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We previously identified the six-transmembrane epithelial antigen of prostate (STEAP) 4 as a novel plasma membrane protein that is up-regulated in obese patients and may play a significant role in the development of human obesity. In this study, a STEAP4-specific antibody was used to characterize the biological functions of the STEAP4 protein in human adipocytes. Cell viability assays (Trypan Blue exclusion), CCK-8 assays and cell cycle analysis showed that the STEAP4 antibody inhibited pre-adipocyte proliferation. Morphological observations by electron microscopy and confocal laser microscopy, annexin V-FITC labeling, caspase-3 and caspase-8 activity assays as well as data from quantitative real-time RT-PCR (qPCR) further determined that the STEAP4 antibody could promote apoptosis in pre-adipocytes. Based on quantitative Oil Red O staining and the expression profiles of specific markers, we demonstrated that the STEAP4 antibody did not affect adipogenesis, but the 2-deoxy-d-[3H]-glucose uptake tests showed that it induced the insulin-stimulated glucose uptake in mature human adipocytes. In conclusion, our results demonstrated that the STEAP4 antibody does not influence human adipocyte differentiation, but it is likely that the STEAP4 protein regulates proliferation and apoptosis and plays an important role in modulating the insulin sensitivity of human adipocytes.

The aim of this study was to investigate whether the early endosome antigen 1 (EEA1) and/or PI3K pathway is involved in the molecular mechanisms underlying the effects of the six-transmembrane protein of prostate 4 (STEAP4; also called STAMP2 and TIARP) on the insulin sensitivity of human adipocytes. Our data demonstrated that siRNA-mediated STEAP4 deficiency significantly decreased insulin-stimulated glucose transport in mature human adipocytes by decreasing GLUT4 translocation to the plasma membrane through attenuated Akt phosphorylation. We further found that EEA1 may not be involved in the mechanisms underlying the effects of STEAP4 on insulin-stimulated glucose uptake and GLUT4 translocation, as indicated by the results that i) STEAP4 does not alter the effects of EEA1 on insulin-stimulated glucose uptake and GLUT4 translocation; ii) STEAP4 does not modify the expression of EEA1 protein; and iii) STEAP4 does not interact with EEA1 according to FRET analysis. In conclusion, this study revealed that the knockdown of STEAP4 inhibits insulin-stimulated glucose transport and GLUT4 translocation via the attenuated phosphorylation of Akt, independent of the effects of EEA1.

To explore the effect of LYRM1 over-expression on basal and insulin-stimulated glucose uptake in rat skeletal muscle cells, and to understand the underlying mechanisms, Rat myoblasts (L6) transfected with either an empty expression vector (pcDNA3.1Myc/His B) or a LYRM1 expression vector were differentiated into myotubes. Glucose uptake was determined by measuring 2-deoxy-D-[(3)H] glucose uptake into L6 myotubes. Western blotting was performed to assess the translocation of insulin-sensitive glucose transporter 4 (GLUT4). It was also used to measure the phosphorylation and total protein contents of insulin-signaling proteins, such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase (PI3K) p85, Akt, ERK1/2, P38, and JNK. LYRM1 over-expression in L6 myotubes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1, PI3K (p85), and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, P38, and JNK. LYRM1 regulates the function of IRS-1, PI3K, and Akt, and decreases GLUT4 translocation and glucose uptake in response to insulin. These observations highlight the potential role of LYRM1 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity.

NYGGF4 is a recently discovered gene that is involved in obesity-associated insulin resistance. It has been suggested that mitochondrial dysfunction might be responsible for the development of insulin resistance induced by NYGGF4 overexpression. In the present study, we aimed to define the impact of down-regulating NYGGF4 expression by RNA interference (RNAi) on the insulin sensitivity and mitochondrial function of 3T3-L1 adipocytes. The results revealed that NYGGF4 knockdown enhanced the glucose uptake of adipocytes, which reconfirmed the regulatory function of NYGGF4 in adipocyte insulin sensitivity. However, an unexpected observation was that knockdown of NYGGF4 reduced intracellular ATP concentration and promoted an increase in mitochondrial transmembrane potential (ΔΨm) and reactive oxygen species (ROS) level without affecting mitochondrial morphology or mtDNA. Therefore, the role of NYGGF4 in mitochondrial function remains unclear, and further animal studies are needed to explore the biological function of this gene.

Resveratrol, a member of a class of polyphenolic compounds known as flavonols, has been extensively studied for its anticancer, antiviral, anti-inflammatory, and neuroprotective roles. Caenorhabidits elegans is a well-established animal for investigating responses to radiation. We found that resveratrol may provide protection against hazardous radiation. Pre-treatment with resveratrol extended both the maximum and mean life span of irradiated C. elegans. Resveratrol acted as a strong radical scavenger and regulated superoxide dismutase (SOD) expression. In addition, resveratrol was shown to be capable of alleviating gamma-ray radiation exposure-induced reduction in mitochondrial SOD expression. Ultimately, a correlation may exist between dietary intake of trace amounts of resveratrol and anti-aging effects. A specific response mechanism may be activated after the administration of resveratrol in irradiated animals. Our results suggest the protective effect of resveratrol is due to its strong ability to protect from oxidative stress and protective effects in mitochondria. Therefore, resveratrol is potentially an effective protecting agent against irradiative damage.

