and induces weight loss in obese animals. In addition to the well-studied functions of FG21, several lines of recent evidence indicate a possible link between FGF21 and nonalcoholic fatty liver disease (NAFLD. High serum levels of FGF21 are associated with NAFLD and its risk factors such as endoplasmic reticulum stress and chronic inflammation. In addition, FGF21 alleviates the major risk factors of NAFLD including obesity, dyslipidemia and insulin insensitivity. Thus, FGF21 is a potential drug candidate for diseases such as NAFLD, dyslipidemia, and type 2 diabetes. In this review, the research perspectives of FGF21 and therapeutic potencies of FGF21 as a modulator of NAFLD are summarized.

Circulating FGF21 in humans is potently induced by short term overfeeding of carbohydrates

DEFF Research Database (Denmark)

Lundsgaard, Annemarie; Fritzen, Andreas Mæchel; Sjøberg, Kim Anker

2017-01-01

OBJECTIVE: Fibroblast-growth factor 21 (FGF21) is thought to be important in metabolic regulation. Recently, low protein diets have been shown to increase circulating FGF21 levels. However, when energy contribution from dietary protein is lowered, other macronutrients, such as carbohydrates, must...... concentration increased 8-fold compared to CON (329 ± 99 vs. 39 ± 9 pg ml(-1), p FAT only a non-significant tendency (p = 0.073) to an increase in plasma FGF21 concentration was found. The increase in FGF21 concentration after CHO correlated closely (r = 0.88, p ... intake and increased plasma FGF21 concentration. CONCLUSION: Excess dietary carbohydrate, but not fat, led to markedly increased FGF21 secretion in humans, notably without protein restriction, and affected glucose and lipid homeostais....

FGF21 as an Endocrine Regulator in Lipid Metabolism: From Molecular Evolution to Physiology and Pathophysiology

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Yusuke Murata

2011-01-01

Full Text Available The FGF family comprises twenty-two structurally related proteins with functions in development and metabolism. The Fgf21 gene was generated early in vertebrate evolution. FGF21 acts as an endocrine regulator in lipid metabolism. Hepatic Fgf21 expression is markedly induced in mice by fasting or a ketogenic diet. Experiments with Fgf21 transgenic mice and cultured cells indicate that FGF21 exerts pharmacological effects on glucose and lipid metabolism in hepatocytes and adipocytes via cell surface FGF receptors. However, experiments with Fgf21 knockout mice indicate that FGF21 inhibits lipolysis in adipocytes during fasting and attenuates torpor induced by a ketogenic diet but maybe not a physiological regulator for these hepatic functions. These findings suggest the pharmacological effects to be distinct from the physiological roles. Serum FGF21 levels are increased in patients with metabolic diseases having insulin resistance, indicating that FGF21 is a metabolic regulator and a biomarker for these diseases.

Association of Fibroblast Growth Factor (Fgf-21) as a Screening ...

African Journals Online (AJOL)

24 control samples by enzyme-linked immunosorbent assay (ELISA) and determined the deletion of mitochondrial genome by multiplex polymerase chain reaction (PCR). Results: FGF-21 concentration in 50 % of CPEO patients showed notable differences from that in control subjects. FGF-21 concentration ratio in patient ...

Relationship between family history of type 2 diabetes and serum FGF21.

Science.gov (United States)

Davis, Greggory R; Deville, Tiffany; Guillory, Joshua; Bellar, David; Nelson, Arnold G

2017-11-01

Determining predictive markers for the development of type 2 diabetes (T2D), particularly in young individuals, offers immense potential benefits in preventative medicine. Previous research examining serum fibroblast growth factor 21 (FGF21) in humans has revealed equivocal relationships with clinical markers of metabolic dysfunction. However, it is unknown to what extent, if any, first-degree family history of T2D (mother or father of the participant diagnosed with T2D) level affects serum FGF21 levels. The aim of this study was to determine whether in healthy individuals with FH+ (n = 18) and without FH- (n = 17) a family history of T2D affects serum FGF21. Fasting serum and clinical, metabolic and anthropometric measures were determined using a cross-sectional design. Differences between groups for FGF21 were not significant (FH+ = 266 pg/mL ± 51·4, FH = 180 pg/mL ± 29; Z = 0·97, P = 0·33). Adiponectin values were lower in FH+ (8·81 μg/mL ± 2·14) compared to FH- (10·65 μg/mL ± 1·44; F = 8·83, P = 0·01). Resistin was negatively correlated with FGF21 for all participants (r = -0·38, P = 0·03), but no other clinical, metabolic, or serum markers were predictive for serum FGF21 in FH+ or FH-. Serum FGF21 is not significantly different between FH+ and FH- in young, healthy individuals. Based upon the data of this pilot study, it is unclear whether serum FGF21 can be used as a stand-alone predictive marker for T2D in healthy subjects. © 2017 Stichting European Society for Clinical Investigation Journal Foundation.

mTORC1 Is a Major Regulatory Node in the FGF21 Signaling Network in Adipocytes

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Annabel Y. Minard

2016-09-01

Full Text Available FGF21 improves the metabolic profile of obese animals through its actions on adipocytes. To elucidate the signaling network responsible for mediating these effects, we quantified dynamic changes in the adipocyte phosphoproteome following acute exposure to FGF21. FGF21 regulated a network of 821 phosphosites on 542 proteins. A major FGF21-regulated signaling node was mTORC1/S6K. In contrast to insulin, FGF21 activated mTORC1 via MAPK rather than through the canonical PI3K/AKT pathway. Activation of mTORC1/S6K by FGF21 was surprising because this is thought to contribute to deleterious metabolic effects such as obesity and insulin resistance. Rather, mTORC1 mediated many of the beneficial actions of FGF21 in vitro, including UCP1 and FGF21 induction, increased adiponectin secretion, and enhanced glucose uptake without any adverse effects on insulin action. This study provides a global view of FGF21 signaling and suggests that mTORC1 may act to facilitate FGF21-mediated health benefits in vivo.

Metformin-induced inhibition of the mitochondrial respiratory chain increases FGF21 expression via ATF4 activation

International Nuclear Information System (INIS)

Kim, Kook Hwan; Jeong, Yeon Taek; Kim, Seong Hun; Jung, Hye Seung; Park, Kyong Soo; Lee, Hae-Youn; Lee, Myung-Shik

2013-01-01

Highlights: •Metformin induces FGF21 expression in an AMPK independent manner. •Metformin enhances FGF21 expression by inhibiting mitochondrial complex I activity. •The PERK-eIF2α-ATF4 axis is required for metformin-induced FGF21 expression. •Metformin activates the ATF4-FGF21 axis in the liver of mouse. •Metformin increases serum FGF21 level in diabetic human subjects. -- Abstract: Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-obesity and anti-diabetes effects. Because metformin is widely used as a glucose-lowering agent in patients with type 2 diabetes (T2D), we investigated whether metformin modulates FGF21 expression in cell lines, and in mice or human subjects. We found that metformin increased the expression and release of FGF21 in a diverse set of cell types, including rat hepatoma FaO, primary mouse hepatocytes, and mouse embryonic fibroblasts (MEFs). Intriguingly, AMP-activated protein kinase (AMPK) was dispensable for the induction of FGF21 by metformin. Mammalian target of rapamycin complex 1 (mTORC1) and peroxisome proliferator-activated receptor α (PPARα), which are additional targets of metformin, were not involved in metformin-induced FGF21 expression. Importantly, inhibition of mitochondrial complex I activity by metformin resulted in FGF21 induction through PKR-like ER kinase (PERK)-eukaryotic translation factor 2α (eIF2α)-activating transcription factor 4 (ATF4). We showed that metformin activated ATF4 and increased FGF21 expression in the livers of mice, which led to increased serum levels of FGF21. We also found that serum FGF21 level was increased in human subjects with T2D after metformin therapy for 6 months. In conclusion, our results indicate that metformin induced expression of FGF21 through an ATF4-dependent mechanism by inhibiting mitochondrial respiration independently of AMPK. Therefore, FGF21 induction by metformin might explain a portion of the beneficial metabolic effects of metformin

Metformin-induced inhibition of the mitochondrial respiratory chain increases FGF21 expression via ATF4 activation

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Kim, Kook Hwan [Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Jeong, Yeon Taek [Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Kim, Seong Hun [Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Jung, Hye Seung; Park, Kyong Soo [Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong Chongno-gu, Seoul 110-744 (Korea, Republic of); Lee, Hae-Youn [Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Lee, Myung-Shik, E-mail: mslee0923@skku.edu [Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of); Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, 50 Irwon-dong Gangnam-gu, Seoul 135-710 (Korea, Republic of)

2013-10-11

Highlights: •Metformin induces FGF21 expression in an AMPK independent manner. •Metformin enhances FGF21 expression by inhibiting mitochondrial complex I activity. •The PERK-eIF2α-ATF4 axis is required for metformin-induced FGF21 expression. •Metformin activates the ATF4-FGF21 axis in the liver of mouse. •Metformin increases serum FGF21 level in diabetic human subjects. -- Abstract: Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-obesity and anti-diabetes effects. Because metformin is widely used as a glucose-lowering agent in patients with type 2 diabetes (T2D), we investigated whether metformin modulates FGF21 expression in cell lines, and in mice or human subjects. We found that metformin increased the expression and release of FGF21 in a diverse set of cell types, including rat hepatoma FaO, primary mouse hepatocytes, and mouse embryonic fibroblasts (MEFs). Intriguingly, AMP-activated protein kinase (AMPK) was dispensable for the induction of FGF21 by metformin. Mammalian target of rapamycin complex 1 (mTORC1) and peroxisome proliferator-activated receptor α (PPARα), which are additional targets of metformin, were not involved in metformin-induced FGF21 expression. Importantly, inhibition of mitochondrial complex I activity by metformin resulted in FGF21 induction through PKR-like ER kinase (PERK)-eukaryotic translation factor 2α (eIF2α)-activating transcription factor 4 (ATF4). We showed that metformin activated ATF4 and increased FGF21 expression in the livers of mice, which led to increased serum levels of FGF21. We also found that serum FGF21 level was increased in human subjects with T2D after metformin therapy for 6 months. In conclusion, our results indicate that metformin induced expression of FGF21 through an ATF4-dependent mechanism by inhibiting mitochondrial respiration independently of AMPK. Therefore, FGF21 induction by metformin might explain a portion of the beneficial metabolic effects of metformin.

Direct effects of FGF21 on glucose uptake in human skeletal muscle

DEFF Research Database (Denmark)

Mashili, Fredirick L; Austin, Reginald L; Deshmukh, Atul S

2011-01-01

21 were determined in normal glucose tolerant (n = 40) and type 2 diabetic (T2D; n = 40) subjects. We determined whether FGF21 has direct effects on glucose metabolism in cultured myotubes (n = 8) and extensor digitorum longus skeletal muscle. RESULTS: Serum FGF21 levels increased 20% in T2D versus...... normal glucose tolerant subjects (p muscle mRNA expression was unaltered. Fasting insulin, homeostatic model assessment of insulin resistance (HOMA-IR), waist circumference, and body mass index (BMI) significantly correlated with serum FGF21 levels in T2D (p ... and insulin-stimulated glucose uptake in human myotubes, coincident with increased glucose transporter 1 mRNA, and enhanced glucose transporter 1 abundance at the plasma membrane. In isolated extensor digitorum longus muscle, FGF21 potentiated insulin-stimulated glucose transport, without altering...

Fibroblast growth factor 21 (FGF21 is robustly induced by ethanol and has a protective role in ethanol associated liver injury

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Bhavna N. Desai

2017-11-01

Conclusions: Acute or binge ethanol consumption significantly increases circulating FGF21 levels in both humans and mice. However, FGF21 does not play a role in acute ethanol clearance. In contrast, chronic ethanol consumption in the absence of FGF21 is associated with significant liver pathology alone or in combination with excess mortality, depending on the type of diet consumed with ethanol. This suggests that FGF21 protects against long term ethanol induced hepatic damage and may attenuate progression of alcoholic liver disease. Further study is required to assess the therapeutic potential of FGF21 in the treatment of alcoholic liver disease.

Fibroblast growth factor (Fgf) 21 is a novel target gene of the aryl hydrocarbon receptor (AhR)

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Cheng, Xingguo, E-mail: chengx@stjohns.edu [Department of Pharmaceutical Sciences, St. John' s University, 8000 Utopia Parkway, Queens, NY 11439 (United States); Vispute, Saurabh G. [Department of Pharmaceutical Sciences, St. John' s University, 8000 Utopia Parkway, Queens, NY 11439 (United States); Liu, Jie [Department of Internal Medicine, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160 (United States); Cheng, Christine; Kharitonenkov, Alexei [Lilly Research Laboratories, Division of Eli Lilly and Co., Indianapolis, IN 46285 (United States); Klaassen, Curtis D., E-mail: curtisklaassenphd@gmail.com [Department of Internal Medicine, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160 (United States)

2014-07-01

The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) of Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (− 105/+ 1 base pair). Fgf21-null mice administered 200 μg/kg of TCDD died within 20 days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver. - Highlights: • TCDD induced Fgf21 expression at both mRNA and protein levels. • Fgf21 induction by TCDD is AhR-dependent. • DEHP attenuated TCDD-induced Fgf21 expression.

Fibroblast growth factor (Fgf) 21 is a novel target gene of the aryl hydrocarbon receptor (AhR)

International Nuclear Information System (INIS)

Cheng, Xingguo; Vispute, Saurabh G.; Liu, Jie; Cheng, Christine; Kharitonenkov, Alexei; Klaassen, Curtis D.

2014-01-01

The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) of Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (− 105/+ 1 base pair). Fgf21-null mice administered 200 μg/kg of TCDD died within 20 days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver. - Highlights: • TCDD induced Fgf21 expression at both mRNA and protein levels. • Fgf21 induction by TCDD is AhR-dependent. • DEHP attenuated TCDD-induced Fgf21 expression

A Specific ChREBP and PPARα Cross-Talk Is Required for the Glucose-Mediated FGF21 Response

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Alison Iroz

2017-10-01

Full Text Available While the physiological benefits of the fibroblast growth factor 21 (FGF21 hepatokine are documented in response to fasting, little information is available on Fgf21 regulation in a glucose-overload context. We report that peroxisome-proliferator-activated receptor α (PPARα, a nuclear receptor of the fasting response, is required with the carbohydrate-sensitive transcription factor carbohydrate-responsive element-binding protein (ChREBP to balance FGF21 glucose response. Microarray analysis indicated that only a few hepatic genes respond to fasting and glucose similarly to Fgf21. Glucose-challenged Chrebp−/− mice exhibit a marked reduction in FGF21 production, a decrease that was rescued by re-expression of an active ChREBP isoform in the liver of Chrebp−/− mice. Unexpectedly, carbohydrate challenge of hepatic Pparα knockout mice also demonstrated a PPARα-dependent glucose response for Fgf21 that was associated with an increased sucrose preference. This blunted response was due to decreased Fgf21 promoter accessibility and diminished ChREBP binding onto Fgf21 carbohydrate-responsive element (ChoRE in hepatocytes lacking PPARα. Our study reports that PPARα is required for the ChREBP-induced glucose response of FGF21.

Circulating FGF21 in humans is potently induced by short term overfeeding of carbohydrates.

Science.gov (United States)

Lundsgaard, Anne-Marie; Fritzen, Andreas M; Sjøberg, Kim A; Myrmel, Lene S; Madsen, Lise; Wojtaszewski, Jørgen F P; Richter, Erik A; Kiens, Bente

2017-01-01

Fibroblast-growth factor 21 (FGF21) is thought to be important in metabolic regulation. Recently, low protein diets have been shown to increase circulating FGF21 levels. However, when energy contribution from dietary protein is lowered, other macronutrients, such as carbohydrates, must be increased to meet eucaloric balance. This raises the possibility that intake of a diet rich in carbohydrates may induce an increase in plasma FGF21 levels per se. Here we studied the role of dietary carbohydrates on the levels of circulating FGF21 and concomitant physiologic effects by feeding healthy men a carbohydrate rich diet without reducing protein intake. A diet enriched in carbohydrates (80 E% carbohydrate; CHO) and a eucaloric control diet (CON) were provided to nine healthy men for three days. The energy intake during the CHO diet was increased (+75% energy) to ensure similar dietary protein intake in CHO and CON. To control for the effect of caloric surplus, we similarly overfed (+75% energy) the same subjects for three days with a fat-rich diet (78 E% fat; FAT), consisting of primarily unsaturated fatty acids. The three diets were provided in random order. After CHO, plasma FGF21 concentration increased 8-fold compared to CON (329 ± 99 vs. 39 ± 9 pg ml -1 , p FAT only a non-significant tendency (p = 0.073) to an increase in plasma FGF21 concentration was found. The increase in FGF21 concentration after CHO correlated closely (r = 0.88, p carbohydrate, but not fat, led to markedly increased FGF21 secretion in humans, notably without protein restriction, and affected glucose and lipid homeostais.

FGF21 Is a Sugar-Induced Hormone Associated with Sweet Intake and Preference in Humans

DEFF Research Database (Denmark)

Søberg, Susanna; Sandholt, Camilla Helene; Z. Jespersen, Naja

2017-01-01

The liking and selective ingestion of palatable foods—including sweets—is biologically controlled, and dysfunction of this regulation may promote unhealthy eating, obesity, and disease. The hepatokine fibroblast growth factor 21 (FGF21) reduces sweet consumption in rodents and primates, whereas...... knockout of Fgf21 increases sugar consumption in mice. To investigate the relevance of these findings in humans, we genotyped variants in the FGF21 locus in participants from the Danish Inter99 cohort (n = 6,514) and examined their relationship with a detailed range of food and ingestive behaviors....... This revealed statistically significant associations between FGF21 rs838133 and increased consumption of candy, as well as nominal associations with increased alcohol intake and daily smoking. Moreover, in a separate clinical study, plasma FGF21 levels increased acutely after oral sucrose ingestion and were...

FGF21 Mediates Endocrine Control of Simple Sugar Intake and Sweet Taste Preference by the Liver

DEFF Research Database (Denmark)

von Holstein-Rathlou, Stephanie; BonDurant, Lucas D; Peltekian, Lila

2016-01-01

The liver is an important integrator of nutrient metabolism, yet no liver-derived factors regulating nutrient preference or carbohydrate appetite have been identified. Here we show that the liver regulates carbohydrate intake through production of the hepatokine fibroblast growth factor 21 (FGF21......), which markedly suppresses consumption of simple sugars, but not complex carbohydrates, proteins, or lipids. Genetic loss of FGF21 in mice increases sucrose consumption, whereas acute administration or overexpression of FGF21 suppresses the intake of both sugar and non-caloric sweeteners. FGF21 does...... not affect chorda tympani nerve responses to sweet tastants, instead reducing sweet-seeking behavior and meal size via neurons in the hypothalamus. This liver-to-brain hormonal axis likely represents a negative feedback loop as hepatic FGF21 production is elevated by sucrose ingestion. We conclude...

Plasma FGF21 displays a circadian rhythm during a 72-h fast in healthy female volunteers

DEFF Research Database (Denmark)

Andersen, Birgitte; Beck-Nielsen, Henning; Højlund, Kurt

2011-01-01

Fibroblast growth factor (FGF21) is a potent regulator of glucose and lipid metabolism. In rodents, the hepatic expression of FGF21 is controlled by fasting and a circadian regulation, but the physiological role and regulation of FGF21 in humans is not well established. Therefore, the objective...

Circulating FGF21 in humans is potently induced by short term overfeeding of carbohydrates

Directory of Open Access Journals (Sweden)

Anne-Marie Lundsgaard

2017-01-01

Full Text Available Objective: Fibroblast-growth factor 21 (FGF21 is thought to be important in metabolic regulation. Recently, low protein diets have been shown to increase circulating FGF21 levels. However, when energy contribution from dietary protein is lowered, other macronutrients, such as carbohydrates, must be increased to meet eucaloric balance. This raises the possibility that intake of a diet rich in carbohydrates may induce an increase in plasma FGF21 levels per se. Here we studied the role of dietary carbohydrates on the levels of circulating FGF21 and concomitant physiologic effects by feeding healthy men a carbohydrate rich diet without reducing protein intake. Methods: A diet enriched in carbohydrates (80 E% carbohydrate; CHO and a eucaloric control diet (CON were provided to nine healthy men for three days. The energy intake during the CHO diet was increased (+75% energy to ensure similar dietary protein intake in CHO and CON. To control for the effect of caloric surplus, we similarly overfed (+75% energy the same subjects for three days with a fat-rich diet (78 E% fat; FAT, consisting of primarily unsaturated fatty acids. The three diets were provided in random order. Results: After CHO, plasma FGF21 concentration increased 8-fold compared to CON (329 ± 99 vs. 39 ± 9 pg ml−1, p < 0.05. In contrast, after FAT only a non-significant tendency (p = 0.073 to an increase in plasma FGF21 concentration was found. The increase in FGF21 concentration after CHO correlated closely (r = 0.88, p < 0.01 with increased leg glucose uptake (62%, p < 0.05 and increased hepatic glucose production (17%, p < 0.01, indicating increased glucose turnover. Plasma fatty acid (FA concentration was decreased by 68% (p < 0.01, supported by reduced subcutaneous adipose tissue HSL Ser660 phosphorylation (p < 0.01 and perilipin 1 protein content (p < 0.01, pointing to a suppression of adipose tissue lipolysis. Concomitantly, a 146% increase in the

FGF21 is not required for glucose homeostasis, ketosis or tumour suppression associated with ketogenic diets in mice.

Science.gov (United States)

Stemmer, Kerstin; Zani, Fabio; Habegger, Kirk M; Neff, Christina; Kotzbeck, Petra; Bauer, Michaela; Yalamanchilli, Suma; Azad, Ali; Lehti, Maarit; Martins, Paulo J F; Müller, Timo D; Pfluger, Paul T; Seeley, Randy J

2015-10-01

Ketogenic diets (KDs) have increasingly gained attention as effective means for weight loss and potential adjunctive treatment of cancer. The metabolic benefits of KDs are regularly ascribed to enhanced hepatic secretion of fibroblast growth factor 21 (FGF21) and its systemic effects on fatty-acid oxidation, energy expenditure (EE) and body weight. Ambiguous data from Fgf21-knockout animal strains and low FGF21 concentrations reported in humans with ketosis have nevertheless cast doubt regarding the endogenous function of FGF21. We here aimed to elucidate the causal role of FGF21 in mediating the therapeutic benefits of KDs on metabolism and cancer. We established a dietary model of increased vs decreased FGF21 by feeding C57BL/6J mice with KDs, either depleted of protein or enriched with protein. We furthermore used wild-type and Fgf21-knockout mice that were subjected to the respective diets, and monitored energy and glucose homeostasis as well as tumour growth after transplantation of Lewis lung carcinoma cells. Hepatic and circulating, but not adipose tissue, FGF21 levels were profoundly increased by protein starvation, independent of the state of ketosis. We demonstrate that endogenous FGF21 is not essential for the maintenance of normoglycaemia upon protein and carbohydrate starvation and is therefore not needed for the effects of KDs on EE. Furthermore, the tumour-suppressing effects of KDs were independent of FGF21 and, rather, driven by concomitant protein and carbohydrate starvation. Our data indicate that the multiple systemic effects of KD exposure in mice, previously ascribed to increased FGF21 secretion, are rather a consequence of protein malnutrition.

Nrf2 represses FGF21 during long-term high-fat diet-induced obesity in mice.

Science.gov (United States)

Chartoumpekis, Dionysios V; Ziros, Panos G; Psyrogiannis, Agathoklis I; Papavassiliou, Athanasios G; Kyriazopoulou, Venetsana E; Sykiotis, Gerasimos P; Habeos, Ioannis G

2011-10-01

Obesity is characterized by chronic oxidative stress. Fibroblast growth factor 21 (FGF21) has recently been identified as a novel hormone that regulates metabolism. NFE2-related factor 2 (Nrf2) is a transcription factor that orchestrates the expression of a battery of antioxidant and detoxification genes under both basal and stress conditions. The current study investigated the role of Nrf2 in a mouse model of long-term high-fat diet (HFD)-induced obesity and characterized its crosstalk to FGF21 in this process. Wild-type (WT) and Nrf2 knockout (Nrf2-KO) mice were fed an HFD for 180 days. During this period, food consumption and body weights were measured. Glucose metabolism was assessed by an intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test. Total RNA was prepared from liver and adipose tissue and was used for quantitative real-time RT-PCR. Fasting plasma was collected and analyzed for blood chemistries. The ST-2 cell line was used for transfection studies. Nrf2-KO mice were partially protected from HFD-induced obesity and developed a less insulin-resistant phenotype. Importantly, Nrf2-KO mice had higher plasma FGF21 levels and higher FGF21 mRNA levels in liver and white adipose tissue than WT mice. Thus, the altered metabolic phenotype of Nrf2-KO mice under HFD was associated with higher expression and abundance of FGF21. Consistently, the overexpression of Nrf2 in ST-2 cells resulted in decreased FGF21 mRNA levels as well as in suppressed activity of a FGF21 promoter luciferase reporter. The identification of Nrf2 as a novel regulator of FGF21 expands our understanding of the crosstalk between metabolism and stress defense.

Contribution of serum FGF21 level to the identification of left ventricular systolic dysfunction and cardiac death.

Science.gov (United States)

Shen, Yun; Zhang, Xueli; Pan, Xiaoping; Xu, Yiting; Xiong, Qin; Lu, Zhigang; Ma, Xiaojing; Bao, Yuqian; Jia, Weiping

2017-08-18

The relationship between fibroblast growth factor 21 (FGF21) and cardiovascular disease has been well established in recent studies. This study aimed to investigate the relationship between FGF21 and left ventricular systolic dysfunction and cardiac death. Two-dimensional echocardiography was used to measure the left ventricular ejection fraction (LVEF) to estimate left ventricular systolic function. The optimal cutoff of FGF21 for identifying left ventricular systolic dysfunction at baseline was analyzed via receiver operating characteristic (ROC) curves. The identification of different serum levels of FGF21 and their association with cardiac death was analyzed via Kaplan-Meier survival curves. Serum FGF21 level was measured by an enzyme-linked immunosorbent assay kit, and serum N-terminal pro-brain natriuretic peptide (NT-pro-BNP) level was determined by a chemiluminescent immunoassay. A total of 253 patients were recruited for this study at baseline. Patients were excluded if they lacked echocardiography or laboratory measurement data, and there were 218 patients enrolled in the final analysis. The average age was 66.32 ± 10.10 years. The optimal cutoff values of FGF21 and NT-pro-BNP for identifying left ventricular systolic dysfunction at baseline were 321.5 pg/mL and 131.3 ng/L, respectively, determined separately via ROC analysis. The areas under the curves were non-significant among FGF21, NT-pro-BNP and FGF21 + NT-pro-BNP as determined by pairwise comparisons. Both a higher serum level of FGF21 and a higher serum level of NT-pro-BNP were independent risk factors for left ventricular systolic dysfunction at baseline (odd ratio (OR) 3.138 [1.037-9.500], P = 0.043, OR 9.207 [2.036-41.643], P = 0.004, separately). Further Kaplan-Meier survival analysis indicated an association between both a higher serum level of FGF21 and a higher serum level of NT-pro-BNP with cardiac death in 5 years [RR 5.000 (1.326-18.861), P = 0.026; RR 9.643 (2

FGF-21 and skeletal remodeling during and after lactation in C57BL/6J mice.

Science.gov (United States)

Bornstein, Sheila; Brown, Sue A; Le, Phuong T; Wang, Xunde; DeMambro, Victoria; Horowitz, Mark C; MacDougald, Ormond; Baron, Roland; Lotinun, Sutada; Karsenty, Gerard; Wei, Wei; Ferron, Mathieu; Kovacs, Christopher S; Clemmons, David; Wan, Yihong; Rosen, Clifford J

2014-09-01

Lactation is associated with significant alterations in both body composition and bone mass. Systemic and local skeletal factors such as receptor activator of nuclear factor κ-B ligand (RANKL), PTHrP, calcitonin, and estrogen are known to regulate bone remodeling during and after lactation. Fibroblast growth factor 21 (FGF-21) may function as an endocrine factor to regulate body composition changes during lactation by inducing gluconeogenesis and fatty acid oxidation. In this study, we hypothesized that the metabolic changes during lactation were due in part to increased circulating FGF-21, which in turn could accentuate bone loss. We longitudinally characterized body composition in C57BL/6J (B6) mice during (day 7 and day 21 of lactation) and after normal lactation (day 21 postlactation). At day 7 of lactation, areal bone density declined by 10% (P < .001), bone resorption increased (P < .0001), percent fat decreased by 20%, energy expenditure increased (P < .01), and markers of brown-like adipogenesis were suppressed in the inguinal depot and in preformed brown adipose tissue. At day 7 of lactation there was a 2.4-fold increase in serum FGF-21 vs baseline (P < .0001), a 8-fold increase in hepatic FGF-21 mRNA (P < .03), a 2-fold increase in undercarboxylated osteocalcin (Glu13 OCn) (P < .01), and enhanced insulin sensitivity. Recovery of total areal bone density was noted at day 21 of lactation, whereas the femoral trabecular bone volume fraction was still reduced (P < .01). Because FGF-21 levels rose rapidly at day 7 of lactation in B6 lactating mice, we next examined lactating mice with a deletion in the Fgf21 gene. Trabecular and cortical bone masses were maintained throughout lactation in FGF-21(-/-) mice, and pup growth was normal. Compared with lactating control mice, lactating FGF-21(-/-) mice exhibited an increase in bone formation, but no change in bone resorption. In conclusion, in addition to changes in calciotropic hormones, systemic FGF-21 plays a

LPS infusion suppresses serum FGF21 levels in healthy adult volunteers

DEFF Research Database (Denmark)

Lauritzen, Esben Stistrup; Rittig, Nikolaj; Bach, Ermina

2017-01-01

circulating levels of FGF21 after lipopolysaccharide (LPS) infusion. DESIGN: Two randomized, single blinded, placebo-controlled crossover trials were used. SETTING: The studies were performed at a university hospital clinical research center. PATIENTS AND INTERVENTIONS: Study 1 (LPS bolus): Eight young......, healthy, lean males were investigated two times: 1) after isotonic saline injection, and 2) after LPS injection (bolus of 1 ng/kg). Each study day lasted 4 hours. Study 2 (continuous LPS infusion): Eight, healthy males were investigated two times: 1) during continuously isotonic saline infusion, and 2......) during continuously LPS infusion (0.06 ng/kg/h). Each study day lasted 4 hours. Circulating FGF21 levels were quantified every second hour by an immunoassay. RESULTS: A LPS bolus resulted in a late suppression (t = 240 minutes) of serum FGF21 (P=0.035). Continuous LPS infusion revealed no significant...

Effects of insulin and exercise training on FGF21, its receptors and target genes in obesity and type 2 diabetes

DEFF Research Database (Denmark)

Sørensen, Rikke Kruse; Vienberg, Sara Gry; Vind, Birgitte F

2017-01-01

obesity with and without type 2 diabetes led to reduced expression of KLB, but increased FGFR1c expression. However, the expression of most FGF21 target genes was unaltered except for reduced CIDEA expression in individuals with type 2 diabetes. CONCLUSIONS....../INTERPRETATION: Insulin-induced expression of muscle FGF21 correlates strongly with a rise in serum FGF21, and this response appears intact in overweight/obesity and type 2 diabetes. FGF21 resistance may involve reduced KLB expression in WAT. However, increased FGFR1c expression or other mechanisms seem to ensure...... that insulin and exercise increase FGF21 in plasma. Obesity and type 2 diabetes are potentially FGF21-resistant states, but to what extent FGF21 responses to insulin and exercise training are preserved, and whether FGF21, its receptors and target genes are altered, remains to be established. METHODS...

FGF21 maintains glucose homeostasis by mediating the cross talk between liver and brain during prolonged fasting.

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Liang, Qingning; Zhong, Ling; Zhang, Jialiang; Wang, Yu; Bornstein, Stefan R; Triggle, Chris R; Ding, Hong; Lam, Karen S L; Xu, Aimin

2014-12-01

Hepatic gluconeogenesis is a main source of blood glucose during prolonged fasting and is orchestrated by endocrine and neural pathways. Here we show that the hepatocyte-secreted hormone fibroblast growth factor 21 (FGF21) induces fasting gluconeogenesis via the brain-liver axis. Prolonged fasting induces activation of the transcription factor peroxisome proliferator-activated receptor α (PPARα) in the liver and subsequent hepatic production of FGF21, which enters into the brain to activate the hypothalamic-pituitary-adrenal (HPA) axis for release of corticosterone, thereby stimulating hepatic gluconeogenesis. Fasted FGF21 knockout (KO) mice exhibit severe hypoglycemia and defective hepatic gluconeogenesis due to impaired activation of the HPA axis and blunted release of corticosterone, a phenotype similar to that observed in PPARα KO mice. By contrast, intracerebroventricular injection of FGF21 reverses fasting hypoglycemia and impairment in hepatic gluconeogenesis by restoring corticosterone production in both FGF21 KO and PPARα KO mice, whereas all these central effects of FGF21 were abrogated by blockage of hypothalamic FGF receptor-1. FGF21 acts directly on the hypothalamic neurons to activate the mitogen-activated protein kinase extracellular signal-related kinase 1/2 (ERK1/2), thereby stimulating the expression of corticotropin-releasing hormone by activation of the transcription factor cAMP response element binding protein. Therefore, FGF21 maintains glucose homeostasis during prolonged fasting by fine tuning the interorgan cross talk between liver and brain. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

A low-protein diet induces body weight loss and browning of subcutaneous white adipose tissue through enhanced expression of hepatic fibroblast growth factor 21 (FGF21).

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Pérez-Martí, Albert; Garcia-Guasch, Maite; Tresserra-Rimbau, Anna; Carrilho-Do-Rosário, Alexandra; Estruch, Ramon; Salas-Salvadó, Jordi; Martínez-González, Miguel Ángel; Lamuela-Raventós, Rosa; Marrero, Pedro F; Haro, Diego; Relat, Joana

2017-08-01

Fibroblast growth factor 21 (FGF21) is considered a promising therapeutic candidate for the treatment of obesity. Since FGF21 production is regulated by various nutritional factors, we analyze the impact of low protein intake on circulating levels of this growth hormone in mice and in a sub cohort of the PREDIMED (Prevención con Dieta Mediterránea) trial. We also describe the role of hepatic FGF21 in metabolic adaptation to a low-protein diet (LPD). We fed control and liver-specific Fgf21 knockout (LFgf21KO) mice a LPD. This diet increased FGF21 production by inducing its overexpression in liver, and this correlated with a body weight decrease without changes in food intake. The LPD also caused FGF21-dependent browning in subcutaneous white adipose tissue (scWAT), as indicated by an increase in the expression of uncoupling protein 1 (UCP1). In a subgroup of 78 individuals from the PREDIMED trial, we observed an inverse correlation between protein intake and circulating FGF21 levels. Our results reinforce the involvement of FGF21 in coordinating energy homeostasis under a range of nutritional conditions. Moreover, here we describe an approach to increase the endogenous production of FGF21, which if demonstrated functional in humans, could generate a treatment for obesity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The alterations of serum FGF-21 levels, metabolic and body composition in early breast cancer patients receiving adjuvant endocrine therapy.

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Akyol, Murat; Alacacioglu, Ahmet; Demir, Leyla; Kucukzeybek, Yuksel; Yildiz, Yasar; Gumus, Zehra; Kara, Mete; Salman, Tarik; Varol, Umut; Taskaynatan, Halil; Oflazoglu, Utku; Bayoglu, Vedat; Tarhan, Mustafa Oktay

2017-01-01

In early breast cancer patients, the effects of hormonal therapy (tamoxifen and aromatase inhibitors) on plasma fibroblast growth factor 21 (FGF-21), lipid levels and body composition have not yet been investigated. Therefore, we aimed to analyze the relationship between FGF-21 and body composition as well as the effects of tamoxifen and aromatase inhibitors on plasma lipid levels, FGF-21, and body composition. A total of 72 patients were treated with either tamoxifen or aromatase inhibitors due to their menopausal status after adjuvant radiotherapy. Each patient was followed-up over a period of 1 year. Changes in body composition and serum lipid profile, glucose and FGF-21 levels were evaluated. We recorded the type of hormonal therapy, body mass index, waist-to-hip ratio, lipid profile, and FGF-21 levels both at the beginning and after 12 months. There was a statistically significant decrease in serum FGF-21 levels after 12 months of adjuvant endocrine therapy (46 ± 19.21 pg/ml vs. 30.99 ± 13.81 pg/ml, pbody water (pbody composition, glucose, lipid profile and FGF-21 were similar in tamoxifen and aromatase inhibitor groups. A positive correlation was found between basal weight, fat mass, fat-free mass and serum FGF-21 levels; however, the correlation was maintained only for the fat-free mass at the 12th month. As part of the present study, we suggest that both tamoxifen and aromatase inhibitors can reduce FGF-21 levels independently of body compositions, and these drugs can provide antihyperlipidemic, antidiabetic and cardio-protective effects. We also recommend that serum FGF-21 level can be utilized as a tumor biomarker in early-stage breast cancer and for monitoring purposes. FGF-21 levels may help physicians estimate prognosis, too. Further studies with larger populations may shed light on the role of FGF-21 in breast cancer.

Serum levels of FGF-21 are increased in coronary heart disease patients and are independently associated with adverse lipid profile.

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Zhuofeng Lin

Full Text Available BACKGROUND: Fibroblast growth factor 21 (FGF-21 is a metabolic regulator with multiple beneficial effects on glucose homeostasis and lipid metabolism in animal models. The relationship between plasma levels of FGF-21 and coronary heart disease (CHD in unknown. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to investigate the correlation of serum FGF-21 levels and lipid metabolism in the patients with coronary heart disease. We performed a logistic regression analysis of the relation between serum levels of FGF-21 and CHD patients with and without diabetes and hypertension. This study was conducted in the Departments of Endocrinology and Cardiovascular Diseases at two University Hospitals. Participants consisted of one hundred and thirty-five patients who have been diagnosed to have CHD and sixty-one control subjects. Serum FGF-21 level and levels of fasting blood glucose; triglyceride; apolipoprotein B100; HOMA-IR; insulin; total cholesterol; HDL-cholesterol; LDL-cholesterol; and C-reactive protein were measured. We found that median serum FGF-21 levels were significantly higher in CHD than that of control subjects (P<0.0001. Serum FGF-21 levels in CHD patients with diabetes, hypertension, or both were higher than that of patients without these comorbidities. Serum FGF-21 levels correlated positively with triglycerides, fasting blood glucose, apolipoprotein B100, insulin and HOMA-IR but negatively with HDL-C and apolipoprotein A1 after adjusting for BMI, diabetes and hypertension. Logistic regression analysis demonstrated that FGF-21 showed an independent association with triglyceride and apolipoprotein A1. CONCLUSIONS/SIGNIFICANCE: High levels of FGF-21 are associated with adverse lipid profiles in CHD patients. The paradoxical increase of serum FGF-21 in CHD patients may indicate a compensatory response or resistance to FGF-21.

Fibroblast Growth Factor 21 (FGF21, Free Fatty Acid (FFA, High Sensitivity C-reactive Protein (hsCRP and Homeostasis Model Assessment of Insulin Resistance (HOMA-IR Among Indonesian Obese Non-Diabetic Males

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Yani Lina

2009-12-01

Full Text Available BACKGROUND: Fibroblast growth factor-21 (FGF21 is known as an important endocrine and paracrine regulator of metabolic homeostasis. Recent studies have shown that FGF21 attenuates lipolysis in human adipocytes, which is suggested as a FGF21's mechanism as anti-hyperlipidemia, anti-hyperglycemia and anti-obesity. The aim of this study was to measure the correlation between FGF21, FFA, hsCRP and HOMA-IR among Indonesian obese non diabetic males. METHODS: The study was observational with cross sectional design. The analysis was done in 137 subjects aged 30-60 years with non diabetic abdominal obesity. We measured the biochemical markers FGF21, FFA, hsCRP, fasting insulin and fasting glucose. We also measured weight, height, waist circumrefence (WC, creatinine, serum glutamin oxaloacetic transaminase (SGOT, and serum glutamic pyruvic transaminase (SGPT, systolic blood pressure (SBP and diastolic blood pressure (DBP. Correlation between markers was measured using Pearson and Spearman's analysis. RESULTS: There were significant positive correlations between FGF21-HOMA-IR (r=0.314, p=0.000; FGF21-WC (r=0.173, p=0.043; FFA=hsCRP r=0.270, p=0.001; and WC-HOMA-IR (r=0.279, p=0.001. There was significant negative correlation between FGF21-FFA (r=-0.038, p=0.657 and FGF21-hsCRP (r=-0.061, p=0.482. CONCLUSIONS: In this study we found that although there was no significant correlation, FGF21 might act as an anti-lipolytic and anti-inflammation agent among Indonesian obese non-diabetic males. Our findings agree with results of previous studies that the positive correlation between FGF21-WC and FGF21-HOMA-IR might occur as a compensatory mechanism or resistance to FGF21 in obesity. KEYWORDS: obesity, FGF21, FFA, hsCRP, HOMA-IR.

A decreased soluble Klotho level with normal eGFR, FGF23, serum phosphate, and FEP in an ADPKD patient with enlarged kidneys due to multiple cysts.

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Kanai, Takahiro; Shiizaki, Kazuhiro; Betsui, Hiroyuki; Aoyagi, Jun; Yamagata, Takanori

2018-05-16

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disorder. ADPKD is characterized clinically by the presence of multiple bilateral renal cysts that lead to chronic renal failure. The cysts evolve from renal tubular epithelial cells that express the Klotho gene. Notably, Klotho acts as a co-receptor for fibroblast growth factor 23 (FGF23); in this context, it induces phosphaturia and maintains serum phosphate at a normal level. Many reports have shown that decreases in the soluble Klotho level and increases in the FGF23 level are associated with glomerular filtration rate (GFR) decline, but a recent study observed these changes in patient with normal eGFR. It remains unclear whether the decrease in the Klotho level precedes the increase in FGF23. Here, we present an ADPKD patient with enlarged kidneys due to multiple cysts who had a decreased soluble Klotho level but a normal eGFR and a normal FGF23 level. The patient's serum phosphate level was normal, as was the fractional excretion of phosphate (FEP). This appears to be the first reported case to show a decreased soluble Klotho level plus normal eGFR, FGF23, and FEP. These results suggest that Klotho decreases before FGF23 increases and further suggest that Klotho is not required to maintain normal serum phosphate levels in ADPKD if the FEP and serum phosphate levels are normal.

Expression and purification of soluble recombinant Hexastatin in E. coli

International Nuclear Information System (INIS)

He Xin; Wen Lei; Song Naling; Wang Dezhi; Zhao Qiren

2012-01-01

Purpose: To construct the expression vector of Hexastatin gene, to express and to purify the recombinant protein for further activity research. Methods: The human Hexastatin gene was isolated by RTPCR from EC9706 cells total RNA and cloned into pMD18-T for sequencing. Then the Hexastatin gene was subcloned into pMAL-c4x expression vector and induced to express by IPTG. The recombinant fusion protein was purified with Amylose Resin Heads. Results: RT-PCR product was about 687 bp and its sequence was the same as that of Hexastatin reported. The recombinant protein was expressed in E. coli BL21 with high level and the soluble protein accounted for 24.8% of the total bacterial protein. The purification of recombinant protein purified with Amylose Resin Heads reached more than 90%. Conclusion: The cloning, expression and purification of human Hexastatin have laid a foundation for its anti-angiogenesis therapy for tumor. (authors)

A Common Allele in FGF21 Associated with Sugar Intake Is Associated with Body Shape, Lower Total Body-Fat Percentage, and Higher Blood Pressure

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Timothy M. Frayling

2018-04-01

Full Text Available Summary: Fibroblast growth factor 21 (FGF21 is a hormone that has insulin-sensitizing properties. Some trials of FGF21 analogs show weight loss and lipid-lowering effects. Recent studies have shown that a common allele in the FGF21 gene alters the balance of macronutrients consumed, but there was little evidence of an effect on metabolic traits. We studied a common FGF21 allele (A:rs838133 in 451,099 people from the UK Biobank study, aiming to use the human allele to inform potential adverse and beneficial effects of targeting FGF21. We replicated the association between the A allele and higher percentage carbohydrate intake. We then showed that this allele is more strongly associated with higher blood pressure and waist-hip ratio, despite an association with lower total body-fat percentage, than it is with BMI or type 2 diabetes. These human phenotypes of variation in the FGF21 gene will inform research into FGF21’s mechanisms and therapeutic potential. : Drugs targeting the hormone FGF21 may have beneficial health effects. Variations in human DNA in the FGF21 gene provide an indication of what those effects may be. Here, we show that variation in the FGF21 gene is associated with higher blood pressure and altered body shape, despite lower total body-fat percentage. Keywords: FGF21, BMI, waist-hip ratio, blood pressure, body fat, allele, genetic variant, UK Biobank

Impact of short-term high-fat feeding and insulin-stimulated FGF21 levels in subjects with low birth weight and controls

DEFF Research Database (Denmark)

Vienberg, Sara Gry; Brøns, Charlotte; Nilsson, Emma

2012-01-01

of type 2 diabetes and 26 control (normal birth weight (NBW)) young men were subjected to 5 days of high-fat (HF) overfeeding (+50%). Basal and clamp insulin-stimulated serum FGF21 levels were examined before and after the diet, and FGF21 mRNA expression was measured in muscle and fat biopsies......OBJECTIVE: Fibroblast growth factor 21 (FGF21) is a metabolic factor involved in glucose and lipid metabolism. However, little is known about the physiological role of FGF21 during a dietary challenge in humans. RESEARCH DESIGN AND METHODS: Twenty healthy low birth weight (LBW) with known risk...

Metabolic hormone FGF21 is induced in ground squirrels during hibernation but its overexpression is not sufficient to cause torpor.

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Bethany T Nelson

Full Text Available Hibernation is a natural adaptation that allows certain mammals to survive physiological extremes that are lethal to humans. Near freezing body temperatures, heart rates of 3-10 beats per minute, absence of food consumption, and depressed metabolism are characteristic of hibernation torpor bouts that are periodically interrupted by brief interbout arousals (IBAs. The molecular basis of torpor induction is unknown, however starved mice overexpressing the metabolic hormone fibroblast growth factor 21 (FGF21 promote fat utilization, reduce body temperature, and readily enter torpor-all hallmarks of mammalian hibernation. In this study we cloned FGF21 from the naturally hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus and found that levels of FGF21 mRNA in liver and FGF21 protein in serum are elevated during hibernation torpor bouts and significantly elevated during IBAs compared to summer active animals. The effects of artificially elevating circulating FGF21 concentrations 50 to 100-fold via adenoviral-mediated overexpression were examined at three different times of the year. This is the first time that a transgenic approach has been used in a natural hibernator to examine mechanistic aspects of hibernation. Surgically implanted transmitters measured various metrics of the hibernation phenotype over a 7-day period including changes in motor activity, heart rate and core body temperature. In April fed-state animals, FGF21 overexpression decreased blood insulin and free fatty acid concentrations, effects similar to those seen in obese mice. However, elevated FGF21 concentrations did not cause torpor in these fed-state animals nor did they cause torpor or affect metabolic parameters in fasted-state animals in March/April, August or October. We conclude that FGF21 is strongly regulated during torpor and IBA but that its overexpression is not sufficient to cause torpor in naturally hibernating ground squirrels.

Hepatic mTORC1 controls locomotor activity, body temperature, and lipid metabolism through FGF21

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Cornu, Marion; Oppliger, Wolfgang; Albert, Verena; Robitaille, Aaron M.; Trapani, Francesca; Quagliata, Luca; Fuhrer, Tobias; Sauer, Uwe; Terracciano, Luigi; Hall, Michael N.

2014-01-01

The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of growth and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α)–dependent fibroblast growth factor 21 (FGF21) expression in the liver. Injection of glutamine or knockdown of PGC-1α or FGF21 in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1α and FGF21. Finally, mTORC1 signaling correlated with FGF21 expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level. PMID:25082895

Additive protection by LDR and FGF21 treatment against diabetic nephropathy in type 2 diabetes model.

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Shao, Minglong; Yu, Lechu; Zhang, Fangfang; Lu, Xuemian; Li, Xiaokun; Cheng, Peng; Lin, Xiufei; He, Luqing; Jin, Shunzi; Tan, Yi; Yang, Hong; Zhang, Chi; Cai, Lu

2015-07-01

The onset of diabetic nephropathy (DN) is associated with both systemic and renal changes. Fibroblast growth factor (FGF)-21 prevents diabetic complications mainly by improving systemic metabolism. In addition, low-dose radiation (LDR) protects mice from DN directly by preventing renal oxidative stress and inflammation. In the present study, we tried to define whether the combination of FGF21 and LDR could further prevent DN by blocking its systemic and renal pathogeneses. To this end, type 2 diabetes was induced by feeding a high-fat diet for 12 wk followed by a single dose injection of streptozotocin. Diabetic mice were exposed to 50 mGy LDR every other day for 4 wk with and without 1.5 mg/kg FGF21 daily for 8 wk. The changes in systemic parameters, including blood glucose levels, lipid profiles, and insulin resistance, as well as renal pathology, were examined. Diabetic mice exhibited renal dysfunction and pathological abnormalities, all of which were prevented significantly by LDR and/or FGF21; the best effects were observed in the group that received the combination treatment. Our studies revealed that the additive renal protection conferred by the combined treatment against diabetes-induced renal fibrosis, inflammation, and oxidative damage was associated with the systemic improvement of hyperglycemia, hyperlipidemia, and insulin resistance. These results suggest that the combination treatment with LDR and FGF21 prevented DN more efficiently than did either treatment alone. The mechanism behind these protective effects could be attributed to the suppression of both systemic and renal pathways. Copyright © 2015 the American Physiological Society.

Hepatic FGF21 mediates sex differences in high-fat high-fructose diet-induced fatty liver.

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Chukijrungroat, Natsasi; Khamphaya, Tanaporn; Weerachayaphorn, Jittima; Songserm, Thaweesak; Saengsirisuwan, Vitoon

2017-08-01

The role of gender in the progression of fatty liver due to chronic high-fat high-fructose diet (HFFD) has not been studied. The present investigation assessed whether HFFD induced hepatic perturbations differently between the sexes and examined the potential mechanisms. Male, female, and ovariectomized (OVX) Sprague-Dawley rats were fed either a control diet or HFFD for 12 wk. Indexes of liver damage and hepatic steatosis were analyzed biochemically and histologically together with monitoring changes in hepatic gene and protein expression. HFFD induced a higher degree of hepatic steatosis in females, with significant increases in proteins involved in hepatic lipogenesis, whereas HFFD significantly induced liver injury, inflammation, and oxidative stress only in males. Interestingly, a significant increase in hepatic fibroblast growth factor 21 (FGF21) protein expression was observed in HFFD-fed males but not in HFFD-fed females. Ovarian hormone deprivation by itself led to a significant reduction in FGF21 with hepatic steatosis, and HFFD further aggravated hepatic fat accumulation in OVX rats. Importantly, estrogen replacement restored hepatic FGF21 levels and reduced hepatic steatosis in HFFD-fed OVX rats. Collectively, our results indicate that male rats are more susceptible to HFFD-induced hepatic inflammation and that the mechanism underlying this sex dimorphism is mediated through hepatic FGF21 expression. Our findings reveal sex differences in the development of HFFD-induced fatty liver and indicate the protective role of estrogen against HFFD-induced hepatic steatosis. Copyright © 2017 the American Physiological Society.