JUSTIFICATIVA E OBJETIVOS: Os anestésicos locais são amplamente utilizados na prevenção ou na reversão de dor aguda e no tratamento de dor crônica. A reação de cardiotoxicidade induzida pelos anestésicos locais é um evento acidental sem terapia farmacológica, exceto a infusão de intralípides relatados recentemente cujo mecanismo de ação ainda não é bem compreendido. CONTEÚDO: A cardiolipina, um fosfolipídio aniônico, desempenha papel relevante na determinação de reação respiratória mitocondrial, metabolismo de ácidos graxos e apoptose celular. A disfunção do metabolismo energético mitocondrial é sugerida em associação com a cardiotoxicidade dos anestésicos locais, a partir de um estudo in vitro de que ela talvez se deva a fortes ligações eletrostáticas entre os anestésicos locais e a cardiolipina na membrana mitocondrial. Não há, contudo, evidência experimental. Portanto, levantamos a hipótese de que as interações anestésico-cardiolipina sejam o principal determinante associado à reação de cardiotoxicidade, o que pode ser estabelecido com a adoção de métodos teóricos e biológicos estruturais. Esse modelo de interação nos daria uma pista sobre o mecanismo da cardiotoxicidade dos anestésicos locais, visando a futuras pesquisas na área de desenvolvimento de fármacos de prevenção a esse evento na prática clínica. CONCLUSÕES: A interação entre a cardiolipina mitocondrial e os anestésicos locais pode ser a principal fonte de sua cardiotoxicidade, em função de seus efeitos sobre o metabolismo energético e o estado eletrostático. BACKGROUND AND OBJECTIVES: Local anesthetics are used broadly to prevent or reverse acute pain and treat symptoms of chronic pain. Local anesthetic-induced cardiotoxic reaction has been considered the accidental event without currently effective therapeutic drugs except for recently reported intralipid infusion whose possible mechanism of action is not well known. CONTENTS: Cardiolipin, an anionic phospholipid, plays a key role in determining mitochondrial respiratory reaction, fatty acid metabolism and cellular apoptosis. Mitochondrial energy metabolism dysfunction is suggested as associated with local anesthetic cardiotoxicity, from an in vitro study report that the local anesthetic cardiotoxicity may be due to the strong electrostatic interaction of local anesthetics and cardiolipin in the mitochondria membrane, although there is a lack for experimental evidence. Herein we hypothesized that local anesthetic-cardiolipin interactions were the major determinant of local anesthetic-associated cardiotoxic reaction, established by means of theoretic and structural biological methods. This interacting model would give an insight on the underlying mechanism of local anesthetic cardiotoxicity and provide clues for further in depth research on designing preventive drugs for such inadvertent accidence in routine clinical practice. CONCLUSIONS: The interaction between local anesthetic and mitochondrial cardiolipin may be the underlying mechanism for cardiotoxicity affecting its energy metabolism and electrostatic status.

Intracellular signalling transduction plays a pivotal role in cell activation,and the regulation of gene transcription needs the involvement of series of nuclear factors. Nuclear transcription factor- kappa B, one of the key factors in mediating gene expression, takes an essential part in a body of physiological and pathological processes. Endotoxin lipopolysaccharides, proinflammatory cytokine tumor necrosis factor-alpha, liganddependent immunocellular B cell receptor and inflammation-associated bradykinin are four typical NF-κB activation pathways. Activating or inhibitory drugs focused on NF-κB maybe strongly related with the therapy of associated diseases, and the exploration of effective and economical drugs concentrated on NF-κB is crucial.KEY WORDS: Nuclear factor-κB; Inhibitor factor-κB; Inflammatory signalling; Drug discovery

Obesity, which is caused by energy uptake being greater than energy expenditure, is widely prevalent today. Currently, only a limited number of efficient interventional strategies are available for the prevention of obesity. Previous studies have shown that UCP4 transcription occurs at a considerable level in mouse skeletal muscle; however, the exact functions of UCP4 remain unclear. In this study, we investigated the effect of UCP4 on mitochondrial function and insulin sensitivity in mature L6 myocytes. UCP4 overexpression in L6 myocytes induced increased mitochondrial carnitine palmitoyltransferase 1A (CPT1A) and decreased citrate synthase (CS) mRNA in the basal condition (i.e., in the absence of insulin). UCP4 overexpression significantly improved insulin sensitivity, increased tyrosine phosphorylation of IRS-1 in the presence of insulin, and significantly reduced intracellular triglyceride (TG). Additionally, intracellular ATP content and mitochondrial membrane potential were downregulated. We also observed that intracellular ROS, mitochondrial morphology, and mitochondrial mtDNA copy number were maintained upon UCP4 expression, with no change in mitochondrial fusion and fission. In summary, our findings provide evidence to show that UCP4 overexpression reduced the insulin sensitivity and mitochondrial fatty acid oxidation of L6 myocytes. These findings support the notion that UCPs are ideal targets for treatment of insulin resistance.