Ketogenic Diet Impairs FGF21 Signaling and Promotes Differential Inflammatory Responses in the Liver and White Adipose Tissue.

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Mohamed Asrih

Full Text Available Beside its beneficial effects on weight loss, ketogenic diet (KD causes dyslipidemia, a pro-inflammatory state involved in the development of hepatic steatosis, glucose intolerance and insulin resistance, although the latter is still being debated. Additionally, KD is known to increase fibroblast growth factor 21 (FGF21 plasma levels. However, FGF21 cannot initiate its beneficial actions on metabolism in these conditions. We therefore hypothesized and tested in the present study that KD may impair FGF21 signaling.Using indirect calorimetry, we found that KD-fed mice exhibited higher energy expenditure than regular chow (RC-fed mice associated with increased Ucp1 levels in white adipose tissue (WAT, along with increased plasma FGF21 levels. We then assessed the effect of KD on FGF21 signaling in both the liver and WAT. We found that Fgfr4 and Klb (β-klotho were downregulated in the liver, while Fgfr1 was downregulated in WAT of KD-fed mice. Because inflammation could be one of the mechanisms linking KD to impaired FGF21 signaling, we measured the expression levels of inflammatory markers and macrophage accumulation in WAT and liver and found an increased inflammation and macrophage accumulation in the liver, but surprisingly, a reduction of inflammation in WAT.We also showed that KD enhances lipid accumulation in the liver, which may explain hepatic inflammation and impaired Fgfr4 and Klb expression. In contrast, import of lipids from the circulation was significantly reduced in WAT of KD-fed mice, as suggested by a downregulation of Lpl and Cd36. This was further associated with reduced inflammation in WAT.Altogether, these results indicate that KD could be beneficial for a given tissue but deleterious for another.

Fibroblast Growth Factor (FGF) 23 Regulates the Plasma Levels of Parathyroid Hormone In Vivo Through the FGF Receptor in Normocalcemia, But Not in Hypocalcemia

DEFF Research Database (Denmark)

Mace, Maria L; Gravesen, Eva; Nordholm, Anders

2018-01-01

hypocalcemia. We demonstrated that FGF23 rapidly inhibited PTH secretion and that this effect was completely blocked by inhibition of the FGF receptor. Furthermore, inhibition of the FGF receptor by itself significantly increased PTH levels, indicating that FGF23 has a suppressive tonus on the parathyroid...... gland's PTH secretion. In acute hypocalcemia, there was no effect of either recombinant FGF23 or FGF receptor inhibition on the physiological response to the low ionized calcium levels. In conclusion, FGF23 has an inhibitory tonus on PTH secretion in normocalcemia and signals through the FGF receptor....... In acute hypocalcemia, when increased PTH secretion is needed to restore the calcium homeostasis, this inhibitory effect of FGF23 is abolished....

Liraglutide increases FGF-21 activity and insulin sensitivity in high fat diet and adiponectin knockdown induced insulin resistance.

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Mengliu Yang

Full Text Available BACKGROUND: Liraglutide is a glucagon-like peptide-1 analogue that stimulates insulin secretion and improves β-cell function. However, it is not clear whether liraglutide achieves its glucose lowering effect only by its known effects or whether other as yet unknown mechanisms are involved. The aim of this study was to examine the effects of liraglutide on Fibroblast growth factor-21 (FGF-21 activity in High-fat diet (HFD fed ApoE(-/- mice with adiponectin (Acrp30 knockdown. METHOD: HFD-fed ApoE(-/- mice were treated with adenovirus vectors expressing shAcrp30 to produce insulin resistance. Hyperinsulinemic-euglycemic clamp studies were performed to evaluate insulin sensitivity of the mouse model. QRT-PCR and Western blot were used to measure the mRNA and protein expression of the target genes. RESULTS: The combination of HFD, ApoE deficiency, and hypoadiponectinemia resulted in an additive effect on insulin resistance. FGF-21 mRNA expressions in both liver and adipose tissues were significantly increased while FGF-21 receptor 1 (FGFR-1 and β-Klotho mRNA levels in adipose tissue, as well as FGFR-1-3 and β-Klotho mRNA levels in liver were significantly decreased in this model. Liraglutide treatment markedly improved insulin resistance and increased FGF-21 expression in liver and FGFR-3 in adipose tissue, restored β-Klotho mRNA expression in adipose tissue as well as FGFR-1-3, β-Klotho levels and phosphorylation of FGFR1 up to the levels observed in control mice in liver. Liraglutide treatment also further increased FGF-21 proteins in liver and plasma. In addition, as shown by hyperinsulinemic-euglycemic clamp, liraglutide treatment also markedly improved glucose metabolism and insulin sensitivity in these animals. CONCLUSION: These findings demonstrate an additive effect of HFD, ApoE deficiency, and adiponectin knockdown on insulin resistance and unveil that the regulation of glucose metabolism and insulin sensitivity by liraglutide may be

Alterations in Hepatic FGF21, Co-Regulated Genes, and Upstream Metabolic Genes in Response to Nutrition, Ketosis and Inflammation in Peripartal Holstein Cows.

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Haji Akbar

Full Text Available In rodents, fibroblast growth factor 21 (FGF21 has emerged as a key metabolic regulator produced by liver. To gather preliminary data on the potential importance of FGF1, co-regulated genes, and upstream metabolic genes, we examined the hepatic mRNA expression in response to nutrition and inflammation in dairy cows. In experiment 1, induction of ketosis through feed restriction on d 5 postpartum upregulated FGF21, its co-receptor KLB, and PPARA but only elicited a numerical increase in serum FGF21 concentration. In experiment 2, cows in control (CON or receiving 50 g/d of L-carnitine (C50 from -14 through 21 d had increased FGF21, PPARA, and NFIL3 on d 10 compared with d 2 postpartum. In contrast, compared with CON and C50, 100 g/d L-carnitine (C100 resulted in lower FGF21, KLB, ANGPTL4, and ARNTL expression on d 10. In experiment 3, cows were fed during the dry period either a higher-energy (OVE; 1.62 Mcal/kg DM or lower-energy (CON; 1.34 Mcal/kg DM diet and received 0 (OVE:N, CON:N or 200 μg of LPS (OVE:Y, CON:Y into the mammary gland at d 7 postpartum. For FGF21 mRNA expression in CON, the LPS challenge (CON:Y prevented a decrease in expression between d 7 and 14 postpartum such that cows in CON:N had a 4-fold lower expression on d 14 compared with d 7. The inflammatory stimulus induced by LPS in CON:Y resulted in upregulation of PPARA on d 14 to a similar level as cows in OVE:N. In OVE:Y, expression of PPARA was lower than CON:N on d 7 and remained unchanged on d 14. On d 7, LPS led to a 4-fold greater serum FGF21 only in OVE but not in CON cows. In fact, OVE:Y reached the same serum FGF21 concentration as CON:N, suggesting a carryover effect of dietary energy level on signaling mechanisms within liver. Overall, results indicate that nutrition, ketosis, and inflammation during the peripartal period can alter hepatic FGF21, co-regulated genes, and upstream metabolic genes to various extents. The functional outcome of these changes merits

Alterations in Hepatic FGF21, Co-Regulated Genes, and Upstream Metabolic Genes in Response to Nutrition, Ketosis and Inflammation in Peripartal Holstein Cows.

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Akbar, Haji; Batistel, Fernanda; Drackley, James K; Loor, Juan J

2015-01-01

In rodents, fibroblast growth factor 21 (FGF21) has emerged as a key metabolic regulator produced by liver. To gather preliminary data on the potential importance of FGF1, co-regulated genes, and upstream metabolic genes, we examined the hepatic mRNA expression in response to nutrition and inflammation in dairy cows. In experiment 1, induction of ketosis through feed restriction on d 5 postpartum upregulated FGF21, its co-receptor KLB, and PPARA but only elicited a numerical increase in serum FGF21 concentration. In experiment 2, cows in control (CON) or receiving 50 g/d of L-carnitine (C50) from -14 through 21 d had increased FGF21, PPARA, and NFIL3 on d 10 compared with d 2 postpartum. In contrast, compared with CON and C50, 100 g/d L-carnitine (C100) resulted in lower FGF21, KLB, ANGPTL4, and ARNTL expression on d 10. In experiment 3, cows were fed during the dry period either a higher-energy (OVE; 1.62 Mcal/kg DM) or lower-energy (CON; 1.34 Mcal/kg DM) diet and received 0 (OVE:N, CON:N) or 200 μg of LPS (OVE:Y, CON:Y) into the mammary gland at d 7 postpartum. For FGF21 mRNA expression in CON, the LPS challenge (CON:Y) prevented a decrease in expression between d 7 and 14 postpartum such that cows in CON:N had a 4-fold lower expression on d 14 compared with d 7. The inflammatory stimulus induced by LPS in CON:Y resulted in upregulation of PPARA on d 14 to a similar level as cows in OVE:N. In OVE:Y, expression of PPARA was lower than CON:N on d 7 and remained unchanged on d 14. On d 7, LPS led to a 4-fold greater serum FGF21 only in OVE but not in CON cows. In fact, OVE:Y reached the same serum FGF21 concentration as CON:N, suggesting a carryover effect of dietary energy level on signaling mechanisms within liver. Overall, results indicate that nutrition, ketosis, and inflammation during the peripartal period can alter hepatic FGF21, co-regulated genes, and upstream metabolic genes to various extents. The functional outcome of these changes merits further study

Effects of EPA and lipoic acid supplementation on circulating FGF21 and the fatty acid profile in overweight/obese women following a hypocaloric diet.

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Escoté, Xavier; Félix-Soriano, Elisa; Gayoso, Lucía; Huerta, Ana Elsa; Alvarado, María Antonella; Ansorena, Diana; Astiasarán, Iciar; Martínez, J Alfredo; Moreno-Aliaga, María Jesús

2018-05-23

FGF21 has emerged as a key metabolism and energy homeostasis regulator. Dietary supplementation with eicosapentaenoic acid (EPA) and/or α-lipoic acid (LIP) has shown beneficial effects on obesity. In this study, we evaluated EPA and/or LIP effects on plasma FGF21 and the fatty acid (FA) profile in overweight/obese women following hypocaloric diets. At the baseline, FGF21 levels were negatively related to the AST/ALT ratio and HMW adiponectin. The weight loss did not cause any significant changes in FGF21 levels, but after the intervention FGF21 increased in EPA-supplemented groups compared to non-EPA-supplemented groups. EPA supplementation decreased the plasma n-6-PUFA content and increased n-3-PUFAs, mainly EPA and DPA, but not DHA. In the LIP-alone supplemented group a decrease in the total SFA and n-6-PUFA content was observed after the supplementation. Furthermore, EPA affected the desaturase activity, lowering Δ4D and raising Δ5/6D. These effects were not observed in the LIP-supplemented groups. Besides, the changes in FGF21 levels were associated with the changes in EPA, n-3-PUFAs, Δ5/6D, and n-6/n-3 PUFA ratio. Altogether, our study suggests that n-3-PUFAs influence FGF21 levels in obesity, although the specific mechanisms implicated remain to be elucidated.

Circulating FGF21 levels are progressively increased from the early to end stages of chronic kidney diseases and are associated with renal function in Chinese.

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Zhuofeng Lin

Full Text Available BACKGROUND: Fibroblast growth factor 21 (FGF21 is a hepatic hormone involved in the regulation of lipid and carbohydrate metabolism. This study aims to test the hypothesis that elevated FGF21 concentrations are associated with the change of renal function and the presence of left ventricular hypertrophy (LVH in the different stages of chronic kidney disease (CKD progression. METHODOLOGY/PRINCIPAL FINDINGS: 240 subjects including 200 CKD patients (146 outpatients and 54 long-term hemodialytic patients and 40 healthy control subjects were recruited. All CKD subjects underwent echocardiograms to assess left ventricular mass index. Plasma FGF21 levels and other clinical and biochemical parameters in all subjects were obtained based on standard clinical examination methods. Plasma FGF21 levels were significantly increased with the development of CKD from early- and end-stage (P<0.001 for trend, and significantly higher in CKD subjects than those in healthy subjects (P<0.001. Plasma FGF21 levels in CKD patients with LVH were higher than those in patients without LVH (P = 0.001. Furthermore, plasma FGF21 level correlated positively with creatinine, blood urea nitrogen (BUN, β2 microglobulin, systolic pressure, adiponectin, phosphate, proteinuria, CRP and triglyceride, but negatively with creatinine clearance rate (CCR, estimated glomerular filtrate rate (eGFR, HDL-c, LDL-c, albumin and LVH after adjusting for BMI, gender, age and the presence of diabetes mellitus. Multiple stepwise regression analyses indicated that FGF21 was independently associated with BUN, Phosphate, LVMI and β2 microglobulin (all P<0.05. CONCLUSION: Plasma FGF21 levels are significantly increased with the development of early- to end-stage CKD and are independently associated with renal function and adverse lipid profiles in Chinese population. Understanding whether increased FGF21 is associated with myocardial hypertrophy in CKD requires further study.

Fibroblast Growth Factor 21 Deficiency Attenuates Experimental Colitis-Induced Adipose Tissue Lipolysis

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Liming Liu

2017-01-01

Full Text Available Aims. Nutrient deficiencies are common in patients with inflammatory bowel disease (IBD. Adipose tissue plays a critical role in regulating energy balance. Fibroblast growth factor 21 (FGF21 is an important endocrine metabolic regulator with emerging beneficial roles in lipid homeostasis. We investigated the impact of FGF21 in experimental colitis-induced epididymal white adipose tissue (eWAT lipolysis. Methods. Mice were given 2.5% dextran sulfate sodium (DSS ad libitum for 7 days to induce colitis. The role of FGF21 was investigated using antibody neutralization or knockout (KO mice. Lipolysis index and adipose lipolytic enzymes were determined. In addition, 3T3-L1 cells were pretreated with IL-6, followed by recombinant human FGF21 (rhFGF21 treatment; lipolysis was assessed. Results. DSS markedly decreased eWAT/body weight ratio and increased serum concentrations of free fatty acid (FFA and glycerol, indicating increased adipose tissue lipolysis. eWAT intracellular lipolytic enzyme expression/activation was significantly increased. These alterations were significantly attenuated in FGF21 KO mice and by circulating FGF21 neutralization. Moreover, DSS treatment markedly increased serum IL-6 and FGF21 levels. IL-6 pretreatment was necessary for the stimulatory effect of FGF21 on adipose lipolysis in 3T3-L1 cells. Conclusions. Our results demonstrate that experimental colitis induces eWAT lipolysis via an IL-6/FGF21-mediated signaling pathway.

GCN2 and FGF21 are likely mediators of the protection from cancer, autoimmunity, obesity, and diabetes afforded by vegan diets.

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McCarty, Mark F

2014-09-01

Third World quasi-vegan cultures have been characterized by low risks for "Western" cancers, autoimmune disorders, obesity, and diabetes. The relatively low essential amino acid contents of many vegan diets may play a role in this regard. It is proposed that such diets modestly activate the kinase GCN2 - a physiological detector of essential amino acid paucity - within the liver, resulting in up-regulated production of fibroblast growth factor 21 (FGF21). FGF21, by opposing the stimulatory effect of growth hormone on hepatic IGF-I production, may be responsible for the down-regulation of plasma IGF-I observed in vegans consuming diets of modest protein content. Decreased IGF-I bioactivity throughout life can be expected to have a favorable impact on cancer risk, as observed in rodents that are calorie restricted or genetically defective in IGF-I activity. Increased FGF21 in vegans might also contribute to their characteristic leanness and low LDL cholesterol by promoting hepatic lipid oxidation while inhibiting lipogenesis. Direct trophic effects of FGF21 on pancreatic beta-cells may help to explain the low risk for diabetes observed in vegans, and the utility of vegan diets in diabetes management. And up-regulation of GCN2 in immune cells, by boosting T regulatory activity, might play some role in the reduced risk for autoimmunity reported in some quasi-vegan cultures. The fact that bone density tends to be no greater in vegans than omnivores, despite consumption of a more "alkaline" diet, might be partially attributable to the fact that FGF21 opposes osteoblastogenesis and decreases IGF-I. If these speculations have merit, it should be possible to demonstrate that adoption of a vegan diet of modest protein content increases plasma FGF21 levels. Copyright © 2014 Elsevier Ltd. All rights reserved.

α-Trinositol inhibits FGF-stimulated growth of smooth muscle and breast cancer cells

International Nuclear Information System (INIS)

Siren, Matti J.; Vainiomaeki, Maija; Vaeaenaenen, Kalervo; Haerkoenen, Pirkko

2004-01-01

α-Trinositol (D-myo-inositol-1,2,6-trisphosphate), an isomer of the intracellular messenger IP 3 , has been studied for its anti-inflammatory and other effects in animal experiments and in human. The mechanisms of action remain unknown. Several human pathologies are associated with uncontrolled production of fibroblast growth factors (FGFs). FGF-2 induces vascular smooth muscle cell proliferation, which contributes to restenosis after coronary balloon angioplasty. The expression of several FGFs is also increased in tumors. We studied the effects of the water- and lipid-soluble derivatives of α-trinositol on the FGF-2- and/or FGF-8-induced proliferation of human pulmonary artery smooth muscle cells (HPASMC) and S115 mouse breast cancer cells. α-Trinositol decreased the FGF-mediated proliferation of HPASMC and S115 cells. Membrane permeability did not seem obligatory since the lipid-soluble form of α-trinositol was less effective than the water-soluble derivative. These results suggest a new biological function for certain phosphoinositides in the modulation of FGF-regulated processes

PF-05231023, a long-acting FGF21 analogue, decreases body weight by reduction of food intake in non-human primates.

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Thompson, W Clayton; Zhou, Yingjiang; Talukdar, Saswata; Musante, Cynthia J

2016-08-01

PF-05231023, a long-acting FGF21 analogue, is a promising potential pharmacotherapy for the treatment of obesity and associated comorbidities. Previous studies have shown the potential of FGF21 and FGF21-like compounds to decrease body weight in mice, non-human primates, and humans; the precise mechanisms of action remain unclear. In particular, there have been conflicting reports on the degree to which FGF21-induced weight loss in non-human primates is attributable to a decrease in food intake versus an increase in energy expenditure. Here, we present a semi-mechanistic mathematical model of energy balance and body composition developed from similar work in mice. This model links PF-05231023 administration and washout to changes in food intake, which in turn drives changes in body weight. The model is calibrated to and compared with recently published data from cynomolgus macaques treated with PF-05231023, demonstrating its accuracy in describing pharmacotherapy-induced weight loss in these animals. The results are consistent with the hypothesis that PF-05231023 decreases body weight in cynomolgus macaques solely by a reduction in food intake, with no direct effect on energy expenditure.

Exercise-Induced Secretion of FGF21 and Follistatin Are Blocked by Pancreatic Clamp and Impaired in Type 2 Diabetes

DEFF Research Database (Denmark)

Hansen, Jakob Schiøler; Pedersen, Bente Klarlund; Xu, Guowang

2016-01-01

blocking the increase in the glucagon to insulin ratio. In addition, we evaluated exercise-induced plasma FGF21 and follistatin in patients with T2D compared with healthy controls in response to 1 hour of bicycle exercise followed by a 3-hour recovery period. RESULTS: In healthy individuals, we observed......CONTEXT: Hepatokines have emerged as liver-derived hormone-like factors. Plasma fibroblast growth factor (FGF)-21 and follistatin increase with a high glucagon to insulin ratio and exercise, and resting levels are elevated in patients with type 2 diabetes (T2D). OBJECTIVE: The objective...... of the study was to investigate the regulatory roles of glucagon to insulin ratio and T2D on exercise-induced FGF21 and follistatin secretion. Design /Interventions: Young healthy males performed a 2-hour bicycle exercise bout followed by 5 hours of rest in supine position with and without a pancreatic clamp...

High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

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Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

2018-02-01

Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

The moderate essential amino acid restriction entailed by low-protein vegan diets may promote vascular health by stimulating FGF21 secretion.

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McCarty, Mark F

2016-02-12

The serum total and LDL cholesterol levels of long-term vegans tend to be very low. The characteristically low ratio of saturated to unsaturated fat in vegan diets, and the absence of cholesterol in such diets, clearly contribute to this effect. But there is reason to suspect that the quantity and composition of dietary protein also play a role in this regard. Vegan diets of moderate protein intake tend to be relatively low in certain essential amino acids, and as a result may increase hepatic activity of the kinase GCN2, which functions as a gauge of amino acid status. GCN2 activation boosts the liver's production of fibroblast growth factor 21 (FGF21), a factor which favorably affects serum lipids and metabolic syndrome. The ability of FGF21 to decrease LDL cholesterol has now been traced to at least two mechanisms: a suppression of hepatocyte expression of sterol response element-binding protein-2 (SREBP-2), which in turn leads to a reduction in cholesterol synthesis; and up-regulated expression of hepatocyte LDL receptors, reflecting inhibition of a mechanism that promotes proteasomal degradation of these receptors. In mice, the vascular benefits of FGF21 are also mediated by favorable effects on adipocyte function - most notably, increased adipocyte secretion of adiponectin, which directly exerts anti-inflammatory effects on the vasculature which complement the concurrent reduction in LDL particles in preventing or reversing atherosclerosis. If, as has been proposed, plant proteins preferentially stimulate glucagon secretion owing to their amino acid composition, this would represent an additional mechanism whereby plant protein promotes FGF21 activity, as glucagon acts on the liver to boost transcription of the FGF21 gene.

Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

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Ha, Jang-Ho; Kim, Ha-Neul; Moon, Ki-Beom; Jeon, Jae-Heung; Jung, Dai-Hyun; Kim, Su-Jung; Mason, Hugh S; Shin, Seo-Yeon; Kim, Hyun-Soon; Park, Kyung-Mok

2017-07-01

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana . Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana . The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana . The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana -derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli -derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana- derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana -derived recombinant human acidic fibroblast growth factor

Macronutrient Intake-Associated FGF21 Genotype Modifies Effects of Weight-Loss Diets on 2-Year Changes of Central Adiposity and Body Composition: The POUNDS Lost Trial.

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Heianza, Yoriko; Ma, Wenjie; Huang, Tao; Wang, Tiange; Zheng, Yan; Smith, Steven R; Bray, George A; Sacks, Frank M; Qi, Lu

2016-11-01

Fibroblast growth factor 21 (FGF21) is involved in the regulation of energy balance and adipose metabolism. Our previous genome-wide association study identified genetic variants in the FGF21 region associated with macronutrient intake preference. We investigated whether the FGF21 genotype modified effects of weight-loss diets varying in macronutrient intake on changes in adiposity in a 2-year randomized diet intervention trial. We genotyped FGF21 rs838147 in 715 overweight or obese individuals who were assigned to one of four diets varying in macronutrient contents. A DEXA scan was performed to evaluate body composition. We observed a significant interaction between the FGF21 genotype and carbohydrate/fat intake on 2-year changes in waist circumference (WC), percentage of total fat mass, and percentage of trunk fat (P = 0.049, P = 0.001, and P = 0.003 for interaction, respectively). In response to the low-carbohydrate/high-fat diet, carrying the carbohydrate intake-decreasing C allele of rs838147 was marginally associated with less reduction in WC (P = 0.08) and significantly associated with less reduction of total fat mass (P = 0.01) and trunk fat (P = 0.02). Opposite genetic associations with these outcomes were observed among the high-carbohydrate/low-fat diet group; carrying the C allele was associated with a greater reduction of WC, total body fat mass, and trunk fat. Our data suggest that FGF21 genotypes may interact with dietary carbohydrate/fat intake on changes in central adiposity and body fat composition. A low-calorie, high-carbohydrate/low-fat diet was beneficial for overweight or obese individuals carrying the carbohydrate intake-decreasing allele of the FGF21 variant to improve body composition and abdominal obesity. © 2016 by the American Diabetes Association.

The FGF21 response to fructose predicts metabolic health and persists after bariatric surgery in obese humans

NARCIS (Netherlands)

ter Horst, Kasper W.; Gilijamse, Pim W.; Demirkiran, Ahmet; van Wagensveld, Bart A.; Ackermans, Mariette T.; Verheij, Joanne; Romijn, Johannes A.; Nieuwdorp, Max; Maratos-Flier, Eleftheria; Herman, Mark A.; Serlie, Mireille J.

2017-01-01

Objective: Fructose consumption has been implicated in the development of obesity and insulin resistance. Emerging evidence shows that fibroblast growth factor 21 (FGF21) has beneficial effects on glucose, lipid, and energy metabolism and may also mediate an adaptive response to fructose ingestion.

OPA1 deficiency promotes secretion of FGF21 from muscle that prevents obesity and insulin resistance.

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Pereira, Renata Oliveira; Tadinada, Satya M; Zasadny, Frederick M; Oliveira, Karen Jesus; Pires, Karla Maria Pereira; Olvera, Angela; Jeffers, Jennifer; Souvenir, Rhonda; Mcglauflin, Rose; Seei, Alec; Funari, Trevor; Sesaki, Hiromi; Potthoff, Matthew J; Adams, Christopher M; Anderson, Ethan J; Abel, E Dale

2017-07-14

Mitochondrial dynamics is a conserved process by which mitochondria undergo repeated cycles of fusion and fission, leading to exchange of mitochondrial genetic content, ions, metabolites, and proteins. Here, we examine the role of the mitochondrial fusion protein optic atrophy 1 (OPA1) in differentiated skeletal muscle by reducing OPA1 gene expression in an inducible manner. OPA1 deficiency in young mice results in non-lethal progressive mitochondrial dysfunction and loss of muscle mass. Mutant mice are resistant to age- and diet-induced weight gain and insulin resistance, by mechanisms that involve activation of ER stress and secretion of fibroblast growth factor 21 (FGF21) from skeletal muscle, resulting in increased metabolic rates and improved whole-body insulin sensitivity. OPA1-elicited mitochondrial dysfunction activates an integrated stress response that locally induces muscle atrophy, but via secretion of FGF21 acts distally to modulate whole-body metabolism. © 2017 The Authors.

A Novel Role of SIRT1/ FGF-21 in Taurine Protection Against Cafeteria Diet-Induced Steatohepatitis in Rats

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Azza H. Abd Elwahab

2017-09-01

Full Text Available Background: Non-alcoholic fatty liver disease (NAFLD is one of the alarmingly rising clinical problems in the 21st century with no effective drug treatment until now. Taurine is an essential amino acid in humans that proved efficacy as a non-pharmacological therapy in a plethora of diseases; however, its impact on NAFLD remains elusive. The aim of the current study is to evaluate the protective mechanism of taurine in experimental steatohepatitis induced by junk food given as cafeteria-diet (CAF-D in male albino rats. Methods: Forty adult male albino rats of local strain between 8-10 weeks old, weighing 150 ± 20 g, were divided into four equal groups: Group I (control group, Group II (Taurine group, Group III (CAF-D for 12 weeks and Group IV (CAF-D +Taurine. CAF-D was given in addition to the standard chow for 12 weeks, where each rat was given one piece of beef burger fried in 15 g of sunflower oil, one teaspoonful of mayonnaise, and one piece of petit pan bread, weighing 60g/ piece. In the serum, liver function tests; ALT, AST, ALP, GGT and the lipid profile; TG, TC, HDL-C added to reduced glutathione (GSH were assessed colorimetrically, while fibroblast growth factor (FGF-21, adiponectin & interleukin (IL-6 via ELISA. The same technique was used for the assays of the hepatic levels of FGF-21, silent information regulator (SIRT1, malondialdehyde (MDA,IL-10, tumor necrosis factor-α (TNF-α as well as the apoptotic markers; caspase-3 and B-cell lymphoma (Bcl-2. Results: The cafeteria-diet induced steatohepatitis was reflected by significantly increased body and liver weight gain, elevation of liver enzymes; ALT, AST, ALP and GGT added to the dyslipidemic panel, presented as increased TC, TG, LDL-C and decreased HDL-C levels. The steatosis-induced inflammatory milieu, marked by elevated serum levels of FGF-21, IL-6, hepatic TNF-α, as well as reduced IL-10 and adiponectin, was associated with steatosis- induced hepatic oxidative stress

A Novel Role of SIRT1/ FGF-21 in Taurine Protection Against Cafeteria Diet-Induced Steatohepatitis in Rats.

Science.gov (United States)

Abd Elwahab, Azza H; Ramadan, Basma K; Schaalan, Mona F; Tolba, Amina M

2017-01-01

Non-alcoholic fatty liver disease (NAFLD) is one of the alarmingly rising clinical problems in the 21st century with no effective drug treatment until now. Taurine is an essential amino acid in humans that proved efficacy as a non-pharmacological therapy in a plethora of diseases; however, its impact on NAFLD remains elusive. The aim of the current study is to evaluate the protective mechanism of taurine in experimental steatohepatitis induced by junk food given as cafeteria-diet (CAF-D) in male albino rats. Forty adult male albino rats of local strain between 8-10 weeks old, weighing 150 ± 20 g, were divided into four equal groups: Group I (control group), Group II (Taurine group), Group III (CAF-D for 12 weeks) and Group IV (CAF-D +Taurine). CAF-D was given in addition to the standard chow for 12 weeks, where each rat was given one piece of beef burger fried in 15 g of sunflower oil, one teaspoonful of mayonnaise, and one piece of petit pan bread, weighing 60g/ piece. In the serum, liver function tests; ALT, AST, ALP, GGT and the lipid profile; TG, TC, HDL-C added to reduced glutathione (GSH) were assessed colorimetrically, while fibroblast growth factor (FGF)-21, adiponectin & interleukin (IL)-6 via ELISA. The same technique was used for the assays of the hepatic levels of FGF-21, silent information regulator (SIRT1), malondialdehyde (MDA),IL-10, tumor necrosis factor-α (TNF-α) as well as the apoptotic markers; caspase-3 and B-cell lymphoma (Bcl-2). The cafeteria-diet induced steatohepatitis was reflected by significantly increased body and liver weight gain, elevation of liver enzymes; ALT, AST, ALP and GGT added to the dyslipidemic panel, presented as increased TC, TG, LDL-C and decreased HDL-C levels. The steatosis-induced inflammatory milieu, marked by elevated serum levels of FGF-21, IL-6, hepatic TNF-α, as well as reduced IL-10 and adiponectin, was associated with steatosis- induced hepatic oxidative stress, reflected by increased hepatic MDA and

FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation

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Pirou, Caroline; Montazer-Torbati, Fatemeh; Jah, Nadège; Delmas, Elisabeth; Lasbleiz, Christelle; Mignotte, Bernard; Renaud, Flore

2017-01-01

Neuroblastoma, a sympathetic nervous system tumor, accounts for 15% of cancer deaths in children. In contrast to most human tumors, p53 is rarely mutated in human primary neuroblastoma, suggesting impaired p53 activation in neuroblastoma. Various studies have shown correlations between fgf1 expression levels and both prognosis severity and tumor chemoresistance. As we previously showed that fibroblast growth factor 1 (FGF1) inhibited p53-dependent apoptosis in neuron-like PC12 cells, we initiated the study of the interaction between the FGF1 and p53 pathways in neuroblastoma. We focused on the activity of either extracellular FGF1 by adding recombinant rFGF1 in media, or of intracellular FGF1 by overexpression in human SH-SY5Y and mouse N2a neuroblastoma cell lines. In both cell lines, the genotoxic drug etoposide induced a classical mitochondrial p53-dependent apoptosis. FGF1 was able to inhibit p53-dependent apoptosis upstream of mitochondrial events in SH-SY5Y cells by both extracellular and intracellular pathways. Both rFGF1 addition and etoposide treatment increased fgf1 expression in SH-SY5Y cells. Conversely, rFGF1 or overexpressed FGF1 had no effect on p53-dependent apoptosis and fgf1 expression in neuroblastoma N2a cells. Using different FGF1 mutants (that is, FGF1K132E, FGF1S130A and FGF1S130D), we further showed that the C-terminal domain and phosphorylation of FGF1 regulate its intracrine anti-apoptotic activity in neuroblastoma SH-SY5Y cells. This study provides the first evidence for a role of an intracrine growth factor pathway on p53-dependent apoptosis in neuroblastoma, and could lead to the identification of key regulators involved in neuroblastoma tumor progression and chemoresistance. PMID:29048426

Diagnostic Value of CK-18, FGF-21, and Related Biomarker Panel in Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis

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Lei He

2017-01-01

Full Text Available Liver biopsy still remains the gold standard for diagnosing nonalcoholic steatohepatitis (NASH, but with limitations. There is an urgent need to develop noninvasive tests that accurately distinguish NASH from simple steatosis. The purpose of this meta-analysis was to evaluate the diagnostic value of serum biomarkers including cytokeratin 18 (CK-18, fibroblast growth factor 21 (FGF-21, and combined biomarker panel (CBP in the diagnosis of NAFLD, especially NASH. A total of 25 studies met the inclusion criteria. Pooled sensitivity and specificity values for chosen serum markers for diagnosing NASH are as follows: CK-18 (M30, 0.75 and 0.77; CK-18 (M65, 0.71 and 0.77; FGF-21, 0.62 and 0.78; and CBP, 0.92 and 0.85. CBP demonstrated better accuracy with higher sensitivity and specificity than those tested individually. Furthermore, the AUROC of CBP was 0.94 (95% CI, 0.92–0.96, compared to CK-18 or FGF-21 assay, which showed the most significant ability to distinguish NASH from simple steatosis. The results suggest that increased circulating CK-18 and FGF-21 are associated with NASH and may be used for initial assessment, but not enough. Importantly, CBP is potentially used as accurate diagnostic tools for NASH. Further prospective designed studies are warranted to confirm our findings.

Rational design of a fibroblast growth factor 21-based clinical candidate, LY2405319.

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Alexei Kharitonenkov

Full Text Available Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21 has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP, which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO mice over 7-14 days resulted in a 25-50% lowering of plasma glucose coupled with a 10-30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.

The Functional Quality of Soluble Recombinant Polypeptides Produced in Escherichia coli Is Defined by a Wide Conformational Spectrum▿

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Martínez-Alonso, Mónica; González-Montalbán, Nuria; García-Fruitós, Elena; Villaverde, Antonio

2008-01-01

We have observed that a soluble recombinant green fluorescent protein produced in Escherichia coli occurs in a wide conformational spectrum. This results in differently fluorescent protein fractions in which morphologically diverse soluble aggregates abound. Therefore, the functional quality of soluble versions of aggregation-prone recombinant proteins is defined statistically rather than by the prevalence of a canonical native structure. PMID:18836021

Curative effect of Sodium hyaluronate and bFGF eye drops after corneal rust foreign body removal operation

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Jin-Xia Li1

2013-10-01

Full Text Available AIM: To observe the combined effect of Sodium hyaluronate eye drops and recombinant bovine basic fibroblast growth factor(bFGFeye drops on cornea epithelial repair after corneal rust foreign body extraction. METHODS: Alinety-eight cases(98 eyesof corneal rust foreign body patients were randomly distributed to combined treatment group(49 cases, 49 eyesand control group(49 cases, 49 eyes. Hyaluronate eye drops, recombinant bFGF eye drops and levofloxacin hydrochloride were applied in combined treatment group after corneal foreign body extraction. Recombinant bFGF eye drops and levofloxacin hydrochloride were applied in control group. Corneal fluorescein stain, cornea epithelial repair and local symptoms were examined thrice weekly for 2 weeks. RESULTS: General effective rate of treatment in combined group reach 96%, significantly higher than that in control group(88%, PCONCLUSION: Combined application of sodium hyaluronate eye drops and recombinant bFGF eye drops can prominently improve cornea epithelial repair after corneal lesion with proven effectiveness and safety.

Ileal Transposition Surgery Decreases Fat Mass and Improves Glucose Metabolism in Diabetic GK Rats: Possible Involvement of FGF21

Directory of Open Access Journals (Sweden)

Kemin Yan

2018-03-01

Full Text Available Objective: Ileal transposition (IT surgery has been reported to improve glucose and lipid metabolism, and fibroblast growth factor 21 (FGF21 is a powerful metabolic regulator. In the present study, we aimed to investigate the effects of IT surgery on metabolism and its possible relationship with the FGF21 signaling pathway in diabetic Goto-Kakizaki (GK rats.Methods: Ten-week-old male GK rats were subjected to IT surgery with translocation of a 10 cm ileal segment to the proximal jejunum (IT group or sham surgery without the ileum transposition (Sham-IT group. Rats in the no surgery group did not receive any surgical intervention. Six weeks later, body weight, fat mass, fasting blood glucose (FBG, and serum levels of FGF21 and leptin were measured. The expression of the FGF21 signaling pathway and white adipose tissue (WAT browning-related genes in the WAT and liver were evaluated by real-time reverse transcription polymerase chain reaction (RT-qPCR and western blot.Results: IT surgery significantly decreased the body weights and FBG levels and increased the insulin sensitivity of GK rats. The total WAT mass of the IT rats showed a 41.5% reduction compared with the Sham-IT rats, and serum levels of FGF21 and leptin of the IT rats decreased by 26.3 and 61.7%, respectively (all P < 0.05. The mRNA levels of fibroblast growth factor receptor 1 (FGFR1 and its co-receptor β klotho (KLB in the perirenal WAT (pWAT of the IT rats were 1.4- and 2.4-fold that of the Sham-IT rats, respectively, and the FGFR1 protein levels were 1.7-fold of the Sham-IT rats (all P < 0.05. In accordance with the pWAT, the protein levels of FGFR1 and KLB in the epididymal WAT (eWAT of the IT rats notably increased to 3.0- and 3.9-fold of the Sham-IT rats (P < 0.05. Furthermore, uncoupling protein 1 (UCP1 protein levels in the eWAT and pWAT of the IT rats also increased to 2.2- and 2.3-fold of the Sham-IT rats (P < 0.05. However, the protein levels of FGFR1 and KLB in the

Interplay between FGF21 and insulin action in the liver regulates metabolism

DEFF Research Database (Denmark)

Emanuelli, Brice; Vienberg, Sara G; Smyth, Graham

2014-01-01

gluconeogenesis in these animals. Improvements in blood sugar were due in part to increased glucose uptake in brown fat, browning of white fat, and overall increased energy expenditure. These effects were preserved even after removal of the main interscapular brown fat pad. In contrast to its retained effects...... of insulin action in the liver by increasing energy metabolism via activation of brown fat and browning of white fat, but intact liver insulin action is required for FGF21 to control hepatic lipid metabolism....

Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

Energy Technology Data Exchange (ETDEWEB)

Liu, Yanlong [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Chunhong [Second Hospital, Jilin University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Yuhua [College of Food Science and Engineering, Jilin Agricultural University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Ma, Zhenhua [First Hospital, Xi' an Jiaotong University, Xi' an (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); McClain, Craig [Department of Medicine, University of Louisville, Louisville, KY (United States); Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States); Robley Rex Veterans Affairs Medical Center, Louisville, KY (United States); Li, Xiaokun [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Feng, Wenke, E-mail: wenke.feng@louisville.edu [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States)

2012-10-15

Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl{sub 2}), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl{sub 2} treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl{sub 2} administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl{sub 2}-induced reactive oxygen species (ROS) formation and completely negated CoCl{sub 2}-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl{sub 2} administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl{sub 2} increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl{sub 2}-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and

Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

International Nuclear Information System (INIS)

Liu, Yanlong; Wang, Chunhong; Wang, Yuhua; Ma, Zhenhua; Xiao, Jian; McClain, Craig; Li, Xiaokun; Feng, Wenke

2012-01-01

Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl 2 ), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl 2 treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl 2 administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl 2 -induced reactive oxygen species (ROS) formation and completely negated CoCl 2 -induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl 2 administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl 2 increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl 2 -induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and absorption. -- Graphical abstract: Physical

Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

Energy Technology Data Exchange (ETDEWEB)

Chen, Qiang [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China); The First Affiliated Hospital of Xiamen University, Xiamen (China); Jiang, Yuan; An, Yuan; Zhao, Na; Zhao, Yang [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China); Yu, Chundong, E-mail: cdyu@xmu.edu.cn [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China)

2011-06-17

Highlights: {yields} Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. {yields} FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. {yields} FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. {yields} FGFR4-ECD reduced tetracycline-induced fatty liver in mice. {yields} FGFR4-ECD partially restored tetracycline-repressed PPAR{alpha} expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whether neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.

Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

International Nuclear Information System (INIS)

Chen, Qiang; Jiang, Yuan; An, Yuan; Zhao, Na; Zhao, Yang; Yu, Chundong

2011-01-01

Highlights: → Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. → FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. → FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. → FGFR4-ECD reduced tetracycline-induced fatty liver in mice. → FGFR4-ECD partially restored tetracycline-repressed PPARα expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whether neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor α (PPARα), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.

Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli

DEFF Research Database (Denmark)

Sørensen, Hans; Mortensen, Kim

2005-01-01

Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed...... molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target...... for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible...

A review of machine learning methods to predict the solubility of overexpressed recombinant proteins in Escherichia coli.

Science.gov (United States)

Habibi, Narjeskhatoon; Mohd Hashim, Siti Z; Norouzi, Alireza; Samian, Mohammed Razip

2014-05-08

Over the last 20 years in biotechnology, the production of recombinant proteins has been a crucial bioprocess in both biopharmaceutical and research arena in terms of human health, scientific impact and economic volume. Although logical strategies of genetic engineering have been established, protein overexpression is still an art. In particular, heterologous expression is often hindered by low level of production and frequent fail due to opaque reasons. The problem is accentuated because there is no generic solution available to enhance heterologous overexpression. For a given protein, the extent of its solubility can indicate the quality of its function. Over 30% of synthesized proteins are not soluble. In certain experimental circumstances, including temperature, expression host, etc., protein solubility is a feature eventually defined by its sequence. Until now, numerous methods based on machine learning are proposed to predict the solubility of protein merely from its amino acid sequence. In spite of the 20 years of research on the matter, no comprehensive review is available on the published methods. This paper presents an extensive review of the existing models to predict protein solubility in Escherichia coli recombinant protein overexpression system. The models are investigated and compared regarding the datasets used, features, feature selection methods, machine learning techniques and accuracy of prediction. A discussion on the models is provided at the end. This study aims to investigate extensively the machine learning based methods to predict recombinant protein solubility, so as to offer a general as well as a detailed understanding for researches in the field. Some of the models present acceptable prediction performances and convenient user interfaces. These models can be considered as valuable tools to predict recombinant protein overexpression results before performing real laboratory experiments, thus saving labour, time and cost.

Potensi Terapeutik Fibroblast Growth Factor 21 terhadap Resistensi Insulin

Directory of Open Access Journals (Sweden)

Kurniasari Kurniasari

2015-12-01

Full Text Available Fibroblast growth factor 21 (FGF21 merupakan salah satu dari anggota FGF yang berperansebagai faktor endokrin. Hepar dan jaringan adiposa merupakan tempat kerja utama FGF21.Ekspresi FGF21 di hepar diatur oleh peroxisome proliferator activated receptor alpha (PPARαsedangkan di jaringan adiposa diatur oleh peroxisome proliferator activated receptor gamma(PPARγ. Kedua faktor transkripsi tersebut terlibat dalam metabolisme karbohidrat dan lipid. Padaresistensi insulin terdapat hiperglikemia, hiperinsulinemia, dan dislipidemia. Pemberian FGF21pada berbagai studi in vivo dan in vitro telah menunjukan potensi FGF21 dalam mengatasi kelainanakibat resistensi insulin sekaligus meningkatkan sensitivitas jaringan terhadap insulin. Kata kunci: FGF21, PPARγ, PPARα, resistensi insulin Fibroblast Growth Factor 21 (FGF21 Potension in InsulinResistance Treatment Abstract Fibroblast growth factor 21 (FGF21 is a member of FGF family that plays a role as endocrinefactor. Liver and adipose tissue are major target of FGF21. The expression of FGF21 in liveris regulated by peroxisome proliferator activated receptor alpha (PPARα, while peroxisomeproliferator activated receptor gamma (PPARγ regulate FGF21 expression in adipose tissue.Both transcription factors are involved in carbohydrate and lipid metabolism. Hyperglycemia,hyperinsulinemia, and dyslipidemia are observed in insulin resistance. Treatment with FGF21 inin vitro and in vivo study showed that FGF21 have the potential to overcome insulin resistance aswell as increasing tissue’s sensitivity towards insulin. Keywords: FGF21, PPARγ, PPARα, insulin resistance Normal 0 false false false IN X-NONE X-NONE

What's So Special about FGF19-Unique Effects Reported on Skeletal Muscle Mass and Function.

Science.gov (United States)

Glass, David J

2017-08-01

In a recent study published in Nature Medicine, Benoit et al. (2017) reported unique effects of FGF19 on mouse skeletal muscle: FGF19 induced skeletal muscle hypertrophy and blocked muscle atrophy, acting via FGF receptors and ßKlotho, while a related FGF21 hormone was ineffective. Copyright © 2017 Elsevier Inc. All rights reserved.

Production of recombinant proteins from Plasmodium falciparum in Escherichia coli.

Science.gov (United States)

Guerra, Ángela Patricia; Calvo, Eliana Patricia; Wasserman, Moisés; Chaparro-Olaya, Jacqueline

2016-02-23

The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies.  To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8).  The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice.  The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies.  The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

Effect of crosslinking chemistry of albumin/heparin multilayers on FGF-2 adsorption and endothelial cell behavior

Energy Technology Data Exchange (ETDEWEB)

Kumorek, Marta, E-mail: kumorek@imc.cas.cz [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic v.v.i., Heyrovského Square 2, Prague 162 06 (Czech Republic); Janoušková, Olga, E-mail: janouskova@imc.cas.cz [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic v.v.i., Heyrovského Square 2, Prague 162 06 (Czech Republic); Höcherl, Anita, E-mail: hocherl@imc.cas.cz [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic v.v.i., Heyrovského Square 2, Prague 162 06 (Czech Republic); Houska, Milan, E-mail: houska@imc.cas.cz [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic v.v.i., Heyrovského Square 2, Prague 162 06 (Czech Republic); Mázl-Chánová, Eliška, E-mail: chanova@imc.cas.cz [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic v.v.i., Heyrovského Square 2, Prague 162 06 (Czech Republic); Kasoju, Naresh, E-mail: naresh.kasoju@eng.ox.ac.uk [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic v.v.i., Heyrovského Square 2, Prague 162 06 (Czech Republic); Cuchalová, Lucie, E-mail: cuchaloval@gmail.com [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic v.v.i., Heyrovského Square 2, Prague 162 06 (Czech Republic); and others

2017-07-31

Highlights: • Amine, carboxyl, and induced aldehyde groups of heparin were used for crosslinking of LbL film. • The carbodiimide-mediated cross-linking was the most efficient fixation method. • FGF-2 bound the most effectively to the glutaraldehyde-cross-linked LbL film. • Films cross-linked through carboxyl and aldehyde groups showed a slow FGF-2 release. • FGF-2-loaded LbL films stimulated the HUVECs growth better than the soluble FGF-2. - Abstract: The biomaterials that efficiently deliver growth factors represent an important challenge in cell-based tissue engineering. Layer-by-layer (LbL) thin films are attractive for incorporating controlled amounts of growth factors and releasing them over time. Herein, we investigated the effect of a method of cross-linking of albumin/heparin layer-by-layer (LbL) assembly ((Alb/Hep){sub 3}) on the loading and release of basic fibroblast growth factor (FGF-2), and subsequent proliferation of human endothelial cells (HUVECs). The (Alb/Hep){sub 3} assemblies were cross-linked using glutaraldehyde, reductive amination or carbodiimide chemistries, and then biofunctionalized with FGF-2. The (Alb/Hep){sub 3} assemblies were characterized by the infrared multi-internal reflection spectroscopy, atomic force microscopy, ellipsometry, and surface plasmon resonance (SPR). The FGF-2 loading was quantified by the SPR in situ analysis. Our results showed that the (Alb/Hep){sub 3} cross-linking affected the amount of the bound heparin (from 150 to 315 ng/cm{sup 2}), amount of FGF-2 loaded (from 75 to 125 ng/cm{sup 2}), FGF-2 release (from 15 to 53% over 8 days), and consequently the HUVEC cell proliferation (from 50 to 80 × 10{sup 3} cells/cm{sup 2} at day 5). All FGF-2 loaded assemblies stimulated the cell growth more than a soluble FGF-2 added into the cell media. In particular, the highest HUVECs proliferation was detected on the carbodiimide-cross-linked assembly. Overall, these biocompatible cross-linked assemblies can fine

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

Science.gov (United States)

Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

2017-05-01

Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

Expression of Fgf23 in activated dendritic cells and macrophages in response to immunological stimuli in mice.

Science.gov (United States)

Masuda, Yuki; Ohta, Hiroya; Morita, Yumiko; Nakayama, Yoshiaki; Miyake, Ayumi; Itoh, Nobuyuki; Konishi, Morichika

2015-01-01

Fibroblast growth factors (Fgfs) are polypeptide growth factors with diverse biological activities. While several studies have revealed that Fgf23 plays important roles in the regulation of phosphate and vitamin D metabolism, the additional physiological roles of Fgf23 remain unclear. Although it is believed that osteoblasts/osteocytes are the main sources of Fgf23, we previously found that Fgf23 mRNA is also expressed in the mouse thymus, suggesting that it might be involved in the immune system. In this study we examined the potential roles of Fgf23 in immunological responses. Mouse serum Fgf23 levels were significantly increased following inoculation with Escherichia coli or Staphylococcus aureus or intraperitoneal injection of lipopolysaccharide. We also identified activated dendritic cells and macrophages that potentially contributed to increased serum Fgf23 levels. Nuclear factor-kappa B (NF-κB) signaling was essential for the induction of Fgf23 expression in dendritic cells in response to immunological stimuli. Moreover, we examined the effects of recombinant Fgf23 protein on immune cells in vitro. Fgfr1c, a potential receptor for Fgf23, was abundantly expressed in macrophages, suggesting that Fgf23 might be involved in signal transduction in these cells. Our data suggest that Fgf23 potentially increases the number in macrophages and induces expression of tumor necrosis factor-α (TNF-α), a proinflammatory cytokine. Collectively, these data suggest that Fgf23 might be intimately involved in inflammatory processes.