Identifying infants that will develop significant hyperbilirubinemia with the risk of kernicterus, and planning appropriate follow-up strategies, is particularly challenging. In this study, 36,921 transcutaneous bilirubin (TcB) measurements were obtained from 6,035 healthy neonates (gestational age ≥ 35 weeks and birth weight ≥ 2,000 g) between January 1 and December 31, 2009. All measurements were performed with the JM-103 bilirubinometer at designated times between 0 and 168 postnatal hours. TcB percentiles were calculated and used to develop an hour-specific nomogram. The rate of increase in TcB was higher during the first 72 h of age, after which levels declined to a plateau by 72-108 h of age. We constructed a TcB nomogram by using the 40th, 75th, and 95th percentile values of TcB for every 12 h of the studied interval. The 75th percentile curve of the nomogram may be an ideal cutoff point for intensive follow-up of the neonate for hyperbilirubinemia as it carries very high sensitivity (78.7%) and negative predictive value (98.5%). The specificity (45.7%) and positive predictive value (15.5%) decreased to reach their lowest levels at the 40th percentile. Of the neonates in the high-risk zone, 167 (48.8%) infants had persistent subsequent hyperbilirubinemia post-discharge, compared with 292 (27.0%) infants in the high-intermediate-risk zone at discharge. One-hundred and seventeen (5.5%) infants in the low-intermediate-risk zone moved into the high-risk zone during follow-up. No newborn infants in the low-risk zone became high-risk during follow-up. We provide an hour-specific TcB nomogram to predict neonatal hyperbilirubinemia in healthy term and late-preterm Chinese infants.

TNF-alpha was the first proinflammatory cytokine identified linking obesity, insulin resistance and chronic inflammation. However, the mechanism of TNF-alpha in the etiology of insulin resistance is still far from clear. Because the mitochondria play an important role in energy metabolism, we investigated whether mitochondrial dysfunction is involved in pathogenesis of TNF-alpha-mediated insulin resistance. First, a fully differentiated insulin-resistant 3T3-L1 adipocyte model was established by incubating with 4 ng/ml TNF-alpha for 4 d, and then the mitochondrial morphology and functions were observed. TNF-alpha treatment induced pronounced morphological changes in the mitochondria, which became smaller and condensed, and some appeared hollow and absent of cristae. Mitochondrial dynamics changes were observed as increased mitofusion protein mfn1 and mitofission protein Drp1 levels compared with controls. No obvious effects on mitochondrial biogenesis were found. PGC-1alpha levels decreased, but no significant changes were found in mtTFA mRNA expression, NRF1mRNA expression and mitochondrial DNA (mtDNA). TNFalpha treatment also led to decreased mitochondrial membrane potential and reduced production of intracellular ATP, as well as accumulation of significant amounts of reactive oxygen species (ROS). Further research is required to determine if mitochondrial dysfunction is involved in the inflammatory mechanism of insulin resistance and may be a potential target for the treatment of insulin resistance.

To observe the changes of NYGGF4 gene expression during human preadipocyte differentiation and to explore the regulative role of tumor necrosis factor alpha (TNFalpha) on NYGGF4 gene expression in matured human adipocytes.Human preadipocytes-visceral (HPA-v) were cultured in vitro and differentiated into the matured adipocytes. Fully differentiated adipocytes were treated with TNFalpha in different concentrations for 16 hours or at 10 ng/mL for various times. Levels of the NYGGF4 mRNA were evaluated by real-time reverse transcription-PCR(RT-PCR).(1) After induction of differentiation, more than 90% of the HPAs-v exhibited typical adipocyte morphology on the 17th day. (2) In human preadipocytes, the level of NYGGF4 mRNA expression remained low. The NYGGF4 mRNA level was gradually increased with the preadipocytes being differentiated into the matured adipocytes, and reached the highest level in the fully differentiated adipocytes. (3) Treatment of the human matured adipocytes with TNFalpha resulted in a significant increase in the level of NYGGF4 mRNA. The up-regulatory effect of TNFalpha on the NYGGF4 gene expression tended to be enhanced with the increasing concentrations and elongation of time.During human preadipocytes differentiation, the level of NYGGF4 gene mRNA expression increases gradually. TNFalpha could upregulate the expression of NYGGF4 mRNA in human matured adipocytes. The regulatory effect of TNFalpha on the NYGGF4 gene expression is of dose-dependent and time-correlated.

Uncoupling proteins (UCPs) located in the inner mitochondrial membrane are involved in the regulation of energy balance. Thus far, 5 UCP isoforms have been identified, but controversies exist in the research focused on the function of the UCPs (except UCP1) in the pathogenesis of obesity. Because of the known cross-reactivity of the antibodies presently available for the detection of UCP proteins, this study systematically analyzed the differential tissue expression profiles of the 5 UCP isoforms in lean control mice and ob/ob mice by using real-time polymerase chain reaction (PCR) analysis. The results show that the tissue-specific expression patterns of individual isoforms in normal and ob/ob mice are considerably different; this will provide new insights into the functions of UCPs in the pathogenesis of genetic obesity.

We previously identified a six-transmembrane epithelial antigen of the prostate (STEAP) 4 as a novel plasma membrane protein that is downregulated in obese patients and may play a significant role in the development of human obesity. This study was designed to identify the biological characteristics of the STEAP4 gene in human adipocytes. On the basis of oil red O staining and the expression profiles of specific markers, we demonstrated that overexpression of STEAP4 did not affect adipogenesis. 2-Deoxy-D-[3H]-glucose uptake tests showed that STEAP4 promoted insulin-stimulated glucose uptake in mature human adipocytes. Further data from quantitative real-time RT-PCR and Western blotting revealed that the adipokines tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), and leptin, which have been implicated in insulin sensitivity regulation, regulate the expression of STEAP4. Our results demonstrate that STEAP4 does not influence human adipocyte differentiation, but it participates in regulating the insulin sensitivity of human adipocytes.