Suramin blocks interaction between human FGF1 and FGFR2 D2 domain and reduces downstream signaling activity

Energy Technology Data Exchange (ETDEWEB)

Wu, Zong-Sian, E-mail: gary810426@hotmail.com [Department of Chemistry, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan (China); Liu, Che Fu, E-mail: s9823002@m98.nthu.edu.tw [Department of Chemistry, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan (China); Fu, Brian, E-mail: brianfu9@gmail.com [Northwood High School, Irvine, CA (United States); Chou, Ruey-Hwang, E-mail: rhchou@mail.cmu.edu.tw [Graduate Institute of Cancer Biology and Center for Molecular Medicine, China Medical University, No.91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Department of Biotechnology, Asia University, Taiwan (China); Yu, Chin, E-mail: cyu.nthu@gmail.com [Department of Chemistry, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan (China)

2016-09-02

The extracellular portion of the human fibroblast growth factor receptor2 D2 domain (FGFR2 D2) interacts with human fibroblast growth factor 1 (hFGF1) to activate a downstream signaling cascade that ultimately affects mitosis and differentiation. Suramin is an antiparasiticdrug and a potent inhibitor of FGF-induced angiogenesis. Suramin has been shown to bind to hFGF1, and might block the interaction between hFGF1 and FGFR2 D2. Here, we titrated hFGF1 with FGFR2 D2 and suramin to elucidate their interactions using the detection of NMR. The docking results of both hFGF1-FGFR2 D2 domain and hFGF1-suramin complex were superimposed. The results indicate that suramin blocks the interaction between hFGF1 and FGFR2 D2. We used the PyMOL software to show the hydrophobic interaction of hFGF1-suramin. In addition, we used a Water-soluble Tetrazolium salts assay (WST1) to assess hFGF1 bioactivity. The results will be useful for the development of new antimitogenic activity drugs. - Highlights: • The interfacial residues on hFGF1-FGFR2 D2 and hFGF1-Suramin contact surface were mapped by {sup 1}H-{sup 15}N HSQC experiments. • hFGF1-FGFR2 D2 and hFGF1-Suramin complex models were generated from NMR restraints by using HADDOCK program. • We analyzed hFGF1-Suramin complex models and found the interaction between hFGF1-Suramin is hydrophobic. • The bioactivity of the hFGF1-FGFR2 D2 and hFGF1-Suramin complex was studied by using WST1 assay.

Suramin blocks interaction between human FGF1 and FGFR2 D2 domain and reduces downstream signaling activity

International Nuclear Information System (INIS)

Wu, Zong-Sian; Liu, Che Fu; Fu, Brian; Chou, Ruey-Hwang; Yu, Chin

2016-01-01

The extracellular portion of the human fibroblast growth factor receptor2 D2 domain (FGFR2 D2) interacts with human fibroblast growth factor 1 (hFGF1) to activate a downstream signaling cascade that ultimately affects mitosis and differentiation. Suramin is an antiparasiticdrug and a potent inhibitor of FGF-induced angiogenesis. Suramin has been shown to bind to hFGF1, and might block the interaction between hFGF1 and FGFR2 D2. Here, we titrated hFGF1 with FGFR2 D2 and suramin to elucidate their interactions using the detection of NMR. The docking results of both hFGF1-FGFR2 D2 domain and hFGF1-suramin complex were superimposed. The results indicate that suramin blocks the interaction between hFGF1 and FGFR2 D2. We used the PyMOL software to show the hydrophobic interaction of hFGF1-suramin. In addition, we used a Water-soluble Tetrazolium salts assay (WST1) to assess hFGF1 bioactivity. The results will be useful for the development of new antimitogenic activity drugs. - Highlights: • The interfacial residues on hFGF1-FGFR2 D2 and hFGF1-Suramin contact surface were mapped by "1H-"1"5N HSQC experiments. • hFGF1-FGFR2 D2 and hFGF1-Suramin complex models were generated from NMR restraints by using HADDOCK program. • We analyzed hFGF1-Suramin complex models and found the interaction between hFGF1-Suramin is hydrophobic. • The bioactivity of the hFGF1-FGFR2 D2 and hFGF1-Suramin complex was studied by using WST1 assay.

Fibroblast growth factor-21 is induced in human skeletal muscles by hyperinsulinemia

DEFF Research Database (Denmark)

Hojman, Pernille; Pedersen, Maria; Nielsen, Anders Rinnov

2009-01-01

DESIGN AND METHODS: We studied muscular FGF-21 expression and plasma FGF-21 after acute insulin stimulation in young healthy men during a hyperinsulinemic-euglycemic clamp. Furthermore, we investigated systemic levels and muscle FGF-21 expression in humans with or without insulin resistance and chronic...... elevated insulin. RESULTS: FGF-21 was barely detectable in young healthy men before insulin infusion. After 3 or 4 h of insulin infusion during a hyperinsulinemic-euglycemic clamp, muscular FGF-21 expression increased significantly. Plasma FGF-21 followed the same pattern. In individuals with chronic...... elevated insulin, muscular FGF-21 expression was associated with hyperinsulinemia in men but not in women. In plasma, hyperinsulinemia and fasting glucose were positively associated with plasma FGF-21 while plasma FGF-21 correlated negatively with HDL cholesterol. No associations between muscle and plasma...

Fibroblast Growth Factor 21 Mediates Glycemic Regulation by Hepatic JNK

Directory of Open Access Journals (Sweden)

Santiago Vernia

2016-03-01

Full Text Available The cJun NH2-terminal kinase (JNK-signaling pathway is implicated in metabolic syndrome, including dysregulated blood glucose concentration and insulin resistance. Fibroblast growth factor 21 (FGF21 is a target of the hepatic JNK-signaling pathway and may contribute to the regulation of glycemia. To test the role of FGF21, we established mice with selective ablation of the Fgf21 gene in hepatocytes. FGF21 deficiency in the liver caused marked loss of FGF21 protein circulating in the blood. Moreover, the protective effects of hepatic JNK deficiency to suppress metabolic syndrome in high-fat diet-fed mice were not observed in mice with hepatocyte-specific FGF21 deficiency, including reduced blood glucose concentration and reduced intolerance to glucose and insulin. Furthermore, we show that JNK contributes to the regulation of hepatic FGF21 expression during fasting/feeding cycles. These data demonstrate that the hepatokine FGF21 is a key mediator of JNK-regulated metabolic syndrome.

Selective synaptic targeting of the excitatory and inhibitory presynaptic organizers FGF22 and FGF7.

Science.gov (United States)

Terauchi, Akiko; Timmons, Kendall M; Kikuma, Koto; Pechmann, Yvonne; Kneussel, Matthias; Umemori, Hisashi

2015-01-15

Specific formation of excitatory and inhibitory synapses is crucial for proper functioning of the brain. Fibroblast growth factor 22 (FGF22) and FGF7 are postsynaptic-cell-derived presynaptic organizers necessary for excitatory and inhibitory presynaptic differentiation, respectively, in the hippocampus. For the establishment of specific synaptic networks, these FGFs must localize to appropriate synaptic locations - FGF22 to excitatory and FGF7 to inhibitory postsynaptic sites. Here, we show that distinct motor and adaptor proteins contribute to intracellular microtubule transport of FGF22 and FGF7. Excitatory synaptic targeting of FGF22 requires the motor proteins KIF3A and KIF17 and the adaptor protein SAP102 (also known as DLG3). By contrast, inhibitory synaptic targeting of FGF7 requires the motor KIF5 and the adaptor gephyrin. Time-lapse imaging shows that FGF22 moves with SAP102, whereas FGF7 moves with gephyrin. These results reveal the basis of selective targeting of the excitatory and inhibitory presynaptic organizers that supports their different synaptogenic functions. Finally, we found that knockdown of SAP102 or PSD95 (also known as DLG4), which impairs the differentiation of excitatory synapses, alters FGF7 localization, suggesting that signals from excitatory synapses might regulate inhibitory synapse formation by controlling the distribution of the inhibitory presynaptic organizer. © 2015. Published by The Company of Biologists Ltd.

Sugar-sweetened beverage intake associations with fasting glucose and insulin concentrations are not modified by selected genetic variants in a ChREBP-FGF21 pathway: A meta-analysis

Science.gov (United States)

Sugar-sweetened beverages (SSBs) are a major dietary contributor to fructose intake. A molecular pathway involving the carbohydrate responsive element-binding protein (ChREBP) and the metabolic hormone fibroblast growth factor 21 (FGF21) may influence sugar metabolism and, thereby, contribute to fru...

Soluble αKlotho as a candidate for the biomarker of aging.

Science.gov (United States)

Koyama, Daisuke; Sato, Yu; Aizawa, Masato; Maki, Takumi; Kurosawa, Masaki; Kuro-o, Makoto; Furukawa, Yusuke

2015-11-27

Although the Klotho gene has been recognized as an aging-suppressor gene, the significance of its soluble product, soluble αKlotho (sKlotho), in aging remains to be elucidated. To address this issue, we conducted a single-centered cross-sectional study in a region with a high prevalence of aging. We compared sKlotho levels with the patient characteristics from medical records and laboratory measurements, including fibroblast growth factor 23 (FGF23), intact parathyroid hormone, activated vitamin D3 and factors associated with mineral bone metabolism, in 52 outpatients with a mean age of 78.2 years. Serum sKlotho levels significantly decreased with age, but were not associated with the stage of chronic kidney disease (CKD). Serum FGF23 levels increased as CKD stages advanced, but were not associated with aging. Univariate analyses revealed that sKlotho levels positively correlated with glomerular filtration rate, and negatively with age and serum levels of FGF23 and phosphorus. In a multivariable linear regression analysis, sKlotho significantly correlated with aging and lower FGF23 levels. Only osteoporosis affected sKlotho and FGF23 levels among the various complications and patient status including medication. In summary, serum sKlotho levels inversely correlated with age and FGF23, and were significantly reduced in patients with osteoporosis. sKlotho may serve as a biomarker of aging independent of renal function. Copyright © 2015 Elsevier Inc. All rights reserved.

Fibroblast growth factor 21 has no direct role in regulating fertility in female mice

Directory of Open Access Journals (Sweden)

Garima Singhal

2016-08-01

Full Text Available Objective: Reproduction is an energetically expensive process. Insufficient calorie reserves, signaled to the brain through peripheral signals such as leptin, suppress fertility. Recently, fibroblast growth factor 21 (FGF21 was implicated as a signal from the liver to the hypothalamus that directly inhibits the hypothalamic–gonadotropin axis during fasting and starvation. However, FGF21 itself increases metabolic rate and can induce weight loss, which suggests that the effects of FGF21 on fertility may not be direct and may reflect changes in energy balance. Methods: To address this important question, we evaluated fertility in several mouse models with elevated FGF21 levels including ketogenic diet fed mice, fasted mice, mice treated with exogenous FGF21 and transgenic mice over-expressing FGF21. Results: We find that ketogenic diet fed mice remain fertile despite significant elevation in serum FGF21 levels. Absence of FGF21 does not alter transient infertility induced by fasting. Centrally infused FGF21 does not suppress fertility despite its efficacy in inducing browning of inguinal white adipose tissue. Furthermore, a high fat diet (HFD can restore fertility of female FGF21-overexpressing mice, a model of growth restriction, even in the presence of supraphysiological serum FGF21 levels. Conclusions: We conclude that FGF21 is not a direct physiological regulator of fertility in mice. The infertility observed in FGF21 overexpressing mice is likely driven by the increased energy expenditure and consequent excess calorie requirements resulting from high FGF21 levels. Keywords: FGF21, Fertility, Leptin, Hypothalamic action

FGF-2 deficiency does not influence FGF ligand and receptor expression during development of the nigrostriatal system.

Science.gov (United States)

Ratzka, Andreas; Baron, Olga; Grothe, Claudia

2011-01-01

Secreted proteins of the fibroblast growth factor (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinson's disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGF-ligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1, -2, -22, FgfR-2c, -3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-17 (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-15 (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-15, FGF-17a) were functionally characterized by plasmid-based over-expression in

Elevated Serum Fibroblast Growth Factor 21 Levels in Patients With Hyperthyroidism.

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Xiao, Fangsen; Lin, Mingzhu; Huang, Peiying; Zeng, Jinyang; Zeng, Xin; Zhang, Huijie; Li, Xiaoying; Yang, Shuyu; Li, Zhibin; Li, Xuejun

2015-10-01

Recent evidence from animal studies indicates that fibroblast growth factor 21 (FGF21), an endocrine hormone that regulates glucose, lipid metabolism, and energy homeostasis, is regulated by T3. However, the role of FGF21 in hyperthyroid patients is unknown. The objective was to study serum FGF21 levels in hyperthyroid patients and the association of serum FGF21 levels with hyperthyroidism. This was a case-control study. A total of 119 hyperthyroid patients and 108 healthy subjects were recruited. Of them, 41 hyperthyroid patients received thionamide treatment for 3 months until euthyroidism was obtained. Serum FGF21 levels were determined using the ELISA method. Serum FGF21 levels were significantly elevated in hyperthyroid patients as compared with normal subjects [median 290.67 (interquartile range, 156.60-502.33) vs 228.10 (169.85.25-320.10) pg/mL; P treatment, serum FGF21 levels in hyperthyroid patients declined markedly from 249.10 (139.10-444.00) to 106.90 (38.70-196.15) pg/mL (P hyperthyroidism. With adjustment for potential confounders, serum FGF21 remained independently associated with hyperthyroidism, with an adjusted odds ratio of 3.123 (95% confidence interval, 1.306-7.468) (P = .010). Serum FGF21 levels were elevated in patients with hyperthyroidism and declined after thionamide treatment. And serum FGF21 level was independently associated with hyperthyroidism.

Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells

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Lazaris, Anthoula; Arcidiacono, Steven; Huang, Yue; Zhou, Jiang-Feng; Duguay, François; Chretien, Nathalie; Welsh, Elizabeth A.; Soares, Jason W.; Karatzas, Costas N.

2002-01-01

Spider silks are protein-based ``biopolymer'' filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to ``biomimic'' the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.

Toxoplasma gondii: Biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6

International Nuclear Information System (INIS)

Bittame, Amina; Effantin, Grégory; Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia; Schoehn, Guy; Weissenhorn, Winfried; Cesbron-Delauw, Marie-France; Gagnon, Jean; Mercier, Corinne

2015-01-01

The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6–8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8–15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. - Highlights: • Toxoplasma gondii: soluble GRA2 forms 2 populations of particles. • T. gondii: the dense granule protein GRA2 folds intrinsically as an alpha-helix. • T. gondii: monomeric soluble GRA6 forms particles of 6–8 nm in diameter. • T. gondii: monomeric soluble GRA6 is random coiled. • Unusual biophysical properties of the dense granule protein GRA2 from T. gondii

Toxoplasma gondii: Biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6

Energy Technology Data Exchange (ETDEWEB)

Bittame, Amina [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Effantin, Grégory [Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble (France); Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Schoehn, Guy; Weissenhorn, Winfried [Université Grenoble Alpes, Institut de Biologie Structurale (IBS), 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Unit for Virus Host-Cell Interactions (UVHCI), UMI 3265 (UJF-EMBL-CNRS), 38027 Grenoble (France); Cesbron-Delauw, Marie-France; Gagnon, Jean [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France); Mercier, Corinne, E-mail: corinne.mercier@ujf-grenoble.fr [CNRS, UMR 5163, 38042 Grenoble (France); Université Grenoble Alpes, 38042 Grenoble (France)

2015-03-27

The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6–8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8–15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. - Highlights: • Toxoplasma gondii: soluble GRA2 forms 2 populations of particles. • T. gondii: the dense granule protein GRA2 folds intrinsically as an alpha-helix. • T. gondii: monomeric soluble GRA6 forms particles of 6–8 nm in diameter. • T. gondii: monomeric soluble GRA6 is random coiled. • Unusual biophysical properties of the dense granule protein GRA2 from T. gondii.

Changes in FGF21 Serum Concentrations and Liver mRNA Expression in an Experimental Model of Complete Lipodystrophy and Insulin-Resistant Diabetes

Czech Academy of Sciences Publication Activity Database

Špolcová, Andrea; Holubová, Martina; Mikulášková, Barbora; Nagelová, Veronika; Štofková, A.; Lacinová, Z.; Jurčovičová, J.; Haluzík, M.; Maletínská, Lenka; Železná, Blanka

2014-01-01

Roč. 63, č. 4 (2014), s. 483-490 ISSN 0862-8408 R&D Projects: GA ČR GAP303/10/1368; GA ČR GAP303/12/0576 Institutional support: RVO:61388963 Keywords : FGF21 * A-ZIP mice * lipodystrophy * insulin resistance * fatty liver * GLUT-1 Subject RIV: CE - Biochemistry Impact factor: 1.293, year: 2014

Stimulation of chondrocytes in vitro by gene transfer with plasmids coding for epidermal growth factor (hEGF) and basic fibroblast growth factor (bFGF)

DEFF Research Database (Denmark)

Schmal, H; Mehlhorn, A T; Zwingmann, J

2005-01-01

Human epidermal growth factor (hEGF) and basic fibroblast growth factor (bFGF) influence critical characteristics of chondrocytes. The effects on metabolism and differentiation were evaluated following transfection using specific plasmids coding for both cytokines. Chondrocytes were isolated from...... of recombinant hEGF and bFGF resulted in a significant increase in cell proliferation and glucosaminoglycan production. Chondrocytes were transfected with vectors coding for either hEGF or bFGF and the production of these proteins was measured in supernatants by ELISA. Expression kinetics showed different...... patterns: hEGF was detectable 2.5 days following transfection and peaked at day 5.5, whereas bFGF-production reached its maximum 1.5 days after transfection, declining thereafter. Chondrocytes endogenously produced significant amounts of bFGF within 5 days following isolation. Proliferation of h...

Fgf8 and Fgf3 are required for zebrafish ear placode induction, maintenance and inner ear patterning.

Science.gov (United States)

Léger, Sophie; Brand, Michael

2002-11-01

The vertebrate inner ear develops from initially 'simple' ectodermal placode and vesicle stages into the complex three-dimensional structure which is necessary for the senses of hearing and equilibrium. Although the main morphological events in vertebrate inner ear development are known, the genetic mechanisms controlling them are scarcely understood. Previous studies have suggested that the otic placode is induced by signals from the chordamesoderm and the hindbrain, notably by fibroblast growth factors (Fgfs) and Wnt proteins. Here we study the role of Fgf8 as a bona-fide hindbrain-derived signal that acts in conjunction with Fgf3 during placode induction, maintenance and otic vesicle patterning. Acerebellar (ace) is a mutant in the fgf8 gene that results in a non-functional Fgf8 product. Homozygous mutants for acerebellar (ace) have smaller ears that typically have only one otolith, abnormal semi-circular canals, and behavioral defects. Using gene expression markers for the otic placode, we find that ace/fgf8 and Fgf-signaling are required for normal otic placode formation and maintenance. Conversely, misexpression of fgf8 or Fgf8-coated beads implanted into the vicinity of the otic placode can increase ear size and marker gene expression, although competence to respond to the induction appears restricted. Cell transplantation experiments and expression analysis suggest that Fgf8 is required in the hindbrain in the rhombomere 4-6 area to restore normal placode development in ace mutants, in close neighbourhood to the forming placode, but not in mesodermal tissues. Fgf3 and Fgf8 are expressed in hindbrain rhombomere 4 during the stages that are critical for placode induction. Joint inactivation of Fgf3 and Fgf8 by mutation or antisense-morpholino injection causes failure of placode formation and results in ear-less embryos, mimicking the phenotype we observe after pharmacological inhibition of Fgf-signaling. Fgf8 and Fgf3 together therefore act during induction

High molecular weight FGF2 isoforms demonstrate canonical receptor-mediated activity and support human embryonic stem cell self-renewal

Directory of Open Access Journals (Sweden)

Denis Kole

2017-05-01

Full Text Available Basic fibroblast growth factor (FGF2 is a highly pleiotropic member of a large family of growth factors with a broad range of activities, including mitogenesis and angiogenesis (Ornitz et al., 1996; Zhang et al., 2006, and it is known to be essential for maintenance of balance between survival, proliferation, and self-renewal in human pluripotent stem cells (Eiselleova et al., 2009; Zoumaro-Djayoon et al., 2011. A single FGF2 transcript can be translated into five FGF2 protein isoforms, an 18 kDa low molecular weight (LMW isoform and four larger high molecular weight (HMW isoforms (Arese et al., 1999; Arnaud et al., 1999. As they are not generally secreted, high molecular weight (HMW FGF2 isoforms have predominantly been investigated intracellularly; only a very limited number of studies have investigated their activity as extracellular factors. Here we report over-expression, isolation, and biological activity of all recombinant human FGF2 isoforms. We show that HMW FGF2 isoforms can support self-renewal of human embryonic stem cells (hESCs in vitro. Exogenous supplementation with HMW FGF2 isoforms also activates the canonical FGFR/MAPK pathway and induces mitogenic activity in a manner similar to that of the 18 kDa FGF2 isoform. Though all HMW isoforms, when supplemented exogenously, are able to recapitulate LMW FGF2 activity to some degree, it appears that certain isoforms tend to do so more poorly, demonstrating a lesser functional response by several measures. A better understanding of isoform-specific FGF2 effects will lead to a better understanding of developmental and pathological FGF2 signaling.

The 21-cm Signal from the cosmological epoch of recombination

Energy Technology Data Exchange (ETDEWEB)

Fialkov, A. [Departement de Physique, Ecole Normale Superieure, CNRS, 24 rue Lhomond, Paris, 75005 (France); Loeb, A., E-mail: anastasia.fialkov@phys.ens.fr, E-mail: aloeb@cfa.harvard.edu [Department of Astronomy, Harvard University, 60 Garden Street, MS-51, Cambridge, MA, 02138 (United States)

2013-11-01

The redshifted 21-cm emission by neutral hydrogen offers a unique tool for mapping structure formation in the early universe in three dimensions. Here we provide the first detailed calculation of the 21-cm emission signal during and after the epoch of hydrogen recombination in the redshift range of z ∼ 500–1,100, corresponding to observed wavelengths of 100–230 meters. The 21-cm line deviates from thermal equilibrium with the cosmic microwave background (CMB) due to the excess Lyα radiation from hydrogen and helium recombinations. The resulting 21-cm signal reaches a brightness temperature of a milli-Kelvin, orders of magnitude larger than previously estimated. Its detection by a future lunar or space-based observatory could improve dramatically the statistical constraints on the cosmological initial conditions compared to existing two-dimensional maps of the CMB anisotropies.

The 21-cm Signal from the cosmological epoch of recombination

International Nuclear Information System (INIS)

Fialkov, A.; Loeb, A.

2013-01-01

The redshifted 21-cm emission by neutral hydrogen offers a unique tool for mapping structure formation in the early universe in three dimensions. Here we provide the first detailed calculation of the 21-cm emission signal during and after the epoch of hydrogen recombination in the redshift range of z ∼ 500–1,100, corresponding to observed wavelengths of 100–230 meters. The 21-cm line deviates from thermal equilibrium with the cosmic microwave background (CMB) due to the excess Lyα radiation from hydrogen and helium recombinations. The resulting 21-cm signal reaches a brightness temperature of a milli-Kelvin, orders of magnitude larger than previously estimated. Its detection by a future lunar or space-based observatory could improve dramatically the statistical constraints on the cosmological initial conditions compared to existing two-dimensional maps of the CMB anisotropies

Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

Science.gov (United States)

Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

2017-11-01

Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells.

Directory of Open Access Journals (Sweden)

Chih-Ping Chung

Full Text Available Endothelial cells (ECs express fibroblast growth factor (FGF receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs and human brain microvascular endothelial cells (HBMECs were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS expression, nitric oxide (NO production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.

Lack of Day/Night variation in fibroblast growth factor 21 levels in young healthy men.

Science.gov (United States)

Foo, J-P; Aronis, K N; Chamberland, J P; Mantzoros, C S

2015-06-01

Fibroblast growth factor (FGF) 21 is an endocrine factor with an emerging role as a metabolic regulator. We previously reported the presence of a significant day/night variation of FGF-21 in energy-replete, healthy female subjects. However the day/night patterns of secretion in male subjects remain to be fully elucidated. To elucidate day/night pattern of FGF-21 levels in male subjects in the energy-replete state, its relationship to FFA and to investigate whether a sexual dimorphism exists in FGF-21 physiology. Eight healthy lean male subjects were studied for up to 5 days while on an isocaloric diet. Blood samples were obtained for measurement of FGF-21 and free fatty acids (FFA) hourly from 0800 AM on day 4 till 0800AM on day 5. FGF-21 did not exhibit any statistically significant day/night variation pattern of circulating FGF-21 levels during the isocaloric fed state in male subjects. FGF-21 levels in male subjects are closely cross-correlated with FFA levels, similar to female subjects. A sexual dimorphism exists in FGF-21 physiology; that as opposed to female subjects, no significant day/night variation exists in FGF-21 rhythm in male subjects in the energy-replete state. Circulating pattern of FGF-21, similar to the female subjects, was highly cross-correlated to the FFA levels in the male subjects, signifying that the sexual dimorphism in FGF-21 physiology may be related to the differing lipid metabolism in both the genders.

A pro-angiogenic degradable Mg-poly(lactic-co-glycolic acid) implant combined with rhbFGF in a rat limb ischemia model.

Science.gov (United States)

Bao, Hanmei; Lv, Feng; Liu, Tianjun

2017-12-01

Site-specific controlled release of exogenous angiogenic growth factors, such as recombinant human basic fibroblast growth factor (rhbFGF), has become a promising approach to improve peripheral vascular disease. Here, we have developed an implant composed of spiral magnesium (Mg) and a coating made using poly(lactic-co-glycolic acid) (PLGA) with encapsulated rhbFGF (Mg-PLGA-rhbFGF). The encapsulated protein could release continually for 4weeks with well preserved bioactivity. We compared the angiogenic effect produced by Mg-PLGA-rhbFGF with that of a PLGA implant loaded with rhbFGF (PLGA-rhbFGF). The incorporation of Mg in the implant raised the microclimate pH in the polymer, which preserved the stability of rhbFGF. Mg-PLGA-rhbFGF exhibited advantages over PLGA-rhbFGF implant in terms of a cytocompatibility evaluation. An in vivo angiogenesis test further confirmed the efficacy of released rhbFGF. HE, CD31 and α-SMA staining revealed that the controlled release of rhbFGF from the Mg-PLGA-rhbFGF implant was superior in promoting angiogenesis compared with that of the PLGA-rhbFGF implant. Four weeks post-implantation, the capillary density of the Mg-PLGA-rhbFGF group was significantly higher than that of the PLGA-rhbFGF, control and the normal group (pspiral magnesium and a coating made using poly(lactic-co-glycolic acid) (PLGA) with encapsulated rhbFGF (Mg-PLGA-rhbFGF). The preparation method does not involve any complex processes and results in a high encapsulation efficiency (approximately 100%). The degradation of metal Mg raise the microclimate pH in the PLGA polymer, which could well preserve the bioactivity of rhbFGF incorporated in the implant. Mg-PLGA-based, sustained local delivery of rhbFGF promotes post-ischemic angiogenesis and blood flow recovery in rat limb ischemic model. This work marks the first report for controlled release of rhbFGF in combination with metal Mg, and suggests potential therapeutic usefulness of Mg-PLGA-rhbFGF for tissue ischemia

Central Fibroblast Growth Factor 21 Browns White Fat via Sympathetic Action in Male Mice.

Science.gov (United States)

Douris, Nicholas; Stevanovic, Darko M; Fisher, Ffolliott M; Cisu, Theodore I; Chee, Melissa J; Nguyen, Ngoc L; Zarebidaki, Eleen; Adams, Andrew C; Kharitonenkov, Alexei; Flier, Jeffrey S; Bartness, Timothy J; Maratos-Flier, Eleftheria

2015-07-01

Fibroblast growth factor 21 (FGF21) has multiple metabolic actions, including the induction of browning in white adipose tissue. Although FGF21 stimulated browning results from a direct interaction between FGF21 and the adipocyte, browning is typically associated with activation of the sympathetic nervous system through cold exposure. We tested the hypothesis that FGF21 can act via the brain, to increase sympathetic activity and induce browning, independent of cell-autonomous actions. We administered FGF21 into the central nervous system via lateral ventricle infusion into male mice and found that the central treatment increased norepinephrine turnover in target tissues that include the inguinal white adipose tissue and brown adipose tissue. Central FGF21 stimulated browning as assessed by histology, expression of uncoupling protein 1, and the induction of gene expression associated with browning. These effects were markedly attenuated when mice were treated with a β-blocker. Additionally, neither centrally nor peripherally administered FGF21 initiated browning in mice lacking β-adrenoceptors, demonstrating that an intact adrenergic system is necessary for FGF21 action. These data indicate that FGF21 can signal in the brain to activate the sympathetic nervous system and induce adipose tissue thermogenesis.

Mifepristone inhibits MPA-and FGF2-induced mammary tumor growth but not FGF2-induced mammary hyperplasia

Directory of Open Access Journals (Sweden)

Juan P. Cerliani

2010-12-01

Full Text Available We have previously demonstrated a crosstalk between fibroblast growth factor 2 (FGF2 and progestins inducing experimental breast cancer growth. The aim of the present study was to compare the effects of FGF2 and of medroxyprogesterone acetate (MPA on the mouse mammary glands and to investigate whether the antiprogestin RU486 was able to reverse the MPA- or FGF2-induced effects on both, mammary gland and tumor growth. We demonstrate that FGF2 administered locally induced an intraductal hyperplasia that was not reverted by RU486, suggesting that FGF2-induced effects are progesterone receptor (PR-independent. However, MPA-induced paraductal hyperplasia was reverted by RU486 and a partial agonistic effect was observed in RU486-treated glands. Using C4-HD tumors which only grow in the presence of MPA, we showed that FGF2 administered intratumorally was able to stimulate tumor growth as MPA. The histology of FGF2-treated tumors showed different degrees of gland differentiation. RU486 inhibited both, MPA or FGF2 induced tumor growth. However, only complete regression was observed in MPA-treated tumors. Our results support the hypothesis that stromal FGF2 activates PR inducing hormone independent tumor growth.

FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

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Ishino, Ruri; Minami, Kaori; Tanaka, Satowa [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Nagai, Mami [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Matsui, Keiji; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Asano, Shigetaka [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan)

2013-10-11

Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1{sup +/+} MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1{sup −/−} MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1{sup +/+} and Med1{sup −/−} MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells.

FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

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Ishino, Ruri; Minami, Kaori; Tanaka, Satowa; Nagai, Mami; Matsui, Keiji; Hasegawa, Natsumi; Roeder, Robert G.; Asano, Shigetaka; Ito, Mitsuhiro

2013-01-01

Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1 +/+ MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1 −/− MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1 +/+ and Med1 −/− MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells

Biological role, clinical significance, and therapeutic possibilities of the recently discovered metabolic hormone fibroblastic growth factor 21.

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Iglesias, Pedro; Selgas, Rafael; Romero, Sara; Díez, Juan J

2012-09-01

Fibroblast growth factor 21 (FGF21), a 181 amino acid circulating protein, is a member of the FGF superfamily, with relevant metabolic actions. It acts through the interaction with specific FGF receptors and a cofactor called β-Klotho, whose expression is predominantly detected in metabolically active organs. FGF21 stimulates glucose uptake in adipocytes via the induction of glucose transporter-1. This action is additive and independent of insulin. β-Cell function and survival are preserved, and glucagon secretion is reduced by this protein, thus decreasing hepatic glucose production and improving insulin sensitivity. Lipid profile has been shown to be improved by FGF21 in several animal models. FGF21 increases energy expenditure in rodents and induces weight loss in diabetic nonhuman primates. It also exerts favorable effects on hepatic steatosis and reduces tissue lipid content in rodents. Adaptive metabolic responses to fasting, including stimulation of ketogenesis and fatty acid oxidation, seem to be partially mediated by FGF21. In humans, serum FGF21 concentrations have been found elevated in insulin-resistant states, such as impaired glucose tolerance and type 2 diabetes. FGF21 levels are correlated with hepatic insulin resistance index, fasting blood glucose, HbA1c, and blood glucose after an oral glucose tolerance test. A relationship between FGF21 levels and long-term diabetic complications, such as nephropathy and carotid atheromatosis, has been reported. FGF21 levels decreased in diabetic patients after starting therapy with insulin or oral agents. Increased FGF21 serum levels have also been found to be associated with obesity. In children, it is correlated with BMI and leptin levels, whereas in adults, FGF21 levels are mainly related to several components of the metabolic syndrome. Serum FGF21 levels have been found to be elevated in patients with ischemic heart disease. In patients with renal disease, FGF21 levels exhibited a progressive increase as

Fibroblast growth factor 21 and its novel association with oxidative stress

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Miguel Ángel Gómez-Sámano

2017-04-01

Full Text Available Fibroblast growth factor 21 (FGF21 is an endocrine-member of the FGF family. It is synthesized mainly in the liver, but it is also expressed in adipose tissue, skeletal muscle, and many other organs. It has a key role in glucose and lipid metabolism, as well as in energy balance. FGF21 concentration in plasma is increased in patients with obesity, insulin resistance, and metabolic syndrome. Recent findings suggest that such increment protects tissue from an increased oxidative stress environment. Different types of physical stress, such as strenuous exercising, lactation, diabetic nephropathy, cardiovascular disease, and critical illnesses, also increase FGF21 circulating concentration. FGF21 is now considered a stress-responsive hormone in humans. The discovery of an essential response element in the FGF21 gene, for the activating transcription factor 4 (ATF4, involved in the regulation of oxidative stress, and its relation with genes such as NRF2, TBP-2, UCP3, SOD2, ERK, and p38, places FGF21 as a key regulator of the oxidative stress cell response. Its role in chronic diseases and its involvement in the treatment and follow-up of these diseases has been recently the target of new studies. The diminished oxidative stress through FGF21 pathways observed with anti-diabetic therapy is another clue of the new insights of this hormone.

Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli.

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Weickert, M J; Pagratis, M; Curry, S R; Blackmore, R

1997-01-01

Accumulation of soluble recombinant hemoglobin (rHb1.1) in Escherichia coli requires proper protein folding, prosthetic group (heme) addition, and subunit assembly. This served as a new model system for the study of the effects of temperature, protein synthesis rates, and protein accumulation rates on protein solubility in E. coli. Fermentation expression of rHb1.1 at 30 degrees C from cultures containing a medium or high globin gene dosage (pBR-based or pUC-based plasmids with rHb1.1 genes under the control of the tac promoter) was compared. A medium gene dosage resulted in rHb1.1 accumulating to approximately 7% of the soluble cell protein, of which 78% was soluble. A high globin gene dosage resulted in a > or = 3-fold increase in total globin to 23 to 24% of the soluble cell protein, but 70% was insoluble. Accumulation of insoluble rHb1.1 began immediately upon induction. The proportion of rHb1.1 from the high globin gene dosage that accumulated as insoluble globin was affected by reducing (i) the inducer concentration and (ii) the temperature. Reducing the inducer concentration reduced globin synthesis up to eightfold but increased the proportion of soluble rHb1.1 to 93%. In contrast, total globin protein synthesis was barely affected by reducing the temperature from 30 to 26 degrees C, while soluble globin accumulation increased > 2-fold to approximately 15% of the soluble cell protein. The contrast between the effects of reducing rates of protein synthesis and accumulation and those of reducing temperature suggests that lower temperature stabilizes one or more folding intermediates. We propose a simplified physical model which integrates protein synthesis, folding, and heme association. This model shows that temperature-dependent apoglobin stability is the most critical factor in soluble rHb1.1 accumulation. PMID:9361418

Fibroblast growth factor 21 participates in adaptation to endoplasmic reticulum stress and attenuates obesity-induced hepatic metabolic stress.

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Kim, Seong Hun; Kim, Kook Hwan; Kim, Hyoung-Kyu; Kim, Mi-Jeong; Back, Sung Hoon; Konishi, Morichika; Itoh, Nobuyuki; Lee, Myung-Shik

2015-04-01

Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-diabetic and anti-obesity activity. FGF21 expression is increased in patients with and mouse models of obesity or nonalcoholic fatty liver disease (NAFLD). However, the functional role and molecular mechanism of FGF21 induction in obesity or NAFLD are not clear. As endoplasmic reticulum (ER) stress is triggered in obesity and NAFLD, we investigated whether ER stress affects FGF21 expression or whether FGF21 induction acts as a mechanism of the unfolded protein response (UPR) adaptation to ER stress induced by chemical stressors or obesity. Hepatocytes or mouse embryonic fibroblasts deficient in UPR signalling pathways and liver-specific eIF2α mutant mice were employed to investigate the in vitro and in vivo effects of ER stress on FGF21 expression, respectively. The in vivo importance of FGF21 induction by ER stress and obesity was determined using inducible Fgf21-transgenic mice and Fgf21-null mice with or without leptin deficiency. We found that ER stressors induced FGF21 expression, which was dependent on a PKR-like ER kinase-eukaryotic translation factor 2α-activating transcription factor 4 pathway both in vitro and in vivo. Fgf21-null mice exhibited increased expression of ER stress marker genes and augmented hepatic lipid accumulation after tunicamycin treatment. However, these changes were attenuated in inducible Fgf21-transgenic mice. We also observed that Fgf21-null mice with leptin deficiency displayed increased hepatic ER stress response and liver injury, accompanied by deteriorated metabolic variables. Our results suggest that FGF21 plays an important role in the adaptive response to ER stress- or obesity-induced hepatic metabolic stress.

Association between circulating fibroblast growth factor 21 and mortality in end-stage renal disease.

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Marina Kohara

Full Text Available Fibroblast growth factor 21 (FGF21 is an endocrine factor that regulates glucose and lipid metabolism. Circulating FGF21 predicts cardiovascular events and mortality in type 2 diabetes mellitus, including early-stage chronic kidney disease, but its impact on clinical outcomes in end-stage renal disease (ESRD patients remains unclear. This study enrolled 90 ESRD patients receiving chronic hemodialysis who were categorized into low- and high-FGF21 groups by the median value. We investigated the association between circulating FGF21 levels and the cardiovascular event and mortality during a median follow-up period of 64 months. A Kaplan-Meier analysis showed that the mortality rate was significantly higher in the high-FGF21 group than in the low-FGF21 group (28.3% vs. 9.1%, log-rank, P = 0.034, while the rate of cardiovascular events did not significantly differ between the two groups (30.4% vs. 22.7%, log-rank, P = 0.312. In multivariable Cox models adjusted a high FGF21 level was an independent predictor of all-cause mortality (hazard ratio: 3.98; 95% confidence interval: 1.39-14.27, P = 0.009. Higher circulating FGF21 levels were associated with a high mortality rate, but not cardiovascular events in patient with ESRD, suggesting that circulating FGF21 levels serve as a predictive marker for mortality in these subjects.

Association between circulating fibroblast growth factor 21 and mortality in end-stage renal disease.

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Kohara, Marina; Masuda, Takahiro; Shiizaki, Kazuhiro; Akimoto, Tetsu; Watanabe, Yuko; Honma, Sumiko; Sekiguchi, Chuji; Miyazawa, Yasuharu; Kusano, Eiji; Kanda, Yoshinobu; Asano, Yasushi; Kuro-O, Makoto; Nagata, Daisuke

2017-01-01

Fibroblast growth factor 21 (FGF21) is an endocrine factor that regulates glucose and lipid metabolism. Circulating FGF21 predicts cardiovascular events and mortality in type 2 diabetes mellitus, including early-stage chronic kidney disease, but its impact on clinical outcomes in end-stage renal disease (ESRD) patients remains unclear. This study enrolled 90 ESRD patients receiving chronic hemodialysis who were categorized into low- and high-FGF21 groups by the median value. We investigated the association between circulating FGF21 levels and the cardiovascular event and mortality during a median follow-up period of 64 months. A Kaplan-Meier analysis showed that the mortality rate was significantly higher in the high-FGF21 group than in the low-FGF21 group (28.3% vs. 9.1%, log-rank, P = 0.034), while the rate of cardiovascular events did not significantly differ between the two groups (30.4% vs. 22.7%, log-rank, P = 0.312). In multivariable Cox models adjusted a high FGF21 level was an independent predictor of all-cause mortality (hazard ratio: 3.98; 95% confidence interval: 1.39-14.27, P = 0.009). Higher circulating FGF21 levels were associated with a high mortality rate, but not cardiovascular events in patient with ESRD, suggesting that circulating FGF21 levels serve as a predictive marker for mortality in these subjects.

Fibroblast growth factor 21 is required for beneficial effects of exercise during chronic high-fat feeding

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Loyd, Christine; Magrisso, I. Jack; Haas, Michael; Balusu, Sowmya; Krishna, Radha; Itoh, Nobuyuki; Sandoval, Darleen A.; Perez-Tilve, Diego; Obici, Silvana

2016-01-01

Exercise is an effective therapy against the metabolic syndrome. However, the molecular pathways underlying the advantageous effects of exercise are elusive. Glucagon receptor signaling is essential for exercise benefits, and recent evidence indicates that a downstream effector of glucagon, fibroblast growth factor 21 (FGF21), is implicated in this response. Therefore, we tested the hypothesis that FGF21 action is necessary in mediating metabolic effects of exercise. We utilized acute exhaustive treadmill exercise in Wistar rats to identify a putative, concomitant increase in plasma glucagon and FGF21 with the increase in glucose and lactate following exercise. To test the necessity of FGF21 action in the exercise response, we exposed FGF21 congenitally deficient mice (Fgf21−/−) and their wild-type (Wt) littermates to chronic high-fat (HF) feeding and inoperable (sedentary) or operable (exercise) voluntary running wheels. Physiological tests were performed to assess the role of FGF21 in the beneficial effect of exercise on glucose metabolism. Wt and Fgf21−/− littermates exhibited similar running behavior, and exercise was effective in suppressing weight and fat mass gain and dyslipidemia independently of genotype. However, exercise failed to positively affect hepatic triglyceride content and glucose tolerance in HF diet-fed Fgf21−/− mice. Furthermore, Fgf21−/− mice exhibited an impaired adaptation to exercise training, including reduced AMP-activated protein kinase activity in skeletal muscle. This study demonstrates that FGF21 action is necessary to achieve the full metabolic benefits of exercise during chronic HF feeding. PMID:27445299

Expression by Streptomyces lividans of the Rat α Integrin CD11b A-Domain as a Secreted and Soluble Recombinant Protein

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Dorra Zouari Ayadi

2007-01-01

Full Text Available We already reported the use of a long synthetic signal peptide (LSSP to secrete the Streptomyces sp. TO1 amylase by Streptomyces lividans strain. We herein report the expression and secretion of the rat CD11b A-domain using the same LSSP and S. lividans as host strain. We have used the Escherichia coli/Streptomyces shuttle vector pIJ699 for the cloning of the A-domain DNA sequence downstream of LSSP and under the control of the constitutive ermE-up promoter of Streptomyces erythraeus. Using this construct and S. lividans as a host strain, we achieved the expression of 8 mg/L of soluble secreted recombinant form of the A-domain of the rat leukocyte β2 integrin CD11/CD18 alpha M subunit (CD11b. This secreted recombinant CD11b A-domain reacted with a function blocking antibody showing that this protein is properly folded and probably functional. These data support the capability of Streptomyces to produce heterologous recombinant proteins as soluble secreted form using the “LSSP” synthetic signal peptide.

Dynamic relationship of the epithelium and mesenchyme during salivary gland initiation: the role of Fgf10

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Wells, Kirsty L.; Gaete, Marcia; Matalova, Eva; Deutsch, Danny; Rice, David; Tucker, Abigail S.

2013-01-01

Summary Salivary glands provide an excellent model for the study of epithelial–mesenchymal interactions. We have looked at the interactions involved in the early initiation and development of murine salivary glands using classic recombination experiments and knockout mice. We show that salivary gland epithelium, at thickening and initial bud stages, is able to direct salivary gland development in non-gland pharyngeal arch mesenchyme at early stages. The early salivary gland epithelium is therefore able to induce gland development in non-gland tissue. This ability later shifts to the mesenchyme, with non-gland epithelium, such as from the limb bud, able to form a branching gland when combined with pseudoglandular stage gland mesenchyme. This shift appears to involve Fgf signalling, with signals from the epithelium inducing Fgf10 in the mesenchyme. Fgf10 then signals back to the epithelium to direct gland down-growth and bud development. These experiments highlight the importance of epithelial–mesenchymal signalling in gland initiation, controlling where, when and how many salivary glands form. PMID:24167707

Muscle atrophy in patients wirh ckd results from fgf23/klotho-mediated supression of insulin/igf-i signaling

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Shinsuke Kido

2012-06-01

Full Text Available Muscle atrophy is a significant consequence of chronic kidney disease (CKD that increases a patient’s risk of mortality and decrease their quality of life. In CKD patients, the circulation levels of FGF23 are significantly increased, but the exact pathological significance of the increase and relationship between FGF23 and muscle atrophy are not clear. Because of Klohto, acts as a co-receptor of FGF23 is detectable in limited tissues including in kidney and brain, but not in skeletal muscles. In contrast, recently reports indicated that the extracellular domain of klohto is cleavage for some reason on the cell surface and detected in the blood in animals. In this study, we attempted to identify the causative factors responsible for the shedding of Klotho, and whether both FGF23 and Klohto induced muscle atrophy via reduction of insulin/IGF-I signaling. We first investigated by treating kidney cells with various factors related in pathological factors in CKD. As a result, we found that advanced glycation endproducts (AGEs, an accumulated in patients with CKD and diabetes mellitus, increases shedding of Klohto in kidney cells. It is common knowledge that insulin/IGF-I signaling is necessary for normal skeletal growth. As a result, we showed that both FGF23 and Klohto inhibited differentiation of cultured skeletal muscle cells through down-regulation of insulin/IGF-I signaling. These observations suggested a divergent role of FGF23 and soluble klohto in the regulation of skeletal muscle differentiation and thereby muscle atrophy under pathological conditioned in CKD patients. Our results further imply that FGF23/Klohto may serve a new therapeutic target for CKD-induced muscle atrophy.

DIFFERENTIAL BINDING OF HUMAN INTERLEUKIN-1 (IL-1) RECEPTOR ANTAGONIST TO NATURAL AND RECOMBINANT SOLUBLE AND CELLULAR IL-1 TYPE-I RECEPTORS

DEFF Research Database (Denmark)

Svenson, Morten; Nedergaard, Susanne; Heegaard, Peter M. H.

1995-01-01

antagonist (IL-1ra). Recombinant soluble human IL-1RI expressed in COS cells (sIL-1RI) consists of the extracellular part of the receptor and binds all three known IL-1 species but preferentially to IL-1ra. We further characterized the sizes and binding of IL-1raBF and sIL-1RI to IL-1ra by polyacrylamide gel...... electrophoresis in the presence of sodium dodecylsulfate, ligand binding interference analyses, N-glycosidase treatment, concanavalin A affinity chromatography, and with the use of monoclonal antibodies (mAb) to human recombinant IL-1ra. We also evaluated the binding of IL-1ra to cellular IL-1RI on MRC5...... binding of both molecules to IL-1ra. Both factors blocked binding of IL-1ra to cellular IL-1RI, as did mAb to IL-1ra, but the sites on IL-1ra which bound to the mAb, and to IL-1raBF and sIL-1RI, differed. We conclude that there are important differences between the natural and recombinant forms of soluble...

Resistance to the Beneficial Metabolic Effects and Hepatic Antioxidant Defense Actions of Fibroblast Growth Factor 21 Treatment in Growth Hormone-Overexpressing Transgenic Mice

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Ravneet K. Boparai

2015-01-01

Full Text Available Fibroblast growth factor 21 (FGF21 modulates a diverse range of biological functions, including glucose and lipid metabolism, adaptive starvation response, and energy homeostasis, but with limited mechanistic insight. FGF21 treatment has been shown to inhibit hepatic growth hormone (GH intracellular signaling. To evaluate GH axis involvement in FGF21 actions, transgenic mice overexpressing bovine GH were used. Expectedly, in response to FGF21 treatment control littermates showed metabolic improvements whereas GH transgenic mice resisted most of the beneficial effects of FGF21, except an attenuation of the innate hyperinsulinemia. Since FGF21 is believed to exert its effects mostly at the transcriptional level, we analyzed and observed significant upregulation in expression of various genes involved in carbohydrate and lipid metabolism, energy homeostasis, and antioxidant defense in FGF21-treated controls, but not in GH transgenics. The resistance of GH transgenic mice to FGF21-induced changes underlines the necessity of normal GH signaling for the beneficial effects of FGF21.

Induction of fibroblast growth factor 21 does not require activation of the hepatic X-box binding protein 1 in mice

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Shantel Olivares

2017-12-01

Full Text Available Objective: Fibroblast growth factor 21 (FGF21, a key regulator of the metabolic response to fasting, is highly induced by endoplasmic reticulum (ER stress. The X-box binding protein 1 (Xbp1 is one of several ER stress proteins that has been shown to directly activate the FGF21 promoter. We aimed to determine whether hepatic Xbp1 is required for induction of hepatic FGF21 in vivo. Methods: Mice bearing a hepatocyte-specific deletion of Xbp1 (Xbp1LKO were subjected to fasting, pharmacologic ER stress, or a ketogenic diet, all potent stimuli of Fgf21 expression. Results: Hepatocyte-specific Xbp1 knockout mice demonstrated normal induction of FGF21 in response to fasting or pharmacologic ER stress and enhanced induction of FGF21 in response to a ketogenic diet. Consistent with preserved induction of FGF21, Xbp1LKO mice exhibited normal induction of FGF21 target genes and normal ketogenesis in response to fasting or a ketogenic diet. Conclusion: Hepatic Xbp1 is not required for induction of FGF21 under physiologic or pathophysiologic conditions in vivo. Keywords: Unfolded protein response, Endoplasmic reticulum stress, Fasting, Fatty acid oxidation, Ketogenic diet

Serum concentrations of fibroblast growth factors 19 and 21 in women with gestational diabetes mellitus: association with insulin resistance, adiponectin, and polycystic ovary syndrome history.

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Dongyu Wang

Full Text Available BACKGROUND: Fibroblast growth factor 19 (FGF19 and FGF21 are considered to be novel adipokines that improve glucose tolerance and insulin sensitivity. In the current study, we investigated serum FGF19 and FGF21 levels in patients with gestational diabetes mellitus (GDM and explored their relationships with anthropometric and endocrine parameters. METHOD: Serum FGF19 and FGF21 levels were determined by enzyme-linked immunosorbent assay (ELISA in patients with GDM (n = 30 and healthy pregnant controls (n = 60 matched for maternal and gestational age. Serum FGF19 and FGF21 levels were correlated with anthropometric, metabolic, and endocrine parameters. RESULTS: Circulating levels of FGF19 were significantly reduced in patients with GDM relative to healthy pregnant subjects, whereas FGF21 levels were increased in GDM patients. Serum FGF19 levels independently and inversely correlated with insulin resistance (increased homeostasis model assessment of insulin resistance, HOMA-IR and were positively related to serum adiponectin in both groups. In contrast, serum FGF21 levels independently and positively correlated with insulin resistance and serum triglycerides and were inversely related to serum adiponectin. In addition, in the combined population of both groups, those women with preconception polycystic ovary syndrome (PCOS history had the lowest levels of FGF19, which were significantly lower than those in GDM patients without PCOS history and those in controls without PCOS history. CONCLUSIONS: Circulating FGF19 levels are reduced in GDM patients, in contrast with FGF21 levels. Both serum FGF19 and FGF21 levels are strongly related to insulin resistance and serum levels of adiponectin. Considering the different situation between FGF19 and FGF21, we suggest that reduced serum FGF19 levels could be involved in the pathophysiology of GDM, while increased serum FGF21 levels could be in a compensatory response to this disease.

Serum concentrations of fibroblast growth factors 19 and 21 in women with gestational diabetes mellitus: association with insulin resistance, adiponectin, and polycystic ovary syndrome history.