The objective of this study was to investigate the effects of vitrification on the preimplantation developmental competence of mouse 2-cell, 4-cell and 8-cell stage embryos.Mouse 2-cell, 4-cell and 8-cell stage embryos were cryopreserved using the cryotop vitrification method and subsequently warmed on a later date. The embryos were then assessed by their morphology, blastocyst formation and hatching rates. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in hatched blastocysts from the control and experimental groups.Vitrified embryos at the 2-cell, 4-cell and 8-cell stages appeared morphologically normal after warming. The overall survival rate of vitrified embryos at various stages after warming was 96.7% and there were no significant differences among 2-cell stage (96.0%), 4-cell stage (96.8%) and 8-cell stage (97.1%) embryos (P0.05). The blastocyst formation rate (69.4%) and hatching rate (52.6%) of vitrified 2-cell embryos were significantly lower than that from the control group and vitrified 8-cell embryos (P0.05). In the vitrified 4-cell embryo group, the blastocyst formation rate (90.3%) was similar to the 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of the non-vitrified control ( 84.1%) and vitrified 8-cell embryo (78.4%) groups (P0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell and 8-cell embryos had significantly lower cell counts both in the ICM and TE, as compared to fresh blastocysts (P0.05). Among the vitrified 2-cell, 4-cell and 8-cell embryo groups, there were no significant differences in the cell counts of ICM and TE (P0.05).Although cryotop vitrification was suitable for the cryopreservation of mouse embryos from the 2-cell stage, 4-cell stage and 8-cell stage without significant loss of survival, vitrification had an adverse effect on the development of 2-cell embryos. Mouse embryos at the 8-cell stage had the best tolerance for vitrification and would yield the highest level of post-vitrification developmental competence among early cleavage stage embryos. Nevertheless, it is unclear how these findings can be extrapolated to human embryos.

Uncoupling proteins (UCPs) belong to a superfamily of mitochondrial transporters that uncouple ATP synthesis from electron transport. We have previously shown that uncoupling protein 4 (UCP4) is differentially expressed in omental adipose tissue in diet-induced obese and normal rats. Overexpression of UCP4 promotes proliferation and inhibits apoptosis and differentiation of preadipocytes. In this work, we further characterized the effect of UCP4 on mitochondrial function in mature 3T3-L1 adipocytes. Transmission electron microscopy (TEM) showed that adipocytes overexpressing UCP4 displayed condensed mitochondria with twisted, condensed, and unclear cristae. Moreover, the loss of the mitochondrial membrane potential and intramitochondrial calcium was found. The adipocytes overexpressing UCP4 also showed decreased mitochondrial copy number (mtDNA) and lower mRNA expression of key factors in mitochondrial biogenesis, including PGC-1alpha and mtTFA. NRF-1 and ERRbeta levels were down-regulated, while NRF-2 levels were upregulated. In addition, UCP4 overexpression impaired mitochondrial fusion and fission, as indicated by decreased mitofusin mfn1, mfn2, and mitofission DRP1. When it came to total adipocytes, the UCP4 overexpressing adipocytes showed higher production reactive oxygen species and diminished levels of intracellular ATP. Furthermore, overexpression of UCP4 brought about impaired insulin sensitivity in adipocytes. UCP4 plays an important role in mitochondrial function and adipocyte insulin resistance. Its function deserves further attention.

Purpose: This randomized, controlled trial was designed to determine whether the DNA cytometry testing is superior to the conventional cytologic testing for mass cervical cancer screening. Experimental Design: After approval by the institutional ethics review boards from three separate screening centers, a total of 23,993 Chinese women ages 20 to 65 years were randomly assigned into one of the two groups: a DNA cytometry testing group (11,999 women) and a cytologic testing group (11,994 women). Each woman underwent the other testing after first attending the assigned screening test. Women with positive results after assigned testing additionally underwent colposcopy and human papillomaviruses detections, and those with cervical precancerous or cancerous lesions received appropriate treatment. Sensitivity and specificity estimates were adjusted for verification bias. Analyses were by intention to treat and per protocol ways. Results: In the cytometric DNA testing group, cervical cancer was diagnosed in 40 subjects, compared with 24 subjects in the cytologic testing group [hazard ratio for the detection of advanced cancer in the DNA cytometry testing group, 0.42; 95% confidence interval (CI), 0.27-0.60]. The sensitivity of the DNA cytometry testing for cervical cancer was 91.7% (95% CI, 64.3-95.8), whereas the sensitivity of cytologic testing was 44.5% (95% CI, 25.2-61.3; P = 0.008). The specificity was 54.1% (95% CI, 31.6-69.0) for DNA cytometry testing and 70.6% (95% CI, 46.8-82.5; P = 0.003) for cytologic testing. The sensitivity of both tests used together was 100%, and the specificity was 91.8%. A total of 187 subjects reported mild to severe adverse events after treatment with positive results in 319 women. Conclusions: Our results highlight the benefit of the DNA cytometry testing strategy in mass cervical cancer screening with greater sensitivity and positive predicted value than the conventional cytologic testing in developing settings. (Clin Cancer Res 2009;15(20):6438–45)

The optimal timing of epidural analgesia has been a controversial issue, and how early women can benefit from epidural analgesia is still debated. The objective of this trial was to test the hypothesis that patient-controlled epidural analgesia given at cervical dilation of 1.0 cm or more does not increase the risk of prolonged labor or Cesarean delivery.After institutional review board approval and patient consent, 12,793 nulliparous patients requesting neuraxial analgesia were enrolled and randomized to an early epidural (cervical dilation at least 1.0 cm) or delayed epidural (cervical dilation at least 4.0 cm) group. A 15-ml epidural analgesic mixture consisting of 0.125% (1.25 mg/ml) ropivacaine plus 0.3 microg/ml sufentanil was given in a single bolus, followed by patient-controlled pump with a 10-ml bolus without background infusion. Repeatable meperidine (25 mg) was prescribed as being the rescue analgesic to patients in the delayed epidural group. The primary outcome was the rate of Cesarean section.The median diameters of cervical dilation were 1.6 cm and 5.1 cm in the early and delayed epidural groups, respectively (P0.0001). The duration of labor from analgesia request to vaginal delivery was equal in both groups (11.3 +/- 4.5 h for early epidural and 11.8 +/- 4.9 h for delayed epidural group women, P = 0.90). No statistically significant difference in the rate of Cesarean section was observed between the two groups on the intention-to-treat analysis (23.2% vs. 22.8% in the early and delayed epidural groups, respectively; P = 0.51).Epidural analgesia in the latent phase of labor at cervical dilation of 1.0 cm or more does not prolong the progression of labor and does not increase the rate of Cesarean in nulliparous women compared with the delayed analgesia at the cervical dilation of 4.0 cm or more.

NYGGF4 is a novel gene that is abundantly expressed in the adipose tissue of obese subjects and is involved in insulin resistance. In the present study, the mRNA expression of NYGGF4 homologous genes was examined in the 3T3-L1 cell line. The NYGGF4 mRNAs were expressed at low levels in the 3T3-L1 preadipocytes. During the conversion of 3T3-L1 preadipocytes to adipocytes, the expression of NYGGF4 mRNA was upregulated. On the 8th day after induction of differentiation, the NYGGF4 mRNA levels peaked and remained high. Free fatty acids (FFA) and tumor necrosis factor-α (TNFα) could upregulate NYGGF4 mRNA expression in 3T3-L1 adipocytes, while interleukin-6 (IL-6), leptin, and resistin exerted an inhibitory effect. The results suggest that the expression of NYGGF4 mRNA is affected by a variety of factors that are related to insulin sensitivity. It is likely that NYGGF4 may be an important mediator in the development of obesity-related insulin resistance.

This paper proposes a new method of automatic registration of urban remote sensing images that has combined bilinear interpolation which is a classic method of image registration and road networks which are the characters of urban remote sensing images. It comprises of extracting the road network in an urban and computing the Ground Control Points (GCP) from the road junctions, then estimating the parameters of the mapping function and transforming the sensed image at last.

Pro-inflammatory cytokine production after nociceptive stimuli is pivotal for hyperalgesia. As macrophage migration inhibitory factor (MIF), a pleiotropic cytokine produced mainly by nonneuronal tissue, has been involved in the regulation of neuronal functions, herein we examined the role for MIF in formalin-induced inflammatory pain model. MIF critically contributed to nociceptive behaviors following formalin injection. Specifically, spinal administration of a MIF inhibitor (ISO-1) prevented and reversed flinching responses in rats. Further examination showed that levels of both MIF and the MIF receptor CD74 were substantially increased within the ipsilateral spinal cord dorsal horn after formalin administration. Mechanistic studies revealed that MIF upregulated the expression of the spinal NMDA receptor subunit NR2B via the MAPK signaling pathway. Moreover, microglial cells were found to be the major source of spinal MIF after formalin administration by fluorescence colocalization. These data highlight spinal MIF plays a critical role in the pathogenesis of formalin-induced inflammatory pain and suggest MIF may be a potential target for therapy of such pathological condition.

Background Attention-deficit/hyperactivity disorder (ADHD) is a pervasive neurobehavioral disorder affecting approximately 5% of children and adolescents and 3% of adults, and the prefrontal cortex (PFC) may play the most critical role in the expression of ADHD. Converging previous studies indicate a potential role of Homer — a scaffolding protein family localized at the postsynaptic density (PSD) of glutamatergic excitatory synapses — in behavioral pathologies associated with neuropsychiatric disorders. Accordingly, we speculate that these Homer isoforms might contribute to the etiology and development of ADHD. Method We investigated the differential mRNA and protein expressions of several Homer isoforms in the PFC of the spontaneous hypertensive rat/Wistar–Kyoto rats (SHR/WKY), the most frequently used animal model of ADHD, using RT-PCR and western blotting. Furthermore, we examined the effects of methylphenidate (MPH) exposure on the behaviors and the expression of different Homer isoforms in the PFC of SHR, using Lat maze, RT-PCR and western blotting, respectively. Results Homer 1a and Homer 2a / b , but not Homer 1b / c , were expressed at a significantly lower levels in the PFC of SHR compared with WKY. MPH exposure decreased the locomotor activity and non-selective attention of SHR, and it up regulated the expression of Homer 1a and Homer 2a / b , but not Homer 1b / c , in the PFC of SHR. Conclusion It is plausible that Homer 1a and Homer 2a / b may be involved in the etiology and pathogenesis of ADHD. Future work will focus on elucidating the specific mechanisms of Homer 1a and Homer 2a / b in ADHD.