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Wang, Dongyu; Zhu, Wenjing; Li, Jieming; An, Chongyou; Wang, Zilian

2013-01-01

Fibroblast growth factor 19 (FGF19) and FGF21 are considered to be novel adipokines that improve glucose tolerance and insulin sensitivity. In the current study, we investigated serum FGF19 and FGF21 levels in patients with gestational diabetes mellitus (GDM) and explored their relationships with anthropometric and endocrine parameters. Serum FGF19 and FGF21 levels were determined by enzyme-linked immunosorbent assay (ELISA) in patients with GDM (n = 30) and healthy pregnant controls (n = 60) matched for maternal and gestational age. Serum FGF19 and FGF21 levels were correlated with anthropometric, metabolic, and endocrine parameters. Circulating levels of FGF19 were significantly reduced in patients with GDM relative to healthy pregnant subjects, whereas FGF21 levels were increased in GDM patients. Serum FGF19 levels independently and inversely correlated with insulin resistance (increased homeostasis model assessment of insulin resistance, HOMA-IR) and were positively related to serum adiponectin in both groups. In contrast, serum FGF21 levels independently and positively correlated with insulin resistance and serum triglycerides and were inversely related to serum adiponectin. In addition, in the combined population of both groups, those women with preconception polycystic ovary syndrome (PCOS) history had the lowest levels of FGF19, which were significantly lower than those in GDM patients without PCOS history and those in controls without PCOS history. Circulating FGF19 levels are reduced in GDM patients, in contrast with FGF21 levels. Both serum FGF19 and FGF21 levels are strongly related to insulin resistance and serum levels of adiponectin. Considering the different situation between FGF19 and FGF21, we suggest that reduced serum FGF19 levels could be involved in the pathophysiology of GDM, while increased serum FGF21 levels could be in a compensatory response to this disease.

High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching.

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Yu, Zhenxiao; Zheng, Hongchen; Zhao, Xingya; Li, Shufang; Xu, Jianyong; Song, Hui

2016-08-01

The effects of induction parameters, osmolytes and ethanol stress on the productivity of the recombinant alkaline catalase (KatA) in Escherichia coli BL21 (pET26b-KatA) were investigated. The yield of soluble KatA was significantly enhanced by 2% ethanol stress. And a certain amount of Triton X-100 supplementation could markedly improved extracellular ratio of KatA. A total soluble catalase activity of 78,762U/mL with the extracellular ratio of 92.5% was achieved by fed-batch fermentation in a 10L fermentor, which was the highest yield so far. The purified KatA showed high stability at 50°C and pH 6-10. Application of KatA for elimination of H2O2 after cotton fabrics bleaching led to less consumption of water, steam and electric power by 25%, 12% and 16.7% respectively without productivity and quality losing of cotton fabrics. Thus, the recombinant KatA is a promising candidate for industrial production and applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immunohistochemical study of the growth factors, aFGF, bFGF, PDGF-AB, VEGF-A and its receptor (Flk-1) during arteriogenesis.

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Wu, Song; Wu, Xiaoqiong; Zhu, Wu; Cai, Wei-Jun; Schaper, Jutta; Schaper, Wolfgang

2010-10-01

Growth factors are viewed as main arteriogenic stimulators for collateral vessel growth. However, the information about their native expression and distribution in collateral vessels is still limited. This study was designed to profile expression of acidic and basic FGF, platelet-derived growth factor (PDGF-AB) and vascular endothelial growth factor (VEGF-A) and its receptor, fetal liver kinase-1 (Flk-1) during arteriogenesis by confocal immunofluorescence in both dog ameroid constrictor model and rabbit arteriovenous shunt model of arteriogenesis. We found that: (1) in normal arteries (NA) in dog heart, aFGF, bFGF, and PDGF-AB all were mainly expressed in endothelial cells (EC) and media smooth muscle cells (SMC), but the expression of aFGF was very weak, with those of the other two being moderate; (2) in collateral arteries (CAs), aFGF, bFGF, and PDGF-AB all were significantly upregulated (P growth factors, aFGF, bFGF, and PDGF-AB are significantly upregulated in collateral vessels in dog heart, and enhanced VEGF-A and its receptor, Flk-1, are associated with rapid and lasting increased shear stress. These findings suggest that endogenous production of growth factors could be an important factor promoting collateral vessel growth.

Expression and purification of recombinant hemoglobin in Escherichia coli

DEFF Research Database (Denmark)

Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela

2011-01-01

BACKGROUND: Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe...... a protocol for expressing Hbs with low intrinsic solubilities. Since the alpha- and beta-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression......-translational modifications. CONCLUSION/SIGNIFICANCE: Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need...

Dimerization effect of sucrose octasulfate on rat FGF1

International Nuclear Information System (INIS)

Kulahin, N.; Kiselyov, V.; Kochoyan, A.; Kristensen, O.; Kastrup, Jette S.; Berezin, V.; Bock, E.; Gajhede, M.

2008-01-01

The work describes the sucrose octasulfate-mediated dimerization of rat FGF1 by gel-filtration experiments and crystal structure determination. Fibroblast growth factors (FGFs) constitute a family of at least 23 structurally related heparin-binding proteins that are involved in regulation of cell growth, survival, differentiation and migration. Sucrose octasulfate (SOS), a chemical analogue of heparin, has been demonstrated to activate FGF signalling pathways. The structure of rat FGF1 crystallized in the presence of SOS has been determined at 2.2 Å resolution. SOS-mediated dimerization of FGF1 was observed, which was further supported by gel-filtration experiments. The major contributors to the sulfate-binding sites in rat FGF1 are Lys113, Lys118, Arg122 and Lys128. An arginine at position 116 is a consensus residue in mammalian FGF molecules; however, it is a serine in rat FGF1. This difference may be important for SOS-mediated FGF1 dimerization in rat

FGF21 does not require adipocyte AMP-activated protein kinase (AMPK) or the phosphorylation of acetyl-CoA carboxylase (ACC) to mediate improvements in whole-body glucose homeostasis

DEFF Research Database (Denmark)

Mottillo, Emilio P; Desjardins, Eric M; Fritzen, Andreas Mæchel

2017-01-01

1β2AKO) and littermate controls were fed a high fat diet (HFD) and treated with native FGF21 or saline for two weeks. Additionally, HFD-fed mice with knock-in mutations on the AMPK phosphorylation sites of acetyl-CoA carboxylase (ACC)1 and ACC2 (DKI mice) along with wild-type (WT) controls received...

Fibroblast growth factor 21 is not required for glucose homeostasis, ketosis and tumour suppression associated to ketogenic diets in mice

Science.gov (United States)

Stemmer, Kerstin; Zani, Fabio; Habegger, Kirk M.; Neff, Christina; Kotzbeck, Petra; Bauer, Michaela; Yalamanchilli, Suma; Azad, Ali; Lehti, Maarit; Martins, Paulo J.F.; Müller, Timo D.; Pfluger, Paul T.; Seeley, Randy J.

2016-01-01

AIMS/HYPOTHESIS Ketogenic diets (KDs) increasingly gained attention as effective means for weight loss and potential adjunctive treatment of cancer. Metabolic benefits of KDs are regularly ascribed towards enhanced hepatic secretion of fibroblast growth factor (FGF) 21, and its systemic effects on fatty acid oxidation, energy expenditure and body weight. Ambiguous data from Fgf21 knockout strains and low FGF21 concentrations reported for humans in ketosis have nevertheless cast doubt regarding the endogenous function of FGF21. We here aimed to elucidate the causal role of FGF21 in mediating therapeutic benefits of KDs on metabolism and cancer. METHODS We established a dietary model of increased vs. decreased FGF21 by feeding C57BL/6J mice with KDs, either depleted or enriched with protein, respectively. We furthermore used wild type and Fgf21 knockout mice that were subjected to the respective diets, and monitored energy and glucose homeostasis as well as tumor growth after transplantation of Lewis-Lung-Carcinoma cells. RESULTS Hepatic and circulating but not adipose tissue FGF21 levels were profoundly increased by protein starvation and independent of the state of ketosis. We demonstrate that endogenous FGF21 is not essential for the maintenance of normoglycemia upon protein and carbohydrate starvation and is dispensable for the effects of KDs on energy expenditure. Furthermore, the tumor-suppressing effects of KDs were independent from FGF21, and rather driven by concomitant protein and carbohydrate starvation. CONCLUSION/INTERPRETATION Our data indicate that multiple systemic effects of KDs exposure in mice that were previously ascribed towards increased FGF21 secretion are rather a consequence of protein malnutrition. PMID:26099854

Fibroblast growth factor 21 improves insulin sensitivity and synergizes with insulin in human adipose stem cell-derived (hASC adipocytes.

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Darwin V Lee

Full Text Available Fibroblast growth factor 21 (FGF21 has evolved as a major metabolic regulator, the pharmacological administration of which causes weight loss, insulin sensitivity and glucose control in rodents and humans. To understand the molecular mechanisms by which FGF21 exerts its metabolic effects, we developed a human in vitro model of adipocytes to examine crosstalk between FGF21 and insulin signaling. Human adipose stem cell-derived (hASC adipocytes were acutely treated with FGF21 alone, insulin alone, or in combination. Insulin signaling under these conditions was assessed by measuring tyrosine phosphorylation of insulin receptor (InsR, insulin receptor substrate-1 (IRS-1, and serine 473 phosphorylation of Akt, followed by a functional assay using 14C-2-deoxyglucose [14C]-2DG to measure glucose uptake in these cells. FGF21 alone caused a modest increase of glucose uptake, but treatment with FGF21 in combination with insulin had a synergistic effect on glucose uptake in these cells. The presence of FGF21 also effectively lowered the insulin concentration required to achieve the same level of glucose uptake compared to the absence of FGF21 by 10-fold. This acute effect of FGF21 on insulin signaling was not due to IR, IGF-1R, or IRS-1 activation. Moreover, we observed a substantial increase in basal S473-Akt phosphorylation by FGF21 alone, in contrast to the minimal shift in basal glucose uptake. Taken together, our data demonstrate that acute co-treatment of hASC-adipocytes with FGF21 and insulin can result in a synergistic improvement in glucose uptake. These effects were shown to occur at or downstream of Akt, or separate from the canonical insulin signaling pathway.

Levothyroxine treatment restored the decreased circulating fibroblast growth factor 21 levels in patients with hypothyroidism.

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Wang, Guang; Liu, Jia; Yang, Ning; Hu, Yanjin; Zhang, Heng; Miao, Li; Yao, Zhi; Xu, Yuan

2016-06-01

Fibroblast growth factor 21 (FGF21) is an important endogenous regulator of energy metabolism. Thyroid hormone has been shown to regulate hepatic FGF21 expression in rodents. The goal of this study was to evaluate the plasma FGF21 levels in participants with normal thyroid function, subclinical hypothyroidism, or overt hypothyroidism and to investigate the change of plasma FGF21 levels in patients with overt hypothyroidism after levothyroxine treatment. A total of 473 drug-naive participants were recruited, including 250 healthy control subjects, 116 patients with subclinical hypothyroidism, and 107 patients with overt hypothyroidism. Thirty-eight patients with overt hypothyroidism were assigned to receive levothyroxine treatment. The overt hypothyroidism group had decreased FGF21 levels compared with the control and subclinical hypothyroidism groups (Ptreatment markedly attenuated the increased circulating levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive protein (hsCRP), and homeostasis model assessment index of insulin resistance (HOMA-IR) in patients with overt hypothyroidism. A significant increase in plasma FGF21 levels was observed after levothyroxine treatment (Ptreatment (FT3: r=0.44; FT4: r=0.53; all Ptreatment ameliorated metabolic disorders and restored the decreased circulating FGF21 levels in patients with overt hypothyroidism. The increase in FGF21 levels after levothyroxine treatment might be partly associated with the amelioration of metabolic disorders in patients with hypothyroidism. Copyright © 2016 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

Significant gender difference in serum levels of fibroblast growth factor 21 in Danish children and adolescents

DEFF Research Database (Denmark)

Bisgaard, Amalie; Sørensen, Kaspar; Johannsen, Trine Holm

2014-01-01

children (21%) had levels below detection limit of assay. Baseline levels of FGF21 showed positive correlation with triglycerides, but no significant correlations were found between FGF21-concentration and body mass index (BMI), DXA-derived fat percentage, LDL- HDL- and non-HDL cholesterol, leptin......INTRODUCTION: Fibroblast Growth Factor 21 (FGF21) is a novel metabolic factor with effect on glucose and lipid metabolism, and shown to be elevated in diseases related to metabolic syndrome. Due to the increasing frequency of metabolic syndrome in the pediatric population, and as FGF21 studies...... in children are limited, we investigated baseline serum levels of FGF21 in healthy children during an oral glucose tolerance test. METHODS: A total of 179 children and adolescents from the COPENHAGEN Puberty Study were included. An OGTT with glucose and insulin measurements, a dual energy X-ray absorptiometry...

Expansion in the presence of FGF-2 enhances the functional development of cartilaginous tissues engineered using infrapatellar fat pad derived MSCs.

Science.gov (United States)

Buckley, C T; Kelly, D J

2012-07-01

MSCs from non-cartilaginous knee joint tissues such as the infrapatellar fat pad (IFP) and synovium possess significant chondrogenic potential and provide a readily available and clinically feasible source of chondroprogenitor cells. Fibroblast growth factor-2 (FGF-2) has been shown to be a potent mitotic stimulator during ex vivo expansion of MSCs, as well as regulating their subsequent differentiation potential. The objective of this study was to investigate the longer term effects of FGF-2 expansion on the functional development of cartilaginous tissues engineered using MSCs derived from the IFP. IFP MSCs were isolated and expanded to passage 2 in a standard media formulation with or without FGF-2 (5 ng/ml) supplementation. Expanded cells were encapsulated in agarose hydrogels, maintained in chondrogenic media for 42 days and analysed to determine their mechanical properties and biochemical composition. Culture media, collected at each feed, was also analysed for biochemical constituents. MSCs expanded in the presence of FGF-2 proliferated more rapidly, with higher cell yields and lower population doubling times. FGF-2 expanded MSCs generated the most mechanically functional tissue. Matrix accumulation was dramatically higher after 21 days for FGF-2 expanded MSCs, but decreased between day 21 and 42. By day 42, FGF-2 expanded MSCs had still accumulated ∼1.4 fold higher sGAG and ∼1.7 fold higher collagen compared to control groups. The total amount of sGAG synthesised (retained in hydrogels and released into the media) was ∼2.4 fold higher for FGF-2 expanded MSCs, with only ∼25% of the total amount generated being retained within the constructs. Further studies are required to investigate whether IFP derived MSCs have a diminished capacity to synthesise other matrix components important in the aggregation, assembly and retention of proteoglycans. In conclusion, expanding MSCs in the presence of FGF-2 rapidly accelerates chondrogenesis in 3D agarose

Fibroblast growth factor 21 attenuates hepatic fibrogenesis through TGF-β/smad2/3 and NF-κB signaling pathways

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Xu, Pengfei; Zhang, Yingjie; Liu, Yunye; Yuan, Qingyan; Song, Liying; Liu, Mingyao; Liu, Zhihang; Yang, Yongbi; Li, Junyan; Li, Deshan, E-mail: deshanli@163.com; Ren, Guiping, E-mail: renguiping@126.com

2016-01-01

Fibroblast growth factor 21 (FGF-21) is a secreted protein, which has anti-diabetic and lipocaic effects, but its ability to protect against hepatic fibrosis has not been studied. In this study, we investigated the ability of FGF-21 to attenuate dimethylnitrosamine (DMN)-induced hepatic fibrogenesis in mice and the mechanism of its action. Hepatic fibrosis was induced by injection of DMN, FGF-21 was administered to the mice once daily in association with DMN injection till the end of the experiment. Histopathological examination, tissue 4-hydroxyproline content and expressions of smooth muscle α-actin (α-SMA) and collagen I were measured to assess hepatic fibrosis. Ethanol/PDGF-BB-activated hepatic stellate cells (HSCs) were used to understand the mechanisms of FGF-21 inhibited hepatic fibrogenesis. Results showed that FGF-21 treatment attenuated hepatic fibrogenesis and was associated with a significant decrease in intrahepatic fibrogenesis, 4-hydroxyproline accumulation, α-SMA expression and collagen I deposition. FGF-21 treatment inhibited the activation of HSCs via down-regulating the expression of TGF-β, NF-κB nuclear translocation, phosphorylation levels of smad2/3 and IκBα. Besides, FGF-21 treatment caused activated HSC apoptosis with increasing expression of Caspase-3, and decreased the ratio of Bcl-2 to Bax. In conclusion, FGF-21 attenuates hepatic fibrogenesis and inhibits the activation of HSC warranting the use of FGF-21 as a potential therapeutic agent in the treatment of hepatic fibrosis. - Highlights: • Fibroblast growth factor 21 attenuates hepatic fibrogenesis. • Fibroblast growth factor 21 attenuates hepatic fibrogenesis via TGF-β/smad2/3 signaling pathways. • Fibroblast growth factor 21 attenuates hepatic fibrogenesis via NF-κB signaling pathways.

Fibroblast growth factor 21, fibroblast growth factor receptor 1, and β-Klotho expression in bovine growth hormone transgenic and growth hormone receptor knockout mice.

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Brooks, Nicole E; Hjortebjerg, Rikke; Henry, Brooke E; List, Edward O; Kopchick, John J; Berryman, Darlene E

Although growth hormone (GH) and fibroblast growth factor 21 (FGF21) have a reported relationship, FGF21 and its receptor, fibroblast growth factor receptor 1 (FGFR1) and cofactor β-Klotho (KLB), have not been analyzed in chronic states of altered GH action. The objective of this study was to quantify circulating FGF21 and tissue specific expression of Fgf21, Fgfr1, and Klb in mice with modified GH action. Based on previous studies, we hypothesized that bovine GH transgenic (bGH) mice will be FGF21 resistant and GH receptor knockout (GHR-/-) mice will have normal FGF21 action. Seven-month-old male bGH mice (n=9) and wild type (WT) controls (n=10), and GHR-/- mice (n=8) and WT controls (n=8) were used for all measurements. Body composition was determined before dissection, and tissue weights were measured at the time of dissection. Serum FGF21 levels were evaluated by ELISA. Expression of Fgf21, Fgfr1, and Klb mRNA in white adipose tissue (AT), brown AT, and liver were evaluated by reverse transcription quantitative PCR. As expected, bGH mice had increased body weight (p=3.70E -8 ) but decreased percent fat mass (p=4.87E -4 ). Likewise, GHR-/- mice had decreased body weight (p=1.78E -10 ) but increased percent fat mass (p=1.52E -9 ), due to increased size of the subcutaneous AT depot when normalized to body weight (p=1.60E -10 ). Serum FGF21 levels were significantly elevated in bGH mice (p=0.041) and unchanged in GHR-/- mice (p=0.88). Expression of Fgf21, Fgfr1, and Klb mRNA in white AT and liver were downregulated or unchanged in both bGH and GHR-/- mice. The only exception was Fgf21 expression in brown AT of GHR-/-, which trended toward increased expression (p=0.075). In accordance with our hypothesis, we provide evidence that circulating FGF21 is increased in bGH animals, but remains unchanged in GHR-/- mice. Downregulation or no change in Fgf21, Fgfr1, and Klb expression are seen in white AT, brown AT, and liver of bGH and GHR-/- mice when compared to their

Expression of fibroblast growth factor-21 in muscle is associated with lipodystrophy, insulin resistance and lipid disturbances in patients with HIV.

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Birgitte Lindegaard

Full Text Available BACKGROUND: Fibroblast growth factor (FGF-21 is a novel regulator of glucose and lipid metabolism. Recently, increased FGF-21 mRNA expression in muscle was found in patients with type 2 diabetes, but the role for FGF-21 in muscle is not well understood. Patients with HIV-infection and lipodystrophy are characterised by various degree of lipid-driven insulin resistance. We hypothesized that muscle FGF-21 mRNA would be altered in HIV patients with lipodystrophy. DESIGN: Twenty-five HIV-infected men with lipodystrophy (LD and 15 age-matched healthy controls, received an oral glucose tolerance test and a euglycemic-hyperinsulinemic clamp (50 mU/m2/min combined with 6,6-H2 glucose infusion. Muscle biopsies were obtained and FGF-21 mRNA and glycogen synthase (GS activity were measured. RESULTS: Subjects with HIV were insulin resistant compared with non-HIV subjects. Compared to controls, HIV subjects demonstrated a twofold increase of plasma FGF-21 from 70.4±56.8 pg/ml vs 109.1±71.8 pg/ml, respectively (p = 0.04 and an eight-fold increase in muscular FGF-21 mRNA expression (p = 0.001. Muscle FGF-21 mRNA correlated inversely with the rate of disappearance of glucose during insulin clamp (r = -0.54, p = 0.0009, and the GS fractional velocity in muscle (r = -0.39, p = 0.03, and directly with fasting insulin (r = 0.50, p = 0.0022, HOMA-IR (r = 0.47, p = 0.004, triglycerides (r = 0.60. P = 0.0001, waist-to-hip ratio (r = 0.51, p = 0.0001 and limb fat mass (-0.46, p = 0.004, but not to plasma FGF-21. CONCLUSION: FGF-21 mRNA is increased in skeletal muscle in HIV patients and correlates to whole-body (primarily reflecting muscle insulin resistance, but not to plasma FGF-21. Those findings add to the evidence that FGF-21 is a myokine and may suggest that muscle FGF-21 is working in a local manner.

Recombinant DNA derived monomeric insulin analogue: comparison with soluble human insulin in normal subjects.

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Vora, J P; Owens, D R; Dolben, J; Atiea, J A; Dean, J D; Kang, S; Burch, A; Brange, J

1988-11-12

To compare the rate of absorption from subcutaneous tissue and the resulting hypoglycaemic effect of iodine-125 labelled soluble human insulin and a monomeric insulin analogue derived by recombinant DNA technology. Single blind randomised comparison of equimolar doses of 125I labelled soluble human insulin and insulin analogue. Study in normal people at a diabetes research unit and a university department of medical physics. Seven healthy male volunteers aged 20-39 not receiving any other drugs. After an overnight fast and a basal period of one hour two doses (0.05 and 0.1 U/kg) of 125I labelled soluble human insulin and insulin analogue were injected subcutaneously into the anterior abdominal wall on four separate days. To find a fast acting insulin for meal related requirements in insulin dependent diabetics. MEASUREMENTS and main results--Residual radioactivity at the injection site was measured continuously for the first two hours after injection of the 125I labelled preparations and thereafter for five minutes simultaneously with blood sampling. Frequent venous blood samples were obtained over six hours for determination of plasma immunoreactive insulin, insulin analogue, glucose, and glucagon values. Time to 50% of initial radioactivity at the injection site for the insulin analogue compared with soluble insulin was 61 v 135 minutes (p less than 0.05) with 0.05 U/kg and 67 v 145 minutes (p less than 0.001) with 0.1 U/kg. Concentrations in plasma increased faster after the insulin analogue compared with soluble insulin, resulting in higher plasma concentrations between 10 and 150 minutes (0.001 less than p less than 0.05) after 0.05 U/kg and between 40 and 360 minutes (0.001 less than p less than 0.05) after 0.1 U/kg. The hypoglycaemic response to insulin analogue was a plasma glucose nadir at 60 minutes with both doses compared with 90 and 120 minutes with soluble insulin at 0.5 and 0.1 U/kg respectively. The response of glucagon substantiated the earlier and

Expression and purification of recombinant hemoglobin in Escherichia coli.

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Chandrasekhar Natarajan

Full Text Available Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus, a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

Expression and purification of recombinant hemoglobin in Escherichia coli.

Science.gov (United States)

Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela; Weber, Roy E; Moriyama, Hideaki; Storz, Jay F

2011-01-01

Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species. As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications. Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

Remodelling of human osteoarthritic cartilage by FGF-2, alone or combined with Sox9 via rAAV gene transfer.

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Cucchiarini, Magali; Terwilliger, Ernest F; Kohn, Dieter; Madry, Henning

2009-08-01

Compensating for the loss of extracellular cartilage matrix, as well as counteracting the alterations of the chondrocyte phenotype in osteoarthritis are of key importance to develop effective therapeutic strategies against this disorder. In the present study, we analysed the benefits of applying a potent gene combination to remodel human osteoarthritic (OA) cartilage. We employed the promising recombinant adeno-associated virus (rAAV) vector to deliver the mitogenic fibroblast growth factor 2 (FGF-2) factor, alone or simultaneously with the transcription factor Sox9 as a key activator of matrix synthesis, to human normal and OA articular chondrocytes. We evaluated the effects of single (FGF-2) or combined (FGF-2/SOX9) transgene expression upon the regenerative activities of chondrocytes in three dimensional cultures in vitro and in cartilage explants in situ. Single overexpression of FGF-2 enhanced the survival and proliferation of both normal and OA chondrocytes, without stimulating the matrix synthetic processes in the increased pools of cells. The mitogenic properties of FGF-2 were maintained when SOX9 was co-overexpressed and concomitant with an increase in the production of proteoglycans and type-II collagen, suggesting that the transcription factor was capable of counterbalancing the effects of FGF-2 on matrix accumulation. Also important, expression of type-X collagen, a marker of hypertrophy strongly decreased following treatment by the candidate vectors. Most remarkably, the levels of activities achieved in co-treated human OA cartilage were similar to or higher than those observed in normal cartilage. The present findings show that combined expression of candidate factors in OA cartilage can re-establish key features of normal cartilage and prevent the pathological shift of metabolic homeostasis. These data provide further motivation to develop coupled gene transfer approaches via rAAV for the treatment of human OA.

Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

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Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

2015-01-01

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins. © 2015 S. Karger AG, Basel.

bFGF influences human articular chondrocyte differentiation

DEFF Research Database (Denmark)

Schmal, H; Zwingmann, J; Fehrenbach, M

2007-01-01

BACKGROUND: The possible functional role of basic fibroblast growth factor (bFGF) in regulating the mitotic and metabolic activity of primary human articular chondrocytes was investigated. METHODS: [EF1]Chondrocytes were enzymatically isolated from femoral head cartilage, and were cultured in vitro......FGF concentrations in supernatants of primary human articular chondrocytes peaked immediately after isolation and then declined. In a dose-dependent manner, bFGF enhanced cell amplification and viability. BFGF induced a decrease in the apoptotic cell population, while the number of proliferating cells remained...... by 53%, which was correlated with diminished mRNA production. Monolayer cultured chondrocytes secreted significant amounts of aggrecan that decreased over time. Secretion of this cartilage-specific marker was further reduced by the addition of bFGF. DISCUSSION: These findings highlight the potential...

Injectable Shear-Thinning CaSO4/FGF-18-Incorporated Chitin-PLGA Hydrogel Enhances Bone Regeneration in Mice Cranial Bone Defect Model.

Science.gov (United States)

Sivashanmugam, A; Charoenlarp, Pornkawee; Deepthi, S; Rajendran, Arunkumar; Nair, Shantikumar V; Iseki, Sachiko; Jayakumar, R

2017-12-13

For craniofacial bone regeneration, shear-thinning injectable hydrogels are favored over conventional scaffolds because of their improved defect margin adaptability, easier handling, and ability to be injected manually into deeper tissues. The most accepted method, after autografting, is the use of recombinant human bone morphogenetic protein-2 (BMP-2); however, complications such as interindividual variations, edema, and poor cost-efficiency in supraphysiological doses have been reported. The endogenous synthesis of BMP-2 is desirable, and a molecule which induces this is fibroblast growth factor-18 (FGF-18) because it can upregulate the BMP-2 expression  by supressing noggin. We developed a chitin-poly(lactide-co-glycolide) (PLGA) composite hydrogel by regeneration chemistry and then incorporated CaSO 4 and FGF-18 for this purpose. Rheologically, a 7-fold increase in the elastic modulus was observed in the CaSO 4 -incorporated chitin-PLGA hydrogels as compared to the chitin-PLGA hydrogel. Shear-thinning Herschel-Bulkley fluid nature was observed for both hydrogels. Chitin-PLGA/CaSO 4 gel showed sustained release of FGF-18. In vitro osteogenic differentiation showed an enhanced alkaline phosphatase (ALP) expression in the FGF-18-containing chitin-PLGA/CaSO 4 gel when compared to cells alone. Further, it was confirmed by studying the expression of osteogenic genes [RUNX2, ALP, BMP-2, osteocalcin (OCN), and osteopontin (OPN)], immunofluorescence staining of BMP-2, OCN, and OPN, and alizarin red S staining. Incorporation of FGF-18 in the hydrogel increased the endothelial cell migration. Further, the regeneration potential of the prepared hydrogels was tested in vivo, and longitudinal live animal μ-CT was performed. FGF-18-loaded chitin-PLGA/CaSO 4 showed early and almost complete bone healing in comparison with chitin-PLGA/CaSO 4 , chitin-PLGA/FGF-18, chitin-PLGA, and sham control systems, as confirmed by hematoxylin and eosin and osteoid tetrachrome stainings

Fgf16 is essential for pectoral fin bud formation in zebrafish

International Nuclear Information System (INIS)

Nomura, Ryohei; Kamei, Eriko; Hotta, Yuuhei; Konishi, Morichika; Miyake, Ayumi; Itoh, Nobuyuki

2006-01-01

Zebrafish pectoral fin bud formation is an excellent model for studying morphogenesis. Fibroblast growth factors (Fgfs) and sonic hedgehog (shh) are essential for pectoral fin bud formation. We found that Fgf16 was expressed in the apical ectodermal ridge (AER) of fin buds. A knockdown of Fgf16 function resulted in no fin bud outgrowth. Fgf16 is required for cell proliferation and differentiation in the mesenchyme and the AER of the fin buds, respectively. Fgf16 functions downstream of Fgf10, a mesenchymal factor, signaling to induce the expression of Fgf4 and Fgf8 in the AER. Fgf16 in the AER and shh in the zone of polarizing activity (ZPA) interact to induce and/or maintain each other's expression. These findings have revealed that Fgf16, a newly identified AER factor, plays a crucial role in pectoral fin bud outgrowth by mediating the interactions of AER-mesenchyme and AER-ZPA

Mapping of FGF1 in the Medulla Oblongata of Macaca fascicularis.

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Bisem, Naomi J; Takeuchi, Shigeko; Imamura, Toru; Abdelalim, Essam M; Tooyama, Ikuo

2012-12-26

FGF1 is highly expressed in neurons and it has been proposed to play a role in the neuroprotection and in regeneration. Low FGF1 expression in neurons has been linked to increased vulnerability in cholinergic neurons. Previous reports have shown that the expression of FGF1 in rat brain is localized to the cholinergic nuclei of the medulla oblongata, with low ratio of neurons positive for FGF1 in the dorsal motor nucleus of the vagus (DMNV). The role of FGF1 in the primate brain has yet to be clarified. In this study, we mapped FGF1 immunoreactivity in the medulla oblongata of cynomolgus monkey brainstems. Our results demonstrated that FGF1 immunoreactivity follows the pattern of distribution of cholinergic nuclei in the medulla oblongata; with strong localization of FGF1 to cholinergic neurons of the hypoglossal nucleus, the facial nucleus and the nucleus ambiguus. In contrast, the DMNV shows markedly lower FGF1 immunoreactivity. Localization of FGF1 to cholinergic neurons was only observed in the lateral region of the DMNV, with higher immunoreactivity in the rostral ventral-lateral region of the DMNV. These findings are consistent with the distribution of FGF1 immunoreactivity in previous studies of the rat brain.

Mapping of FGF1 in the Medulla Oblongata of Macaca fascicularis

International Nuclear Information System (INIS)

Bisem, Naomi J.; Takeuchi, Shigeko; Imamura, Toru; Abdelalim, Essam M.; Tooyama, Ikuo

2012-01-01

FGF1 is highly expressed in neurons and it has been proposed to play a role in the neuroprotection and in regeneration. Low FGF1 expression in neurons has been linked to increased vulnerability in cholinergic neurons. Previous reports have shown that the expression of FGF1 in rat brain is localized to the cholinergic nuclei of the medulla oblongata, with low ratio of neurons positive for FGF1 in the dorsal motor nucleus of the vagus (DMNV). The role of FGF1 in the primate brain has yet to be clarified. In this study, we mapped FGF1 immunoreactivity in the medulla oblongata of cynomolgus monkey brainstems. Our results demonstrated that FGF1 immunoreactivity follows the pattern of distribution of cholinergic nuclei in the medulla oblongata; with strong localization of FGF1 to cholinergic neurons of the hypoglossal nucleus, the facial nucleus and the nucleus ambiguus. In contrast, the DMNV shows markedly lower FGF1 immunoreactivity. Localization of FGF1 to cholinergic neurons was only observed in the lateral region of the DMNV, with higher immunoreactivity in the rostral ventral-lateral region of the DMNV. These findings are consistent with the distribution of FGF1 immunoreactivity in previous studies of the rat brain

A SEP tag enhances the expression, solubility and yield of recombinant TEV protease without altering its activity.

Science.gov (United States)

Nautiyal, Kalpana; Kuroda, Yutaka

2018-05-25

Tobacco Etch Virus (TEV) protease is used in the purification of recombinant proteins, but its usage is often hampered by solubility issues. Here, we report a short, 12-residue solubility enhancing peptide (SEP) tag attached at the C-terminus of TEV (TEV-C9R). We assessed the effects of the C9R tag on the biophysical and biochemical characteristics of TEV. The yield of HPLC purified TEV-C9R expressed in E. coli grown in 200 mL LB or TB media was between 10 and 13 mg, which was up to 6.5 times higher than the yield of the untagged TEV (untagged-TEV). TEV-C9R was active over a pH range of 5-8, which was wider than that of the commonly used thrombin, and it remained active upon incubation at 60 °C much longer than the untagged-TEV, which aggregated at this temperature. Static and dynamic light scattering demonstrated the higher solubility of purified TEV-C9R. Furthermore, the thermal unfolding of TEV-C9R, as assessed by circular dichroism at pH 4.7, was almost perfectly reversible, in contrast to that of untagged-TEV, which aggregated at high temperature. These results demonstrate the improved biophysical and biochemical characteristics of TEV-C9R originating from higher solubility and provide another example of how SEP tags can enhance enzyme solubility without altering its activity. Copyright © 2018 Elsevier B.V. All rights reserved.

Application of VEGFA and FGF-9 enhances angiogenesis, osteogenesis and bone remodeling in type 2 diabetic long bone regeneration.

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Christoph Wallner

Full Text Available Although bone regeneration is typically a reliable process, type 2 diabetes is associated with impaired or delayed healing processes. In addition, angiogenesis, a crucial step in bone regeneration, is often altered in the diabetic state. In this study, different stages of bone regeneration were characterized in an unicortical bone defect model comparing transgenic type 2 diabetic (db-/db- and wild type (WT mice in vivo. We investigated angiogenesis, callus formation and bone remodeling at early, intermediate and late time points by means of histomorphometry as well as protein level analyses. In order to enhance bone regeneration, defects were locally treated with recombinant FGF-9 or VEGFA. Histomorphometry of aniline blue stained sections indicated that bone regeneration is significantly decreased in db-/db- as opposed to WT mice at intermediate (5 days post operation and late stages (7 days post operation of bone regeneration. Moreover, immunohistochemical analysis revealed significantly decreased levels of RUNX-2, PCNA, Osteocalcin and PECAM-1 in db-/db- defects. In addition, osteoclastogenesis is impaired in db-/db- indicating altered bone remodeling. These results indicate significant impairments in angiogenesis and osteogenesis in type 2 diabetic bones. Importantly, angiogenesis, osteogenesis and bone remodeling could be reconstituted by application of recombinant FGF-9 and, in part, by VEGFA application. In conclusion, our study demonstrates that type 2 diabetes affects angiogenesis, osteogenesis and subsequently bone remodeling, which in turn leads to decreased bone regeneration. These effects could be reversed by local application of FGF-9 and to a lesser degree VEGFA. These data could serve as a basis for future therapeutic applications aiming at improving bone regeneration in the type 2 diabetic patient population.

IHH and FGF8 coregulate elongation of digit primordia.

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Zhou, Jian; Meng, Junwei; Guo, Shengzhen; Gao, Bo; Ma, Gang; Zhu, Xuming; Hu, Jianxin; Xiao, Yue; Lin, Chuwen; Wang, Hongsheng; Ding, Lusheng; Feng, Guoyin; Guo, Xizhi; He, Lin

2007-11-23

In the developing limb bud, digit pattern arises from anterior-posterior (A-P) positional information which is provided by the concentration gradient of SHH. However, the mechanisms of translating early asymmetry into morphological form are still unclear. Here, we examined the ability of IHH and FGF8 signaling to regulate digital chondrogenesis, by implanting protein-loaded beads in the interdigital space singly and in combination. We found that IHH protein induced an elongated digit and that FGF8 protein blocked the terminal phalange formation. Molecular marker analysis showed that IHH expanded Sox9 expression in mesenchymal cells possibly through up-regulated FGF8 expression. Application of both IHH and FGF8 protein induced a large terminal phalange. These results suggest that both enhanced IHH and FGF8 signaling are required for the development of additional cartilage element in limbs. IHH and FGF8 maybe play different roles and act synergistically to promote chondrogenesis during digit primordia elongation.

Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (Fgf23) concentrations and hyperphosphatemia despite increased Fgf23 expression.

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Ichikawa, Shoji; Sorenson, Andrea H; Austin, Anthony M; Mackenzie, Donald S; Fritz, Timothy A; Moh, Akira; Hui, Siu L; Econs, Michael J

2009-06-01

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia. The disease is caused by inactivating mutations in fibroblast growth factor 23 (FGF23), Klotho (KL), and uridine diphosphate-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In vitro studies indicate that GALNT3 O-glycosylates a phosphaturic hormone, FGF23, and prevents its proteolytic processing, thereby allowing secretion of intact FGF23. In this study we generated mice lacking the Galnt3 gene, which developed hyperphosphatemia without apparent calcifications. In response to hyperphosphatemia, Galnt3-deficient mice had markedly increased Fgf23 expression in bone. However, compared with wild-type and heterozygous littermates, homozygous mice had only about half of circulating intact Fgf23 levels and higher levels of C-terminal Fgf23 fragments in bone. Galnt3-deficient mice also exhibited an inappropriately normal 1,25-dihydroxyvitamin D level and decreased alkaline phosphatase activity. Furthermore, renal expression of sodium-phosphate cotransporters and Kl were elevated in Galnt3-deficient mice. Interestingly, there were sex-specific phenotypes; only Galnt3-deficient males showed growth retardation, infertility, and significantly increased bone mineral density. In summary, ablation of Galnt3 impaired secretion of intact Fgf23, leading to decreased circulating Fgf23 and hyperphosphatemia, despite increased Fgf23 expression. Our findings indicate that Galnt3-deficient mice have a biochemical phenotype of tumoral calcinosis and provide in vivo evidence that Galnt3 plays an essential role in proper secretion of Fgf23 in mice.

Effect of rb-bFGF eye drops and hydroxyl indican eye drops on tear film stability and dry eye symptoms after age-related cataract surgery

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Jing Chen

2017-10-01

Full Text Available AIM: To compare and analyze the effect of recombinant bovine basic fibroblast growth factor(bFGFeye drops and hydroxyl indican eye drops on the tear film stability and dry eye symptoms after age-related cataract surgery. METHODS: A total of 115 patients(115 affected eyeswith dry eyes after age-related cataract surgery were divided into the bFGF group, the hydroxyl indican group and the blank control group by the random number table method. The blank control group was only given routine anti-inflammatory treatment, and on the basis, the bFGF group and the hydroxyl indican group were treated with bFGF eye drops and hydroxyl indican eye drops respectively. The clinical efficacy, adverse reactions, changes in scores of dry eye symptoms, Schirmer test(SⅠt, tear film break-up time(BUTand corneal fluorescein staining(CFSscores at different time points were compared among the three groups. RESULTS: The total markedly effective rates in the bFGF group(89.5%was higher than that in the hydroxyl indican group(70.3%or the blank control group(47.5%(PPPCONCLUSION: The tear film is unstable in early stage after age-related cataract surgery, and there are dry eye symptoms. The intervention with bFGF eye drops and hydroxyl indican eye drops can effectively restore the tear film stability and significantly relieve dry eye symptoms, and the effect of bFGF eye drops is more significant.

Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

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Adam Bajinting

2017-06-01

Full Text Available Fibroblast growth factor receptors (FGFRs are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells.

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Arii, Yasuhiro; Yamaguchi, Hidenori; Fukuoka, Shin-Ichi

2007-10-01

The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 degrees C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88+/-0.07 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.

Fibroblast growth factor 15/19 (FGF15/19) protects from diet-induced hepatic steatosis: development of an FGF19-based chimeric molecule to promote fatty liver regeneration.

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Alvarez-Sola, Gloria; Uriarte, Iker; Latasa, M Ujue; Fernandez-Barrena, Maite G; Urtasun, Raquel; Elizalde, Maria; Barcena-Varela, Marina; Jiménez, Maddalen; Chang, Haisul C; Barbero, Roberto; Catalán, Victoria; Rodríguez, Amaia; Frühbeck, Gema; Gallego-Escuredo, José M; Gavaldà-Navarro, Aleix; Villarroya, Francesc; Rodriguez-Ortigosa, Carlos M; Corrales, Fernando J; Prieto, Jesus; Berraondo, Pedro; Berasain, Carmen; Avila, Matias A

2017-10-01

Fibroblast growth factor 15/19 (FGF15/19), an enterokine that regulates synthesis of hepatic bile acids (BA), has been proposed to influence fat metabolism. Without FGF15/19, mouse liver regeneration after partial hepatectomy (PH) is severely impaired. We studied the role of FGF15/19 in response to a high fat diet (HFD) and its regulation by saturated fatty acids. We developed a fusion molecule encompassing FGF19 and apolipoprotein A-I, termed Fibapo, and evaluated its pharmacological properties in fatty liver regeneration. Fgf15 -/- mice were fed a HFD. Liver fat and the expression of fat metabolism and endoplasmic reticulum (ER) stress-related genes were measured. Influence of palmitic acid (PA) on FGF15/19 expression was determined in mice and in human liver cell lines. In vivo half-life and biological activity of Fibapo and FGF19 were compared. Hepatoprotective and proregenerative activities of Fibapo were evaluated in obese db/db mice undergoing PH. Hepatosteatosis and ER stress were exacerbated in HFD-fed Fgf15 -/- mice. Hepatic expression of Pparγ2 was elevated in Fgf15 -/- mice, being reversed by FGF19 treatment. PA induced FGF15/19 expression in mouse ileum and human liver cells, and FGF19 protected from PA-mediated ER stress and cytotoxicity. Fibapo reduced liver BA and lipid accumulation, inhibited ER stress and showed enhanced half-life. Fibapo provided increased db/db mice survival and improved regeneration upon PH. FGF15/19 is essential for hepatic metabolic adaptation to dietary fat being a physiological regulator of Pparγ2 expression . Perioperative administration of Fibapo improves fatty liver regeneration. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

FGF-dependent metabolic control of vascular development

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Yu, Pengchun; Alves, Tiago C.; Fang, Jennifer S.; Xie, Yi; Zhu, Jie; Chen, Zehua; De Smet, Frederik; Zhang, Jiasheng; Jin, Suk-Won; Sun, Lele; Sun, Hongye; Kibbey, Richard G.; Hirschi, Karen K.; Hay, Nissim; Carmeliet, Peter; Chittenden, Thomas W.; Eichmann, Anne; Potente, Michael; Simons, Michael

2017-01-01

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are of importance to these processes1. While much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism2,3, little is understood about the role of fibroblast growth factors (FGFs) in this context4. Here we identify FGF receptor (FGFR) signaling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signaling inputs results in decreased glycolysis leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/r3 double mutant mice while HK2 overexpression partially rescues the defects caused by suppression of FGF signaling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development. PMID:28467822

Genetic rescue of glycosylation-deficient Fgf23 in the Galnt3 knockout mouse.

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Ichikawa, Shoji; Gray, Amie K; Padgett, Leah R; Allen, Matthew R; Clinkenbeard, Erica L; Sarpa, Nicole M; White, Kenneth E; Econs, Michael J

2014-10-01

Fibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence ((176)RHTR(179)↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineate the role of glycosylation in the Fgf23 function, we generated an inducible FGF23 transgenic mouse expressing human mutant FGF23 (R176Q and R179Q) found in patients with autosomal dominant hypophosphatemic rickets (ADHR) and bred this animal to Galnt3 knockout mice, a model of familial tumoral calcinosis. Due to the low intact Fgf23 level, Galnt3 knockout mice with wild-type Fgf23 alleles were hyperphosphatemic. In contrast, carriers of the mutant FGF23 transgene, regardless of Galnt3 mutation status, had significantly higher serum intact FGF23, resulting in severe hypophosphatemia. Importantly, serum phosphorus and FGF23 were comparable between transgenic mice with or without normal Galnt3 alleles. To determine whether the presence of the ADHR mutation could improve biochemical and skeletal abnormalities in Galnt3-null mice, these mice were also mated to Fgf23 knock-in mice, carrying heterozygous or homozygous R176Q ADHR Fgf23 mutations. The knock-in mice with functional Galnt3 had normal Fgf23 but were slightly hypophosphatemic. The stabilized Fgf23 ADHR allele reversed the Galnt3-null phenotype and normalized total Fgf23, serum phosphorus, and bone Fgf23 mRNA. However, the skeletal phenotype was unaffected. In summary, these data demonstrate that O-glycosylation by ppGaINAc-T3 is only necessary for proper secretion of intact Fgf23 and, once secreted, does not affect Fgf23 function. Furthermore, the more stable Fgf23 ADHR mutant protein could normalize serum phosphorus

Fibroblast growth factor 21 protects mouse brain against D-galactose induced aging via suppression of oxidative stress response and advanced glycation end products formation.

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Yu, Yinhang; Bai, Fuliang; Wang, Wenfei; Liu, Yaonan; Yuan, Qingyan; Qu, Susu; Zhang, Tong; Tian, Guiyou; Li, Siming; Li, Deshan; Ren, Guiping

2015-06-01

Fibroblast growth factor 21 (FGF21) is a hormone secreted predominantly in the liver, pancreas and adipose tissue. Recently, it has been reported that FGF21-Transgenic mice can extend their lifespan compared with wild type counterparts. Thus, we hypothesize that FGF21 may play some roles in aging of organisms. In this study d-galactose (d-gal)-induced aging mice were used to study the mechanism that FGF21 protects mice from aging. The three-month-old Kunming mice were subcutaneously injected with d-gal (180mg·kg(-1)·d(-1)) for 8weeks and administered simultaneously with FGF21 (1, 2 or 5mg·kg(-1)·d(-1)). Our results showed that administration of FGF21 significantly improved behavioral performance of d-gal-treated mice in water maze task and step-down test, reduced brain cell damage in the hippocampus, and attenuated the d-gal-induced production of MDA, ROS and advanced glycation end products (AGEs). At the same time, FGF21 also markedly renewed the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total anti-oxidation capability (T-AOC), and decreased the enhanced total cholinesterase (TChE) activity in the brain of d-gal-treated mice. The expression of aldose reductase (AR), sorbitol dehydrogenase (SDH) and member-anchored receptor for AGEs (RAGE) declined significantly after FGF21 treatment. Furthermore, FGF21 suppressed inflamm-aging by inhibiting IκBα degradation and NF-κB p65 nuclear translocation. The expression levels of pro-inflammatory cytokines, such as TNF-α and IL-6, decreased significantly. In conclusion, these results suggest that FGF21 protects the aging mice brain from d-gal-induced injury by attenuating oxidative stress damage and decreasing AGE formation. Copyright © 2015 Elsevier Inc. All rights reserved.

Soluble expression of recomb inant cMyc, Klf4, Oct4, and Sox2 proteins in bacteria and transduction into living cells

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Guo-Dan Liu

2017-04-01

Full Text Available AIM: To develop a new method to produce recombinant reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, in soluble format with low cost for the generation of induced pluripotent stem cells (iPSCs. METHODS: A short polypeptide sequence derived from the HIV trans-activator of transcription protein (TAT and the nucleus localization signal (NLS polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into pCold-SUMO vector which can extremely improve the solubility of recombinant proteins. Then these vector plasmids were transformed into E. coli BL21 (DE3 Chaperone competent cells for amplification. The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining. The recombinant proteins were purified by Ni-NTA resin and identified by Western blot. The transduction of these proteins into HEK 293T cells were evaluated by immunofluorescence staining. RESULTS: These four reprogramming proteins could be produced in soluble format in pCold-SUMO expression vector system with the assistance of chaperone proteins in bacteria. The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells. CONCLUSION: The results in the present study indicate the four important reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, can be produced in soluble format in bacteria with low cost. Our new method thus might be expected to greatly contribute to the future study of iPSCs.

Dietary moderately oxidized oil induces expression of fibroblast growth factor 21 in the liver of pigs

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Varady Juliane

2012-03-01

Full Text Available Abstract Background Fibroblast growth factor 21 (FGF21, whose expression is induced by peroxisome proliferator-activated receptor α (PPARα, has been recently identified as a novel metabolic regulator which plays a crucial role in glucose homeostasis, lipid metabolism, insulin sensitivity and obesity. Previous studies have shown that administration of oxidized fats leads to an activation of PPARα in the liver. Therefore, the present study investigated the hypothesis that feeding of oxidized fats causes an induction of FGF21 in the liver. Methods Twenty four crossbred pigs were allocated to two groups of 12 pigs each and fed nutritionally adequate diets with either fresh rapeseed oil or oxidized rapeseed oil prepared by heating at a temperature of 175°C for 72 h. Results In pigs fed the oxidized fat mRNA abundance and protein concentrations of FGF21 in liver were significantly increased (P P P Conclusion The present study shows for the first time that administration of an oxidized fat induces the expression of FGF21 in the liver, probably mediated by activation of PPARα. Induction of FGF21 could be involved in several effects observed in animals administered an oxidized fat.

A Comparative Analysis of Recombinant Expression and Solubility Screening of Two Phytases in E. coli

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Ashok Pandey

2011-01-01

Full Text Available Microbial phytases, especially from fungal and bacterial sources, have received much attention as food additives in human nutrition and as feed supplements for monogastric animals. An effective expression screening method for recombinant production of this enzyme on a small scale is industrially desirable. An effort has been made in this work to clone and express phytase genes from Aspergillus sp. and Escherichia coli with the selected host, vector and inducer combination. Albeit the formation of insoluble inclusion bodies by fungal phytase, recombinant E. coli appA was effectively expressed in a cost-effective manner in the periplasm of BL21plysS using an inducer concentration of 0.01 mM in 4 h of growth. Enzyme was purified in three consecutive steps and functional studies were carried out.

Fibroblast growth factor-21 and omentin-1 hepatic mRNA expression and serum levels in morbidly obese women with non-alcoholic fatty liver disease.