The differentiation of pancreatic ductal epithelial cells into beta-cells has been considered as an alternative method for increasing the number of islets for transplantation. Critical factors have been introduced into the in vitro differentiation protocol for pancreatic duct cells in order to enhance the production of beta-cells. Islet neogenesis-associated protein (INGAP) is an initiator of islet neogenesis and the peptide sequence 104-118 of INGAP has been shown to stimulate an increase in beta-cell mass in animals and also found in human pathological states involving islet neogenesis. To establish a novel method for the differentiation of beta-cells from human pancreatic duct cells with INGAP-related pentadecapeptide (INGAP-PP), the pancreatic duct cells were isolated, purified and expanded in vitro and differentiated using a four-step protocol that included nicotinamide, exendin-4, transforming growth factor beta(1) and INGAP-PP/Scrambled peptide (Scrambled-P). The production of islet-like clusters (ILCs) in the INGAP-PP group was significantly higher than that in the Scrambled-P control group after differentiation from an equal number of duct cells. The duct cells showed positive staining and expression for cytokeratin 19, pancreatic duodenal homeobox-1, nestin, and were negative for insulin and glucagon, as detected by both immunofluorescence and RT-PCR. Following differentiation the cells became insulin and glucagon positive. In addition, the ILCs from the INGAP-PP group secreted higher levels of insulin and C-peptide than the Scrambled-P group under a high glucose challenge. We conclude that INGAP peptide enhances the in vitro differentiation of pancreatic duct cells into islet-like clusters.

A growing body of evidence suggests that reactive oxygen species (ROS) play an important role in human cancers. Manganese superoxide dismutase (MnSOD) is the major antioxidant in the mitochondria, catalysing the dismutation of superoxide radicals to form hydrogen peroxide. Since the identification of a well-characterised functional polymorphism, Val-9Ala of MnSOD, a number of molecular epidemiological studies have evaluated the association between Val-9Ala and cancer risk. However, the results remain conflicting rather than conclusive. This meta-analysis on 15,320 cancer cases and 19,534 controls from 34 published case-control studies shows no significant overall main effect of MnSOD Val-9Ala on cancer risk. However, we found that the MnSOD 9Ala allele was associated with an increased prostate cancer risk (Val/Ala versus Val/Val: odds ratio (OR)=1.1; 95% confidence intervals (CI): 1.0-1.3; Ala/Ala versus Val/Val: OR=1.3; 95% CI: 1.0-1.6; Val/Ala+Ala/Ala versus Val/Val: OR=1.2; 95% CI, 1.0-1.3). In addition, we found that the MnSOD Ala-9Ala genotype contributed to an increased breast cancer risk in premenopausal women who had low consumption of antioxidants (Ala/Ala versus Val/Ala+Val/Val: OR=2.6, 95% CI: 1.0-6.4 with low vitamin C consumption; OR=2.1, 95%CI: 1.3-3.4 with low vitamin E consumption and OR=2.9, 95%CI: 1.5-5.7 with low carotenoid consumption). These results suggest that the MnSOD Val-9Ala polymorphism may contribute to cancer development through a disturbed antioxidant balance.

ObjectiveTo characterize a novel gene, Homo sapiens LYR motif containing 1 (LYRM1), that is highly expressed in omental adipose tissue of obese subjects.Methods and resultsRT-PCR and western blot analysis confirmed that both mRNA and protein levels of LYRM1 were higher in omental adipose tissue of obese subjects than in normal weight subjects. RT-PCR analysis demonstrated that LYRM1 expression is widely distributed, with the highest levels of expression occurring in adipose tissue. A fusion protein of LYRM1 and green fluorescent protein as well as western blot analysis were used to identify the subcellular localization of LYRM1 in the nucleus. Based on Oil red O staining and the expression profile of specific differentiation markers, ectopic LYRM1 expression was not found to significantly affect adipogenesis. MTT assays and cell cycle analysis showed that LYRM1 promotes preadipocyte proliferation, and data from annexin V-FITC and caspase-3 activity assays further determined that LYRM1 can inhibit apoptosis of preadipocytes.ConclusionsBy increasing cell proliferation and lowering the rate of apoptosis, LYRM1 has the potential to modulate the size of the preadipocyte pool and influence adipose tissue homeostasis.

Beta-cell apoptosis induced by adipokines may result in beta-cell dysfunction in type 2 diabetes. Resistin, an adipokine-linked obesity with type 2 diabetes, impairs glucose-stimulated insulin secretion (GSIS) in beta-cells. Presently, the effects of resistin on rat insulinoma cells RINm5F were examined. Treatment of RINm5F with resistin induced cell damage. Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) protected resistin-mediated cytotoxicity in RINm5F. Incubation with resistin up-regulated caspase-3 activity and induced the formation of a DNA ladder. TIMP-1 attenuated these effects. The molecular mechanism of TIMP-1 inhibition of resistin-mediated cytotoxicity appeared to involve Akt phosphorylation and activation of IkB-alpha phosphorylation. Resistin treatment suppressed Akt phosphorylation and activated IkB-alpha phosphorylation, which could be attenuated by TIMP-1. We conclude that resistin can induce beta-cell apoptosis and that resistin-related beta-cell apoptosis can be prevented by TIMP-1.