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Waluga, M; Kukla, M; Zorniak, M; Kajor, M; Liszka, L; Dyaczynski, M; Kowalski, G; Zadlo, D; Waluga, E; Olczyk, P; Buldak, R J; Berdowska, A; Hartleb, M

2017-06-01

Fibroblast growth factor-21 (FGF21) and omentin-1 have been recognized as potent antidiabetic agents with potential hepatoprotective activity. The aim of this study was to evaluate hepatic FGF21 and omentin-1 mRNA expression as well as their serum levels as predictive markers of liver injury and insulin resistance in morbidly obese women with non-alcoholic fatty liver disease (NAFLD). This study included 56 severely obese women who underwent intraoperative wedge liver biopsy during the bariatric surgery. Hepatic FGF21 and omentin-1 mRNA were assessed by quantitative real-time PCR, while their serum concentrations were measured with commercially available enzyme-linked immunosorbent assays. The FGF21 serum level was significantly higher in patients with a greater extent of steatosis (grade 2 and 3) compared to those without or with mild steatosis (grade 0 and 1) (P = 0.049). Receiver Operating Characteristic analysis, however, showed poor discriminant power for the FGF21 serum levels in differentiating between more and less extensive steatosis with an AUC = 0.666. There was a tendency towards higher levels of hepatic FGF21 mRNA in patients with lobular inflammation and fibrosis and towards lower levels in the case of hepatocyte ballooning and steatosis. There was a positive mutual correlation between hepatic FGF21 and omentin-1 mRNA levels (r = 0.78; P hepatic omentin-1 mRNA levels showed a tendency to be lower in patients with advanced steatosis and hepatocyte ballooning. In conclusion, our study, which focused on hepatic FGF21 and omentin-1 mRNA expression, confirmed marked expression of both molecules in the liver of morbidly obese patients with NAFLD. More extensive steatosis was associated with evident changes in the serum FGF21 concentration in morbidly obese women with NAFLD, but the difference did not reach statistical significance. The vast amount of fat, both visceral and subcutaneous, in severely obese patients may be the additional source and influence

FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation

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Marie, Melanie; Hafner, Sophie; Moratille, Sandra; Vaigot, Pierre; Rigaud, Odile; Martin, Michele T.; Mine, Solene

2012-01-01

Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair. The side population (SP) with cancer stem cell-like properties and the main population (MP) were isolated from human A431 squamous carcinoma cells. Radiation-induced DNA damage and repair were assessed using the alkaline comet assay. FGF2 expression was quantified by enzyme linked immunosorbent assay (ELISA). SP cells exhibited rapid repair of radiation induced DNA damage and a high constitutive level of nuclear FGF2. Blocking FGF2 signaling abrogated the rapid DNA repair. In contrast, in MP cells, a slower repair of damage was associated with low basal expression of FGF2. Moreover, the addition of exogenous FGF2 accelerated DNA repair in MP cells. When irradiated, SP cells secreted FGF2, whereas MP cells did not. FGF2 was found to mediate DNA repair in epidermoid carcinoma cells. We postulate that carcinoma stem cells would be intrinsically primed to rapidly repair DNA damage by a high constitutive level of nuclear FGF2. In contrast, the main population with a low FGF2 content exhibits a lower repair rate which can be increased by exogenous FGF2. (authors)

Role of PPARα in the Control of Torpor through FGF21-NPY Pathway: From Circadian Clock to Seasonal Change in Mammals

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Norio Ishida

2009-01-01

Full Text Available In nature, hibernating animals encounter fasting, cold temperature and short day seasonally. Torpor is a state of decreased physiological activity in an animal, usually characterized by a reduced body temperature and rate of metabolism to adapt such a severe environment. Ablation of the central clock synchronizer, the suprachiasmatic nucleus in brain, abolishes torpor, a hibernation-like state, implicating the circadian clock involved in this seasonal change. Biologists knows well the energy source of daily heterotherms/hibernators changed from glucose to lipids in winter. Here we review several lines of evidence of a master transcriptional regulator in lipid catabolism, PPARα, in the control of torpor through FGF21-NPY pathway. This indicate the importance of circadian—and photoperiod—regulation of PPARα to tell seasons in our body.

A successful strategy for the recovering of active P21, an insoluble recombinant protein of Trypanosoma cruzi

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Santos, Marlus Alves Dos; Teixeira, Francesco Brugnera; Moreira, Heline Hellen Teixeira; Rodrigues, Adele Aud; Machado, Fabrício Castro; Clemente, Tatiana Mordente; Brigido, Paula Cristina; Silva, Rebecca Tavares E.; Purcino, Cecílio; Gomes, Rafael Gonçalves Barbosa; Bahia, Diana; Mortara, Renato Arruda; Munte, Claudia Elisabeth; Horjales, Eduardo; da Silva, Claudio Vieira

2014-03-01

Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. In these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D 1H nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.

Fibroblast Growth Factor 23 (FGF23 and Disorders of Phosphate Metabolism

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Tasuku Saito

2009-01-01

Full Text Available Derangements in serum phosphate level result in rickets/osteomalacia or ectopic calcification indicating that healthy people without these abnormalities maintain serum phosphate within certain ranges. These results indicate that there must be a regulatory mechanism of serum phosphate level. Fibroblast growth factor 23 (FGF23 was identified as the last member of FGF family. FGF23 is produced by bone and reduces serum phosphate level by suppressing phosphate reabsorption in proximal tubules and intestinal phosphate absorption through lowering 1,25-dihydroxyvitamin D level. It has been shown that excess and deficient actions of FGF23 result in hypophosphatemic rickets/osteomalacia and hyperphosphatemic tumoral calcinosis, respectively. These results indicate that FGF23 works as a hormone, and several disorders of phosphate metabolism can be viewed as endocrine diseases. It may become possible to treat patients with abnormal phosphate metabolism by pharmacologically modifying the activity of FGF23.

Iron Modifies Plasma FGF23 Differently in Autosomal Dominant Hypophosphatemic Rickets and Healthy Humans

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Peacock, Munro; Gray, Amie K.; Padgett, Leah R.; Hui, Siu L.; Econs, Michael J.

2011-01-01

Context: In autosomal dominant hypophosphatemic rickets (ADHR), fibroblast growth factor 23 (FGF23) resists cleavage, causing increased plasma FGF23 levels. The clinical phenotype includes variable onset during childhood or adulthood and waxing/waning of hypophosphatemia. Delayed onset after puberty in females suggests iron status may be important. Objective: Studies were performed to test the hypothesis that plasma C-terminal and intact FGF23 concentrations are related to serum iron concentrations in ADHR. Design and Setting: Cross-sectional and longitudinal studies of ADHR and a cross-sectional study in healthy subjects were conducted at an academic medical center. Participants: Participants included 37 subjects with ADHR mutations from four kindreds and 158 healthy adult controls. Main Outcome Measure: The relationships of serum iron concentrations with plasma C-terminal and intact FGF23 concentrations were evaluated. Results: Serum phosphate and 1,25-dihydroxyvitamin D correlated negatively with C-terminal FGF23 and intact FGF23 in ADHR but not in controls. Serum iron was negatively correlated to both C-terminal FGF23 (r = −0.386; P < 0.05) and intact FGF23 (r = −0.602; P < 0.0001) in ADHR. However, control subjects also demonstrated a negative relationship of serum iron with C-terminal FGF23 (r = −0.276; P < 0.001) but no relationship with intact FGF23. Longitudinally in ADHR subjects, C-terminal FGF23 and intact FGF23 concentrations changed negatively with iron concentrations (P < 0.001 and P = 0.055, respectively), serum phosphate changed negatively with C-terminal FGF23 and intact FGF23 (P < 0.001), and there was a positive relationship between serum iron and phosphate (P < 0.001). Conclusions: Low serum iron is associated with elevated FGF23 in ADHR. However, in controls, low serum iron was also associated with elevated C-terminal FGF23, but not intact FGF23, suggesting cleavage maintains homeostasis despite increased FGF23 expression. PMID:21880793

Clinical efficacy of basic fibroblast growth factor (bFGF) for diabetic ulcer.

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UCHI, Hiroshi; IGARASHI, Atsuyuki; URABE, Kazunori; KOGA, Tetsuya; NAKAYAMA, Juichiro; KAWAMORI, Ryuzo; TAMAKI, Kunihiko; HIRAKATA, Hideki; OHURA, Takehiko; FURUE, Masutaka

2009-01-01

Basic fibroblast growth factor (bFGF) has been shown to promote wound healing. The present trial evaluated the clinical efficacy of bFGF for diabetic ulcer, a type of refractory skin ulcer, and the dose-response relationship. This was designed as a randomized, double-blind, dose-ranging, placebo-controlled trial. A total of 150 patients with non-ischaemic diabetic ulcers measuring 900 mm2 or less were randomized into a placebo group (n = 51), a 0.001% bFGF group (n = 49) and a 0.01% bFGF group (n = 50), and 148 of these patients received treatment for 8 weeks or less. The efficacy evaluation was carried out on 139 patients who met the protocol in this trial. The primary outcome was the percentage of patients showing 75% or greater reductions in the area of ulcer. The area of ulcer decreased by 75% or more in 57.5% (27/47), 72.3% (34/47), and 82.2% (37/45) in the placebo, 0.001% bFGF and 0.01% bFGF groups, respectively, and differences were significant between the 0.01% bFGF and placebo groups (p = 0.025). The cure rate was 46.8% (22/47), 57.4% (27/47), and 66.7% (30/45) in the placebo, 0.001% bFGF and 0.01% bFGF groups, respectively. The findings obtained in this trial showed wound healing accelerating effects of bFGF on diabetic ulcers.

Soluble expression and purification of the recombinant bioactive peptide precursor BPP-1 in Escherichia coli using a cELP-SUMO dual fusion system.

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Rao, Shengqi; Zang, Xiangyu; Yang, Zhenquan; Gao, Lu; Yin, Yongqi; Fang, Weiming

2016-02-01

A bioactive peptide precursor (BPP-1, 14.3 kDa/115AA), a newly designed polypeptide that may exert a potential antihypertensive effect in vivo, is composed of many different ACE inhibitory peptides and antioxidant peptides tandemly linked according to the restriction sites of gastrointestinal proteases. In this report, we present a novel method to obtain soluble BPP-1 in Escherichia coli using cationic elastin-like polypeptide and SUMO (cELP-SUMO) tags. The cELP-SUMO-tagged fusion protein was expressed in soluble form at 20 °C for 20 h. After purification based on the inverse transition cycling (ITC) method, the purified cELP-SUMO-CFPP fusion protein was subsequently cleaved by a SUMO protease to release the mature BPP-1. After a subsequent simple salt precipitation process, approximately 167.2 mg of recombinant BPP-1 was obtained from 1 l of bacterial culture with at least 92% purity. The molecular mass (Mr) of the recombinant BPP-1 was confirmed by MALDI-TOF MS to equal 14,347. The purified BPP-1 was subjected to simulated gastrointestinal digestion, and the resulting hydrolysates exhibited notable ACE inhibitory and antioxidant activities in vitro. This report provides the first description of the soluble production of a bioactive peptide multimer with potential ACE inhibitory and antioxidant activities in E. coli using a cELP-SUMO tag. Copyright © 2015 Elsevier Inc. All rights reserved.

[Anti-FGF23 antibody therapy for patients with tumor-induced osteomalacia].

Science.gov (United States)

Kinoshita, Yuka; Fukumoto, Seiji

2014-08-01

Tumor-induced osteomalacia (TIO) is a disease caused by fibroblast growth factor 23 (FGF23) secreted from the causative tumor. This disease is cured by complete surgical removal of the tumor. However, there are several difficult cases in which the responsible tumors cannot be found, are incompletely removed, or relapse after the surgery. Anti-FGF23 antibody is being studied as a novel therapy for FGF23-related hypophosphatemic diseases. The efficacy of anti-FGF23 antibodies were confirmed using a murine model of X-linked hypophosphatemic rickets (XLHR) , which is the most common heritable form of FGF23-related hypophosphatemic disease. In addition, results of phase I study of single injection of humanized anti-FGF23 antibody for adult patients with XLHR were recently published and the safety and effectiveness of this antibody was shown. This antibody therapy may be useful for patients with TIO with similar pathogenesis to that of XLHR.

Allelic heterogeneity of FGF5 mutations causes the long-hair phenotype in dogs.

Science.gov (United States)

Dierks, C; Mömke, S; Philipp, U; Distl, O

2013-08-01

Hitherto, the only known mutant gene leading to the long-hair phenotype in mammals is the fibroblast growth factor 5 (FGF5). In many dog breeds, the previously discovered FGF5:p.Cys95Phe mutation appeared completely concordant with the long-hair phenotype, but for some breeds, the long-hair phenotype could not be resolved. First, we studied the role of the FGF5:p.Cys95Phe and FGF5:g.145_150dupACCAGC mutations in 268 dogs descending from 27 breeds and seven wolves. As these mutations did not explain all the long-hair phenotypes, all exons and their neighbouring regions of FGF5 were re-sequenced. We detected three novel mutations in the coding sequence and one novel non-coding splice-site mutation in FGF5 associated with the long-hair phenotype. The FGF5:p.Ala193Val polymorphism was perfectly consistent with long hair in Akitas and probably in Siberian huskies, too. Dogs of the long-hair breed Samoyed were either homozygous or compound heterozygous for the FGF5:p.Ala193Val or the FGF5:p.Cys95Phe polymorphisms respectively. The two newly detected polymorphisms FGF5:c.559_560dupGG and FGF5:g.8193T>A and the known mutation FGF5:p.Cys95Phe explained the long-hair phenotype of all Afghan hounds analysed. An FGF5:c.556_571del16 mutation was found in one longhaired Eurasier. All long-hair-associated mutations follow a recessive mode of inheritance, and allelic heterogeneity was a common finding in breeds other than Akita. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.

Fibroblast growth factor 21 levels in young healthy females display day and night variations and are increased in response to short-term energy deprivation through a leptin-independent pathway.

Science.gov (United States)

Foo, Joo-Pin; Aronis, Konstantinos N; Chamberland, John P; Paruthi, Jason; Moon, Hyun-Seuk; Mantzoros, Christos S

2013-04-01

Fibroblast growth factor (FGF)-21 is an endocrine factor with potent metabolic effects. Its day-night patterns of secretion and/or its physiological response to energy deprivation and relationship to free fatty acids (FFAs) and/or leptin remain to be fully elucidated. We aim to elucidate day-night pattern of FGF-21 levels and its relationship to FFA, to assess whether energy deprivation alters its circulating patterns, and to examine whether leptin may mediate these changes. Six healthy lean females were studied for 72 h in a cross-over interventional study under three different conditions: on isocaloric diet and in a fasting state with administration of either placebo or metreleptin in physiological replacement doses. Blood samples were obtained hourly from 8:00 a.m. on day 4 until 8:00 a.m. on day 5. FGF-21 exhibited day-night variation pattern during the isocaloric fed state. Fasting significantly increased FGF-21 levels (P Day-night variation pattern in the fed state was lost on fasting. Leptin replacement in the hypoleptinemic state restored approximate entropy of FGF-21 time series but did not alter circulating levels. FGF-21 levels were closely cross-correlated with FFA levels in all three states. A day-night variation in the levels of FGF-21 exists in young lean females in the fed state. Energy deprivation increases FGF-21 levels via a leptin-independent pathway. The interaction between FGF-21 and starvation-induced lipolysis, as indicated by its close cross-correlations with FFA in both fed state and energy deprivation, needs to be studied further.

FGF10: A multifunctional mesenchymal-epithelial signaling growth factor in development, health, and disease.

Science.gov (United States)

Itoh, Nobuyuki

2016-04-01

The FGF family comprises 22 members with diverse functions in development and health. FGF10 specifically activates FGFR2b in a paracrine manner with heparan sulfate as a co-factor. FGF10and FGFR2b are preferentially expressed in the mesenchyme and epithelium, respectively. FGF10 is a mesenchymal signaling molecule in the epithelium. FGF10 knockout mice die shortly after birth due to the complete absence of lungs as well as fore- and hindlimbs. FGF10 is also essential for the development of multiple organs. The phenotypes of Fgf10 knockout mice are very similar to those of FGFR2b knockout mice, indicating that FGF10 acts as a ligand that is specific to FGFR2b in mouse multi-organ development. FGF10 also plays roles in epithelial-mesenchymal transition, the repair of tissue injury, and embryonic stem cell differentiation. In humans, FGF10 loss-of-function mutations result in inherited diseases including aplasia of lacrimal and salivary gland, lacrimo-auriculo-dento-digital syndrome, and chronic obstructive pulmonary disease. FGF10 is also involved in the oncogenicity of pancreatic and breast cancers. Single nucleotide polymorphisms in FGF10 are also potential risk factors for limb deficiencies, cleft lip and palate, and extreme myopia. These findings indicate that FGF10 is a crucial paracrine signal from the mesenchyme to epithelium for development, health, and disease. Copyright © 2015. Published by Elsevier Ltd.

Hepatic SIRT1 attenuates hepatic steatosis and controls energy balance in mice by inducing fibroblast growth factor 21.

Science.gov (United States)

Li, Yu; Wong, Kimberly; Giles, Amber; Jiang, Jianwei; Lee, Jong Woo; Adams, Andrew C; Kharitonenkov, Alexei; Yang, Qin; Gao, Bin; Guarente, Leonard; Zang, Mengwei

2014-02-01

The hepatocyte-derived hormone fibroblast growth factor 21 (FGF21) is a hormone-like regulator of metabolism. The nicotinamide adenine dinucleotide-dependent deacetylase SIRT1 regulates fatty acid metabolism through multiple nutrient sensors. Hepatic overexpression of SIRT1 reduces steatosis and glucose intolerance in obese mice. We investigated mechanisms by which SIRT1 controls hepatic steatosis in mice. Liver-specific SIRT1 knockout (SIRT1 LKO) mice and their wild-type littermates (controls) were divided into groups that were placed on a normal chow diet, fasted for 24 hours, or fasted for 24 hours and then fed for 6 hours. Liver tissues were collected and analyzed by histologic examination, gene expression profiling, and real-time polymerase chain reaction assays. Human HepG2 cells were incubated with pharmacologic activators of SIRT1 (resveratrol or SRT1720) and mitochondrion oxidation consumption rate and immunoblot analyses were performed. FGF21 was overexpressed in SIRT1 LKO mice using an adenoviral vector. Energy expenditure was assessed by indirect calorimetry. Prolonged fasting induced lipid deposition in livers of control mice, but severe hepatic steatosis in SIRT1 LKO mice. Gene expression analysis showed that fasting up-regulated FGF21 in livers of control mice but not in SIRT1 LKO mice. Decreased hepatic and circulating levels of FGF21 in fasted SIRT1 LKO mice were associated with reduced hepatic expression of genes involved in fatty acid oxidation and ketogenesis, and increased expression of genes that control lipogenesis, compared with fasted control mice. Resveratrol or SRT1720 each increased the transcriptional activity of the FGF21 promoter (-2070/+117) and levels of FGF21 messenger RNA and protein in HepG2 cells. Surprisingly, SIRT1 LKO mice developed late-onset obesity with impaired whole-body energy expenditure. Hepatic overexpression of FGF21 in SIRT1 LKO mice increased the expression of genes that regulate fatty acid oxidation, decreased

[Updates on rickets and osteomalacia: FGF23-mediated hypophosphatemic rickets/osteomalacia].

Science.gov (United States)

Michigami, Toshimi

2013-10-01

Some of the hypophosphatemic rickets/osteomalacia are caused by the increased bioactivity of FGF23, and classified into FGF23-mediated hypophosphatemic rickets/osteomalacia. This group includes various disorders such as X-linked, autosomal dominant and autosomal recessive hypophosphatemic rickets/osteomalacia, tumor-induced osteomalacia, and rickets/osteomalacia caused by the administration of iron polymaltose or saccharated ferric oxide. Measurement of serum levels of FGF23 is useful for diagnosis of these conditions. In the adult patients with FGF23-mediated hypophosphatemic rickets/osteomalacia, mineralizing enthesoopathy is an often observed complication.

Postnatal soluble FGFR3 therapy rescues achondroplasia symptoms and restores bone growth in mice.

Science.gov (United States)

Garcia, Stéphanie; Dirat, Béatrice; Tognacci, Thomas; Rochet, Nathalie; Mouska, Xavier; Bonnafous, Stéphanie; Patouraux, Stéphanie; Tran, Albert; Gual, Philippe; Le Marchand-Brustel, Yannick; Gennero, Isabelle; Gouze, Elvire

2013-09-18

Achondroplasia is a rare genetic disease characterized by abnormal bone development, resulting in short stature. It is caused by a single point mutation in the gene coding for fibroblast growth factor receptor 3 (FGFR3), which leads to prolonged activation upon ligand binding. To prevent excessive intracellular signaling and rescue the symptoms of achondroplasia, we have developed a recombinant protein therapeutic approach using a soluble form of human FGFR3 (sFGFR3), which acts as a decoy receptor and prevents FGF from binding to mutant FGFR3. sFGFR3 was injected subcutaneously to newborn Fgfr3(ach/+) mice-the mouse model of achondroplasia-twice per week throughout the growth period during 3 weeks. Effective maturation of growth plate chondrocytes was restored in bones of treated mice, with a dose-dependent enhancement of skeletal growth in Fgfr3(ach/+) mice. This resulted in normal stature and a significant decrease in mortality and associated complications, without any evidence of toxicity. These results describe a new approach for restoring bone growth and suggest that sFGFR3 could be a potential therapy for children with achondroplasia and related disorders.

Endostatin competes with bFGF for binding to heparin-like glycosaminoglycans

International Nuclear Information System (INIS)

Reis, Renata C.M.; Schuppan, Detlef; Barreto, Aline C.; Bauer, Michael; Bork, Jens P.; Hassler, Gerda; Coelho-Sampaio, Tatiana

2005-01-01

Endostatin is a potent inhibitor of angiogenesis and tumor growth. Here, we used human endothelial cells from lung capillaries to investigate if endostatin competes with the proangiogenic growth factors, bFGF and VEGF, for binding to costimulatory heparan sulfate molecules. Endostatin inhibited 79% and 95% of the increase in proliferation induced by bFGF and VEGF 165 , respectively. The stimulatory effect of VEGF 165 was not affected by the presence of exogenous heparin, while that of bFGF was further enhanced in the presence of up to 0.1 μg/ml heparin. The heparin-binding protein protamine completely blocked bFGF-stimulated proliferation, while it did not affect the response to VEGF 165 . Simultaneous addition of endostatin and protamine led to additive effects both in inhibition of proliferation and induction of apoptosis. Although bFGF was found to bind more strongly to heparin-Sepharose than endostatin, the latter, but not the former, displaced protamine from heparin in solution, which supports the notion that endostatin can compete with bFGF for binding to heparan sulfate in vivo. Taken as a whole, our results demonstrate that there is a direct connection between the dependence of endostatin activity on heparin-like glycosaminoglycans and its ability to antagonize bFGF

Role of Circulating Fibroblast Growth Factor 21 Measurement in Primary Prevention of Coronary Heart Disease Among Chinese Patients With Type 2 Diabetes Mellitus.

Science.gov (United States)

Lee, Chi Ho; Woo, Yu Cho; Chow, Wing Sun; Cheung, Chloe Yu Yan; Fong, Carol Ho Yi; Yuen, Michele Mae Ann; Xu, Aimin; Tse, Hung Fat; Lam, Karen Siu Ling

2017-06-06

Fibroblast growth factor 21 (FGF21) has demonstrated beneficial effects on lipid and carbohydrate metabolism. In cross-sectional studies, an association of raised circulating FGF21 levels with coronary heart disease (CHD) was found in some but not all studies. Here we investigated prospectively whether baseline serum FGF21 levels could predict incident CHD in subjects with type 2 diabetes mellitus and no known cardiovascular diseases. Baseline serum FGF21 levels were measured in 3528 Chinese subjects with type 2 diabetes mellitus recruited from the Hong Kong West Diabetes Registry. The role of baseline serum FGF21 levels in predicting incident CHD over a median follow-up of 3.8 years was analyzed using Cox regression analysis. Among 3528 recruited subjects without known cardiovascular diseases, 147 (4.2%) developed CHD over a mean follow-up of 4 years. Baseline serum log-transformed FGF21 levels were significantly higher in those who had incident CHD than those who did not (222.7 pg/mL [92.8-438.4] versus 151.1 pg/mL [75.6-274.6]; P 1). On multivariable Cox regression analysis, baseline serum FGF21 levels, using an optimal cutoff of 206.22 pg/mL derived from our study, independently predicted incident CHD (hazard ratio, 1.55; 95% CI, 1.10-2.19; P =0.013) and significantly improved net reclassification index and integrated discrimination improvement after adjustment for conventional cardiovascular risk factors. We have demonstrated, for the first time, that serum FGF21 level is an independent predictor of incident CHD and might be usefully utilized as a biomarker for identifying type 2 diabetes mellitus subjects with raised CHD risk, for primary prevention. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Circulating FGF23 levels in response to acute changes in plasma Ca(2+)

DEFF Research Database (Denmark)

Gravesen, E; Mace, M.L.; Hofman-Bang, J.

2014-01-01

The regulation of fibroblast growth factor 23 (FGF23) synthesis and secretion is still incompletely understood. FGF23 is an important regulator of renal phosphate excretion and has regulatory effects on the calciotropic hormones calcitriol and parathyroid hormone (PTH). Calcium (Ca) and phosphate...... FGF23 levels and whether a close relationship, similar that known for Ca and PTH, exists between Ca and FGF23. Thus, the aim of the present study was to examine whether acute hypercalcemia and hypocalcemia regulate FGF23 levels in the rat. Acute hypercalcemia was induced by an intravenous Ca infusion...... and hypocalcemia by infusion of ethylene glycol tetraacetic acid (EGTA) in normal and acutely parathyroidectomized rats. Intact plasma FGF23 and intact plasma PTH and plasma Ca(2+) and phosphate were measured. Acute hypercalcemia and hypocalcemia resulted as expected in adequate PTH secretory responses. Plasma FGF...

Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

Science.gov (United States)

Jeong, Young-Hee; Kim, Yeong Ji; Kim, Eun Young; Kim, Se Eun; Kim, Jiwoo; Park, Min Jee; Lee, Hong-Gu; Park, Se Pill; Kang, Man-Jong

2016-06-01

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.

Ablation of the Galnt3 Gene Leads to Low-Circulating Intact Fibroblast Growth Factor 23 (Fgf23) Concentrations and Hyperphosphatemia Despite Increased Fgf23 Expression

OpenAIRE

Ichikawa, Shoji; Sorenson, Andrea H.; Austin, Anthony M.; Mackenzie, Donald S.; Fritz, Timothy A.; Moh, Akira; Hui, Siu L.; Econs, Michael J.

2009-01-01

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia. The disease is caused by inactivating mutations in fibroblast growth factor 23 (FGF23), Klotho (KL), and uridine diphosphate-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In vitro studies indicate that GALNT3 O-glycosylates a phosphaturic hormone, FGF23, and prevents its proteolytic processing, thereby allowing secretion of intact FGF23. In this study we generat...

Patients with FGF23-related hypophosphatemic rickets/osteomalacia do not present with left ventricular hypertrophy.

Science.gov (United States)

Takashi, Yuichi; Kinoshita, Yuka; Hori, Michiko; Ito, Nobuaki; Taguchi, Manabu; Fukumoto, Seiji

2017-05-01

Fibroblast growth factor 23 (FGF23) is a hormone regulating phosphate metabolism. Excessive actions of FGF23 cause several types of FGF23-related hypophosphatemic rickets/osteomalacia. Recently, it was reported that FGF23 levels were independently correlated with left ventricular hypertrophy (LVH) in patients with chronic kidney disease (CKD). In addition, FGF23 was also shown to cause cardiac hypertrophy directly acting on cardiomyocytes. However, there is no study indicating the correlation between FGF23 and LVH in adult patients with FGF23-related hypophosphatemic rickets/osteomalacia. Therefore, we examined the existence of LVH in these patients. We recruited consecutive 24 patients with FGF23-related hypophosphatemic diseases. Their serum intact FGF23 levels and the parameters associated with LVH, including left ventricular mass index (LVMI), relative wall thickness (RWT), Sokolow-Lyon voltage, and Cornell product, were measured. The correlations between FGF23 and these parameters were examined. The participants did not show LVH on the whole. In addition, no significant correlation was observed by these examinations. It seems unlikely that FGF23 levels are the apparent determinant of the cardiac mass in patients with FGF23-related hypophosphatemic rickets/osteomalacia.

Thrombin induces rapid PAR1-mediated non-classical FGF1 release

International Nuclear Information System (INIS)

Duarte, Maria; Kolev, Vihren; Soldi, Raffaella; Kirov, Alexander; Graziani, Irene; Oliveira, Silvia Marta; Kacer, Doreen; Friesel, Robert; Maciag, Thomas; Prudovsky, Igor

2006-01-01

Thrombin induces cell proliferation and migration during vascular injury. We report that thrombin rapidly stimulated expression and release of the pro-angiogenic polypeptide fibroblast growth factor 1 (FGF1). Thrombin failed to induce FGF1 release from protease-activated receptor 1 (PAR1) null fibroblasts, indicating that this effect was dependent on PAR1. Similarly to thrombin, FGF1 expression and release were induced by TRAP, a specific oligopeptide agonist of PAR1. These results identify a novel aspect of the crosstalk between FGF and thrombin signaling pathways which both play important roles in tissue repair and angiogenesis

Chronic high-sucrose diet increases fibroblast growth factor 21 production and energy expenditure in mice.

Science.gov (United States)

Maekawa, Ryuya; Seino, Yusuke; Ogata, Hidetada; Murase, Masatoshi; Iida, Atsushi; Hosokawa, Kaori; Joo, Erina; Harada, Norio; Tsunekawa, Shin; Hamada, Yoji; Oiso, Yutaka; Inagaki, Nobuya; Hayashi, Yoshitaka; Arima, Hiroshi

2017-11-01

Excess carbohydrate intake causes obesity in humans. On the other hand, acute administration of fructose, glucose or sucrose in experimental animals has been shown to increase the plasma concentration of anti-obesity hormones such as glucagon-like peptide 1 (GLP-1) and Fibroblast growth factor 21 (FGF21), which contribute to reducing body weight. However, the secretion and action of GLP-1 and FGF21 in mice chronically fed a high-sucrose diet has not been investigated. To address the role of anti-obesity hormones in response to increased sucrose intake, we analyzed mice fed a high-sucrose diet, a high-starch diet or a normal diet for 15 weeks. Mice fed a high-sucrose diet showed resistance to body weight gain, in comparison with mice fed a high-starch diet or control diet, due to increased energy expenditure. Plasma FGF21 levels were highest among the three groups in mice fed a high-sucrose diet, whereas no significant difference in GLP-1 levels was observed. Expression levels of uncoupling protein 1 (UCP-1), FGF receptor 1c (FGFR1c) and β-klotho (KLB) mRNA in brown adipose tissue were significantly increased in high sucrose-fed mice, suggesting increases in FGF21 sensitivity and energy expenditure. Expression of carbohydrate responsive element binding protein (ChREBP) mRNA in liver and brown adipose tissue was also increased in high sucrose-fed mice. These results indicate that FGF21 production in liver and brown adipose tissue is increased in high-sucrose diet and participates in resistance to weight gain. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Klotho expression in long bones regulates FGF23 production during renal failure.

Science.gov (United States)

Kaludjerovic, Jovana; Komaba, Hirotaka; Sato, Tadatoshi; Erben, Reinhold G; Baron, Roland; Olauson, Hannes; Larsson, Tobias E; Lanske, Beate

2017-05-01

Circulating levels of bone-derived fibroblast growth factor 23 (FGF23) increase early during acute and chronic kidney disease and are associated with adverse outcomes. Membrane-bound Klotho acts as a permissive coreceptor for FGF23, and its expression was recently found in osteoblasts/osteocytes. We hypothesized that Klotho in bone cells is part of an autocrine feedback loop that regulates FGF23 expression during renal failure. Thus, we induced renal failure in mice with targeted deletion of Klotho in long bones. Uremic wild-type ( KL fl/fl ) and knockout ( Prx1-Cre;KL fl/fl ) mice both responded with reduced body weight, kidney atrophy, hyperphosphatemia, and increased bone turnover. Importantly, long bones of Prx1-Cre;KL fl/fl mice but not their axial skeleton failed to increase FGF23 expression as observed in uremic KL fl/fl mice. Consequently, Prx1-Cre;KL fl/fl mice had significantly lower serum FGF23 and parathyroid hormone levels, and higher renal 1-α-hydroxylase expression, serum 1,25-dihydroxyvitamin D, and calcium levels than KL fl/fl mice. These results were confirmed in two independent models of renal failure, adenine diet induced and 5/6 nephrectomy. Moreover, FGF23-treated bone cells required Klotho to increase FGF23 mRNA and ERK phosphorylation. In summary, our novel findings show that Klotho in bone is crucial for inducing FGF23 production upon renal failure. We propose the presence of an autocrine feedback loop in which Klotho senses the need for FGF23.-Kaludjerovic, J., Komaba, H., Sato, T., Erben, R. G., Baron, R., Olauson, H., Larsson, T. E., Lanske, B. Klotho expression in long bones regulates FGF23 production during renal failure. © FASEB.

FGF-23 dysregulates calcium homeostasis and electrophysiological properties in HL-1 atrial cells.

Science.gov (United States)

Kao, Yu-Hsun; Chen, Yao-Chang; Lin, Yung-Kuo; Shiu, Rong-Jie; Chao, Tze-Fan; Chen, Shih-Ann; Chen, Yi-Jen

2014-08-01

Fibroblast growth factor (FGF)-23 is a key regulator of phosphate homeostasis. Higher FGF-23 levels are correlated with poor outcomes in cardiovascular diseases. FGF-23 can produce cardiac hypertrophy and increase intracellular calcium, which can change cardiac electrical activity. However, it is not clear whether FGF-23 possesses arrhythmogenic potential through calcium dysregulation. Therefore, the purposes of this study were to evaluate the electrophysiological effects of FGF-23 and identify the underlying mechanisms. Patch clamp, confocal microscope with Fluo-4 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, calcium homeostasis and calcium regulatory proteins in HL-1 atrial myocytes with and without FGF-23 (10 and 25 ng/mL) incubation for 24 h. FGF-23 (25 ng/mL) increased L-type calcium currents, calcium transient and sarcoplasmic reticulum Ca(2+) contents in HL-1 cells. FGF-23 (25 ng/mL)-treated cells (n = 14) had greater incidences (57%, 17% and 15%, P calcium/calmodulin-dependent protein kinase IIδ and phospholamban (PLB) at threonine 17 but had similar phosphorylation extents of PLB at serine 16, total PLB and sarcoplasmic reticulum Ca(2+) -ATPase protein. Moreover, the FGF receptor inhibitor (PD173074, 10 nM), calmodulin inhibitor (W7, 5 μM) and phospholipase C inhibitor (U73122, 1 μM) attenuated the effects of FGF-23 on calcium/calmodulin-dependent protein kinase II phosphorylation. FGF-23 increases HL-1 cells arrhythmogenesis with calcium dysregulation through modulating calcium-handling proteins. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

FGF-2 promotes osteocyte differentiation through increased E11/podoplanin expression.

Science.gov (United States)

Ikpegbu, Ekele; Basta, Lena; Clements, Dylan N; Fleming, Robert; Vincent, Tonia L; Buttle, David J; Pitsillides, Andrew A; Staines, Katherine A; Farquharson, Colin

2018-07-01

E11/podoplanin is critical in the early stages of osteoblast-to-osteocyte transitions (osteocytogenesis), however, the upstream events which regulate E11 expression are unknown. The aim of this study was to examine the effects of FGF-2 on E11-mediated osteocytogenesis and to reveal the nature of the underlying signaling pathways regulating this process. Exposure of MC3T3 osteoblast-like cells and murine primary osteoblasts to FGF-2 (10 ng/ml) increased E11 mRNA and protein expression (p 70% reduction of basal E11 mRNA expression (p < 0.05) and effectively abrogated FGF-2-related changes in E11 expression and dendrite formation. FGF-2 strongly activated the ERK signaling pathway in osteoblast-like cells but inhibition of this pathway did not block the ability of FGF-2 to enhance E11 expression or to promote acquisition of the osteocyte phenotype. The results of this study highlight a novel mechanism by which FGF-2 can regulate osteoblast differentiation and osteocyte formation. Specifically, the data suggests that FGF-2 promotes osteocytogenesis through increased E11 expression and further studies will identify if this regulatory pathway is essential for bone development and maintenance in health and disease. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

The Regulation of the Angiogenic Factor FGF Binding Protein (FGF-BP) by the APC/Beta-Catenin Signaling Pathway in the Progression of Breast Cancer

National Research Council Canada - National Science Library

Stylianou, Dora

2004-01-01

...) to study the expression of FGF-BP in mammary tumorigenesis progression of the APC/+ mouse and 2) to determine the mechanism of regulation of FGF-BP b the APC/beta-catenin signaling pathway in breast cancer...

The Regulation of the Angiogenic Factor FGF Binding Protein (FGF-BP) by the APC/Beta-Catenin Signaling Pathway in the Progression of Breast Cancer

National Research Council Canada - National Science Library

Stylianou, Dora

2003-01-01

...) to study the expression of FGF-BP in mammary tumorigenesis% progression of the APC/+ mouse and 2) to determine the mechanism of regulation of FGF-BP by the APC/beta- catenin signaling pathway in breast cancer...

Polyguluronate sulfate and its oligosaccharides but not heparin promotes FGF19/FGFR1c signaling

Science.gov (United States)

Lan, Ying; Zeng, Xuan; Guo, Zhihua; Zeng, Pengjiao; Hao, Cui; Zhao, Xia; Yu, Guangli; Zhang, Lijuan

2017-06-01

Fibroblast growth factor 19(FGF19) functions as a hormone by affecting glucose metabolism. FGF19 improves glucose tolerance when overexpressed in mice with impaired glucose tolerance or diabetes. A functional cellular FGF19 receptor consists of FGF receptor (FGFR) and glycosaminoglycan complexed with either α Klotho or β Klotho. Interestingly, in mice with diet-induced diabetes, a single injection of FGF1 is enough to restore blood sugar levels to a healthy range. FGF1 binds heparin with high affinity whereas FGF19 does not, indicating that polysaccharides other than heparin might enhance FGF19/FGFR signaling. Using a FGFs/FGFR1c signaling-dependent BaF3 cell proliferation assay, we discovered that polyguluronate sulfate (PGS) and its oligosaccharides, PGS12 and PGS25, but not polyguluronate (PG), a natural marine polysaccharide, enhanced FGF19/FGFR1c signaling better than that of heparin based on 3H-thymidine incorporation. Interestingly, PGS6, PGS8, PGS10, PGS12, PGS25, and PGS, but not PG, had comparable FGF1/FGFR1c signal-stimulating activity compared to that of heparin. These results indicated that PGS and its oligosaccharides were excellent FGF1/FGFR1c and FGF19/FGFR1c signaling enhancers at cellular level. Since the inexpensive PGS and PGS oligosaccharides can be absorbed through oral route, these seaweed-derived compounds merit further investigation as novel agents for the treatment of type 2 diabetes through enhancing FGF1/FGFR1c and FGF19/FGFR1c signaling in future.

Diverse FGF receptor signaling controls astrocyte specification and proliferation

Energy Technology Data Exchange (ETDEWEB)

Kang, Kyungjun [School of Life Sciences, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of); Song, Mi-Ryoung, E-mail: msong@gist.ac.kr [School of Life Sciences, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of); Bioimaging Research Center and Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of)

2010-05-07

During CNS development, pluripotency neuronal progenitor cells give rise in succession to neurons and glia. Fibroblast growth factor-2 (FGF-2), a major signal that maintains neural progenitors in the undifferentiated state, is also thought to influence the transition from neurogenesis to gliogenesis. Here we present evidence that FGF receptors and underlying signaling pathways transmit the FGF-2 signals that regulate astrocyte specification aside from its mitogenic activity. Application of FGF-2 to cortical progenitors suppressed neurogenesis whereas treatment with an FGFR antagonist in vitro promoted neurogenesis. Introduction of chimeric FGFRs with mutated tyrosine residues into cortical progenitors and drug treatments to specifically block individual downstream signaling pathways revealed that the overall activity of FGFR rather than individual autophosphorylation sites is important for delivering signals for glial specification. In contrast, a signal for cell proliferation by FGFR was mainly delivered by MAPK pathway. Together our findings indicate that FGFR activity promotes astrocyte specification in the developing CNS.

Diverse FGF receptor signaling controls astrocyte specification and proliferation

International Nuclear Information System (INIS)

Kang, Kyungjun; Song, Mi-Ryoung

2010-01-01

During CNS development, pluripotency neuronal progenitor cells give rise in succession to neurons and glia. Fibroblast growth factor-2 (FGF-2), a major signal that maintains neural progenitors in the undifferentiated state, is also thought to influence the transition from neurogenesis to gliogenesis. Here we present evidence that FGF receptors and underlying signaling pathways transmit the FGF-2 signals that regulate astrocyte specification aside from its mitogenic activity. Application of FGF-2 to cortical progenitors suppressed neurogenesis whereas treatment with an FGFR antagonist in vitro promoted neurogenesis. Introduction of chimeric FGFRs with mutated tyrosine residues into cortical progenitors and drug treatments to specifically block individual downstream signaling pathways revealed that the overall activity of FGFR rather than individual autophosphorylation sites is important for delivering signals for glial specification. In contrast, a signal for cell proliferation by FGFR was mainly delivered by MAPK pathway. Together our findings indicate that FGFR activity promotes astrocyte specification in the developing CNS.

Combination therapy with sivelestat and recombinant human soluble thrombomodulin for ARDS and DIC patients

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Miyoshi S

2014-09-01

Full Text Available Seigo Miyoshi,1 Ryoji Ito,1 Hitoshi Katayama,1 Kentaro Dote,2 Mayuki Aibiki,3 Hironobu Hamada,1,4 Takafumi Okura,1 Jitsuo Higaki1 1Department of Cardiology, Pulmonology, Hypertension and Nephrology, Ehime University Graduate School of Medicine, 2Intensive Care Division, Ehime University Hospital, 3Department of Emergency and Critical Care Medicine, School of Medicine, Ehime University, Shitsukawa, Toon, Ehime, 4Department of Physical Analysis and Therapeutic Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi, Minami-ku, Hiroshima, Japan Background: Neutrophil elastase, alveolar thrombin generation, and fibrin deposition play crucial roles in the development of acute respiratory distress syndrome (ARDS and disseminated intravascular coagulation (DIC. However, the usefulness of combination therapy with a selective neutrophil elastase inhibitor, sivelestat, and recombinant human soluble thrombomodulin (rhTM for patients with ARDS and DIC remains unknown. Methods: We conducted a retrospective data analysis of 142 ARDS patients with DIC to assess the effects of sivelestat combined with rhTM. Patients were divided into four groups: control (no sivelestat or rhTM treatment, sivelestat treatment alone, rhTM treatment alone, and combined treatment with sivelestat and rhTM. A Cox proportional hazard model was used to assess subject mortality rates. The efficacy of these drugs was evaluated based on survival rate, number of ventilator-free days, and change in PaO2/FIO2 (P/F ratios and DIC scores before and at 7 days after a diagnosis of ARDS with DIC. Results: Multivariate analysis showed that patient age, combination therapy, gas exchange, organ failure, cause, associated disease score, and serum C-reactive protein levels were predictors of mortality for patients with ARDS and DIC. As compared with untreated controls, combination therapy significantly improved the 60-day survival rate of patients with ARDS and DIC

[HPV DNA vaccines expressing recombinant CRT/HPV6bE7 fusion protein inhibit tumor growth and angiogenic activity].

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Xu, Yan; Cheng, Hao; Zhao, Ke-Jia; Zhu, Ke-Jian; Zhang, Xing

2007-11-01

This paper was to study the angiogenic inhibitory effect and the potential antitumor effect of the constructed recombinant DNA vaccine CRT/HPV6bE7 in vivo. The C57BL/6 mice were vaccinated respectively with recombinant CRT/HPV6bE7 DNA plamids. The inhibitory effects on angiogenesis of generated vaccines in vivo were evaluated by a bFGF-induced angiogenesis assay using the Matrigel kit. To investigate the potential antitumor effect, the mean tumor weights, sizes and tumor appearing times were measured in C57BL/6 mice treated with HPV6bE7-expressing B16 cells. The results indicated that the recombinants CRT180/HPV6bE7 and CRT180 showed strong anti-angiogenic effects in bFGF-induced angiogenesis in vivo. Moreover, CRT180/HPV6bE7 and CRT180 DNA vaccines could significantly inhibit the tumor growth in tumor challenge experiment, and CRT180/HPV6bE7 was superior to other vaccines in delaying tumor formation time, limiting tumor size and weight in tumor protection experiment. In conclusion, recombinant CRT180/HPV6bE7 DNA could elicit a most efficient anti-angiogenic effect and inhibit tumor growth in mice inoculated with DNA vaccines. The antiangiogenic activity of CRT were suggested residing in a domain between CRT 120-180 aa.

FGF signaling is required for brain left-right asymmetry and brain midline formation.

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Neugebauer, Judith M; Yost, H Joseph

2014-02-01

Early disruption of FGF signaling alters left-right (LR) asymmetry throughout the embryo. Here we uncover a role for FGF signaling that specifically disrupts brain asymmetry, independent of normal lateral plate mesoderm (LPM) asymmetry. When FGF signaling is inhibited during mid-somitogenesis, asymmetrically expressed LPM markers southpaw and lefty2 are not affected. However, asymmetrically expressed brain markers lefty1 and cyclops become bilateral. We show that FGF signaling controls expression of six3b and six7, two transcription factors required for repression of asymmetric lefty1 in the brain. We found that Z0-1, atypical PKC (aPKC) and β-catenin protein distribution revealed a midline structure in the forebrain that is dependent on a balance of FGF signaling. Ectopic activation of FGF signaling leads to overexpression of six3b, loss of organized midline adherins junctions and bilateral loss of lefty1 expression. Reducing FGF signaling leads to a reduction in six3b and six7 expression, an increase in cell boundary formation in the brain midline, and bilateral expression of lefty1. Together, these results suggest a novel role for FGF signaling in the brain to control LR asymmetry, six transcription factor expressions, and a midline barrier structure. Copyright © 2013 Elsevier Inc. All rights reserved.

Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins.

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David J Leibly

Full Text Available Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2, proline, xylitol, NDSB 201, CTAB and K(2PO(4 solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40% were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

FGF23 modulates the effects of erythropoietin on gene expression in renal epithelial cells

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Yashiro M

2018-04-01

Full Text Available Mitsuru Yashiro,1 Masaki Ohya,1 Toru Mima,1 Yumi Ueda,2 Yuri Nakashima,1 Kazuki Kawakami,1 Yohei Ishizawa,2 Shuto Yamamoto,1 Sou Kobayashi,1 Takurou Yano,1 Yusuke Tanaka,1 Kouji Okuda,1 Tomohiro Sonou,1 Tomohiro Shoshihara,1 Yuko Iwashita,1 Yu Iwashita,1 Kouichi Tatsuta,1 Ryo Matoba,2 Shigeo Negi,1 Takashi Shigematsu1 1Department of Nephrology, Wakayama Medical University, Wakayama, Japan; 2DNA Chip Research Inc., Minato, Japan Background: FGF23 plays an important role in calcium–phosphorus metabolism. Other roles of FGF23 have recently been reported, such as commitment to myocardium enlargement and immunological roles in the spleen. In this study, we aimed to identify the roles of FGF23 in the kidneys other than calcium–phosphorus metabolism. Methods: DNA microarrays and bioinformatics tools were used to analyze gene expression in mIMCD3 mouse renal tubule cells following treatment with FGF23, erythropoietin and/or an inhibitor of ERK. Results: Three protein-coding genes were upregulated and 12 were downregulated in response to FGF23. Following bioinformatics analysis of these genes, PPARγ and STAT3 were identified as candidate transcript factors for mediating their upregulation, and STAT1 as a candidate for mediating their downregulation. Because STAT1 and STAT3 also mediate erythropoietin signaling, we investigated whether FGF23 and erythropoietin might show interactive effects in these cells. Of the 15 genes regulated by FGF23, 11 were upregulated by erythropoietin; 10 of these were downregulated following cotreatment with FGF23. Inhibition of ERK, an intracellular mediator of FGF23, reversed the effects of FGF23. However, FGF23 did not influence STAT1 phosphorylation, suggesting that it impinges on erythropoietin signaling through other mechanisms. Conclusion: Our results suggest cross talk between erythropoietin and FGF23 signaling in the regulation of renal epithelial cells. Keywords: FGF23, STAT1, PPARγ, DNA microarray

Repression of the DCL2 and DCL4 genes in Nicotiana benthamiana plants for the transient expression of recombinant proteins.

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Matsuo, Kouki; Matsumura, Takeshi

2017-08-01

The production of recombinant proteins in plants has many advantages, including safety and reduced costs. However, this technology still faces several issues, including low levels of production. The repression of RNA silencing seems to be particularly important for improving recombinant protein production because RNA silencing effectively degrades transgene-derived mRNAs in plant cells. Therefore, to overcome this, we used RNA interference technology to develop DCL2- and DCL4-repressed transgenic Nicotiana benthamiana plants (ΔD2, ΔD4, and ΔD2ΔD4 plants), which had much lower levels of NbDCL2 and/or NbDCL4 mRNAs than wild-type plants. A transient gene expression assay showed that the ΔD2ΔD4 plants accumulated larger amounts of green fluorescent protein (GFP) and human acidic fibroblast growth factor (aFGF) than ΔD2, ΔD4, and wild-type plants. Furthermore, the levels of GFP and aFGF mRNAs were also higher in ΔD2ΔD4 plants than in ΔD2, ΔD4, and wild-type plants. These findings demonstrate that ΔD2ΔD4 plants express larger amounts of recombinant proteins than wild-type plants, and so would be useful for recombinant protein production. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A simple vector system to improve performance and utilisation of recombinant antibodies

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Vincent Karen J

2006-12-01

Full Text Available Abstract Background Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. Results We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs. Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3. The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3 was investigated and found to be inferior to periplasmic expression in BL21 (DE3 cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner, bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule, or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and

Vitamin D treatment attenuates cardiac FGF23/FGFR4 signaling and hypertrophy in uremic rats.

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Leifheit-Nestler, Maren; Grabner, Alexander; Hermann, Laura; Richter, Beatrice; Schmitz, Karin; Fischer, Dagmar-Christiane; Yanucil, Christopher; Faul, Christian; Haffner, Dieter

2017-09-01

Vitamin D deficiency and excess of circulating fibroblast growth factor 23 (FGF23) contribute to cardiovascular mortality in patients with chronic kidney disease (CKD). FGF23 activates FGF receptor 4 and (FGFR4) calcineurin/nuclear factor of activated T cells (NFAT) signaling in cardiac myocytes, thereby causing left ventricular hypertrophy (LVH). Here, we determined if 1,25-dihydroxyvitamin D (calcitriol) inhibits FGF23-induced cardiac signaling and LVH. 5/6 nephrectomized (5/6 Nx) rats were treated with different doses of calcitriol for 4 or 10 weeks and cardiac expression of FGF23/FGFR4 and activation of calcineurin/NFAT as well as LVH were analyzed. FGFR4 activation and hypertrophic cell growth were studied in cultured cardiac myocytes that were co-treated with FGF23 and calcitriol. In 5/6Nx rats with LVH, we detected elevated FGF23 expression in bone and myocardium, increased cardiac expression of FGFR4 and elevated cardiac activation of calcineurin/NFAT signaling. Cardiac expression levels of FGF23 and FGFR4 significantly correlated with the presence of LVH in uremic rats. Treatment with calcitriol reduced LVH as well as cardiac FGFR4 expression and calcineurin/NFAT activation. Bone and cardiac FGF23 expression were further stimulated by calcitriol in a dose-dependent manner, but levels of intact cardiac FGF23 protein were suppressed by high-dose calcitriol. In cultured cardiac myocytes, co-treatment with calcitriol blocked FGF23-induced activation of FGFR4 and hypertrophic cell growth. Our data suggest that in CKD, cardioprotective effects of calcitriol stem from its inhibitory actions on the cardiac FGF23/FGFR4 system, and based on their counterbalancing effects on cardiac myocytes, high FGF23 and low calcitriol synergistically contribute to cardiac hypertrophy. © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Differentiation of stem cells upon deprivation of exogenous FGF2

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Kjartansdóttir, Kristín Rós; Gabrielsen, Anette; Reda, Ahmed

2012-01-01

Establishing a model for in vitro differentiation of human embryonic stem cells (hESCs) towards the germ cell lineage could be used to identify molecular mechanisms behind germ cell differentiation that may help in understanding human infertility. Here, we evaluate whether a lack of exogenous...... fibroblast growth factor 2 (FGF2) is supporting spontaneous differentiation of hESCs cultured on human foreskin fibroblast (hFF) monolayers towards germ cell lineage. Additionally to depriving the hESCs of exogenous FGF2, cells were stimulated with all-trans retinoic acid (ATRA). To get a more comprehensive...... impression on effects of removal of FGF2 and stimulation with ATRA, we combined the results of three cell lines for each experimental setting. When combining gene expression profiles of three cell lines for 96 genes, only 6 genes showed a significant up-regulation in all cell lines, when no FGF2 was added...

TGF-β2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

International Nuclear Information System (INIS)

Lu Jiawei; Lu Zhenyu; Reinach, Peter

2006-01-01

The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-β2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-β2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-β2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-β2 and FGF-2 oppositely affect BCE cell proliferation and TGF-β2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-β2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-β2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-β2-induced suppression of the PI3-kinase/AKT signaling pathway

PILOTSTUDY TO EVALUATE THE EFFECT OF PHOSPHORUSBINDERS ON FGF23

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A.Bouma de Krijger

2012-06-01

Results are as follows; phosphorus binding by sevelamer significantly lowered urinary phosphate excretion, from a median of 26.25 mmol/24 h to 17.5 mmol/l. Other parameters showed no sgnificant association with urinary phosphate excretion. The serum phosphate was unchanged during treatment. Creatinin clearance was significantly associated with FGF23 (p0.03. FGF23 did not change significant following phosphorus binding therapy. In conclusion; although 8 weeks sevelamer treatment significantly lowered 24 h urinary phosphate excretion, there was no reduction in FGF23 levels in this group of CKD stage 3 patients.

TROUBLESHOOTING IN EXPRESSION AND PURIFICATION OF RECOMBINANT SEVERE ACUTE RESPIRATORY SYNDROME-ASSOCIATED CORONAVIRUS NUCLEOCAPSID PROTEIN IN Escherichia coli BL21

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Budiman Bela

2010-11-01

Full Text Available Considering importance of N protein for study of viral pathogenesis or development of immunodiagnostic assay, wereported effects of several conditions on purity and homogeneity of recombinant SARS-CoV N protein expressed in E.coli BL21. The SARS-CoV N gene was reverse transcribed and amplified by the reverse transcription-polymerase chainreaction (RT-PCR technique. The amplicons were cloned into pGEX-6P1 and followed by subcloning of the targetgene into pQE-80L. After inserting the recombinant plasmid (pQE80-N into E. coli, the recombinant protein (6 x Histag-N protein fusion was expressed by inducing the bacterial cells with 0.1-0.5 mM isopropyl-1-thio-Dgalactopyranoside(IPTG for 1-5 h. The protein recombinant were extracted from the bacterial cells by NTT buffercontaining 0-20 mM imidazol, and followed by Ni-NTA affinity resin purification. The results showed that induction ofE. coli BL21 with 0.2 mM IPTG for 4 h and followed with lysis of bacterial cells in NTT buffer containing 10 mMimidazol were optimal conditions to obtain the pure recombinant SARS-CoV N protein.

Factors affecting the solubility of Bacillus halmapalus alpha-amylase

DEFF Research Database (Denmark)

Faber, Cornelius; Hobley, Timothy John; Mollerup, Jørgen

2008-01-01

A detailed study of the solubility of recombinant Bacillus halmapalus alpha-amylase has been conducted. A semi-purified preparation from a bulk crystallisation was chos en that contained six isoforms with pI-values of between 5.5 and 6.1. The solubility was strongly affected by pH and could...

Stage-specific effects of FGF2 on the differentiation of dental pulp cells

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Sagomonyants, Karen; Mina, Mina

2015-01-01

Dentinogenesis is a complex and multistep process, which is regulated by various growth factors, including members of the Fibroblast Growth Factor (FGF) family. Both positive and negative effects of FGFs on dentinogenesis have been reported but the underlying mechanisms of these conflicting results are still unclear. To gain better insight into the role of FGF2 in dentinogenesis, we used dental pulp cells from various transgenic mice, in which fluorescent protein expression identifies cells at different stages of odontoblast differentiation. Our results showed that continuous exposure of pulp cells to FGF2 inhibited mineralization and revealed both stimulatory and inhibitory effects of FGF2 on expression of markers of dentinogenesis and various transgenes. During the proliferation phase of in vitro growth FGF2 increased expression of markers of dentinogenesis and the percentages of DMP1-GFP+ functional odontoblasts and DSPP-Cerulean+ odontoblasts. Additional exposure to FGF2 during the differentiation/mineralization phase of in vitro growth decreased the extent of mineralization, expression of markers of dentinogenesis, and expression of DMP1-GFP and DSPP-Cerulean transgenes. Recovery experiments showed that the inhibitory effects of FGF2 on dentinogenesis were related to the blocking of differentiation of cells into mature odontoblasts. These observations together showed stage-specific effects of FGF2 on dentinogenesis by dental pulp cells and provide critical information for the development of improved treatments for vital pulp therapy and dentin regeneration. PMID:25823776

Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix.

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Gigout, A; Guehring, H; Froemel, D; Meurer, A; Ladel, C; Reker, D; Bay-Jensen, A C; Karsdal, M A; Lindemann, S

2017-11-01

Fibroblast growth factor (FGF) 18 has been shown to increase cartilage volume when injected intra-articularly in animal models of osteoarthritis (OA) and in patients with knee OA (during clinical development of the recombinant human FGF18, sprifermin). However, the exact nature of this effect is still unknown. In this study, we aimed to investigate the effects of sprifermin at the cellular level. A combination of different chondrocyte culture systems was used and the effects of sprifermin on proliferation, the phenotype and matrix production were evaluated. The involvement of MAPKs in sprifermin signalling was also studied. In monolayer, we observed that sprifermin promoted a round cell morphology and stimulated both cellular proliferation and Sox9 expression while strongly decreasing type I collagen expression. In 3D culture, sprifermin increased the number of matrix-producing chondrocytes, improved the type II:I collagen ratio and enabled human OA chondrocytes to produce a hyaline extracellular matrix (ECM). Furthermore, we found that sprifermin displayed a 'hit and run' mode of action, with intermittent exposure required for the compound to fully exert its anabolic effect. Finally, sprifermin appeared to signal through activation of ERK. Our results indicate that intermittent exposure to sprifermin leads to expansion of hyaline cartilage-producing chondrocytes. These in vitro findings are consistent with the increased cartilage volume observed in the knees of OA patients after intra-articular injection with sprifermin in clinical studies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Scheduled transplantation of bone marrow cells preincubated with acidic fibroblast growth factor (aFGF)

International Nuclear Information System (INIS)

Xiang Yingsong; Yang Rujun; Cai Jianming; Li Bailong

1999-01-01

Objective: To develop a new method of bone marrow scheduled transplantation (BMST) by making use of the effects of acidic fibroblast growth factor (aFGF) on improving hematopoiesis. Methods: The scheduled transplantation of bone marrow cells preincubated with aFGF (aFGF-BMST) was carried out to study the effects of aFGF on hematopoietic reconstitution and reducing acute graft versus host disease (GVHD) in acute radiation disease model of Kunming mice. Results: The survival rate of the group of aFGF-BMST mice with 4 x 10 6 BMXs was 40%, which was higher than the survival of the group of BMT with 1 x 10 7 BMCs alone (30%), but was lower than the survival of the group of BMST with 4 x 10 6 BMCs. On the other hand, the recovery rates in numbers of leucocytes, nucleated cells and CFU-E, CFU-GM, CFU-S were faster than those in the group of BMT with 1 x 10 7 BMCs alone and in the group of BMST with 4 x 10 6 BMCs. In addition, the severity of GVHD in the group of aFGF-BMST mice with 4 x 10 6 BMCs was lower than that in the group of BMT with 1 x 10 7 BMCs alone but was higher than that in the group of BMST with 4 x 10 6 BMCs. Conclusion: Although aFGF can activate heterogeneous T cells to cause GVHD, there is prospect of making full use of the effects of aFGF on improving hematopoiesis and reducing the side effects of aFGF leading to GVHD through scheduled transplantation of bone marrow cells preincubated with aFGF

FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting

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Riminucci, Mara; Collins, Michael T.; Fedarko, Neal S.; Cherman, Natasha; Corsi, Alessandro; White, Kenneth E.; Waguespack, Steven; Gupta, Anurag; Hannon, Tamara; Econs, Michael J.; Bianco, Paolo; Gehron Robey, Pamela

2003-01-01

FGF-23, a novel member of the FGF family, is the product of the gene mutated in autosomal dominant hypophosphatemic rickets (ADHR). FGF-23 has been proposed as a circulating factor causing renal phosphate wasting not only in ADHR (as a result of inadequate degradation), but also in tumor-induced osteomalacia (as a result of excess synthesis by tumor cells). Renal phosphate wasting occurs in approximately 50% of patients with McCune-Albright syndrome (MAS) and fibrous dysplasia of bone (FD), which result from postzygotic mutations of the GNAS1 gene. We found that FGF-23 is produced by normal and FD osteoprogenitors and bone-forming cells in vivo and in vitro. In situ hybridization analysis of FGF-23 mRNA expression identified “fibrous” cells, osteogenic cells, and cells associated with microvascular walls as specific cellular sources of FGF-23 in FD. Serum levels of FGF-23 were increased in FD/MAS patients compared with normal age-matched controls and significantly higher in FD/MAS patients with renal phosphate wasting compared with those without, and correlated with disease burden bone turnover markers commonly used to assess disease activity. Production of FGF-23 by FD tissue may play an important role in the renal phosphate–wasting syndrome associated with FD/MAS. PMID:12952917

Functions and Mechanisms of Fibroblast Growth Factor (FGF Signalling in Drosophila melanogaster

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Hans-Arno J. Müller

2013-03-01

Full Text Available Intercellular signalling via growth factors plays an important role in controlling cell differentiation and cell movements during the development of multicellular animals. Fibroblast Growth Factor (FGF signalling induces changes in cellular behaviour allowing cells in the embryo to move, to survive, to divide or to differentiate. Several examples argue that FGF signalling is used in multi-step morphogenetic processes to achieve and maintain a transitional state of the cells required for the control of cell fate. In the genetic model Drosophila melanogaster, FGF signalling via the receptor tyrosine kinases Heartless (Htl and Breathless (Btl is particularly well studied. These FGF receptors affect gene expression, cell shape and cell–cell interactions during mesoderm layer formation, caudal visceral muscle (CVM formation, tracheal morphogenesis and glia differentiation. Here, we will address the current knowledge of the biological functions of FGF signalling in the fly on the tissue, at a cellular and molecular level.

FGF19 regulates cell proliferation, glucose and bile acid metabolism via FGFR4-dependent and independent pathways.

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Ai-Luen Wu

Full Text Available Fibroblast growth factor 19 (FGF19 is a hormone-like protein that regulates carbohydrate, lipid and bile acid metabolism. At supra-physiological doses, FGF19 also increases hepatocyte proliferation and induces hepatocellular carcinogenesis in mice. Much of FGF19 activity is attributed to the activation of the liver enriched FGF Receptor 4 (FGFR4, although FGF19 can activate other FGFRs in vitro in the presence of the coreceptor βKlotho (KLB. In this report, we investigate the role of FGFR4 in mediating FGF19 activity by using Fgfr4 deficient mice as well as a variant of FGF19 protein (FGF19v which is specifically impaired in activating FGFR4. Our results demonstrate that FGFR4 activation mediates the induction of hepatocyte proliferation and the suppression of bile acid biosynthesis by FGF19, but is not essential for FGF19 to improve glucose and lipid metabolism in high fat diet fed mice as well as in leptin-deficient ob/ob mice. Thus, FGF19 acts through multiple receptor pathways to elicit pleiotropic effects in regulating nutrient metabolism and cell proliferation.

Diagnostic Modalities for FGF23-Producing Tumors in Patients with Tumor-Induced Osteomalacia

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Seiji Fukumoto

2014-06-01

Full Text Available Fibroblast growth factor 23 (FGF23 is a hormone that is produced by osteocytes and regulates phosphate and vitamin D metabolism through binding to the Klotho-FGF receptor complex. Excessive actions of FGF23 cause several kinds of hypophosphatemic rickets/osteomalacia. Tumor-induced rickets/osteomalacia (TIO is a paraneoplastic syndrome caused by overproduction of FGF23 from the responsible tumors. Because TIO is cured by complete resection of the causative tumors, it is of great clinical importance to locate these tumors. Several imaging methods including skeletal survey by magnetic resonance imaging and octreotide scintigraphy have been used to identify the tumors that cause TIO. However, none of these imaging studies indicate that the detected tumors are actually producing FGF23. Recently, systemic venous sampling was conducted for locating FGF23-producing tumor in suspected patients with TIO and demonstrated that this test might be beneficial to a subset of patient. Further studies with more patients are necessary to establish the clinical utility of venous sampling in patients with TIO.

Impact of Annexin A 7 Deficiency on FGF23 Plasma Concentrations

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Anja T. Umbach

2016-11-01

Full Text Available Background/Aims: The release of fibroblast growth factor FGF23, a powerful regulator of 1,25(OH2D3 formation and mineral metabolism, is stimulated by store-operated Ca2+ entry (SOCE, which is accomplished by the pore forming Ca2+ release activated channel protein Orai1. Regulators of Orai1 and thus FGF23 release include serum & glucocorticoid inducible kinase SGK1, a kinase up-regulated by glucocorticosteroids. Some effects of glucocorticoids require the presence of annexin A7, such as suppression of prostaglandin E2 in gastric glands. The present study thus explored whether annexin A7 impacts on FGF23 plasma levels. Methods: Comparisons were made between gene targeted mice lacking functional annexin A7 (Anx7-/- and their wild type littermates (Anx7+/+. Serum C-terminal-FGF23, intact FGF23, 1,25(OH2D3 and PTH concentrations were measured by ELISA or EIA. The serum and urinary phosphate concentrations were measured by colorimetry, the serum Ca2+ concentration and the urinary Ca2+ concentration by flame photometry. Results: Serum C-terminal FGF23 levels and corticosterone levels were significantly higher and serum 1,25(OH2 D3 and PTH levels were significantly lower in Anx7-/- than in Anx7+/+ mice. Water intake was slightly but significantly higher in Anx7-/- mice than in Anx7+/+ mice. No significant difference was observed between Anx7-/- and Anx7+/+ mice in urinary fluid excretion, plasma Ca2+ concentration, plasma phosphate concentration and urinary Ca2+ output. The urinary phosphate output was significantly lower in Anx7-/- mice than in Anx7+/+ mice. Conclusion: Annexin A7 deficiency upregulates FGF23 plasma levels, an effect paralleled by increased corticosterone plasma levels, as well as decreased 1,25(OH2 D3 and PTH plasma levels.

Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry.

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Meisser Redeuil, Karine; Longet, Karin; Bénet, Sylvie; Munari, Caroline; Campos-Giménez, Esther

2015-11-27

This manuscript reports a validated analytical approach for the quantification of 21 water soluble vitamins and their main circulating forms in human plasma. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. Instrumental lower limits of detection and quantification reached water soluble vitamins in human plasma single donor samples. The present report provides a sensitive and reliable approach for the quantification of water soluble vitamins and main circulating forms in human plasma. In the future, the application of this analytical approach will give more confidence to provide a comprehensive assessment of water soluble vitamins nutritional status and bioavailability studies in humans. Copyright © 2015 Elsevier B.V. All rights reserved.

Collaborative interplay between FGF-2 and VEGF-C promotes lymphangiogenesis and metastasis

DEFF Research Database (Denmark)

Cao, Renhai; Ji, Hong; Feng, Ninghan

2012-01-01

Interplay between various lymphangiogenic factors in promoting lymphangiogenesis and lymphatic metastasis remains poorly understood. Here we show that FGF-2 and VEGF-C, two lymphangiogenic factors, collaboratively promote angiogenesis and lymphangiogenesis in the tumor microenvironment, leading...... endothelial cell tip cell formation is a prerequisite for FGF-2-stimulated lymphangiogenesis. In the tumor microenvironment, the reciprocal interplay between FGF-2 and VEGF-C collaboratively stimulated tumor growth, angiogenesis, intratumoral lymphangiogenesis, and metastasis. Thus, intervention and targeting...

Sustained levels of FGF2 maintain undifferentiated stem cell cultures with biweekly feeding.

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Steven Lotz

Full Text Available An essential aspect of stem cell culture is the successful maintenance of the undifferentiated state. Many types of stem cells are FGF2 dependent, and pluripotent stem cells are maintained by replacing FGF2-containing media daily, while tissue-specific stem cells are typically fed every 3rd day. Frequent feeding, however, results in significant variation in growth factor levels due to FGF2 instability, which limits effective maintenance due to spontaneous differentiation. We report that stabilization of FGF2 levels using controlled release PLGA microspheres improves expression of stem cell markers, increases stem cell numbers and decreases spontaneous differentiation. The controlled release FGF2 additive reduces the frequency of media changes needed to maintain stem cell cultures, so that human embryonic stem cells and induced pluripotent stem cells can be maintained successfully with biweekly feedings.

Generation of FGF reporter transgenic zebrafish and their utility in chemical screens

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Tsang Michael

2007-06-01

Full Text Available Abstract Background Fibroblast Growth Factors (FGFs represent a large family of secreted proteins that are required for proper development and physiological processes. Mutations in mouse and zebrafish FGFs result in abnormal embryogenesis and lethality. A key to understanding the precise role for these factors is to determine their spatial and temporal activity during embryogenesis. Results Expression of Dual Specificity Phosphatase 6 (dusp6, also known as Mkp3 is controlled by FGF signalling throughout development. The Dusp6 promoter was isolated from zebrafish and used to drive expression of destabilized green fluorescent protein (d2EGFP in transgenic embryos (Tg(Dusp6:d2EGFP. Expression of d2EGFP is initiated as early as 4 hours post-fertilization (hpf within the future dorsal region of the embryo, where fgf3 and fgf8 are initially expressed. At later stages, d2EGFP is detected within structures that correlate with the expression of Fgf ligands and their receptors. This includes the mid-hindbrain boundary (MHB, pharyngeal endoderm, otic vesicle, hindbrain, and Kupffer's vesicle. The expression of d2EGFP is under the control of FGF signalling as treatment with FGF Receptor (FGFR inhibitors results in the suppression of d2EGFP expression. In a pilot screen of commercially available small molecules we have evaluated the effectiveness of the transgenic lines to identify specific FGF inhibitors within the class of indolinones. These compounds were counter screened with the transgenic line Tg(Fli1:EGFPy1, that serves as an indirect read-out for Vascular Endothelial Growth Factor (VEGF signalling in order to determine the specificity between related receptor tyrosine kinases (RTKs. From these assays it is possible to determine the specificity of these indolinones towards specific RTK signalling pathways. This has enabled the identification of compounds that can block specifically the VEGFR or the FGFR signalling pathway. Conclusion The generation of

VEGF and bFGF Gene Polymorphisms in Patients with Non-Hodgkin's Lymphoma

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Tomasz Wróbel

2013-01-01

Full Text Available Angiogenesis and lymphangiogenesis are important in the proliferation and survival of the malignant hematopoietic neoplasms, including non-Hodgkin’s lymphomas (NHLs. Vascular endothelial growth factor (VEGF and basic fibroblast growth factor (bFGF play an important role in the initiation of angiogenesis. Both VEGF and bFGF have been reported to have prognostic significance in NHL. The present study aimed to determine an association between the VEGF and bFGF gene polymorphisms and disease susceptibility and progression. VEGF (rs3025039; 936 C>T and bFGF (rs308395, −921 G>C variants were determined in 78 NHL patients and 122 healthy individuals by PCR-RFLP technique. The presence of the VEGF 936T allele was found to significantly associate with worse prognosis of the disease (expressed by the highest International Prognostic Index (IPI (0.41 versus 0.20, for IPI 4 among patients having and lacking the T allele. The VEGF 936T variant was also more frequent among patients with IPI 4 than in controls (OR = 3.37, . The bFGF −921G variant was more frequently detected among patients with aggressive as compared to those with indolent histological subtype (0.37 versus 0.18, and healthy individuals (0.37 versus 0.19, OR = 2.51, . These results imply that VEGF and bFGF gene polymorphisms have prognostic significance in patients with NHL.

Oncogenic osteomalacia due to FGF23-expressing colon adenocarcinoma.

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Leaf, David E; Pereira, Renata C; Bazari, Hasan; Jüppner, Harald

2013-03-01

Oncogenic osteomalacia, a paraneoplastic syndrome associated with hypophosphatemia due to increased urinary phosphate excretion, is caused by excessive synthesis and secretion of fibroblast growth factor 23 (FGF23), a phosphaturic hormone that is normally produced by osteocytes. Most cases of oncogenic osteomalacia have been associated with benign tumors of bone or soft tissue; however, whether malignant neoplasms can also produce and secrete FGF23 is currently unknown. The aim was to determine whether a malignant neoplasm could cause oncogenic osteomalacia through excessive production and secretion of FGF23. We describe an 80-year-old woman with stage IV colon adenocarcinoma who presented with severe hypophosphatemia (0.4 mg/dL; reference, 2.6-4.5 mg/dL). Fractional excretion of phosphate was 34% (reference, osteomalacia should be considered in the differential diagnosis for patients with a malignant tumor who present with hypophosphatemia.

Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination.

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Yuna Kim

2006-06-01

Full Text Available The genes encoding members of the wingless-related MMTV integration site (WNT and fibroblast growth factor (FGF families coordinate growth, morphogenesis, and differentiation in many fields of cells during development. In the mouse, Fgf9 and Wnt4 are expressed in gonads of both sexes prior to sex determination. Loss of Fgf9 leads to XY sex reversal, whereas loss of Wnt4 results in partial testis development in XX gonads. However, the relationship between these signals and the male sex-determining gene, Sry, was unknown. We show through gain- and loss-of-function experiments that fibroblast growth factor 9 (FGF9 and WNT4 act as opposing signals to regulate sex determination. In the mouse XY gonad, Sry normally initiates a feed-forward loop between Sox9 and Fgf9, which up-regulates Fgf9 and represses Wnt4 to establish the testis pathway. Surprisingly, loss of Wnt4 in XX gonads is sufficient to up-regulate Fgf9 and Sox9 in the absence of Sry. These data suggest that the fate of the gonad is controlled by antagonism between Fgf9 and Wnt4. The role of the male sex-determining switch--Sry in the case of mammals--is to tip the balance between these underlying patterning signals. In principle, sex determination in other vertebrates may operate through any switch that introduces an imbalance between these two signaling pathways.

A pilot study of regenerative therapy using controlled release of recombinant human fibroblast growth factor for patients with pre-collapse osteonecrosis of the femoral head.

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Kuroda, Yutaka; Asada, Ryuta; So, Kazutaka; Yonezawa, Atsushi; Nankaku, Manabu; Mukai, Kumi; Ito-Ihara, Toshiko; Tada, Harue; Yamamoto, Michio; Murayama, Toshinori; Morita, Satoshi; Tabata, Yasuhiko; Yokode, Masayuki; Shimizu, Akira; Matsuda, Shuichi; Akiyama, Haruhiko

2016-08-01

We evaluated the safety and clinical outcomes of a single local administration of gelatin hydrogel impregnated with recombinant human fibroblast growth factor (rhFGF)-2 for the treatment of the precollapse stage of osteonecrosis of the femoral head (ONFH). Patients with ONFH (precollapse stage ≤2) received a single local administration of 800 μg of rhFGF-2-impregnated gelatin hydrogel and were followed up for one year. The surgery was performed using a minimally invasive technique involving a 1-cm skin incision, and walking was allowed from day one postoperatively. The primary outcomes included occurrence of adverse events and complications. The secondary outcomes included changes in the Harris hip scores, visual analog scale for pain scores, University of California, Los Angeles (UCLA) activity scores, and radiological images. We included ten patients, of which five experienced 14 adverse events, including one complication from spinal anesthesia. However, patients completely recovered from all adverse events. The mean clinical scores significantly improved by one year postoperatively compared with the pre-operative scores (before vs. after: visual analog score for pain, 21.2 vs. 5.3 mm; UCLA activity score, 5.5 vs. 6.6; Harris hip score, 81.0 vs. 96.9 points). There was only one case of femoral head collapse; however, this occurred in a hip with extensive necrosis. Stage progression and collapse did not occur in the other nine cases. Computed tomography confirmed bone regeneration in the femoral heads. Clinical application of rhFGF-2-impregnated gelatin hydrogel for patients with precollapse ONFH was feasible and safe.

Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis.

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Mohammadi, M; Dionne, C A; Li, W; Li, N; Spivak, T; Honegger, A M; Jaye, M; Schlessinger, J

1992-08-20

Stimulation of growth factor receptors with tyrosine kinase activity is followed by rapid receptor dimerization, tyrosine autophosphorylation and phosphorylation of signalling molecules such as phospholipase C gamma (PLC gamma) and the ras GTPase-activating protein. PLC gamma and GTPase-activating protein bind to specific tyrosine-phosphorylated regions in growth factor receptors through their src-homologous SH2 domains. Growth factor-induced tyrosine phosphorylation of PLC gamma is essential for stimulation of phosphatidylinositol hydrolysis in vitro and in vivo. We have shown that a short phosphorylated peptide containing tyrosine at position 766 from a conserved region of the fibroblast growth factor (FGF) receptor is a binding site for the SH2 domain of PLC gamma (ref. 8). Here we show that an FGF receptor point mutant in which Tyr 766 is replaced by a phenylalanine residue (Y766F) is unable to associate with and tyrosine-phosphorylate PLC gamma or to stimulate hydrolysis of phosphatidylinositol. Nevertheless, the Y766F FGF receptor mutant can be autophosphorylated, and can phosphorylate several cellular proteins and stimulate DNA synthesis. Our data show that phosphorylation of the conserved Tyr 766 of the FGF receptor is essential for phosphorylation of PLC gamma and for hydrolysis of phosphatidylinositol, but that elimination of this hydrolysis does not affect FGF-induced mitogenesis.

Recombination in the evolution of enterovirus C species sub-group that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99.

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Teemu Smura

Full Text Available Genetic recombination is considered to be a very frequent phenomenon among enteroviruses (Family Picornaviridae, Genus Enterovirus. However, the recombination patterns may differ between enterovirus species and between types within species. Enterovirus C (EV-C species contains 21 types. In the capsid coding P1 region, the types of EV-C species cluster further into three sub-groups (designated here as A-C. In this study, the recombination pattern of EV-C species sub-group B that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99 was determined using partial 5'UTR and VP1 sequences of enterovirus strains isolated during poliovirus surveillance and previously published complete genome sequences. Several inter-typic recombination events were detected. Furthermore, the analyses suggested that inter-typic recombination events have occurred mainly within the distinct sub-groups of EV-C species. Only sporadic recombination events between EV-C species sub-group B and other EV-C sub-groups were detected. In addition, strict recombination barriers were inferred for CVA-21 genotype C and CVA-24 variant strains. These results suggest that the frequency of inter-typic recombinations, even within species, may depend on the phylogenetic position of the given viruses.

Quantitative image analysis of intra-tumoral bFGF level as a molecular marker of paclitaxel resistance

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Wientjes M Guillaume

2008-01-01

Full Text Available Abstract Background The role of basic fibroblast growth factor (bFGF in chemoresistance is controversial; some studies showed a relationship between higher bFGF level and chemoresistance while other studies showed the opposite finding. The goal of the present study was to quantify bFGF levels in archived tumor tissues, and to determine its relationship with chemosensitivity. Methods We established an image analysis-based method to quantify and convert the immunostaining intensity of intra-tumor bFGF to concentrations; this was accomplished by generating standard curves using human xenograft tumors as the renewable tissue source for simultaneous image analysis and ELISA. The relationships between bFGF concentrations and tumor chemosensitivity of patient tumors (n = 87 to paclitaxel were evaluated using linear regression analysis. Results The image analysis results were compared to our previous results obtained using a conventional, semi-quantitative visual scoring method. While both analyses indicated an inverse relationship between bFGF level and tumor sensitivity to paclitaxel, the image analysis method, by providing bFGF levels in individual tumors and therefore more data points (87 numerical values as opposed to four groups of staining intensities, further enabled the quantitative analysis of the relationship in subgroups of tumors with different pathobiological properties. The results show significant correlation between bFGF level and tumor sensitivity to the antiproliferation effect, but not the apoptotic effect, of paclitaxel. We further found stronger correlations of bFGF level and paclitaxel sensitivity in four tumor subgroups (high stage, positive p53 staining, negative aFGF staining, containing higher-than-median bFGF level, compared to all other groups. These findings suggest that the relationship between intra-tumoral bFGF level and paclitaxel sensitivity was context-dependent, which may explain the previous contradictory findings

Attenuation of hyperlipidemia- and diabetes-induced early-stage apoptosis and late-stage renal dysfunction via administration of fibroblast growth factor-21 is associated with suppression of renal inflammation.

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Chi Zhang

Full Text Available BACKGROUND: Lipotoxicity is a key feature of the pathogenesis of diabetic kidney disease, and is attributed to excessive lipid accumulation (hyperlipidemia. Increasing evidence suggests that fibroblast growth factor (FGF21 has a crucial role in lipid metabolism under diabetic conditions. OBJECTIVE: The present study investigated whether FGF21 can prevent hyperlipidemia- or diabetes-induced renal damage, and if so, the possible mechanism. METHODS: Mice were injected with free fatty acids (FFAs, 10 mg/10 g body weight or streptozotocin (150 mg/kg to establish a lipotoxic model or type 1 diabetic model, respectively. Simultaneously the mice were treated with FGF21 (100 µg/kg for 10 or 80 days. The kidney weight-to-tibia length ratio and renal function were assessed. Systematic and renal lipid levels were detected by ELISA and Oil Red O staining. Renal apoptosis was examined by TUNEL assay. Inflammation, oxidative stress, and fibrosis were assessed by Western blot. RESULTS: Acute FFA administration and chronic diabetes were associated with lower kidney-to-tibia length ratio, higher lipid levels, severe renal apoptosis and renal dysfunction. Obvious inflammation, oxidative stress and fibrosis also observed in the kidney of both mice models. Deletion of the fgf21 gene further enhanced the above pathological changes, which were significantly prevented by administration of exogenous FGF21. CONCLUSION: These results suggest that FFA administration and diabetes induced renal damage, which was further enhanced in FGF21 knock-out mice. Administration of FGF21 significantly prevented both FFA- and diabetes-induced renal damage partially by decreasing renal lipid accumulation and suppressing inflammation, oxidative stress, and fibrosis.

Variable expressivity of FGF3 mutations associated with deafness and LAMM syndrome

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Griffith Andrew J

2011-02-01

Full Text Available Abstract Background Recessive mutations of fibroblast growth factor 3 (FGF3 can cause LAMM syndrome (OMIM 610706, characterized by fully penetrant complete labyrinthine aplasia, microtia and microdontia. Methods We performed a prospective molecular genetic and clinical study of families segregating hearing loss linked to FGF3 mutations. Ten affected individuals from three large Pakistani families segregating FGF3 mutations were imaged with CT, MRI, or both to detect inner ear abnormalities. We also modeled the three dimensional structure of FGF3 to better understand the structural consequences of the three missense mutations. Results Two families segregated reported mutations (p.R104X and p.R95W and one family segregated a novel mutation (p.R132GfsX26 of FGF3. All individuals homozygous for p.R104X or p.R132GfsX26 had fully penetrant features of LAMM syndrome. However, recessive p.R95W mutations were associated with nearly normal looking auricles and variable inner ear structural phenotypes, similar to that reported for a Somali family also segregating p.R95W. This suggests that the mild phenotype is not entirely due to genetic background. Molecular modeling result suggests a less drastic effect of p.R95W on FGF3 function compared with known missense mutations detected in fully penetrant LAMM syndrome. Since we detected significant intrafamilial variability of the inner ear structural phenotype in the family segregating p.R95W, we also sequenced FGF10 as a likely candidate for a modifier. However, we did not find any sequence variation, pointing out that a larger sample size will be needed to map and identify a modifier. We also observed a mild to moderate bilateral conductive hearing loss in three carriers of p.R95W, suggesting either a semi-dominant effect of this mutant allele of FGF3, otitis media, or a consequence of genetic background in these three family members. Conclusions We noted a less prominent dental and external ear phenotype in

Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL

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Douette, Pierre; Navet, Rachel; Gerkens, Pascal; Galleni, Moreno; Levy, Daniel; Sluse, Francis E.

2005-01-01

Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein

High-resolution recombination patterns in a region of human chromosome 21 measured by sperm typing.

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Irene Tiemann-Boege

2006-05-01

Full Text Available For decades, classical crossover studies and linkage disequilibrium (LD analysis of genomic regions suggested that human meiotic crossovers may not be randomly distributed along chromosomes but are focused instead in "hot spots." Recent sperm typing studies provided data at very high resolution and accuracy that defined the physical limits of a number of hot spots. The data were also used to test whether patterns of LD can predict hot spot locations. These sperm typing studies focused on several small regions of the genome already known or suspected of containing a hot spot based on the presence of LD breakdown or previous experimental evidence of hot spot activity. Comparable data on target regions not specifically chosen using these two criteria is lacking but is needed to make an unbiased test of whether LD data alone can accurately predict active hot spots. We used sperm typing to estimate recombination in 17 almost contiguous ~5 kb intervals spanning 103 kb of human Chromosome 21. We found two intervals that contained new hot spots. The comparison of our data with recombination rates predicted by statistical analyses of LD showed that, overall, the two datasets corresponded well, except for one predicted hot spot that showed little crossing over. This study doubles the experimental data on recombination in men at the highest resolution and accuracy and supports the emerging genome-wide picture that recombination is localized in small regions separated by cold areas. Detailed study of one of the new hot spots revealed a sperm donor with a decrease in recombination intensity at the canonical recombination site but an increase in crossover activity nearby. This unique finding suggests that the position and intensity of hot spots may evolve by means of a concerted mechanism that maintains the overall recombination intensity in the region.

Genetic Rescue of Glycosylation-deficient Fgf23 in the Galnt3 Knockout Mouse

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Ichikawa, Shoji; Gray, Amie K.; Padgett, Leah R.; Allen, Matthew R.; Clinkenbeard, Erica L.; Sarpa, Nicole M.; White, Kenneth E.; Econs, Michael J.

2014-01-01

Fibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence (176RHTR179↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineat...

Positive effects of bFGF modified rat amniotic epithelial cells transplantation on transected rat optic nerve.

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Jia-Xin Xie

Full Text Available Effective therapy for visual loss caused by optic nerve injury or diseases has not been achieved even though the optic nerve has the regeneration potential after injury. This study was designed to modify amniotic epithelial cells (AECs with basic fibroblast growth factor (bFGF gene, preliminarily investigating its effect on transected optic nerve.A human bFGF gene segment was delivered into rat AECs (AECs/hbFGF by lentiviral vector, and the gene expression was examined by RT-PCR and ELISA. The AECs/hbFGF and untransfected rat AECs were transplanted into the transected site of the rat optic nerve. At 28 days post transplantation, the survival and migration of the transplanted cells was observed by tracking labeled cells; meanwhile retinal ganglion cells (RGCs were observed and counted by employing biotin dextran amine (BDA and Nissl staining. Furthermore, the expression of growth associated protein 43 (GAP-43 within the injury site was examined with immunohistochemical staining.The AECs/hbFGF was proven to express bFGF gene and secrete bFGF peptide. Both AECs/hbFGF and AECs could survive and migrate after transplantation. RGCs counting implicated that RGCs numbers of the cell transplantation groups were significantly higher than that of the control group, and the AECs/hbFGF group was significantly higher than that of the AECs group. Moreover GAP-43 integral optical density value in the control group was significantly lower than that of the cell transplantation groups, and the value in the AECs/hbFGF group was significantly higher than that of the AECs group.AECs modified with bFGF could reduce RGCs loss and promote expression of GAP-43 in the rat optic nerve transected model, facilitating the process of neural restoration following injury.

Rare codons effect on expression of recombinant gene cassette in Escherichia coli BL21(DE3

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Aghil Esmaeili-Bandboni

2017-11-01

Full Text Available Objective: To demonstrate the sensitivity of expression of fusion genes to existence of a large number of rare codons in recombinant gene sequenced. Methods: Primers for amplification of cholera toxin B, Shiga toxin B and gfp genes were designed by Primer3 software and synthesized. All of these 3 genes were cloned. Then the genes were fused together by restriction sites and enzymatic method. Two linkers were used as a flexible bridge in connection of these genes. Results: Cloning and fusion of cholera toxin B, Shiga toxin B and gfp genes were done correctly. After that, expression of the recombinant gene construction was surveyed. Conclusions: According to what was seen, because of the accumulation of 12 rare codons of Shiga toxin B and 19 rare codons of cholera toxin B in this gene cassette, the expression of the recombinant gene cassette, in Escherichia coli BL21, failed.

Biotransformation of rutin to isoquercitrin using recombinant α-L-rhamnosidase from Bifidobacterium breve.

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Zhang, Ru; Zhang, Bian-Ling; Xie, Tao; Li, Gu-Cai; Tuo, Yi; Xiang, Yu-Ting

2015-06-01

To biotransform rutin into isoquercitrin. A α-L-rhamnosidase from Bifidobacterium breve was produced by using Escherichia coli BL21 for biotransformation of rutin to isoquercitrin. The enzyme was purified by Ni(2+)-NTA chromatography to yield a soluble protein with a specific activity of 56 U protein mg(-1). The maximum enzyme activities were at pH 6.5, 55 °C, 20 mM rutin, and 1.2 U enzyme ml(-1). Under optimal conditions, the half-life of the enzyme was 96 h. The K m and V max values were 2.2 mM, 56.4 μmol mg(-1) min(-1) and 2.1 mM, 57.5 μmol mg(-1) min(-1) using pNP-Rha and rutin as substrates, respectively. The kinetic behavior indicated that the recombinant α-L-rhamnosidase has good catalytic performance for producing isoquercitrin. 20 mM rutin was biotransformed into 18.25 and 19.87 mM isoquercitrin after 60 and 240 min. The specific biotransformation of rutin to isoquercitrin using recombinant α-L-rhamnosidase from B. breve is a feasible method for use in industrial processes.

Renal expression of FGF23 in progressive renal disease of diabetes and the effect of ACE inhibitor.

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Cristina Zanchi

Full Text Available Fibroblast growth factor 23 (FGF23 is a phosphaturic hormone mainly produced by bone that acts in the kidney through FGF receptors and Klotho. Here we investigated whether the kidney was an additional source of FGF23 during renal disease using a model of type 2 diabetic nephropathy. Renal expression of FGF23 and Klotho was assessed in Zucker diabetic fatty (ZDF and control lean rats at 2, 4, 6, 8 months of age. To evaluate whether the renoprotective effect of angiotensin converting enzyme (ACE inhibitor in this model was associated with changes in FGF23 and Klotho, ZDF rats received ramipril from 4, when proteinuric, to 8 months of age. FGF23 mRNA was not detectable in the kidney of lean rats, nor of ZDF rats at 2 months of age. FGF23 became measurable in the kidney of diabetic rats at 4 months and significantly increased thereafter. FGF23 protein localized in proximal and distal tubules. Renal Klotho mRNA and protein decreased during time in ZDF rats. As renal disease progressed, serum phosphate levels increased in parallel with decline of fractional phosphorus excretion. Ramipril limited proteinuria and renal injury, attenuated renal FGF23 upregulation and ameliorated Klotho expression. Ramipril normalized serum phosphate levels and tended to increase fractional phosphorus excretion. These data indicate that during progressive renal disease the kidney is a site of FGF23 production which is limited by ACE inhibition. Interfering pharmacologically with the delicate balance of FGF23 and phosphorus in diabetes may have implications in clinics.

Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system

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Wang, Xiaolong; Yu, Honghao; Lei, Anmin; Zhou, Jiankui; Zeng, Wenxian; Zhu, Haijing; Dong, Zhiming; Niu, Yiyuan; Shi, Bingbo; Cai, Bei; Liu, Jinwang; Huang, Shuai; Yan, Hailong; Zhao, Xiaoe; Zhou, Guangxian; He, Xiaoling; Chen, Xiaoxu; Yang, Yuxin; Jiang, Yu; Shi, Lei; Tian, Xiue; Wang, Yongjun; Ma, Baohua; Huang, Xingxu; Qu, Lei; Chen, Yulin

2015-01-01

Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed. The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding. PMID:26354037

Deletion of fibroblast growth factor 22 (FGF22) causes a depression-like phenotype in adult mice.

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Williams, Aislinn J; Yee, Patricia; Smith, Mitchell C; Murphy, Geoffrey G; Umemori, Hisashi

2016-07-01

Specific growth factors induce formation and differentiation of excitatory and inhibitory synapses, and are essential for brain development and function. Fibroblast growth factor 22 (FGF22) is important for specifying excitatory synapses during development, including in the hippocampus. Mice with a genetic deletion of FGF22 (FGF22KO) during development subsequently have fewer hippocampal excitatory synapses in adulthood. As a result, FGF22KO mice are resistant to epileptic seizure induction. In addition to playing a key role in learning, the hippocampus is known to mediate mood and anxiety. Here, we explored whether loss of FGF22 alters affective, anxiety or social cognitive behaviors in mice. We found that relative to control mice, FGF22KO mice display longer duration of floating and decreased latency to float in the forced swim test, increased immobility in the tail suspension test, and decreased preference for sucrose in the sucrose preference test, which are all suggestive of a depressive-like phenotype. No differences were observed between control and FGF22KO mice in other behavioral assays, including motor, anxiety, or social cognitive tests. These results suggest a novel role for FGF22 specifically in affective behaviors. Copyright © 2016 Elsevier B.V. All rights reserved.

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle.

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Peterson, Soren J; Krasnow, Mark A

2015-01-15

To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here, we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains, including inside the cell. Copyright © 2015 Elsevier Inc. All rights reserved.

A FGF3 mutation associated with differential inner ear malformation, microtia, and microdontia.

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Ramsebner, Reinhard; Ludwig, Martin; Parzefall, Thomas; Lucas, Trevor; Baumgartner, Wolf-Dieter; Bodamer, Olaf; Cengiz, Filiz Basak; Schoefer, Christian; Tekin, Mustafa; Frei, Klemens

2010-02-01

Analysis of association between genotype and phenotype. Prospective genetic study in a family. Auditory investigations, computer tomography, and genetic sequencing of the fibroblast growth factor 3 (FGF3) gene were performed on a Somali family presenting with autosomal recessive, hearing impairment, microdontia, and outer ear morphologies ranging from normal auricle development to microtia assessed as type 1 Weerda dysplasia in affected individuals. Computed tomography imaging identified differential inter- and intraindividual malformations of the inner ear including labyrinth aplasia, development of a common cavity to the presence of a cochlear with 1.5 windings (Mondini malformation) in affected individuals, symptoms similar to those described as labyrinth aplasia, microtia, and microdontia (LAMM) syndrome, caused by mutations in FGF3. Genetic sequencing revealed the presence of a novel p.R95W missense mutation in FGF3 segregating with pathology. The p.R95W mutation substitutes a positively charged arginine for a polar tryptophan in the highly conserved RYLAM consensus of the beta 6 sheet of FGF3 that interacts with FGFR2. These findings describe, for the first time, variable inner ear malformations and outer ear dysplasia in the presence of constant microdontia, associated with homozygous inheritance of the p.R95W mutation in FGF3, mirroring phenotypes observed in mouse models ablating FGF3/FGFR2 signaling.

FGF23 inhibits extra-renal synthesis of 1,25-dihydroxyvitamin D in human monocytes

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Bacchetta, Justine; Sea, Jessica L; Chun, Rene F; Lisse, Thomas S; Wesseling-Perry, Katherine; Gales, Barbara; Adams, John S.; Salusky, Isidro B; Hewison, Martin

2012-01-01

Vitamin D is a potent stimulator of monocyte innate immunity, with this effect being mediated via intracrine conversion of 25-hydroxyvitamin D (25OHD) to 1,25-dihydroxyvitamin D (1,25(OH)2D). In the kidney synthesis of 1,25(OH)2D is suppressed by fibroblast growth factor 23 (FGF23), via transcriptional suppression of the vitamin D-activating enzyme 1α-hydroxylase (CYP27B1). We hypothesized that FGF23 also suppresses CYP27B1 in monocytes, with concomitant effects on intracrine responses to 1,25(OH)2D. Monocytes from healthy donor peripheral blood mononuclear cells (PBMCm) and from peritoneal dialysate effluent from kidney disease patients (PDm) were assessed at baseline to confirm the presence of mRNA for FGF23 receptors (FGFRs), with Klotho and FGFR1 being more strongly expressed than FGFR2/3/4 in both cell types. Immunohistochemistry showed co-expression of Klotho and FGFR1 in PBMCm and PDm, with this effect being enhanced following treatment with FGF23 in PBMCm but not PDm. Treatment with FGF23 activated MAP kinase (MAPK) and Akt pathways in PBMCm, demonstrating functional FGFR signaling in these cells. FGF23 treatment of PBMCm and PDm decreased expression of mRNA for CYP27B1. In PBMCm this was associated with downregulation of 25OHD to 1,25(OH)2D metabolism, and concomitant suppression of intracrine induced 24-hydroxylase (CYP24A1) and antibacterial cathelicidin (LL37). FGF23 suppression of CYP27B1 was particularly pronounced in PBMCm treated with interleukin-15 to stimulate synthesis of 1,25(OH)2D. These data indicate that FGF23 can inhibit extra-renal expression of CYP27B1 and subsequent intracrine responses to 1,25(OH)2D in two different human monocyte models. Elevated expression of FGF23 may therefore play a crucial role in defining immune responses to vitamin D and this, in turn, may be a key determinant of infection in patients with CKD. PMID:22886720

Effect of bFGF on radiation-induced apoptosis of vascular endothelial cells

International Nuclear Information System (INIS)

Gu Qingyang; Wang Dewen; Li Yuejuan; Peng Ruiyun; Dong Bo; Wang Zhaohai; Liu Jie; Deng Hua; Jiang Tao

2003-01-01

Objective: To study the effect of bFGF on radiation-induced apoptosis vascular endothelial cells. Methods: A cell line PAE (porcine aortic endothelial cells) and primary cultured HUVEC (human umbilical vein endothelial cells) were irradiated with 60 Co γ-rays to establish cell apoptosis models. Flow cytometry with annexin-V-FITC + PI labeling was used to evaluate cell apoptosis. Different amounts of bFGF were used to study their effects on radiation-induced endothelial cell apoptosis. Results and Conclusions: It is found that bFGF could inhibit radiation-induced endothelial cell apoptosis in a considerable degree

Myc and Fgf Are Required for Zebrafish Neuromast Hair Cell Regeneration.

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Lee, Sang Goo; Huang, Mingqian; Obholzer, Nikolaus D; Sun, Shan; Li, Wenyan; Petrillo, Marco; Dai, Pu; Zhou, Yi; Cotanche, Douglas A; Megason, Sean G; Li, Huawei; Chen, Zheng-Yi

2016-01-01

Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration.

Fibroblast growth factor 21 is elevated in metabolically unhealthy obesity and affects lipid deposition, adipogenesis, and adipokine secretion of human abdominal subcutaneous adipocytes

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Lucia Berti

2015-07-01

Conclusions: The hepatokine FGF21 exerts weak lipogenic and anti-adipogenic actions and marked adiponectin-suppressive and leptin and interleukin-6 release-promoting effects in human differentiating preadipocytes. Together with the higher serum concentrations in MUHO subjects, our findings reveal FGF21 as a circulating factor promoting the development of metabolically unhealthy adipocytes.

An Fgf-Shh signaling hierarchy regulates early specification of the zebrafish skull.

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McCarthy, Neil; Sidik, Alfire; Bertrand, Julien Y; Eberhart, Johann K

2016-07-15

The neurocranium generates most of the craniofacial skeleton and consists of prechordal and postchordal regions. Although development of the prechordal is well studied, little is known of the postchordal region. Here we characterize a signaling hierarchy necessary for postchordal neurocranial development involving Fibroblast growth factor (Fgf) signaling for early specification of mesodermally-derived progenitor cells. The expression of hyaluron synthetase 2 (has2) in the cephalic mesoderm requires Fgf signaling and Has2 function, in turn, is required for postchordal neurocranial development. While Hedgehog (Hh)-deficient embryos also lack a postchordal neurocranium, this appears primarily due to a later defect in chondrocyte differentiation. Inhibitor studies demonstrate that postchordal neurocranial development requires early Fgf and later Hh signaling. Collectively, our results provide a mechanistic understanding of early postchordal neurocranial development and demonstrate a hierarchy of signaling between Fgf and Hh in the development of this structure. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Interpretation of the FGF8 morphogen gradient is regulated by endocytic trafficking.

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Nowak, Matthias; Machate, Anja; Yu, Shuizi Rachel; Gupta, Mansi; Brand, Michael

2011-02-01

Forty years ago, it was proposed that during embryonic development and organogenesis, morphogen gradients provide positional information to the individual cells within a tissue leading to specific fate decisions. Recently, much insight has been gained into how such morphogen gradients are formed and maintained; however, which cellular mechanisms govern their interpretation within target tissues remains debated. Here we used in vivo fluorescence correlation spectroscopy and automated image analysis to assess the role of endocytic sorting dynamics on fibroblast growth factor 8 (Fgf8) morphogen gradient interpretation. By interfering with the function of the ubiquitin ligase Cbl, we found an expanded range of Fgf target gene expression and a delay of Fgf8 lysosomal transport. However, the extracellular Fgf8 morphogen gradient remained unchanged, indicating that the observed signalling changes are due to altered gradient interpretation. We propose that regulation of morphogen signalling activity through endocytic sorting allows fast feedback-induced changes in gradient interpretation during the establishment of complex patterns.