To investigate the gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides (ODNs), Y5 receptor antisense, mismatched ODNs or vehicle was intracerebroventricularly injected and cDNA microarrays were undertaken. Central administration of NPY-Y5 receptor antisense ODNs decreased food intake, body weight and serum insulin compared with both vehicle and mismatched ODNs. The average area of adipocytes both at retroperitoneal and epididymal adipose tissue were fall in antisense group while only the weight of the retroperitoneal fat pats was reduced in antisense group. cDNA microarrays containing 18,000 genes/Ests were used to investigate gene expression of adipose tissue. Autoradiographic analysis showed that 404, 81, and 34 genes were differently expressed over twofold, threefold, and fivefold, respectively. The analysis of gene expression profiles indicated that 332 genes were up-regulated and 187 genes were down-regulated in response to Y5 receptor antisense ODNs treatment. Different clusters of genes associated with apoptosis, signal transduction, energy metabolism, lipid metabolism, etc., such as FXR1, PHLDA1, MAEA, PIK3R1, ICAM2, PITPN, CALM2, CAMK2D, PKIA, DRD2, SLC25A14, CKB, AADAC, LIPA, ACOX3, FADS1, were concerned. Analysis of differentially expressed genes will help to understand the effects of Y5 receptor antisense ODNs therapy.

Background: While early kidney development has been studied exhaustively, the later stages of nephrogenesis that occur after birth in the rodent are relatively poorly understood. To gain insight into this process, we detected the alterations in gene expression in rat kidney at two postnatal stages, P0 (0 day after birth), the time at which nephrogensis is still active, and P21 (21 days after birth), when nephrogenesis is complete. Methods: Sprague-Dawley rats were mated, and appearance of a vaginal plug was designated as E0. Kidneys were dissected from embryos at E13, E15, E17 and E19, and from postnatal days P0, P7, P14, P21 and adult rats. Suppression subtractive hybridization (SSH) analysis was performed and highly expressed genes were evaluated as molecular markers by real-time reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immunofluorescence, and Western blot. Results: Several differentially expressed genes were identified, including rPEA3, a member of the PEA3 subfamily of Ets domain transcription factors. Real time RT-PCR analysis revealed that rPEA3 exhibited dynamic developmental regulation, with high levels of expression from embryonic day E15 until birth, and declining levels thereafter. By in situ hybridization, rPEA3 mRNA was detected in the ureteric bud (UB) and surrounding metanephric mesenchyme of the kidneys from E15 until birth, but was undetectable in mature kidneys. Double-immunofluorescence staining showed that both rPEA3 and WT1 expressed in the condensed mesenchymal cells at E15 and E17; and later from E19 to P7, both expressed in the epithelial cells of ureteric bud and their branches. Conclusions: These studies provide compelling evidence that SSH is an effective method for identifying genes that are regulated during renal development, and suggest that the newly identified gene rPEA3 may play an important role in kidney development and differentiation.

Aim: To assess the effects and mechanisms of the action of resistin on basal and insulin-stimulated glucose uptake in rat skeletal muscle cells. Methods: Rat myo-blasts (L6) were cultured and differentiated into myotubes followed by stimulation with single commercial resistin (130 ng/mL, 0-24 h) or cultured supernatant from 293-T cells transfected with resistin-expressing vectors (130 ng/mL, 0-24 h). Liquid scintillation counting was used to quantitate [ 3 H] 2-deoxyglucose uptake. The translocation of insulin-sensitive glucose transporters GLUT4 and GLUT1, synaptosomal-associated protein 23 (SNAP23) and GLUT protein content, as well as the tyrosine phosphorylation status and protein content of insulin receptor substrate (IRS) -1, were assessed by Western blotting. Results: Treatment of L6 myotubes with single resistin or cultured supernatant containing recombinant resistin reduced basal and insulin-stimulated 2-deoxyglucose uptake and impaired insulin-stimulated GLUT4 translocation. While SNAP23 protein content was decreased, no effects were noted in GLUT4 or GLUT1 protein content. Resistin also diminished insulin-stimulated IRS-1 tyrosine phosphorylation levels without affecting its protein content. The effects of recombinant resistin from 293-T cells transfected with resistin-expressing vectors were greater than that of single resistin treatment. Conclusion: Resistin regulated IRS-1 function and decreased GLUT4 translocation and glucose uptake in response to insulin. The downregulated expression of SNAP23 may have been partly attributed to the decrease of glucose uptake by resistin treatment. These observations highlight the potential role of resistin in the pathophysiology of type 2 diabetes related to obesity.

A novel gene named NYGGF4, which was expressed at a higher level in obese subjects, was isolated and characterized. It is a 1527-bp cDNA, containing 753 nucleotides of an ORF (open reading frame) predicting 250 amino acids with a molecular mass of 28.27 kDa. Amino acid sequence analysis revealed NYGGF4 has a phosphotyrosine-binding (PTB) domain. Northern blot analysis revealed NYGGF4 is expressed primarily in adipose tissue, heart, and skeletal muscle but not in any other tissue examined. Confocal imagery analyses with green fluorescent protein-tagged protein transiently expressed in 3T3-L1 preadipocytes and 293-T cells show that NYGGF4 localizes in the cytoplasm. Furthermore, ectopic expression of NYGGF4 dramatically increases the proliferation of 3T3-L1 peadipocytes without affecting adipocytic differentiation. And the NYGGF4 expression 3T3-L1 cells had a greater number of cells in S-phase. Our data suggest that NYGGF4 might be a signaling molecule and could play a role in cell growth and adipogenesis process.