Peptides derived from specific interaction sites of the fibroblast growth factor 2 - FGF receptor complexes induce receptor activation and signaling

DEFF Research Database (Denmark)

Manfè, Valentina; Kochoyan, Artur; Bock, Elisabeth

2010-01-01

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06718.x Abstract Basic fibroblast growth factor (FGF2, bFGF) is the most extensively studied member of the FGF family and is involved in neurogenesis, differentiation, neuroprotection, and synaptic plasticity in the CNS. FGF2 executes its pleiotropic...... biologic actions by binding, dimerizing, and activating FGF receptors (FGFRs). The present study reports the physiologic impact of various FGF2-FGFR1 contact sites employing three different synthetic peptides, termed canofins, designed based on structural analysis of the interactions between FGF2 and FGFR1...

FGF8 activates proliferation and migration in mouse post-natal oligodendrocyte progenitor cells.

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Pablo Cruz-Martinez

Full Text Available Fibroblast growth factor 8 (FGF8 is a key molecular signal that is necessary for early embryonic development of the central nervous system, quickly disappearing past this point. It is known to be one of the primary morphogenetic signals required for cell fate and survival processes in structures such as the cerebellum, telencephalic and isthmic organizers, while its absence causes severe abnormalities in the nervous system and the embryo usually dies in early stages of development. In this work, we have observed a new possible therapeutic role for this factor in demyelinating disorders, such as leukodystrophy or multiple sclerosis. In vitro, oligodendrocyte progenitor cells were cultured with differentiating medium and in the presence of FGF8. Differentiation and proliferation studies were performed by immunocytochemistry and PCR. Also, migration studies were performed in matrigel cultures, where oligodendrocyte progenitor cells were placed at a certain distance of a FGF8-soaked heparin bead. The results showed that both migration and proliferation was induced by FGF8. Furthermore, a similar effect was observed in an in vivo demyelinating mouse model, where oligodendrocyte progenitor cells were observed migrating towards the FGF8-soaked heparin beads where they were grafted. In conclusion, the results shown here demonstrate that FGF8 is a novel factor to induce oligodendrocyte progenitor cell activation, migration and proliferation in vitro, which can be extrapolated in vivo in demyelinated animal models.

Regulatory module involving FGF13, miR-504, and p53 regulates ribosomal biogenesis and supports cancer cell survival

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Bublik, Débora R.; Bursać, Slađana; Sheffer, Michal; Oršolić, Ines; Shalit, Tali; Tarcic, Ohad; Kotler, Eran; Mouhadeb, Odelia; Hoffman, Yonit; Fuchs, Gilad; Levin, Yishai; Volarević, Siniša; Oren, Moshe

2017-01-01

The microRNA miR-504 targets TP53 mRNA encoding the p53 tumor suppressor. miR-504 resides within the fibroblast growth factor 13 (FGF13) gene, which is overexpressed in various cancers. We report that the FGF13 locus, comprising FGF13 and miR-504, is transcriptionally repressed by p53, defining an additional negative feedback loop in the p53 network. Furthermore, we show that FGF13 1A is a nucleolar protein that represses ribosomal RNA transcription and attenuates protein synthesis. Importantly, in cancer cells expressing high levels of FGF13, the depletion of FGF13 elicits increased proteostasis stress, associated with the accumulation of reactive oxygen species and apoptosis. Notably, stepwise neoplastic transformation is accompanied by a gradual increase in FGF13 expression and increased dependence on FGF13 for survival (“nononcogene addiction”). Moreover, FGF13 overexpression enables cells to cope more effectively with the stress elicited by oncogenic Ras protein. We propose that, in cells in which activated oncogenes drive excessive protein synthesis, FGF13 may favor survival by maintaining translation rates at a level compatible with the protein quality-control capacity of the cell. Thus, FGF13 may serve as an enabler, allowing cancer cells to evade proteostasis stress triggered by oncogene activation. PMID:27994142

Overexpression of high molecular weight FGF-2 forms inhibits glioma growth by acting on cell-cycle progression and protein translation

International Nuclear Information System (INIS)

Lemiere, Sylvie; Azar, Rania; Belloc, Francis; Guersel, Demir; Pyronnet, Stephane; Bikfalvi, Andreas; Auguste, Patrick

2008-01-01

In order to clarify the role of HMW FGF-2 in glioma development and angiogenesis, we over-expressed different human FGF-2 isoforms in C6 rat glioma cell line using a tetracycline-regulated expression system. Phenotypic modifications were analyzed in vitro and compared to untransfected cells or to cells over-expressing 18 kDa FGF-2 or all FGF-2 isoforms. In particular, we demonstrate that HMW FGF-2 has unique features in inhibiting glioma cell proliferation. HMW FGF-2 expressing cells showed a cell-cycle arrest at the G2M, demonstrating a role of HMW FGF-2 in controlling the entry in mitosis. Moreover, hydroxyurea was ineffective in blocking cells at the G1S boundary when HMW FGF-2 was expressed. We also show that the HMW FGF-2 isoforms inhibit 4E-BP1 phosphorylation at critical sites restoring the translation inhibitory activity of 4E-BP1. In vivo, inhibition of tumor growth was observed when cells expressed HMW FGF-2. This indicates that HMW FGF-2 inhibits tumor growth in glioma cells by acting on cell-cycle progression and protein translation

Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies.

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Kumorek, Marta; Kubies, Dana; Filová, Elena; Houska, Milan; Kasoju, Naresh; Mázl Chánová, Eliška; Matějka, Roman; Krýslová, Markéta; Bačáková, Lucie; Rypáček, František

2015-01-01

In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm(2). The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm(2)) than on surfaces with a higher concentration of FGF-2 (120 ng/cm(2)).

The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

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Qingwen Jin

Full Text Available Insertion of T4 lysozyme (T4L into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1 infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

Decline in Proliferation and Immature Neuron Markers in the Human Subependymal Zone during Aging: Relationship to EGF- and FGF-related Transcripts

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Christin Weissleder

2016-11-01

Full Text Available Neuroblasts exist within the human subependymal zone (SEZ; however, it is debated to what extent neurogenesis changes during normal aging. It is also unknown how precursor proliferation may correlate with the generation of neuronal and glial cells or how expression of growth factors and receptors may change throughout the adult lifespan. We provided evidence of dividing cells in the human SEZ in conjunction with a dramatic age-related decline (n=50; 21-103 years of mRNAs indicative of proliferating cells (Ki67 and immature neurons (doublecortin. Microglia mRNA (ionized calcium-binding adapter molecule 1 increased during aging, whereas transcript levels of stem/precursor cells (glial fibrillary acidic protein delta and achaete-scute homolog 1, astrocytes (vimentin and glial fibrillary acidic protein and oligodendrocytes (oligodendrocyte lineage transcription factor 2 remained stable. Epidermal growth factor receptor (EGFR and fibroblast growth factor 2 (FGF2 mRNAs increased throughout adulthood, while transforming growth factor alpha (TGFα, EGF, Erb-B2 receptor tyrosine kinase 4 (ErbB4 and FGF receptor 1 (FGFR1 mRNAs were unchanged across adulthood. Cell proliferation mRNA positively correlated with FGFR1 transcripts. Immature neuron and oligodendrocyte expression positively correlated with TGFα and ErbB4 mRNAs, whilst astrocyte transcripts positively correlated with EGF, FGF2 and FGFR1 mRNAs. Microglia mRNA positively correlated with EGF and FGF2 expression. Our findings indicate that neurogenesis in the human SEZ continues well into adulthood, although proliferation and neuronal differentiation may decline across adulthood. We suggest that mRNA expression of EGF- and FGF-related family members do not become limited during aging and may modulate neuronal and glial fate determination in the SEZ throughout human life.

IGF-I AND FGF-2 RESPONSES TO WINGATE ANAEROBIC TEST IN OLDER MEN

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Ruthie Amir

2007-06-01

Full Text Available Reduced activity of the potent anabolic effectors: insulin-like growth factor-I (IGF-I and fibroblast growth factor-2 (FGF-2, play a role in aging associated muscle loss. The effect of fitness level on IGF-I and FGF-2 responses to all-out anaerobic exercise in older men was studied. Twenty four healthy older males: 12 higher fit (58 ± 1y and 12 lower fit (59 ± 1y underwent the Wingate anaerobic test. Serum levels of IGF-I and FGF-2 were measured before, immediately after exercise, and 50 min into recovery. Immediately post exercise, the average peak power output and serum lactate were higher (p < 0.05 in the higher fit (446.0 ± 14. 9 kgm·min-1 for mean (± SD peak power and 12.6 ± 1.1 mml·l-1 for lactate compared with the lower fit individuals (284.0 ± 6.5 kgm·min-1 and 8.5 ± 0.7 mml·l-1, respectively. Pre-exercise IGF-I was lower and FGF-2 was higher in the higher fit (335.0 ± 54.0 ng·ml-1 and 1.6 ± 0.1 ng·ml-1, respectively compared with lower fit individuals (402.0 ± 50.0 ng·ml-1 and 1.4 ± 0.2 ng·ml-1, respectively. Following the anaerobic exercise, in both groups, FGF-2 decreased dramatically (p < 0.05; in the higher fit individuals FGF-2 level was 0.4 ± 0.1 pg·ml-1 compared to 0.1 ± 0.02 pg·ml-1 in the lower fit individuals. In contrast to FGF-2, IGF-I increased transiently to levels of 405.0 ± 62.0 ng·ml-1 in the higher fit individuals and to levels of 436 ± 57.0 ng·ml-1 in the lower fit individuals. However, the IGF-I elevation was significant (p < 0. 05 only in the higher fit individuals. In conclusion, the present study demonstrates that during aging, fitness level can alter circulating levels of IGF-I and FGF-2. Furthermore, fitness level can affect the response of both mediators to all-out anaerobic exercise.

Functional Proteomics Defines the Molecular Switch Underlying FGF Receptor Trafficking and Cellular Outputs

DEFF Research Database (Denmark)

Francavilla, Chiara; Rigbolt, Kristoffer T.G.; Emdal, Kristina B

2013-01-01

The stimulation of fibroblast growth factor receptors (FGFRs) with distinct FGF ligands generates specific cellular responses. However, the mechanisms underlying this paradigm have remained elusive. Here, we show that FGF-7 stimulation leads to FGFR2b degradation and, ultimately, cell proliferation...

[The effect of bFGF and sucralfate on cell proliferation during continuous tissue expansion].

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Hu, Ya-lan; Guo, Shu-zhong; Lu, Kai-hua

2003-05-01

To investigate the effect of local application of bFGF combined with sucralfate on the cell proliferation during continuous tissue expansion (CTE). Nine white pigs were selected to undergo the continuous tissue expansion in this study and treated with bFGF and sucralfate, respectively as the following groups: group 1 with both bFGF and sucralfate, group 2 only with bFGF, group 3 with only sucralfate, and group 4 with saline as control. Fifteen samples were taken in each pig for immunohistochemistry analysis 1-14 days and 6 weeks after the operation. In the group with both bFGF and sucralfate, the epidermic basal cells proliferated significantly after the operation and reached top level in 3 days, which was statistical higher than the control group, but the multiplication of basal cell was the lowest 14 days after the operation, still more than the control group. In dermal layer, proliferation of fibroblasts, vessel endothelial cells, hair follicles epidermic cells and sweat gland epicytes was also significant higher in the group with both bFGF and sucralfate than that the control group and reached top level 7 day after the operation, but the proliferation of cells decreased obviously 14 days after the operation, still higher than the control group. The mitotic activity of cells returned to the basal level in 42 days. There were no significant differences among the group 2, group 3 and group 4. Local application of both bFGF and sucralfate could be more effect to induce cells multiplication during early skin expansion to facilitate the growth of neoformed skin soft tissue.

FGF: A web tool for Fishing Gene Family in a whole genome database

DEFF Research Database (Denmark)

Zheng, Hongkun; Shi, Junjie; Fang, Xiaodong

2007-01-01

to efficiently search for and identify gene families. The FGF output displays the results as visual phylogenetic trees including information on gene structure, chromosome position, duplication fate and selective pressure. It is particularly useful to identify pseudogenes and detect changes in gene structure. FGF...

Disruption of Fgf13 causes synaptic excitatory-inhibitory imbalance and genetic epilepsy and febrile seizures plus.

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Puranam, Ram S; He, Xiao Ping; Yao, Lijun; Le, Tri; Jang, Wonjo; Rehder, Catherine W; Lewis, Darrell V; McNamara, James O

2015-06-10

We identified a family in which a translocation between chromosomes X and 14 was associated with cognitive impairment and a complex genetic disorder termed "Genetic Epilepsy and Febrile Seizures Plus" (GEFS(+)). We demonstrate that the breakpoint on the X chromosome disrupted a gene that encodes an auxiliary protein of voltage-gated Na(+) channels, fibroblast growth factor 13 (Fgf13). Female mice in which one Fgf13 allele was deleted exhibited hyperthermia-induced seizures and epilepsy. Anatomic studies revealed expression of Fgf13 mRNA in both excitatory and inhibitory neurons of hippocampus. Electrophysiological recordings revealed decreased inhibitory and increased excitatory synaptic inputs in hippocampal neurons of Fgf13 mutants. We speculate that reduced expression of Fgf13 impairs excitability of inhibitory interneurons, resulting in enhanced excitability within local circuits of hippocampus and the clinical phenotype of epilepsy. These findings reveal a novel cause of this syndrome and underscore the powerful role of FGF13 in control of neuronal excitability. Copyright © 2015 the authors 0270-6474/15/358866-16$15.00/0.

Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis

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Pieper Rembert

2011-05-01

Full Text Available Abstract Background Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host. Results In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3 pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP, SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA and Glutathione S-transferase (GST improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed. Conclusions Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

[Effect of concomitant use of dental drug on the properties of recombinant human basic fibroblast growth factor formulation for periodontal disease].

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Sato, Yasuhiko; Oba, Takuma; Danjo, Kazumi

2013-01-01

We have discussed the essential property for periodontal disease medication using protein, such as recombinant human basic fibroblast growth factor (rhbFGF). In our previous study, the criteria of thickener for the medication, viscosity, flowability etc., were set. The aim of this study was to evaluate the physical and chemical effect of concomitant use of general dental drug or device on thickener properties for the clinical use of viscous rhbFGF formulation. Viscous formulation was prepared with six cellulose derivatives, two types hydroxy propyl cellulose (HPC), three types hydroxy ethyl cellulose (HEC) and methyl cellulose (MC). Antibiotic ointment, local anesthetic, bone graft substitute, agent for gargle and mouthwashes, were chosen as general dental drug and device. These drugs and device were mixed with the viscous formulations and the change of viscosity and flowability, the remaining ratio of rhbFGF were evaluated. When the various thickener solutions were mixed with the liquid drugs, viscosity and flowability did not changed much. However, in the case of MC solution, viscous property declined greatly when MC solution was mixed with cationic surfactant for gargle. The flowabilities of thickener solutions were declined with insoluble bone graft. The stabilities of rhbFGF in thickener solutions were no problem for 24 hours even in the case of mixing with dental drug or device. Our findings suggested that the viscous rhbFGF formulations prepared in this research were not substantially affected by the concomitant use of dental drug or device, especially the formulation with HPC or HEC was useful.

Theoretical models for recombination in expanding gas

International Nuclear Information System (INIS)

Avron, Y.; Kahane, S.

1978-09-01

In laser isotope separation of atomic uranium, one is confronted with the theoretical problem of estimating the concentration of thermally ionized uranium atoms. To investigate this problem theoretical models for recombination in an expanding gas and in the absence of local thermal equilibrium have been constructed. The expansion of the gas is described by soluble models of the hydrodynamic equation, and the recombination by rate equations. General results for the freezing effect for the suitable ranges of the gas parameters are obtained. The impossibility of thermal equilibrium in expanding two-component systems is proven

The Effect of Covalently Immobilized FGF-2 on Biphasic Calcium Phosphate Bone Substitute on Enhanced Biological Compatibility and Activity

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Kyung-Suk Moon

2015-01-01

Full Text Available The purpose of this research was to covalently graft fibroblast growth factor 2 (FGF-2 onto biphasic calcium phosphate (BCP via a bifunctional cross-linker technique and to estimate the optimal dose of FGF-2 resulting in the best osteogenic differentiation of human mesenchymal stem cells (hMSCs. SEM observation revealed that the surface of the 100 ng FGF-2 coated BCP was completely covered with the nanoparticles expected to be from the silane coupling agent. XRD, FT-IR, and XPS analysis showed that silane treatment, bifunctional cross-linker coating, and FGF-2 covalent grafts were conducted successfully without deforming the crystalline structure of BCP. An MTT assay demonstrated that FGF-2 coated BCP had good biocompatibility, regardless of the concentration of FGF-2, after 24 or 48 h of incubation. An alkaline phosphatase (ALP activity assay (14 days of incubation and the ALP gene expression level of real-time PCR analysis (7 days of incubation revealed that 50, 100, and 200 ng FGF-2 coated BCP induced the highest activities among all experimental groups and control group (P<0.05. Thus, low concentrations of FGF-2 facilitated excellent osteogenesis and were effective at enhancing osteogenic potential. Also, the bifunctional cross-linker technique is expected to be a more feasible way to induce osteogenic differentiation while minimizing the risk of FGF-2 overdose.

A requirement for FGF signalling in the formation of primitive streak-like intermediates from primitive ectoderm in culture.

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Zhiqiang Zheng

Full Text Available BACKGROUND: Embryonic stem (ES cells hold considerable promise as a source of cells with therapeutic potential, including cells that can be used for drug screening and in cell replacement therapies. Differentiation of ES cells into the somatic lineages is a regulated process; before the promise of these cells can be realised robust and rational methods for directing differentiation into normal, functional and safe cells need to be developed. Previous in vivo studies have implicated fibroblast growth factor (FGF signalling in lineage specification from pluripotent cells. Although FGF signalling has been suggested as essential for specification of mesoderm and endoderm in vivo and in culture, the exact role of this pathway remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a culture model based on early primitive ectoderm-like (EPL cells we have investigated the role of FGF signalling in the specification of mesoderm. We were unable to demonstrate any mesoderm inductive capability associated with FGF1, 4 or 8 signalling, even when the factors were present at high concentrations, nor any enhancement in mesoderm formation induced by exogenous BMP4. Furthermore, there was no evidence of alteration of mesoderm sub-type formed with addition of FGF1, 4 or 8. Inhibition of endogenous FGF signalling, however, prevented mesoderm and favoured neural differentiation, suggesting FGF signalling was required but not sufficient for the differentiation of primitive ectoderm into primitive streak-like intermediates. The maintenance of ES cell/early epiblast pluripotent marker expression was also observed in cultures when FGF signalling was inhibited. CONCLUSIONS/SIGNIFICANCE: FGF signalling has been shown to be required for the differentiation of primitive ectoderm to neurectoderm. This, coupled with our observations, suggest FGF signalling is required for differentiation of the primitive ectoderm into the germ lineages at gastrulation.

Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

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Dai, Xiaoyong; Cai, Cuizan [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China); Xiao, Fei [Department of Pharmacology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong (China); Xiong, Yaoling [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China); Huang, Yadong; Zhang, Qihao [Department of Biopharmaceutical Research and Development Centre, Institute of Biomedicine, Jinan University, Guangzhou 510632, Guangdong (China); Xiang, Qi [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China); Lou, Guofeng [Department of Biopharmaceutical Research and Development Centre, Institute of Biomedicine, Jinan University, Guangzhou 510632, Guangdong (China); Lian, Mengyang [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China); Su, Zhijian, E-mail: tjnuszj@jnu.edu.cn [Department of Biopharmaceutical Research and Development Centre, Institute of Biomedicine, Jinan University, Guangzhou 510632, Guangdong (China); Zheng, Qing, E-mail: tzhengq@jnu.edu.cn [College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong (China)

2014-03-21

Highlights: • A specific aFGF-binding peptide AP8 was identified from a phage display library. • AP8 could inhibit aFGF-stimulated cell proliferation in a dose-dependent manner. • AP8 arrested the cell cycle at the G0/G1 phase by suppressing Cyclin D1. • AP8 could block the activation of Erk1/2 and Akt kinase. • AP8 counteracted proliferation and cell cycle via influencing PA2G4 and PCNA. - Abstract: It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.

Two recessive mutations in FGF5 are associated with the long-hair phenotype in donkeys.

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Legrand, Romain; Tiret, Laurent; Abitbol, Marie

2014-09-25

Seven donkey breeds are recognized by the French studbook. Individuals from the Pyrenean, Provence, Berry Black, Normand, Cotentin and Bourbonnais breeds are characterized by a short coat, while those from the Poitou breed (Baudet du Poitou) are characterized by a long-hair phenotype. We hypothesized that loss-of-function mutations in the FGF5 (fibroblast growth factor 5) gene, which are associated with a long-hair phenotype in several mammalian species, may account for the special coat feature of Poitou donkeys. To the best of our knowledge, mutations in FGF5 have never been described in Equidae. We sequenced the FGF5 gene from 35 long-haired Poitou donkeys, as well as from a panel of 67 short-haired donkeys from the six other French breeds and 131 short-haired ponies and horses. We identified a recessive c.433_434delAT frameshift deletion in FGF5, present in Poitou and three other donkey breeds and a recessive nonsense c.245G > A substitution, present in Poitou and four other donkey breeds. The frameshift deletion was associated with the long-hair phenotype in Poitou donkeys when present in two copies (n = 31) or combined with the nonsense mutation (n = 4). The frameshift deletion led to a stop codon at position 159 whereas the nonsense mutation led to a stop codon at position 82 in the FGF5 protein. In silico, the two truncated FGF5 proteins were predicted to lack the critical β strands involved in the interaction between FGF5 and its receptor, a mandatory step to inhibit hair growth. Our results highlight the allelic heterogeneity of the long-hair phenotype in donkeys and enlarge the panel of recessive FGF5 loss-of-function alleles described in mammals. Thanks to the DNA test developed in this study, breeders of non-Poitou breeds will have the opportunity to identify long-hair carriers in their breeding stocks.

Roles of different IRES-dependent FGF2 isoforms in the acquisition of the major aggressive features of human metastatic melanoma.

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Andreucci, Elena; Bianchini, Francesca; Biagioni, Alessio; Del Rosso, Mario; Papucci, Laura; Schiavone, Nicola; Magnelli, Lucia

2017-01-01

Fibroblast growth factor 2 (FGF2) is involved in many physiological and pathological processes. Fgf2 deregulation contributes to the acquisition of malignant features of melanoma and other cancers. FGF2 is an alternative translation product expressed as five isoforms, a low-molecular-weight (18 KDa) and four high-molecular-weight (22, 22.5, 24, 34 KDa) isoforms, with different subcellular distributions. An internal ribosomal entry site (IRES) in its mRNA controls the translation of all the isoforms with the exception for the cap-dependent 34 KDa. The 18-KDa isoform has been extensively studied, while very few is known about the roles of high molecular weight isoforms. FGF2 is known to promote melanoma development and progression. To disclose the differential contribution of FGF2 isoforms in melanoma, we forced the expression of IRES-dependent low-molecular-weight (LMW, 18 KDa) and high-molecular-weight (HMW, 22, 22.5, 24 KDa) isoforms in a human metastatic melanoma cell line. This comparative study highlights that, while LMW isoform confers stem-like features to melanoma cells and promotes angiogenesis, HMW isoforms induce higher migratory ability and contribute to tumor perfusion by promoting vasculogenic mimicry (VM) when endothelial cell-driven angiogenesis is lacking. To conclude, FGF2 isoforms mainly behave in specific, antithetical manners, but can cooperate in different steps of tumor progression, providing melanoma cells with major malignant features. FGF2 is an alternative translation product expressed as different isoforms termed LMW and HMW. FGF2 is involved in melanoma development and progression. HMW FGF2 isoforms enhance in vitro motility of melanoma cells. LMW FGF2 confers stem-like features and increases in vivo metastasization. LMW FGF2 promotes angiogenesis while HMW FGF2 induces vasculogenic mimicry.

FGF: A web tool for Fishing Gene Family in a whole genome database

DEFF Research Database (Denmark)

Zheng, Hongkun; Shi, Junjie; Fang, Xiaodong

2007-01-01

Gene duplication is an important process in evolution. The availability of genome sequences of a number of organisms has made it possible to conduct comprehensive searches for duplicated genes enabling informative studies of their evolution. We have established the FGF (Fishing Gene Family) progr...... is freely available on a web server at http://fgf.genomics.org.cn/...

Klotho-related Molecules Upregulated by Smoking Habit in Apparently Healthy Men: A Cross-sectional Study.

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Nakanishi, Kaori; Nishida, Makoto; Harada, Masaya; Ohama, Tohru; Kawada, Noritaka; Murakami, Masaaki; Moriyama, Toshiki; Yamauchi-Takihara, Keiko

2015-09-18

While aging is unavoidable, the aging mechanism is still unclear because of its complexity. Smoking causes premature death and is considered as an environmental aging accelerator. In the present study, we focused on the influence of smoking to the serum concentration of anti-aging protein α-klotho (αKl) and the β-klotho-associated protein fibroblast growth factor (FGF)-21 in men. Subjects consisted of apparently healthy men over 40 years of age who underwent health examination. Physical and biochemical parameters, including the levels of several cytokines and growth factors, were obtained from the subjects. Among middle-aged men (46.1 ± 5.1 years), serum levels of FGF-21, soluble αKl (sαKl), and inflammation-related cytokine interleukin (IL)-6 were significantly higher in smokers than in never-smokers. Serum levels of FGF-21 increased and correlated with alanine transaminase, γ guanosine-5'-triphosphate, and total cholesterol only in smokers, suggesting FGF-21 as a metabolic disorder-related factor in smokers. In aged men (60.3 ± 1.7 years), although the serum levels of sαKl in never-smokers were low, smokers showed highly increased serum levels of sαKl. Serum levels of sαKl was correlated with IL-6 in middle-aged never-smokers, suggesting sαKl regulates IL-6. However, this correlation was disrupted in smokers and aged men.

Expression and purification of recombinant truncated human keratinocyte growth factor-1

International Nuclear Information System (INIS)

Deng Lin; Ma Jisheng; Liu Xiaoju; Wang Xiaojie; Li Xiaokun; Gong Shouliang; Wang Huiyan; Tian Haishan

2010-01-01

Objective: To construct the genetic engineering bacteria highly expressing 23 amino acids human keratinocyte growth factor-1 (rhKGF1 dest23 ) missing N terminal, and provide experimental data for development of new drug for treatment of oral mucositis after radiotherapy and chemotherapy. Methods: PCR was used to synthese 23 amino acids rhKGF1 dest23 missing N terminal and sumo gene fragments, and construct four kinds of recombinant prokaryotic expression vectors: pET22b-rhKGF1 dest23 , pET22b-sumo-rhKGF1 dest23 , pET3c-rhKGF1 dest23 and pET3c-sumo-rhKGF1 dest23 , then they were transformed into prokaryotic expression host bacteria: Rosetta (DE3) plysS, BL21 (DE3), BL21 (DE3) Star plysS, origima(DE3) and BL21AI, the best expression combination of plasmid and host strain of rhKGF1 dest23 protein was screened and purified by CM ion-exchange and heparin affinity chromatography and identified with Western blotting. Results: pET22b-rhKGF1 dest23 plasmid and the BL21AI host bacteria was the best combination of expression, after induced by IPTG and arabinose, the majority of recombinant protein was expressed in soluble form, accounting for about 12% of the total bacterial proteins. Its purity reached to more than 95% of the protein after two steps chromatography, then conformed with Western blotting. Conclusion: Human genetic engineering bacteria of KGF1 dest23 is successfully constructed and induced by IPTG and arabinose, then after CM weak cation exchange and heparin affinity chromatography, the purified rhKGF1 dest23 protein is obtained. (authors)

Angiogenic activity of bFGF and VEGF suppressed by proteolytic cleavage by neutrophil elastase

International Nuclear Information System (INIS)

Ai, Shingo; Cheng Xianwu; Inoue, Aiko; Nakamura, Kae; Okumura, Kenji; Iguchi, Akihisa; Murohara, Toyoaki; Kuzuya, Masafumi

2007-01-01

Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation

FGF9 can induce endochondral ossification in cranial mesenchyme

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Overbeek Paul A

2006-02-01

Full Text Available Abstract Background The flat bones of the skull (i.e., the frontal and parietal bones normally form through intramembranous ossification. At these sites cranial mesenchymal cells directly differentiate into osteoblasts without the formation of a cartilage intermediate. This type of ossification is distinct from endochondral ossification, a process that involves initial formation of cartilage and later replacement by bone. Results We have analyzed a line of transgenic mice that expresses FGF9, a member of the fibroblast growth factor family (FGF, in cranial mesenchymal cells. The parietal bones in these mice show a switch from intramembranous to endochondral ossification. Cranial cartilage precursors are induced to proliferate, then hypertrophy and are later replaced by bone. These changes are accompanied by upregulation of Sox9, Ihh, Col2a1, Col10a1 and downregulation of CbfaI and Osteocalcin. Fate mapping studies show that the cranial mesenchymal cells in the parietal region that show a switch in cell fate are likely to be derived from the mesoderm. Conclusion These results demonstrate that FGF9 expression is sufficient to convert the differentiation program of (at least a subset of mesoderm-derived cranial mesenchyme cells from intramembranous to endochondral ossification.

Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF).

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Abdian, Narges; Ghasemi-Dehkordi, Payam; Hashemzadeh-Chaleshtori, Morteza; Ganji-Arjenaki, Mahbobe; Doosti, Abbas; Amiri, Beheshteh

2015-12-01

Basic fibroblast growth factor (bFGF or FGF-2) is a member of the FGF family secreted by different kinds of cells like HDFs and it is an important nutritional factor for cell growth and differentiation. The HDFs release bFGF in culture media at very low. The present study aims to investigate the HDFs growth rate in culture media supplemented either with or without bFGF. In brief, HDFs were isolated from human foreskin sample and were cultured in vitro in media containing bFGF and lack of this factor. The cells growth rate was calculated by trypan blue. The karyotyping was performed using G-banding to investigate the chromosomal abnormality of HDFs in both groups. Total RNA of each groups were extracted and cDNA samples were synthesized then, real-time Q-PCR was used to measure the expression level of p27kip1 and cyclin D1 genes normalized to internal control gene (GAPDH). The karyotype analysis showed that HDFs cultured in media or without bFGF had normal karyotype (46 chromosomes, XY) and chromosomal abnormalities were not observed. The cell growth rates in both groups were normal with proliferated exponentially but the slope of growth curve in HDFs cultured in media containing bFGF was increased. Karyotyp test showed that bFGF does not affect on cytogenetic stability of cells. The survey of p27kip1 and cyclin D1 genes by real-time Q-PCR showed that the expression level of these genes were up-regulated when adding bFGF in culture media (p culture media with growth factor like bFGF could enhance the proliferation and differentiation capacity of cells and improve cells growth rate. Similarly, fibroblast growth factors did not induce any chromosomal abnormality in cells. Furthermore, in HDFs cultured in bFGF supplemented media, the p27kip1 and cyclin D1 genes were up-regulated and suggesting an important role for bFGF in cell-cycle regulation and progression and fibroblast division stimulation. It also suggests that the effects of bFGF on different cell types with

Activation of human natural killer cells by the soluble form of cellular prion protein

International Nuclear Information System (INIS)

Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

2015-01-01

Cellular prion protein (PrP C ) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP C in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP C protein on human natural killer (NK) cells. Recombinant soluble PrP C protein was generated by fusion of human PrP C with the Fc portion of human IgG 1 (PrP C -Fc). PrP C -Fc binds to the surface of human NK cells, particularly to CD56 dim NK cells. PrP C -Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP C -Fc facilitated the IL-15-induced proliferation of NK cells. PrP C -Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP C -Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP C -Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP C (PrP C -Fc) was generated by fusion of human PrP C with IgG1 Fc portion. • PrP C -Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP C -Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP C -Fc protein activates human NK cells via the ERK and JNK signaling pathways

Effects of structurally stabilized EGF and bFGF on wound healing in type I and type II diabetic mice.

Science.gov (United States)

Choi, Seong Mi; Lee, Kyoung-Mi; Kim, Hyun Jung; Park, Ik Kyu; Kang, Hwi Ju; Shin, Hang-Cheol; Baek, Dawoon; Choi, Yoorim; Park, Kwang Hwan; Lee, Jin Woo

2018-01-15

Diabetes mellitus comprises a multiple metabolic disorder that affects millions of people worldwide and consequentially poses challenges for clinical treatment. Among the various complications, diabetic ulcer constitutes the most prevalent associated disorder and leads to delayed wound healing. To enhance wound healing capacity, we developed structurally stabilized epidermal growth factor (ST-EGF) and basic fibroblast growth factor (ST-bFGF) to overcome limitations of commercially available EGF (CA-EGF) and bFGF (CA-bFGF), such as short half-life and loss of activity after loading onto a matrix. Neither ST-EGF nor ST-bFGF was toxic, and both were more stable at higher temperatures than CA-EGF and CA-bFGF. We loaded ST-EGF and ST-bFGF onto a hyaluronate-collagen dressing (HCD) matrix, a biocompatible carrier, and tested the effectiveness of this system in promoting wound healing in a mouse model of diabetes. Wounds treated with HCD matrix loaded with 0.3 μg/cm 2 ST-EGF or 1 μg/cm 2 ST-bFGF showed a more rapid rate of tissue repair as compared to the control in type I and II diabetes models. Our results indicate that an HDC matrix loaded with 0.3 μg/cm 2 ST-EGF or 1 μg/cm 2 ST-bFGF can promote wound healing in diabetic ulcers and are suitable for use in wound dressings owing to their stability for long periods at room temperature. Various types of dressing materials loaded with growth factors, such as VEGF, EGF, and bFGF, are widely used to effect wound repair. However, such growth factor-loaded materials have several limitations for use as therapeutic agents in healing-impaired diabetic wounds. To overcome these limitations, we have developed new materials containing structurally stabilized EGF (ST-EGF) and bFGF (ST-bFGF). To confirm the wound healing capacity of newly developed materials (ST-EGF and ST-bFGF-loaded hyaluronate-collagen dressing [HCD] matrix), we applied these matrices in type I and type II diabetic wounds. Notably, these matrices were

FGF signaling regulates the number of posterior taste papillae by controlling progenitor field size.

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Camille I Petersen

2011-06-01

Full Text Available The sense of taste is fundamental to our ability to ingest nutritious substances and to detect and avoid potentially toxic ones. Sensory taste buds are housed in papillae that develop from epithelial placodes. Three distinct types of gustatory papillae reside on the rodent tongue: small fungiform papillae are found in the anterior tongue, whereas the posterior tongue contains the larger foliate papillae and a single midline circumvallate papilla (CVP. Despite the great variation in the number of CVPs in mammals, its importance in taste function, and its status as the largest of the taste papillae, very little is known about the development of this structure. Here, we report that a balance between Sprouty (Spry genes and Fgf10, which respectively antagonize and activate receptor tyrosine kinase (RTK signaling, regulates the number of CVPs. Deletion of Spry2 alone resulted in duplication of the CVP as a result of an increase in the size of the placode progenitor field, and Spry1(-/-;Spry2(-/- embryos had multiple CVPs, demonstrating the redundancy of Sprouty genes in regulating the progenitor field size. By contrast, deletion of Fgf10 led to absence of the CVP, identifying FGF10 as the first inductive, mesenchyme-derived factor for taste papillae. Our results provide the first demonstration of the role of epithelial-mesenchymal FGF signaling in taste papilla development, indicate that regulation of the progenitor field size by FGF signaling is a critical determinant of papilla number, and suggest that the great variation in CVP number among mammalian species may be linked to levels of signaling by the FGF pathway.

Implantable porous gelatin microspheres sustained release of bFGF and improved its neuroprotective effect on rats after spinal cord injury.

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Li Lan

Full Text Available In this study, porous gelatin microspheres (GMSs were constructed to improve the neuroprotective effect of basic fibroblast growth factor (bFGF on spinal cord injury. GMSs were prepared by a W/O emulsion template, followed by cross-linking, washing and drying. The particle sizes and surface porosity of the blank GMSs were carefully characterized by scan electronic microscopy. The blank GMSs have a mean particle size of 35μm and theirs surface was coarse and porous. bFGF was easily encapsulated inside the bulk GMSs through diffusion along the porous channel. 200μg of bFGF was completely encapsulated in 100mg of GMSs. The bFGF-loaded GMSs displayed a continuous drug release pattern without an obvious burst release over two weeks in vitro. Moreover, the therapeutic effects of bFGF-loaded GMSs were also evaluated in spinal cord injury rat model. After implantation of bFGF-loaded GMSs, the recovery of the motor function of SCI rats were evaluated by behavioral score and foot print experiment. The motor function of SCI rats treated with bFGF-loaded GMSs was more obvious than that treated with free bFGF solution (P<0.05. At the 28th days after treatment, rats were sacrificed and the injured spinal were removed for histopathological and apoptosis examination. Compared with treatment with free bFGF solution, treatment with bFGF-loaded GMSs resulted in a less necrosis, less infiltration of leukocytes, and a reduced the cavity ratio and less apoptotic cells in injured spinal(P<0.01, indicating its better therapeutic effect. Implantable porous GMSs may be a potential carrier to deliver bFGF for therapy of spinal cord injury.

Screening a phage display library for a novel FGF8b-binding peptide with anti-tumor effect on prostate cancer

International Nuclear Information System (INIS)

Wang, Wenhui; Chen, Xilei; Li, Tao; Li, Yanmei; Wang, Ruixue; He, Dan; Luo, Wu; Li, Xiaokun; Wu, Xiaoping

2013-01-01

Fibroblast growth factor 8b (FGF8b) is the major isoform of FGF8 expressed in prostate cancer and it correlates with the stage and grade of the disease. FGF8b has been considered as a potential target for prostate cancer therapy. Here we isolated 12 specific FGF8b-binding phage clones by screening a phage display heptapeptide library with FGF8b. The peptide (HSQAAVP, named as P12) corresponding to one of these clones showed high homology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor (FGFR3c), contained 3 identical amino acids (AVP) to the authentic FGFR3 D2 sequence aa 163–169 (LLAVPAA) directly participating in ligand binding, carried the same charges as its corresponding motif (aa163–169) in FGFR3c, suggesting that P12 may have a greater potential to interrupt FGF8b binding to its receptors than other identified heptapeptides do. Functional analysis indicated that synthetic P12 peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrest cell cycle at the G0/G1 phase via suppression of Cyclin D1 and PCNA, and blockade of the activations of Erk1/2 and Akt cascades in both prostate cancer cells and vascular endothelial cells. The results demonstrated that the P12 peptide acting as an FGF8b antagonist may have therapeutic potential in prostate cancer. - Highlights: ► A novel FGF8b-binding peptide P12 was isolated from a phage display library. ► The mechanisms for P12 peptide inhibiting cell proliferation were proposed. ► P12 caused cell cycle arrest at G0/G1 phase via suppression of Cyclin D1 and PCNA. ► P12 suppressed FGF8b-induced activations of Akt and MAP kinases. ► P12 acting as an FGF8b antagonist may have therapeutic potential in prostate cancer

Screening a phage display library for a novel FGF8b-binding peptide with anti-tumor effect on prostate cancer

Energy Technology Data Exchange (ETDEWEB)

Wang, Wenhui; Chen, Xilei; Li, Tao; Li, Yanmei; Wang, Ruixue; He, Dan; Luo, Wu [Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632 (China); Li, Xiaokun [Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632 (China); School of Pharmaceutical Science, Wenzhou Medical College, Wenzhou 325035 (China); Wu, Xiaoping, E-mail: twxp@jnu.edu.cn [Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632 (China); School of Pharmaceutical Science, Wenzhou Medical College, Wenzhou 325035 (China)

2013-05-01

Fibroblast growth factor 8b (FGF8b) is the major isoform of FGF8 expressed in prostate cancer and it correlates with the stage and grade of the disease. FGF8b has been considered as a potential target for prostate cancer therapy. Here we isolated 12 specific FGF8b-binding phage clones by screening a phage display heptapeptide library with FGF8b. The peptide (HSQAAVP, named as P12) corresponding to one of these clones showed high homology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor (FGFR3c), contained 3 identical amino acids (AVP) to the authentic FGFR3 D2 sequence aa 163–169 (LLAVPAA) directly participating in ligand binding, carried the same charges as its corresponding motif (aa163–169) in FGFR3c, suggesting that P12 may have a greater potential to interrupt FGF8b binding to its receptors than other identified heptapeptides do. Functional analysis indicated that synthetic P12 peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrest cell cycle at the G0/G1 phase via suppression of Cyclin D1 and PCNA, and blockade of the activations of Erk1/2 and Akt cascades in both prostate cancer cells and vascular endothelial cells. The results demonstrated that the P12 peptide acting as an FGF8b antagonist may have therapeutic potential in prostate cancer. - Highlights: ► A novel FGF8b-binding peptide P12 was isolated from a phage display library. ► The mechanisms for P12 peptide inhibiting cell proliferation were proposed. ► P12 caused cell cycle arrest at G0/G1 phase via suppression of Cyclin D1 and PCNA. ► P12 suppressed FGF8b-induced activations of Akt and MAP kinases. ► P12 acting as an FGF8b antagonist may have therapeutic potential in prostate cancer.

miR-194 Inhibits Innate Antiviral Immunity by Targeting FGF2 in Influenza H1N1 Virus Infection

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Keyu Wang

2017-11-01

Full Text Available Fibroblast growth factor 2 (FGF2 or basic FGF regulates a wide range of cell biological functions including proliferation, angiogenesis, migration, differentiation, and injury repair. However, the roles of FGF2 and the underlying mechanisms of action in influenza A virus (IAV-induced lung injury remain largely unexplored. In this study, we report that microRNA-194-5p (miR-194 expression is significantly decreased in A549 alveolar epithelial cells (AECs following infection with IAV/Beijing/501/2009 (BJ501. We found that miR-194 can directly target FGF2, a novel antiviral regulator, to suppress FGF2 expression at the mRNA and protein levels. Overexpression of miR-194 facilitated IAV replication by negatively regulating type I interferon (IFN production, whereas reintroduction of FGF2 abrogated the miR-194-induced effects on IAV replication. Conversely, inhibition of miR-194 alleviated IAV-induced lung injury by promoting type I IFN antiviral activities in vivo. Importantly, FGF2 activated the retinoic acid-inducible gene I signaling pathway, whereas miR-194 suppressed the phosphorylation of tank binding kinase 1 and IFN regulatory factor 3. Our findings suggest that the miR-194-FGF2 axis plays a vital role in IAV-induced lung injury, and miR-194 antagonism might be a potential therapeutic target during IAV infection.

A dual protease approach for expression and affinity purification of recombinant proteins.

Science.gov (United States)

Raran-Kurussi, Sreejith; Waugh, David S

2016-07-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

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Shaohui Yuan

2013-01-01

Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

Functional and pathological improvements of the hearts in diabetes model by the combined therapy of bFGF-loaded nanoparticles with ultrasound-targeted microbubble destruction.

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Zhao, Ying-Zheng; Tian, Xin-Qiao; Zhang, Ming; Cai, Lu; Ru, Ao; Shen, Xiao-Tong; Jiang, Xi; Jin, Rong-Rong; Zheng, Lei; Hawkins, Kyle; Charkrabarti, Subrata; Li, Xiao-Kun; Lin, Qian; Yu, Wen-Ze; Ge, Shuping; Lu, Cui-Tao; Wong, Ho Lun

2014-07-28

Diabetic cardiomyopathy (DCM) is the leading cause of morbidity and mortality among the diabetic patients and currently there is no effective means to reverse its pathological progress. Basic fibroblast growth factor (bFGF) has shown promise as a molecular therapy for DCM, but its delivery is inefficient and non-specific. In the present study, a therapy combining nanoparticle (NP) carrier and ultrasound-targeted microbubble destruction (UTMD) was reported the first time for bFGF delivery to the heart of diabetic rats. bFGF-loaded NP (bFGF-NP) were prepared with Poloxamer 188-grafted heparin copolymer using water-in-water technique, and the morphology, encapsulation efficiency, and bioactivity of bFGF-NP were studied. The cellular uptake and cytotoxicity of bFGF-NP were evaluated with primary cultures of the left ventricular (LV) cardiomyocytes in vitro. Therapeutic effects of bFGF-NP/UTMD on the heart of DCM rats were studied by measuring LV systolic and diastolic functions, hemodynamic characteristics and indicators of cardiac remodeling including myocardial collagen volume fraction and capillary density. Results demonstrated that bFGF-NP showed good round morphology, efficient bFGF encapsulation and stable bioactivity of bFGF in vitro. bFGF-NP/UTMD combined treatment significantly enhanced the efficiency of bFGF cellular uptake (Pfunctions and tissue morphology in the DCM rats were observed in bFGF-NP/UTMD group. These were not achievable using free bFGF, bFGF-NP or UTMD treatment alone. Our results show that combining a non-viral vector with UTMD technique is an effective strategy to deliver bFGF to the heart, and the resulting growth factor therapy has demonstrated potential to reverse the progress of DCM by restoring the cardiac functions and even the structure of damaged cardiac tissues. Copyright © 2014 Elsevier B.V. All rights reserved.

Three new hydrochlorothiazide cocrystals: Structural analyses and solubility studies

Science.gov (United States)

Ranjan, Subham; Devarapalli, Ramesh; Kundu, Sudeshna; Vangala, Venu R.; Ghosh, Animesh; Reddy, C. Malla

2017-04-01

Hydrochlorothiazide (HCT) is a diuretic BCS class IV drug with poor aqueous solubility and low permeability leading to poor oral absorption. The present work explores the cocrystallization technique to enhance the aqueous solubility of HCT. Three new cocrystals of HCT with water soluble coformers phenazine (PHEN), 4-dimethylaminopyridine (DMAP) and picolinamide (PICA) were prepared successfully by solution crystallization method and characterized by single crystal X-ray diffraction (SCXRD), powder X-ray diffraction (PXRD), fourier transform -infraredspectroscopy (FT-IR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Structural characterization revealed that the cocrystals with PHEN, DMAP and PICA exists in P21/n, P21/c and P21/n space groups, respectively. The improved solubility of HCT-DMAP (4 fold) and HCT-PHEN (1.4 fold) cocrystals whereas decreased solubility of HCT-PICA (0.5 fold) as compared to the free drug were determined after 4 h in phosphate buffer, pH 7.4, at 25 °C by using shaking flask method. HCT-DMAP showed a significant increase in solubility than all previously reported cocrystals of HCT suggest the role of a coformer. The study demonstrates that the selection of coformer could have pronounced impact on the physicochemical properties of HCT and cocrystallization can be a promising approach to improve aqueous solubility of drugs.

WNT7A/β-catenin signaling induces FGF1 and influences sensitivity to niclosamide in ovarian cancer.

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King, M L; Lindberg, M E; Stodden, G R; Okuda, H; Ebers, S D; Johnson, A; Montag, A; Lengyel, E; MacLean Ii, J A; Hayashi, K

2015-06-01

We previously characterized the link between WNT7A and the progression of ovarian cancer. Other groups have identified FGF1 as a relevant risk factor in ovarian cancer. Here, we show a linkage between these two signaling pathways that may be exploited to improve treatment and prognosis of patients with ovarian cancer. High expression of WNT7A and FGF1 are correlated in ovarian carcinomas and poor overall patient survival. A chromatin immunoprecipitation assay demonstrated that WNT7A/β-catenin signaling directly regulates FGF1 expression via TCF binding elements in the FGF1-1C promoter locus. In vitro gene manipulation studies revealed that FGF1 is sufficient to drive the tumor-promoting effects of WNT7A. In vivo xenograft studies confirmed that the stable overexpression of WNT7A or FGF1 induced a significant increase in tumor incidence, whereas FGF1 knockdown in WNT7A overexpressing cells caused a significant reduction in tumor size. Niclosamide most efficiently abrogated WNT7A/β-catenin signaling in our model, inhibited β-catenin transcriptional activity and cell viability, and increased cell death. Furthermore, niclosamide decreased cell migration following an increase in E-cadherin subsequent to decreased levels of SLUG. The effects of niclosamide on cell functions were more potent in WNT7A-overexpressing cells. Oral niclosamide inhibited tumor growth and progression in an intraperitoneal xenograft mouse model representative of human ovarian cancer. Collectively, these results indicate that FGF1 is a direct downstream target of WNT7A/β-catenin signaling and this pathway has potential as a therapeutic target in ovarian cancer. Moreover, niclosamide is a promising inhibitor of this pathway and may have clinical relevance.

A Shh-Foxf-Fgf18-Shh Molecular Circuit Regulating Palate Development.

Directory of Open Access Journals (Sweden)

Jingyue Xu

2016-01-01

Full Text Available Cleft palate is among the most common birth defects in humans. Previous studies have shown that Shh signaling plays critical roles in palate development and regulates expression of several members of the forkhead-box (Fox family transcription factors, including Foxf1 and Foxf2, in the facial primordia. Although cleft palate has been reported in mice deficient in Foxf2, whether Foxf2 plays an intrinsic role in and how Foxf2 regulates palate development remain to be elucidated. Using Cre/loxP-mediated tissue-specific gene inactivation in mice, we show that Foxf2 is required in the neural crest-derived palatal mesenchyme for normal palatogenesis. We found that Foxf2 mutant embryos exhibit altered patterns of expression of Shh, Ptch1, and Shox2 in the developing palatal shelves. Through RNA-seq analysis, we identified over 150 genes whose expression was significantly up- or down-regulated in the palatal mesenchyme in Foxf2-/- mutant embryos in comparison with control littermates. Whole mount in situ hybridization analysis revealed that the Foxf2 mutant embryos exhibit strikingly corresponding patterns of ectopic Fgf18 expression in the palatal mesenchyme and concomitant loss of Shh expression in the palatal epithelium in specific subdomains of the palatal shelves that correlate with where Foxf2, but not Foxf1, is expressed during normal palatogenesis. Furthermore, tissue specific inactivation of both Foxf1 and Foxf2 in the early neural crest cells resulted in ectopic activation of Fgf18 expression throughout the palatal mesenchyme and dramatic loss of Shh expression throughout the palatal epithelium. Addition of exogenous Fgf18 protein to cultured palatal explants inhibited Shh expression in the palatal epithelium. Together, these data reveal a novel Shh-Foxf-Fgf18-Shh circuit in the palate development molecular network, in which Foxf1 and Foxf2 regulate palatal shelf growth downstream of Shh signaling, at least in part, by repressing Fgf18

[The administration of interleukin-1beta during early postnatal develop ment impairs FGF2, but not TIMP1, mRNA expression in brain structures of adult rats].

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Trofimov, A N; Zubareva, O E; Shvarts, A P; Ishchenko, A M; Klimenko, V M

2014-09-01

According to the Neurodevelopmental hypothesis, the long-lasting cognitive deficit in schizophrenia and other types of neuropathology may occur by injurious factors, such as hypoxia, traumas, infections that take place during pre- and postnatal development, at least at early stages. These pathological conditions are often associated with the high production of pro-inflammatory cytokine interleukin-1B (IL-1B) by the cells of immune and nervous systems. We investigated the expression of genes involved in the neuroplastic regulation (Fgf2 and Timp2) in medial prefrontal cortex and dorsal and ventral regions of hippocampus of adult rats that were treated with IL-1beta between P15 and P21. The learning impairment in IL-1beta-treated rats is accompanied by lower FGF-2 mRNA levels in medial prefrontal cortex and ventral (not dorsal) hippocampus, but TIMP-1 was not affected. No differences in TIMP-1 and FGF-2 mRNA expressions were observed in untrained IL-1beta-treated when compared to control rats.