OBJECTIVE: To investigate the relationship between resistin (a potential link between obesity and type 2 diabetes) and preadipocyte differentiation. DESIGN: A rat resistin expression vector was transfected into 3T3-L1 preadipocytes and differentiation was compared between normal 3T3-L1 cells, rat resistin-transfected cells and non-transfected cells grown in conditioned medium taken from resistin-expressing cultures. METHODS: The rat resistin gene was inserted into the pDual GC and pEFGP-N2 expression vectors for examination of the effects of resistin overexpression in 3T3-L1 cells before and after differentiation was stimulated with 3-isobutyl-1-methyxanthine (MIX), insulin and dexamethasone (DEX). Smaller conserved fragments were inserted into short interference RNA (siRNA) expression vectors, for examination of the effect of targeted resistin inhibition on differentiation of resistin-overexpressing 3T3-L1 cells. RESULTS: Prior to stimulation, the resistin-transfected 3T3-L1 cells contained many more small lipid droplets than did non-transfected 3T3-L1 cells. Following stimulation, differentiation in the resistin-transfected 3T3-L1 cells was dramatically promoted, especially in the early stages. Stimulation of differentiation was also observed in non-transfected 3T3-L1 cells grown in resistin protein-containing conditioned medium. The expression of adipocyte differentiation-associated markers such as CCAAT enhancer binding protein (C/EBPalpha), retinoid X receptor (RXRalpha) and lipoprotein lipase (LPL) was upregulated in resistin-overexpressing cells, whereas expression of preadipocyte factor-1 (Pref-1), an inhibitor of preadipocyte differentiation, was downregulated. In addition, expression of two of the three tested siRNAs inhibited the adipoconversion process, providing further evidence that resistin promotes the differentiation of preadipocytes to adipocytes. CONCLUSION: Resistin can promote preadipocyte differentiation. Based on this, we propose that resistin may be an important candidate mediator of obesity-induced insulin resistance.

In their Article on the epidemiology of multimorbidity in Scotland (July 7, p 37), Karen Barnett and colleagues do not stratify the population studied according to ethnic group. We think that there could be important diff erences between such groups. Additionally, Barnett and col leagues analysed 40 long-term disorders, but they did not include obesity. Did the dataset from which Barnett and colleagues retrieved information for analysis contain basic demographic information such as weight and height? If it did, why did Barnett and colleagues not calculate body-mass index as an indicator of obesity and include it in their list of diseases? Given the increasing prevalence of obesity worldwide, we strongly believe it is a serious health problem in itself and contributes to the morbidity of other diseases. Finally, Barnett and colleagues showed that women had higher rates of multimorbidity than did men. This ratio might be artifi cially skewed if gynaecological diseases were included. So it would be better for physicians and policy makers if the results were analysed for men and women separately. Therefore, we propose that future studies should focus on a more complete list of diseases and stratify by race and sex.

This study assessed the growth trends and reference ranges of the ultrasound parameters, fetal abdominal subcutaneous tissue thickness (ASTT) and subscapular subcutaneous tissue thickness (SSTT), in the last two trimesters of normal pregnancy in a Chinese population. We recruited 744 healthy women with singleton pregnancies. The ASTT and SSTT were evaluated at different times between 21 and 36 weeks of gestation. The correlations between these parameters and fetal gestational weeks were assessed using linear regression analysis. Both ASTT and SSTT increased with gestation, and both parameters showed a strong correlation with gestation (ASTT vs. GA, R(2) = 0.792; P

RING box protein-1 (RBX1) is an essential component of Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase and participates in diverse cellular processes by targeting various substrates for degradation. However, the physiological function of RBX1 in mouse oocyte maturation remains unknown. Here, we examined the expression, localization and function of RBX1 during mouse oocyte meiotic maturation. Immunofluorescence analysis showed that RBX1 displayed dynamic distribution during the maturation process: it localized around and migrated along with the spindle and condensed chromosomes. Rbx1 knockdown with the appropriate siRNAs led to a decreased rate of first polar body extrusion and most oocytes were arrested at metaphase I. Moreover, downregulation of Rbx1 caused accumulation of Emi1, an inhibitor of the anaphase-promoting complex/cyclosome (APC/C), which is required for mouse meiotic maturation. In addition, we found apparently increased expression of the homologue disjunction-associated protein securin and cyclin B1, which are substrates of APC/C E3 ligase and need to be degraded for meiotic progression. These results indicate the essential role of the SCF(βTrCP)-EMI1-APC/C axis in mouse oocyte meiotic maturation. In conclusion, we provide evidence for the indispensable role of RBX1 in mouse oocyte meiotic maturation.

Etminan and colleagues reported on an interesting study in which they found merely a little association between the use of oral contraceptives and gallbladder disease.[1][1] This resembled the results of our recently completed observational study (unpublished data: 2011) in which the cumulative

AIM: To confirm whether insulin regulates resistin expression and secretion during differentiation of 3T3-L1 preadipocytes and the relationship of resistin with insulin resistance both in vivo and in vitro. METHODS: Supernatant resistin was measured during differentiation of 3T3-L1 preadipocytes. L6 rat myoblasts and hepatoma cell line H4IIE were used to confirm the cellular function of resistin. Diet-induced obese rats were used as an insulin resistance model to study the relationship of resistin with insulin resistance. RESULTS: Resistin expression and secretion were enhanced during differentiation 3T3-L1 preadipocytes. This cellular differentiation stimulated resistin expression and secretion, but was suppressed by insulin. Resistin also induced insulin resistance in H4IIE hepatocytes and L6 myoblasts. In diet-induced obese rats, serum resistin levels were negatively correlated with insulin sensitivity, but not with serum insulin. CONCLUSION: Insulin can inhibit resistin expression and secretion in vitro, but insulin is not a major regulator of resistin in vivo. Fat tissue mass affects insulin sensitivity by altering the expression and secretion of resistin.