Formation of the embryonic organizer is restricted by the competitive influences of Fgf signaling and the SoxB1 transcription factors.

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Cheng-Liang Kuo

Full Text Available The organizer is one of the earliest structures to be established during vertebrate development and is crucial to subsequent patterning of the embryo. We have previously shown that the SoxB1 transcription factor, Sox3, plays a central role as a transcriptional repressor of zebrafish organizer gene expression. Recent data suggest that Fgf signaling has a positive influence on organizer formation, but its role remains to be fully elucidated. In order to better understand how Fgf signaling fits into the complex regulatory network that determines when and where the organizer forms, the relationship between the positive effects of Fgf signaling and the repressive effects of the SoxB1 factors must be resolved. This study demonstrates that both fgf3 and fgf8 are required for expression of the organizer genes, gsc and chd, and that SoxB1 factors (Sox3, and the zebrafish specific factors, Sox19a and Sox19b can repress the expression of both fgf3 and fgf8. However, we also find that these SoxB1 factors inhibit the expression of gsc and chd independently of their repression of fgf expression. We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression. These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling. The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.

A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

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Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

2012-01-01

In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906

Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag.

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Raran-Kurussi, Sreejith; Waugh, David S

2017-01-01

Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His 6 - or a dual His 6 -MBP tagged fusion protein by Gateway ® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His 6 tag or a His 6 -MBP tag can be made on the basis of this solubility test.

Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells

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Li, Ju; Han, Suhyoun; Cousin, Wendy; Conboy, Irina M.

2014-01-01

The regenerative capacity of muscle dramatically decreases with age because old muscle stem cells fail to proliferate in response to tissue damage. Here we uncover key age-specific differences underlying this proliferative decline: namely, the genetic loci of CDK inhibitors (CDKI) p21 and p16 are more epigenetically silenced in young muscle stem cells, as compared to old, both in quiescent cells and those responding to tissue injury. Interestingly, phosphorylated ERK (pERK) induced in these cells by ectopic FGF-2 is found in association with p21 and p16 promoters, and moreover, only in the old cells. Importantly, in the old satellite cells FGF-2/pERK silences p21 epigenetically and transcriptionally, which leads to reduced p21 protein levels and enhanced cell proliferation. In agreement with the epigenetic silencing of the loci, young muscle stem cells do not depend as much as old on ectopic FGF/pERK for their myogenic proliferation. In addition, other CDKIs, such asp15INK4B and p27KIP1, become elevated in satellite cells with age, confirming and explaining the profound regenerative defect of old muscle. This work enhances our understanding of tissue aging, promoting strategies for combating age-imposed tissue degeneration. PMID:25447026

Activation of human natural killer cells by the soluble form of cellular prion protein

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Seong, Yeon-Jae [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Hafis Clinic, Seoul (Korea, Republic of); Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Bum-Chan [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Park, Su-Hyung [Laboratory of Translational Immunology and Vaccinology, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Young Woo [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Shin, Eui-Cheol, E-mail: ecshin@kaist.ac.kr [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of)

2015-08-21

Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

High dose intravenous iron, mineral homeostasis and intact FGF23 in normal and uremic rats

DEFF Research Database (Denmark)

Gravesen, Eva; Hofman-Bang, Jacob; Mace, Maria L.

2013-01-01

High iron load might have a number of toxic effects in the organism. Recently intravenous (iv) iron has been proposed to induce elevation of fibroblast growth factor 23 (FGF23), hypophosphatemia and osteomalacia in iron deficient subjects. High levels of FGF23 are associated with increased...

A calculation of the surface recombination rate constant for hydrogen isotopes on metals

International Nuclear Information System (INIS)

Baskes, M.J.

1980-01-01

The surface recombination rate constant for hydrogen isotopes on a metal has been calculated using a simple model whose parameters may be determined by direct experimental measurements. Using the experimental values for hydrogen diffusivity, solubility, and sticking coefficient at zero surface coverage a reasonable prediction of the surface recombination constant may be made. The calculated recombination constant is in excellent agreement with experiment for bcc iron. A heuristic argument is developed which, along with the rate constant calculation, shows that surface recombination is important in those metals in which hydrogen has an exothermic heat of solution. (orig.)

Molecular characterization of the llama FGF5 gene and identification of putative loss of function mutations.

Science.gov (United States)

Daverio, M S; Vidal-Rioja, L; Frank, E N; Di Rocco, F

2017-12-01

Llama, the most numerous domestic camelid in Argentina, has good fiber-production ability. Although a few genes related to other productive traits have been characterized, the molecular genetic basis of fiber growth control in camelids is still poorly understood. Fibroblast growth factor 5 (FGF5) is a secreted signaling protein that controls hair growth in humans and other mammals. Mutations in the FGF5 gene have been associated with long-hair phenotypes in several species. Here, we sequenced the llama FGF5 gene, which consists of three exons encoding 813 bp. cDNA analysis from hair follicles revealed the expression of two FGF5 alternative spliced transcripts, in one of which exon 2 is absent. DNA variation analysis showed four polymorphisms in the coding region: a synonymous SNP (c.210A>G), a single base deletion (c.348delA), a 12-bp insertion (c.351_352insCATATAACATAG) and a non-sense mutation (c.499C>T). The deletion was always found together with the insertion forming a haplotype and producing a putative truncated protein of 123 amino acids. The c.499C>T mutation also leads to a premature stop codon at position 168. In both cases, critical functional domains of FGF5, including one heparin binding site, are lost. All animals analyzed were homozygous for one of the deleterious mutations or compound heterozygous for both (i.e. c.348delA, c.351_352insCATATAACATAG/c.499T). Sequencing of guanaco samples showed that the FGF5 gene encodes a full-length 270-amino acid protein. These results suggest that FGF5 is likely functional in short-haired wild species and non-functional in the domestic fiber-producing species, the llama. © 2017 Stichting International Foundation for Animal Genetics.

Short daily hemodialysis is associated with lower plasma FGF23 levels when compared with conventional hemodialysis.

Science.gov (United States)

Zaritsky, Joshua; Rastogi, Anjay; Fischmann, George; Yan, Jieshi; Kleinman, Kenneth; Chow, Georgina; Gales, Barbara; Salusky, Isidro B; Wesseling-Perry, Katherine

2014-02-01

The utilization of short-term daily hemodialysis has increased over the last few years, but little is known on its effects on the control of serum phosphate and fibroblast growth factor 23 (FGF23) levels. We therefore performed a cross-sectional study to compare FGF23 levels as well as other biochemical variables between 24 patients undergoing short daily hemodialysis using the NxStage System® and 54 patients treated with conventional in-center hemodialysis. FGF23 levels were measured using the second-generation Immutopics® C-terminal assay. Short daily hemodialysis patients were younger than patients on conventional hemodialysis but there were no differences between groups in the duration of end-stage renal disease nor in the number of patients with residual renal function. A greater number of short daily hemodialysis patients received vitamin D sterol therapy than did conventional in-center hemodialysis patients while there were no differences in the use of different phosphate binders and calcimimetic therapy between groups. Overall serum calcium, phosphorus and intact parathyroid hormone levels were similar between groups. While serum phosphorus levels correlated with FGF23 concentrations in each group separately [r=0.522 (P<0.01) and r=0.42 (P<0.01) in short daily and conventional in-center hemodialysis, respectively], FGF23 levels were lower [823 RU/mL (263, 2169)] in the patients receiving short daily hemodialysis than in patients treated with conventional hemodialysis [2521 RU/mL (909, 5556)] (P<0.01 between groups). These findings demonstrate that FGF23 levels are significantly lower in short daily hemodialysis patients and suggest that FGF23 levels may be a more sensitive biomarker of cumulative phosphate burden than single or multiple serum phosphorus determinations in patients treated with hemodialysis.

Salivary Gland Dysplasia in Fgf10 Heterozygous Mice: A New Mouse Model of Xerostomia

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May, A.J.; Chatzeli, L.; Proctor, G.B.; Tucker, A.S.

2017-01-01

Xerostomia, or chronic dry mouth, is a common syndrome caused by a lack of saliva that can lead to severe eating difficulties, dental caries and oral candida infections. The prevalence of xerostomia increases with age and affects approximately 30% of people aged 65 or older. Given the large numbers of sufferers, and the potential increase in incidence given our aging population, it is important to understand the complex mechanisms that drive hyposalivation and the consequences for the dentition and oral mucosa. From this study we propose the Fgf10 +/- mouse as a model to investigate xerostomia. By following embryonic salivary gland development, in vivo and in vitro, we show that a reduction in Fgf10 causes a delay in branching of salivary glands. This leads to hypoplasia of the glands, a phenotype that is not rescued postnatally or by adulthood in both male and female Fgf10 +/- mice. Histological analysis of the glands showed no obvious defect in cellular differentiation or acini/ductal arrangements, however there was a significant reduction in their size and weight. Analysis of saliva secretion showed that hypoplasia of the glands led to a significant reduction in saliva production in Fgf10 +/- adults, giving rise to a reduced saliva pellicle in the oral cavity of these mice. Mature mice were shown to drink more and in many cases had severe tooth wear. The Fgf10 +/- mouse is therefore a useful model to explore the causes and effects of xerostomia. PMID:26321752

Salivary Gland Dysplasia in Fgf10 Heterozygous Mice: A New Mouse Model of Xerostomia.

Science.gov (United States)

May, A J; Chatzeli, L; Proctor, G B; Tucker, A S

2015-01-01

Xerostomia, or chronic dry mouth, is a common syndrome caused by a lack of saliva that can lead to severe eating difficulties, dental caries and oral candida infections. The prevalence of xerostomia increases with age and affects approximately 30% of people aged 65 or older. Given the large numbers of sufferers, and the potential increase in incidence given our aging population, it is important to understand the complex mechanisms that drive hyposalivation and the consequences for the dentition and oral mucosa. From this study we propose the Fgf10 +/- mouse as a model to investigate xerostomia. By following embryonic salivary gland development, in vivo and in vitro, we show that a reduction in Fgf10 causes a delay in branching of salivary glands. This leads to hypoplasia of the glands, a phenotype that is not rescued postnatally or by adulthood in both male and female Fgf10 +/- mice. Histological analysis of the glands showed no obvious defect in cellular differentiation or acini/ductal arrangements, however there was a significant reduction in their size and weight. Analysis of saliva secretion showed that hypoplasia of the glands led to a significant reduction in saliva production in Fgf10 +/- adults, giving rise to a reduced saliva pellicle in the oral cavity of these mice. Mature mice were shown to drink more and in many cases had severe tooth wear. The Fgf10 +/- mouse is therefore a useful model to explore the causes and effects of xerostomia.

Cloning of Soluble Human Stem Cell Factor in pET-26b(+) Vector.

Science.gov (United States)

Asghari, Salman; Shekari Khaniani, Mahmoud; Darabi, Masood; Mansoori Derakhshan, Sima

2014-01-01

Stem cell factor (SCF) plays an important role in the survival, proliferation and differentiation of hematopoietic stem cells and progenitor cells. Potential therapeutic applications of SCF include hematopoietic stem cell mobilization, exvivo stem/progenitor cell expansion, gene therapy, and immunotherapy. Considering the cost and problem in accessibility of this product in Iran, clears the importance of indigenizing production of rhSCF. In the present work, we describe the construction of the soluble rhSCF expression vector in pET-26b (+) with periplasmic localization potential. Following PCR amplification of human SCF ORF, it is cloned in pET-26b (+) vector in NcoI and XhoI sites. The recombinant construct was transformed into BL21 (DE3) Ecoli strains. The construction of recombinant vector was verified by colony PCR and sequence analysis of pET26b-hSCF vector. Sequence analyses proved that human SCF ORF has been inserted into NcoI and XhoI site with correct orientation downstream of strong T7 promotor and showed no nucleotide errors. The SCF ORF was successfully cloned in pET-26b (+) expression vector and is ready for future production of SCF protein.

The pathologic effect of a novel neomorphic Fgf9(Y162C allele is restricted to decreased vision and retarded lens growth.

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Oliver Puk

Full Text Available Fibroblast growth factor (Fgf signalling plays a crucial role in many developmental processes. Among the Fgf pathway ligands, Fgf9 (UniProt: P54130 has been demonstrated to participate in maturation of various organs and tissues including skeleton, testes, lung, heart, and eye. Here we establish a novel Fgf9 allele, discovered in a dominant N-ethyl-N-nitrosourea (ENU screen for eye-size abnormalities using the optical low coherence interferometry technique. The underlying mouse mutant line Aca12 was originally identified because of its significantly reduced lens thickness. Linkage studies located Aca12 to chromosome 14 within a 3.6 Mb spanning interval containing the positional candidate genes Fgf9 (MGI: 104723, Gja3 (MGI: 95714, and Ift88 (MGI: 98715. While no sequence differences were found in Gja3 and Ift88, we identified an A→G missense mutation at cDNA position 770 of the Fgf9 gene leading to an Y162C amino acid exchange. In contrast to previously described Fgf9 mutants, Fgf9(Y162C carriers were fully viable and did not reveal reduced body-size, male-to-female sexual reversal or skeletal malformations. The histological analysis of the retina as well as its basic functional characterization by electroretinography (ERG did not show any abnormality. However, the analysis of head-tracking response of the Fgf9(Y162C mutants in a virtual drum indicated a gene-dosage dependent vision loss of almost 50%. The smaller lenses in Fgf9(Y162C suggested a role of Fgf9 during lens development. Histological investigations showed that lens growth retardation starts during embryogenesis and continues after birth. Young Fgf9(Y162C lenses remained transparent but developed age-related cataracts. Taken together, Fgf9(Y162C is a novel neomorphic allele that initiates microphakia and reduced vision without effects on organs and tissues outside the eye. Our data point to a role of Fgf9 signalling in primary and secondary lens fiber cell growth. The results

Discovery of recombining plasma from the faintest GeV supernova remnant HB 21 and a possible scenario for cosmic rays escaping from supernova remnant shocks

Science.gov (United States)

Suzuki, Hiromasa; Bamba, Aya; Nakazawa, Kazuhiro; Furuta, Yoshihiro; Sawada, Makoto; Yamazaki, Ryo; Koyama, Katsuji

2018-06-01

We present an X-ray study of the GeV gamma-ray supernova remnant (SNR) HB 21 with Suzaku. HB 21 is interacting with molecular clouds, and is the faintest in the GeV band among known GeV SNRs. We discovered strong radiative recombination continua of Si and S from the center of the remnant, which provide direct evidence of a recombining plasma (RP). The total emission can be explained with the RP and ionizing plasma components. The electron temperature and recombination timescale of the RP component were estimated as 0.17 (0.15-0.18) keV and 3.2 (2.0-4.8) × 1011 s cm-3, respectively. The estimated age of the RP (˜170 kyr) is the longest among known recombining GeV SNRs, because of a very low density of electrons (˜0.05 cm-3). We have examined the dependencies of GeV spectral indices on each of RP ages and SNR diameters for nine recombining GeV SNRs. Both showed possible positive correlations, indicating that both the parameters can be good indicators of properties of accelerated protons, for instance the degree of escape from SNR shocks. A possible scenario for a process of proton escape is introduced: interaction with molecular clouds makes weaker magnetic turbulence and cosmic-ray protons escape, simultaneously cooling down the thermal electrons and generating an RP.

Efficient expression of a soluble lipid transfer protein (LTP) of Platanus orientalis using short peptide tags and structural comparison with the natural form.

Science.gov (United States)

Salari, Farhad; Vahedi, Fatemeh; Chamani, Jamshidkhan; Varasteh, Abdolreza; Ketabdar, Hanieh; Sankian, Mojtaba

2015-01-01

Successful recombinant allergen-based immunotherapy has drawn a great deal of attention to use recombinant allergens for new therapeutic and/or diagnostic strategies. The Escherichia coli expression system is frequently used to produce recombinant allergens; however, protein expression in E. coli often results in inclusion bodies. Here, we focused on the expression of two recombinant soluble forms of Pla or 3 using solubility-enhancing peptide tags, human immune deficiency virus type 1 transactivator of transcription core domain and poly-arginine-lysine: rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3. Structural characteristics and IgE reactivity of purified recombinant proteins were compared with natural Pla or 3 (nPla or 3) isolated from Platanus orientalis using circular dichroism spectra, fluorescence spectroscopy, and immunoblotting. Likewise, intrinsic viscosity and Stokes radius of the natural and recombinant Pla or 3 allergens were determined to analyze structural compactness in aqueous media. The results indicate high-level solubility and efficient expression of the fusion proteins (rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3) compared with the wild-type recombinant. Furthermore, the similar structural characteristics and IgE-binding activities of the fusion proteins to nPla or 3 provide a promising tool for allergy diagnosis and treatment. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

IGF-II is up-regulated and myofibres are hypertrophied in regenerating soleus of mice lacking FGF6

International Nuclear Information System (INIS)

Armand, Anne-Sophie; Lecolle, Sylvie; Launay, Thierry; Pariset, Claude; Fiore, Frederic; Della Gaspera, Bruno; Birnbaum, Daniel; Chanoine, Christophe; Charbonnier, Frederic

2004-01-01

Important functions in myogenesis have been proposed for FGF6, a member of the fibroblast growth factor family accumulating almost exclusively in the myogenic lineage. However, the use of FGF6(-/-) mutant mice gave contradictory results and the role of FGF6 during myogenesis remains largely unclear. Using FGF6(-/-) mice, we first analysed the morphology of the regenerated soleus following cardiotoxin injection and showed hypertrophied myofibres in soleus of the mutant mice as compared to wild-type mice. Secondly, to examine the function of the IGF family in the hypertrophy process, we used semiquantitative and real-time RT-PCR assays and Western blots to monitor the expression of the insulin-like growth factors (IGF-I and IGF-II), their receptors [type I IGF receptor (IGF1R) and IGF-II receptor (IGF2R)], and of a binding protein IGFBP-5 in regenerating soleus muscles of FGF6(-/-) knockout mice vs. wild-type mice. In the mutant, both IGF-II and IGF2R, but not IGF-I and IGF1R, were strongly up-regulated, whereas IGFBP5 was down-regulated, strongly suggesting that, in the absence of FGF6, the mechanisms leading to myofibre hypertrophy were mediated specifically by an IGF-II/IGF2R signalling pathway distinct from the classic mechanism involving IGF-I and IGF1R previously described for skeletal muscle hypertrophy. The potential regulating role of IGFBP5 on IGF-II expression is also discussed. This report shows for the first time a specific role for FGF6 in the regulation of myofibre size during a process of in vivo myogenesis

Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

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Jiafei Xi

Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

Endogenous Retrovirus ev21 Dose Not Recombine with ALV-J and Induces the Expression of ISGs in the Host.

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Feng, Min; Tan, Yan; Dai, Manman; Li, Yuanfang; Xie, Tingting; Li, Hongmei; Shi, Meiqing; Zhang, Xiquan

2016-01-01

Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression. Endogenous viruses integrate into host genomes and can recombine with exogenous avian leukosis virus (ALV). In this study, we analyzed the interaction of endogenous retrovirus 21 ( ev21 ) with the ALV-J in late-feathering Chinese yellow chicken. Two ALV-J strains M180 and K243 were isolated from late-feathering and fast-feathering Chinese yellow chicken flocks, respectively. The env gene of the two strains showed 94.2-94.8% nucleotide identity with reference ALV-J strains. Compared with the env gene and the LTR of ev21 and M180, the nucleotide identity of LTR was 69.7% and env gene was 58.4%, respectively, especially the amino acid identity of env gene as low as 14.2%. Phylogenetic analysis of the nucleotide sequence of the env gene and the 3'LTR showed that M180 was closely related to ALV-J, and was located in a distinct group with ev21 in the phylogenetic tree. Using co-immunoprecipitation (co-IP), we next demonstrate that the envelope protein of ev21 does not interact with the M180 envelope protein. We further show that the envelope protein of ev21 cannot activate ALV-J LTR promoter activity using luciferase-reporter assays. qPCR and western blot analysis revealed that envelope protein of endogenous ev21 can facilitate the expression of PKR at 6h post ALV-J infection (hpi) and facilitate the expression of ISG12 and CH25H at 24 hpi. However, the expression of the env gene of M180 strain was not significantly at 6 and 24 hpi. We conclude that there is no evidence of recombination between endogenous retrovirus ev21 and ALV-J strain M180 in late-feathering Chinese yellow chicken, and envelope protein of ev21 can affect the expression of host ISGs, but appears not to influence the replication of ALV-J strain M180. This is the first report of interaction among the endogenous retrovirus ev21, ALV-J and the late-feathering chicken.

Simplified Method for Rapid Purification of Soluble Histones

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Nives Ivić

2016-06-01

Full Text Available Functional and structural studies of histone-chaperone complexes, nucleosome modifications, their interactions with remodelers and regulatory proteins rely on obtaining recombinant histones from bacteria. In the present study, we show that co-expression of Xenopus laevis histone pairs leads to production of soluble H2AH2B heterodimer and (H3H42 heterotetramer. The soluble histone complexes are purified by simple chromatographic techniques. Obtained H2AH2B dimer and H3H4 tetramer are proficient in histone chaperone binding and histone octamer and nucleosome formation. Our optimized protocol enables rapid purification of multiple soluble histone variants with a remarkable high yield and simplifies histone octamer preparation. We expect that this simple approach will contribute to the histone chaperone and chromatin research. This work is licensed under a Creative Commons Attribution 4.0 International License.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the soluble domain of PPA0092, a putative nitrite reductase from Propionibacterium acnes

International Nuclear Information System (INIS)

Nojiri, Masaki; Shirota, Felicia; Hira, Daisuke; Suzuki, Shinnichiro

2009-01-01

The soluble domain of a putative copper-containing nitrite reductase from P. acnes has been overexpressed, purified and crystallized. The crystal belonged to space group P2 1 3 and diffracted to 2.4 Å resolution. The soluble domain (residues 483–913) of PPA0092, a putative copper-containing nitrite reductase from Propionibacterium acnes KPA171202, has been overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 2.4 Å. The crystal belonged to space group P2 1 3, with unit-cell parameters a = b = c = 108.63 Å. Preliminary diffraction data show that one molecule is present in the asymmetric unit; this corresponds to a V M of 2.1 Å 3 Da −1

Differentiation of the lateral compartment of the cochlea requires a temporally restricted FGF20 signal.

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Sung-Ho Huh

2012-01-01

Full Text Available A large proportion of age-related hearing loss is caused by loss or damage to outer hair cells in the organ of Corti. The organ of Corti is the mechanosensory transducing apparatus in the inner ear and is composed of inner hair cells, outer hair cells, and highly specialized supporting cells. The mechanisms that regulate differentiation of inner and outer hair cells are not known. Here we report that fibroblast growth factor 20 (FGF20 is required for differentiation of cells in the lateral cochlear compartment (outer hair and supporting cells within the organ of Corti during a specific developmental time. In the absence of FGF20, mice are deaf and lateral compartment cells remain undifferentiated, postmitotic, and unresponsive to Notch-dependent lateral inhibition. These studies identify developmentally distinct medial (inner hair and supporting cells and lateral compartments in the developing organ of Corti. The viability and hearing loss in Fgf20 knockout mice suggest that FGF20 may also be a deafness-associated gene in humans.

Measurement of biologically active interleukin-1 by a soluble receptor binding assay

International Nuclear Information System (INIS)

Riske, F.; Chizzonite, R.; Nunes, P.; Stern, A.S.

1990-01-01

A soluble receptor binding assay has been developed for measuring human interleukin-1 alpha (IL-1 alpha), human IL-1 beta, and mouse IL-1 alpha. The assay is based on a competition between unlabeled IL-1 and 125I-labeled mouse recombinant IL-1 alpha for binding to soluble IL-1 receptor prepared from mouse EL-4 cells. The assay measures only biologically active IL-1 folded in its native conformation. The ratio of human IL-1 alpha to human IL-1 beta can be measured in the same sample by a pretreatment step which removes human IL-1 beta from samples prior to assay. This technique has been used to monitor the purification of recombinant IL-1, and may be utilized to specifically and accurately measure bioactive IL-1 in human serum and cell culture supernatants

FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

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Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki, E-mail: yasukiishizaki@gunma-u.ac.jp

2015-08-07

The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytes and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture.

FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

International Nuclear Information System (INIS)

Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki

2015-01-01

The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytes and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture

Biphasic effects of FGF2 on odontoblast differentiation involve changes in the BMP and Wnt signaling pathways.

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Sagomonyants, Karen; Mina, Mina

2014-08-01

Odontoblast differentiation during physiological and reparative dentinogenesis is dependent upon multiple signaling molecules, including fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs) and Wingless/Integrated (Wnt) ligands. Recent studies in our laboratory showed that continuous exposure of primary dental pulp cultures to FGF2 exerted biphasic effects on the expression of markers of dentinogenesis. In the present study, we examined the possible involvement of the BMP and Wnt signaling pathways in mediating the effects of FGF2 on dental pulp cells. Our results showed that stimulatory effects of FGF2 on dentinogenesis during the proliferation phase of growth were associated with increased expression of the components of the BMP (Bmp2, Dlx5, Msx2, Osx) and Wnt (Wnt10a, Wisp2) pathways, and decreased expression of an inhibitor of the Wnt signaling, Nkd2. Further addition of FGF2 during the differentiation/mineralization phase of growth resulted in decreased expression of components of the BMP signaling (Bmp2, Runx2, Osx) and increased expression of inhibitors of the Wnt signaling (Nkd2, Dkk3). This suggests that both BMP and Wnt pathways may be involved in mediating the effects of FGF2 on dental pulp cells.

Long-term Observation of Regenerated Periodontium Induced by FGF-2 in the Beagle Dog 2-Wall Periodontal Defect Model.

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Jun Anzai

Full Text Available The long-term stability and qualitative characteristics of periodontium regenerated by FGF-2 treatment were compared with normal physiological healing tissue controls in a Beagle dog 2-wall periodontal defect model 13 months after treatment by assessing tissue histology and three-dimensional microstructure using micro-computed tomography (μCT. After FGF-2 (0.3% or vehicle treatment at the defect sites, serial changes in the bone mineral content (BMC were observed using periodic X-ray imaging. Tissues were harvested at 13 months, evaluated histomorphometrically, and the cortical bone volume and trabecular bone structure of the newly formed bone were analyzed using μCT. FGF-2 significantly increased the BMC of the defect area at 2 months compared with that of the control group, and this difference was unchanged through 13 months. The cortical bone volume was significantly increased by FGF-2, but there was no difference between the groups in trabecular bone structure. Bone maturation was occurring in both groups because of the lower cortical volume and denser trabecular bone than what is found in intact bone. FGF-2 also increased the area of newly formed bone as assessed histomorphometrically, but the ratios of trabecular bone in the defect area were similar between the control and FGF-2 groups. These results suggest that FGF-2 stimulates neogenesis of alveolar bone that is of similar quality to that of the control group. The lengths of the regenerated periodontal ligament and cementum, measured as the distance from the defect bottom to the apical end of the gingival epithelium, and height and area of the newly formed bone in the FGF-2 group were larger than those in the control group. The present study demonstrated that, within the limitation of artificial periodontal defect model, the periodontal tissue regenerated by FGF-2 was maintained for 13 months after treatment and was qualitatively equivalent to that generated through the physiological

CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli

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Kusdianawati Kusdianawati

2015-09-01

Full Text Available Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from Lactobacillus plantarum S34 in Escherichia coli. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. plantarum S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3 pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. plantarum S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His6tag had successfully been expressed in E. coli BL21 (DE3 pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1. Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.

Cohnella amylopullulanases: Biochemical characterization of two recombinant thermophilic enzymes.

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Fatemeh Zebardast Roodi

Full Text Available Some industries require newer, more efficient recombinant enzymes to accelerate their ongoing biochemical reactions in harsh environments with less replenishment. Thus, the search for native enzymes from extremophiles that are suitable for use under industrial conditions is a permanent challenge for R & D departments. Here and toward such discoveries, two sequences homologous to amylopullulanases (EC 3.2.1.41, GH57 from an endogenous Cohnella sp., [Coh00831 (KP335161; 1998 bp and Coh01133 (KP335160: 3678 bp] were identified. The genes were heterologously expressed in E. coli to both determine their type and further characterize their properties. The isolated DNA was PCR amplified with gene specific primers and cloned in pET28a, and the recombinant proteins were expressed in E. coli BL21 (DE3. The temperatures and pH optima of purified recombinants Coh 01133 and Coh 00831 enzymes were 70°C and 8, and 60°C and 6, respectively. These enzymes are stable more than 90% in 60°C and 50°C for 90 min respectively. The major reactions released sugars which could be fractionated by HPLC analysis, from soluble starch were mainly maltose (G2, maltotriose (G3 and maltotetraose (G4. The enzymes hydrolyzed pullulan to maltotriose (G3 only. Enzyme activities for both proteins were improved in the availability of Mn2+, Ba2+, Ca2+, and Mg2+ and reduced in the presence of Fe2+, Li2+, Na2+, Triton X100 and urea. Moreover, Co2+, K+, and Cu2+ had a negative effect only on Coh 01133 enzyme.

Cohnella amylopullulanases: Biochemical characterization of two recombinant thermophilic enzymes.

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Zebardast Roodi, Fatemeh; Aminzadeh, Saeed; Farrokhi, Naser; Karkhane, AliAsghar; Haghbeen, Kamahldin

2017-01-01

Some industries require newer, more efficient recombinant enzymes to accelerate their ongoing biochemical reactions in harsh environments with less replenishment. Thus, the search for native enzymes from extremophiles that are suitable for use under industrial conditions is a permanent challenge for R & D departments. Here and toward such discoveries, two sequences homologous to amylopullulanases (EC 3.2.1.41, GH57) from an endogenous Cohnella sp., [Coh00831 (KP335161; 1998 bp) and Coh01133 (KP335160: 3678 bp)] were identified. The genes were heterologously expressed in E. coli to both determine their type and further characterize their properties. The isolated DNA was PCR amplified with gene specific primers and cloned in pET28a, and the recombinant proteins were expressed in E. coli BL21 (DE3). The temperatures and pH optima of purified recombinants Coh 01133 and Coh 00831 enzymes were 70°C and 8, and 60°C and 6, respectively. These enzymes are stable more than 90% in 60°C and 50°C for 90 min respectively. The major reactions released sugars which could be fractionated by HPLC analysis, from soluble starch were mainly maltose (G2), maltotriose (G3) and maltotetraose (G4). The enzymes hydrolyzed pullulan to maltotriose (G3) only. Enzyme activities for both proteins were improved in the availability of Mn2+, Ba2+, Ca2+, and Mg2+ and reduced in the presence of Fe2+, Li2+, Na2+, Triton X100 and urea. Moreover, Co2+, K+, and Cu2+ had a negative effect only on Coh 01133 enzyme.

Polysaccharides purified from wild Cordyceps activate FGF2/FGFR1c signaling

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Zeng, Yangyang; Han, Zhangrun; Yu, Guangli; Hao, Jiejie; Zhang, Lijuan

2015-02-01

Land animals as well as all organisms in ocean synthesize sulfated polysaccharides. Fungi split from animals about 1.5 billion years ago. As fungi make the evolutionary journey from ocean to land, the biggest changes in their living environment may be a sharp decrease in salt concentration. It is established that sulfated polysaccharides interact with hundreds of signaling molecules and facilitate many signaling transduction pathways, including fibroblast growth factor (FGF) and FGF receptor signaling pathway. The disappearance of sulfated polysaccharides in fungi and plants on land might indicate that polysaccharides without sulfation might be sufficient in facilitating protein ligand/receptor interactions in low salinity land. Recently, it was reported that plants on land start to synthesize sulfated polysaccharides in high salt environment, suggesting that fungi might be able to do the same when exposed in such environment. Interestingly, Cordyceps, a fungus habituating inside caterpillar body, is the most valued traditional Chinese Medicine. One of the important pharmaceutical active ingredients in Cordyceps is polysaccharides. Therefore, we hypothesize that the salty environment inside caterpillar body might allow the fungi to synthesize sulfated polysaccharides. To test the hypothesis, we isolated polysaccharides from both lava and sporophore of wild Cordyceps and also from Cordyceps militaris cultured without or with added salts. We then measured the polysaccharide activity using a FGF2/FGFR1c signaling-dependent BaF3 cell proliferation assay and found that polysaccharides isolated from wild Cordyceps activated FGF2/FGFR signaling, indicating that the polysaccharides synthesized by wild Cordyceps are indeed different from those by the cultured mycelium.

Perlecan is required for FGF-2 signaling in the neural stem cell niche

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Aurelien Kerever

2014-03-01

Full Text Available In the adult subventricular zone (neurogenic niche, neural stem cells double-positive for two markers of subsets of neural stem cells in the adult central nervous system, glial fibrillary acidic protein and CD133, lie in proximity to fractones and to blood vessel basement membranes, which contain the heparan sulfate proteoglycan perlecan. Here, we demonstrate that perlecan deficiency reduces the number of both GFAP/CD133-positive neural stem cells in the subventricular zone and new neurons integrating into the olfactory bulb. We also show that FGF-2 treatment induces the expression of cyclin D2 through the activation of the Akt and Erk1/2 pathways and promotes neurosphere formation in vitro. However, in the absence of perlecan, FGF-2 fails to promote neurosphere formation. These results suggest that perlecan is a component of the neurogenic niche that regulates FGF-2 signaling and acts by promoting neural stem cell self-renewal and neurogenesis.

BMP4 and FGF3 haplotypes increase the risk of tendinopathy in volleyball athletes.

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Salles, José Inácio; Amaral, Marcus Vinícius; Aguiar, Diego Pinheiro; Lira, Daisy Anne; Quinelato, Valquiria; Bonato, Letícia Ladeira; Duarte, Maria Eugenia Leite; Vieira, Alexandre Rezende; Casado, Priscila Ladeira

2015-03-01

To investigate whether genetic variants can be correlated with tendinopathy in elite male volleyball athletes. Case-control study. Fifteen single nucleotide polymorphisms within BMP4, FGF3, FGF10, FGFR1 genes were investigated in 138 elite volleyball athletes, aged between 18 and 35 years, who undergo 4-5h of training per day: 52 with tendinopathy and 86 with no history of pain suggestive of tendinopathy in patellar, Achilles, shoulder, and hip abductors tendons. The clinical diagnostic criterion was progressive pain during training, confirmed by magnetic resonance image. Genomic DNA was obtained from saliva samples. Genetic markers were genotyped using TaqMan real-time PCR. Chi-square test compared genotypes and haplotype differences between groups. Multivariate logistic regression analyzed the significance of covariates and incidence of tendinopathy. Statistical analysis revealed participant age (p=0.005) and years of practice (p=0.004) were risk factors for tendinopathy. A significant association between BMP4 rs2761884 (p=0.03) and tendinopathy was observed. Athletes with a polymorphic genotype have 2.4 times more susceptibility to tendinopathy (OR=2.39; 95%CI=1.10-5.19). Also, association between disease and haplotype TTGGA in BMP4 (p=0.01) was observed. The FGF3 TGGTA haplotype showed a tendency of association with tendinopathy (p=0.05), and so did FGF10 rs900379. FGFR1 showed no association with disease. These findings indicate that haplotypes in BMP4 and FGF3 genes may contribute to the tendon disease process in elite volleyball athletes. Copyright © 2014 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

FGF8 signaling sustains progenitor status and multipotency of cranial neural crest-derived mesenchymal cells in vivo and in vitro

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Shao, Meiying; Liu, Chao; Song, Yingnan; Ye, Wenduo; He, Wei; Yuan, Guohua; Gu, Shuping; Lin, Congxin; Ma, Liang; Zhang, Yanding; Tian, Weidong; Hu, Tao; Chen, YiPing

2015-01-01

The cranial neural crest (CNC) cells play a vital role in craniofacial development and regeneration. They are multi-potent progenitors, being able to differentiate into various types of tissues. Both pre-migratory and post-migratory CNC cells are plastic, taking on diverse fates by responding to different inductive signals. However, what sustains the multipotency of CNC cells and derivatives remains largely unknown. In this study, we present evidence that FGF8 signaling is able to sustain progenitor status and multipotency of CNC-derived mesenchymal cells both in vivo and in vitro. We show that augmented FGF8 signaling in pre-migratory CNC cells prevents cell differentiation and organogenesis in the craniofacial region by maintaining their progenitor status. CNC-derived mesenchymal cells with Fgf8 overexpression or control cells in the presence of exogenous FGF8 exhibit prolonged survival, proliferation, and multi-potent differentiation capability in cell cultures. Remarkably, exogenous FGF8 also sustains the capability of CNC-derived mesenchymal cells to participate in organogenesis such as odontogenesis. Furthermore, FGF8-mediated signaling strongly promotes adipogenesis but inhibits osteogenesis of CNC-derived mesenchymal cells in vitro. Our results reveal a specific role for FGF8 in the maintenance of progenitor status and in fate determination of CNC cells, implicating a potential application in expansion and fate manipulation of CNC-derived cells in stem cell-based craniofacial regeneration. PMID:26243590

Androgen receptor-negative human prostate cancer cells induce osteogenesis in mice through FGF9-mediated mechanisms.

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Li, Zhi Gang; Mathew, Paul; Yang, Jun; Starbuck, Michael W; Zurita, Amado J; Liu, Jie; Sikes, Charles; Multani, Asha S; Efstathiou, Eleni; Lopez, Adriana; Wang, Jing; Fanning, Tina V; Prieto, Victor G; Kundra, Vikas; Vazquez, Elba S; Troncoso, Patricia; Raymond, Austin K; Logothetis, Christopher J; Lin, Sue-Hwa; Maity, Sankar; Navone, Nora M

2008-08-01

In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor-negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer-induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor-null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells.

Study on the effect of Huo Xue Bo Gu Pill on secretion of endogenous bFGF after fracture

International Nuclear Information System (INIS)

Li Qiang; Wang Xiping; Guo E; Ye Liyan; Liu Yingjie

2006-01-01

Objective: To study the effect of a Chinese traditional preparation Huo Xue Bo Gu Pill on the serum contents of basic fibroblast growth factor (bFGF) after forearm fractures. Method: Serum bFGF contents were measured (with ELISA) in 62 patients with forearm fractures on d1, d8, d14 and d20 after the accident. Thirty-two of the patients were treated with Huo Xue Bo Gu Pills and thirty patients were not. Results: The serum contents of bFGF on d8 and d14 were significantly higher in the patients treated with the pills than those in patients not treated with the pills (P<0.05). Conclusion: This pill could promote the secretion of endogenous bFGF with proliferation of capillaries at the fracture site. (authors)

Postprandial changes in glucose oxidation and insulin sensitivity in metabolic syndrome: Influence of fibroblast growth factor 21 and vitamin D status.

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Pathak, Kaveri; Soares, Mario J; Zhao, Yun; James, Anthony P; Sherriff, Jillian L; Newsholme, Philip

2017-05-01

Metabolic inflexibility due to insulin resistance has been reported in metabolic syndrome (MetS). Fibroblast growth factor 21 (FGF21) and vitamin D status may improve insulin sensitivity. The aim of this study was to investigate glucose-induced thermogenesis and oxidation in MetS, and to examine whether changes in FGF21 or prevailing vitamin D status modulated defined metabolic parameters. Forty-eight overweight and obese older adults (14 men, 34 women; ages 51 ± 15 y) were studied. Resting metabolic rate (RMR) and respiratory quotient (RQ) were measured before and intermittently for 2 h after an oral glucose tolerance test (OGTT). The total area under the curve (TAUC) was calculated. Insulin sensitivity index (ISI) was determined as 10 4 /(insulin × glucose) for fasting and 2 h venous blood. Fat mass (FM) and fat free mass (FFM) were measured by dual-energy x-ray absorptiometry. Participants were grouped by metabolic syndrome (MetS+ for disease presence; MetS- when no disease was present) and by median 25 hydroxyvitamin D (OHD) concentration as VD_low and VD_high. 25 OHD was also tested as a continuous variable. A parsimonious 2 × 2 analysis of variance included age, FM, FFM and MetS × sex interaction. Adjusted RMR was similar between groups but an interactive effect of MetS and sex was noted. Fasting RQ was significantly different between vitamin groups (VD_low: 0.835 ± 0.008 versus VD_high: 0.810 ± 0.008; P = 0.024) and fasting ISI was significantly greater in MetS- compared with MetS+ (P = 0.037). Postglucose increases in thermogenesis, RQ, and FGF21 were significant, but ISI decreased. Adjusted postprandial TAUC_RQ (VD_low: 1.71 ± 0.01; VD_high: 1.74 ± 0.001; P = 0.041) and ISI_2 h (VD_low: 35.41 ± 0.21; VD_high: 101.90 ± 0.21; P = 0.001) were significantly different. Adjusted FGF21 was similar across all comparisons before and after OGTT. Higher vitamin D status, but not FGF21, was associated with greater postprandial

Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

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Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

Compensatory Paracrine Mechanisms That Define The Urothelial Response to Injury in Partial Bladder Outlet Obstruction

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Bassuk, James; Lendvay, Thomas S.; Sweet, Robert; Han, Chang-Hee; Soygur, Tarkan; Cheng, Jan-Fang; Plaire, J. Chadwick; Charleston, Jay S.; Charleston, Lynne B.; Bagai, Shelly; Cochrane, Kimberly; Rubio, Eric; Bassuk, James A.; Fuchs, Elaine

2007-06-21

Diseases and conditions affecting the lower urinary tract are a leading cause of dysfunctional sexual health, incontinence, infection, and kidney failure. The growth, differentiation, and repair of the bladder's epithelial lining are regulated, in part, by fibroblast growth factor (FGF)-7 and -10 via a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the receptor for FGF-7 and -10 within the transitional epithelium (urothelium). The FGF-7 gene is located at the 15q15-q21.1 locus on chromosome 15 and four exons generate a 3.852-kb mRNA. Five duplicated FGF-7 gene sequences that localized to chromosome 9 were predicted not to generate functional protein products, thus validating the use of FGF-7-null mice as an experimental model. Recombinant FGF-7 and -10 induced proliferation of human urothelial cells in vitro and transitional epithelium of wild-type and FGF-7-null mice in vivo.To determine the extent that induction of urothelial cell proliferation during the bladder response to injury is dependent on FGF-7, an animal model of partial bladder outlet obstruction was developed. Unbiased stereology was used to measure the percentage of proliferating urothelial cells between obstructed groups of wild-type and FGF-7-null mice. The stereological analysis indicated that a statistical significant difference did not exist between the two groups, suggesting that FGF-7 is not essential for urothelial cell proliferation in response to partial outlet obstruction. In contrast, a significant increase in FGF-10 expression was observed in the obstructed FGF-7-null group, indicating that the compensatory pathway that functions in this model results in urothelial repair.

ANTXR2 is a potential causative gene in the genome-wide association study of the blood pressure locus 4q21.

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Park, So Yon; Lee, Hyeon-Ju; Ji, Su-Min; Kim, Marina E; Jigden, Baigalmaa; Lim, Ji Eun; Oh, Bermseok

2014-09-01

Hypertension is the most prevalent cardiovascular disease worldwide, but its genetic basis is poorly understood. Recently, genome-wide association studies identified 33 genetic loci that are associated with blood pressure. However, it has been difficult to determine whether these loci are causative owing to the lack of functional analyses. Of these 33 genome-wide association studies (GWAS) loci, the 4q21 locus, known as the fibroblast growth factor 5 (FGF5) locus, has been linked to blood pressure in Asians and Europeans. Using a mouse model, we aimed to identify a causative gene in the 4q21 locus, in which four genes (anthrax toxin receptor 2 (ANTXR2), PR domain-containing 8 (PRDM8), FGF5 and chromosome 4 open reading frame 22 (C4orf22)) were near the lead single-nucleotide polymorphism (rs16998073). Initially, we examined Fgf5 gene by measuring blood pressure in Fgf5-knockout mice. However, blood pressure did not differ between Fgf5 knockout and wild-type mice. Therefore, the other candidate genes were studied by in vivo small interfering RNA (siRNA) silencing in mice. Antxr2 siRNA was pretreated with polyethylenimine and injected into mouse tail veins, causing a significant decrease in Antxr2 mRNA by 22% in the heart. Moreover, blood pressure measured under anesthesia in Antxr2 siRNA-injected mice rose significantly compared with that of the controls. These results suggest that ANTXR2 is a causative gene in the human 4q21 GWAS-blood pressure locus. Additional functional studies of ANTXR2 in blood pressure may identify a novel genetic pathway, thus increasing our understanding of the etiology of essential hypertension.

A decrease in fasting FGF19 levels is associated with the development of non-alcoholic fatty liver disease in obese adolescents.

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Wojcik, Malgorzata; Janus, Dominika; Dolezal-Oltarzewska, Katarzyna; Kalicka-Kasperczyk, Anna; Poplawska, Karolina; Drozdz, Dorota; Sztefko, Krystyna; Starzyk, Jerzy B

2012-01-01

Fibroblast growth factor 19 (FGF19) is a hormone released from the small intestine; recently, it has emerged as an endocrine regulator of glucose and lipid metabolism. The aim of this study was to investigate the role of FGF19 in the development of nonalcoholic fatty liver disease (NAFLD). This study included 23 (17 boys) obese adolescents (mean age of 14.1 years) with NAFLD. The control group consisted of 34 (13 boys) obese peers with normal ultrasonographic imaging and normal liver function tests. The definition of NAFLD was based on clinical criteria: elevated alanine aminotransferase (>35 U/L) and liver steatosis features on ultrasound imaging. Serum FGF19 levels were measured in a fasting blood sample. The definition of insulin resistance was based on the homeostasis model assessment (HOMA) threshold: >2.5. There was a significant difference between mean FGF19 levels in patients with NAFLD and controls (142.2 vs. 206 pg/mL, p=0.04). Mean fasting FGF19 levels were decreased in insulin-resistant patients in comparison with the non-insulin-resistant group (155.0 vs. 221.0 pg/mL, p=0.05). There was an inverse correlation between FGF19 and alanine aminotransferase levels (R=-0.3, pexogenous delivery of FGF19 might be therapeutically beneficial.

Activation of Aurora A kinase through the FGF1/FGFR signaling axis sustains the stem cell characteristics of glioblastoma cells

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Hsu, Yi-Chao [Division of Regenerative Medicine, Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan (China); Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan (China); Kao, Chien-Yu [Division of Regenerative Medicine, Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan (China); Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan (China); Chung, Yu-Fen; Lee, Don-Ching [Division of Regenerative Medicine, Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan (China); Liu, Jen-Wei [Division of Regenerative Medicine, Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan (China); Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chiu, Ing-Ming, E-mail: ingming@nhri.org.tw [Division of Regenerative Medicine, Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan (China); Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan (China); Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan (China)

2016-06-10

Fibroblast growth factor 1 (FGF1) binds and activates FGF receptors, thereby regulating cell proliferation and neurogenesis. Human FGF1 gene 1B promoter (−540 to +31)-driven SV40 T antigen has been shown to result in tumorigenesis in the brains of transgenic mice. FGF1B promoter (−540 to +31)-driven green fluorescent protein (F1BGFP) has also been used in isolating neural stem cells (NSCs) with self-renewal and multipotency from developing and adult mouse brains. In this study, we provide six lines of evidence to demonstrate that FGF1/FGFR signaling is implicated in the expression of Aurora A (AurA) and the activation of its kinase domain (Thr288 phosphorylation) in the maintenance of glioblastoma (GBM) cells and NSCs. First, treatment of FGF1 increases AurA expression in human GBM cell lines. Second, using fluorescence-activated cell sorting, we observed that F1BGFP reporter facilitates the isolation of F1BGFP(+) GBM cells with higher expression levels of FGFR and AurA. Third, both FGFR inhibitor (SU5402) and AurA inhibitor (VX680) could down-regulate F1BGFP-dependent AurA activity. Fourth, inhibition of AurA activity by two different AurA inhibitors (VX680 and valproic acid) not only reduced neurosphere formation but also induced neuronal differentiation of F1BGFP(+) GBM cells. Fifth, flow cytometric analyses demonstrated that F1BGFP(+) GBM cells possessed different NSC cell surface markers. Finally, inhibition of AurA by VX680 reduced the neurosphere formation of different types of NSCs. Our results show that activation of AurA kinase through FGF1/FGFR signaling axis sustains the stem cell characteristics of GBM cells. Implications: This study identified a novel mechanism for the malignancy of GBM, which could be a potential therapeutic target for GBM. - Highlights: • We report that FGF1 treatment can stimulate AurA kinase expression in human GBM cells. • FGF1/FGFR signaling is involved in the activation of AurA kinase. • FGF1 sustains the self

Activation of Aurora A kinase through the FGF1/FGFR signaling axis sustains the stem cell characteristics of glioblastoma cells

International Nuclear Information System (INIS)

Hsu, Yi-Chao; Kao, Chien-Yu; Chung, Yu-Fen; Lee, Don-Ching; Liu, Jen-Wei; Chiu, Ing-Ming

2016-01-01

Fibroblast growth factor 1 (FGF1) binds and activates FGF receptors, thereby regulating cell proliferation and neurogenesis. Human FGF1 gene 1B promoter (−540 to +31)-driven SV40 T antigen has been shown to result in tumorigenesis in the brains of transgenic mice. FGF1B promoter (−540 to +31)-driven green fluorescent protein (F1BGFP) has also been used in isolating neural stem cells (NSCs) with self-renewal and multipotency from developing and adult mouse brains. In this study, we provide six lines of evidence to demonstrate that FGF1/FGFR signaling is implicated in the expression of Aurora A (AurA) and the activation of its kinase domain (Thr288 phosphorylation) in the maintenance of glioblastoma (GBM) cells and NSCs. First, treatment of FGF1 increases AurA expression in human GBM cell lines. Second, using fluorescence-activated cell sorting, we observed that F1BGFP reporter facilitates the isolation of F1BGFP(+) GBM cells with higher expression levels of FGFR and AurA. Third, both FGFR inhibitor (SU5402) and AurA inhibitor (VX680) could down-regulate F1BGFP-dependent AurA activity. Fourth, inhibition of AurA activity by two different AurA inhibitors (VX680 and valproic acid) not only reduced neurosphere formation but also induced neuronal differentiation of F1BGFP(+) GBM cells. Fifth, flow cytometric analyses demonstrated that F1BGFP(+) GBM cells possessed different NSC cell surface markers. Finally, inhibition of AurA by VX680 reduced the neurosphere formation of different types of NSCs. Our results show that activation of AurA kinase through FGF1/FGFR signaling axis sustains the stem cell characteristics of GBM cells. Implications: This study identified a novel mechanism for the malignancy of GBM, which could be a potential therapeutic target for GBM. - Highlights: • We report that FGF1 treatment can stimulate AurA kinase expression in human GBM cells. • FGF1/FGFR signaling is involved in the activation of AurA kinase. • FGF1 sustains the self

An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding.

Science.gov (United States)

Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad

2017-09-01

The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.

The MAPKERK-1,2 pathway integrates distinct and antagonistic signals from TGF alpha and FGF7 in morphogenesis of mouse mammary epithelium

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